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About the cover: Mitotic PtK2 cellsin late anaphasestainedblue for DNA and greenfor tubulin. Courtesyof Torsten\gittman. Library of CongressCataloging-in-PublicationData Molecular cell biology I Harvey Lodish . . . [et al.]. -6th ed. p. cm. Includesbibliographicalreferencesand index. 1. Cytology. 2. Molecular biology. I. Lodish, Harvey F. QH581.2.M6552007 57L5-dc22 2007006188 ISBN-13: 97 8-0-7167-7601-7 ISBN-10:0-7167-7 601-4 @ 1986,1.990,1995,2000,2004,2008 by tJf.H. Freemanand Company All rights reserved. Printed in the United Statesof America Secondprinting W. H. Freemanand Company 41 Madison Avenue,New York, NY 10010 Houndmills, Basingstoke RG21 6XS, England

www.whfreeman.com

To our studentsand to our teachers, from whom we continueto learn, and to our families,for their support, and love encouragement,

PREFACE

I n writing the sixth edition of Molecular Cell Biology I we have incorporated many of the spectacularadvances I made over the past four years in biomedical science,driven in part by new experimental technologiesthat have revolutionized many fields. High-velocity techniquesfor sequencing DNA, for example,have generatedthe completesequence of dozens of eukaryotic genomes;these in turn have led to important discoveriesabout the organization of the human genome and regulation of gene expression,as well as novel insights into the evolution of life-forms and the functions of individual members of multiprotein families. New imaging techniqueshave generated profound revelations about cell organization and movement, and new molecular structures have greatly increased our understanding of life processes such as cell-cell signaling, photosynthesis, gene transcription, and chromatin structure.

what we know. A number of experimental organisms,from yeaststo worms to mice, are used throughout so the student can seehow discoveriesmade with a "lower organism" can Iead directly to insights even about human biology and disease.This experimental approach, evident in the text itself, has also been thoroughly integrated into the pedagogical framework. For example:

New Author Team

r Updated Perspectives for the Future essays explore potential applications of future discoveriesand unanswered questions that lie ahead in research.

Two new authors have been instrumental in refocusing this book toward these exciting new developments. Anthony Bretscher of Cornell University is known for identifying and characterizing new components of the actin cytoskeleton and elucidating their biological functions in relation to cell polarity and membrane traffic. Hidde Ploegh, of the Massachusetts Institute of Technologg has made major contributions to our understanding of immune system behavior, particularly in regard to the various tactics that viruses employ to evade our immune responsesand the ways our immune systemresponds. Both authors are widely recognized for their researchas well as their classroomteachingabilities. 'We are grateful to Paul Matsudaira, Jim Darnell, Larry ZipurskS and David Baltimore for their exceptional contributions to the previous editions of Molecular Cell Biology. Much of their vision and insight is apparent at many places in this book.

Experimental Emphasis The hallmark of Molecular Cell Biology has always been the use of experiments to teach students how we have learned

r Experimental Figures lead students through important experimental results. r Classic Experiments essaysfocus on historically important and Nobel Prize-winningexperiments. r New and revised Analyze the Data problems at the end of each chapter require the student to synthesizereal experimental data to answer a seriesof questions.

showsthe locationof DNAand multiple microscopy Fluorescence et al, 2006,Science BNG Giepmans proteins withinthe samecell.lFrom 3'12:217 |

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New Discoveries, New Methodologies

Coiled-coilstalk

Motor head Microtubule

Necklinkers

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Methodological advances continue to expand and enrich our knowledge of molecular cell biology and lead to new understanding. Following are just a selection of the new experimental methodologiesand cutting-edgescienceintroduced in this edition: r Expanded coverage of proteomics, including organelle proteome profiling and advances in mass spectroscopy (Chapter 3)

I ForwardmotorbindsB-tubulin, ADP ^ releasing

p ForwardheadbindsATP

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r Expanded coverageof RNAi, including the use of shRNAs to inhibit any gene of interest in a cultured cell or organism (Chapters5, 8) r Updated discussionsof chromatin, including structure and condensation(Chapter 6), control of geneexpressionby chromatin remodeling (Chapter 71, and chromatin-remodeling proteins and tumor development(Chapter 25) r Evolution (Chapter 6)

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Conformationalchangein necklinkercausesrear head to swing forward

of chromosomes and the mitochondrion

r New molecular models, including pre-initiation complex and mediator complex (Chapter 7); annular phospholipids (Chapter 1,0);Caz* ATPase (Chapter 11); rhodopsin, transducin, and protein kinase A (Chapter 15); and myosin ATPase (Chapter 17) r Latest advancesin light and electron microscopy, including cryoelectron tomography (Chapter 9) r Reactive oxygen species(ROS) (Chapter 12)

ADB [ rue* forwardheadreleases ^ trailingheadhydrolyzesATP V and releases P.

r Role of supercomplexesin electron transport (Chapter 12) r Human epidermal growth factor receptors (HERs) and treatment of cancer (Chapter 16) r Myosin ATPasecycle (Chapter 17) r Kinesin-1MPase cycle (Chapter 18) r Use of retrovirus infection for tracing cell lineage (Chapter 21) r Axon guidance molecules (Chapter 23) r Somaticgenerearrangementin immune cells (Chapter 24) r Cancer stem cells (Chapter25) r Use of DNA (Chapter25)

Figure18-22 Kinesin-1 usesATPto "walk" downa microtubule

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PREFACE

microarray analysis in tumor typing

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For Students Companion Web Site www.whfreeman. com,/lodish5e NEW: Podcastsnarrated by the authors give students a deeperunderstanding of key figures in the text and a senseof the thrill of discovery. r NEW: Now available for your MP3 player or personal computer, more than 1,25animations and researchvideos show the dynamic nature of key cellular processesand important experimental techniques.The animations were storyboarded by the textbook authors in conjunction with BioStudio, Inc., and programmed by Sumanas,Inc. r Classic Experiment essaysfocus on classicgroundbreaking experiments and explore the investigativeprocess. r Online Quizzing is provided, including multiple-choice and short answer questions. -4), written Student SolutionsManual (ISBN:1-4292-01.27 'Wong, Richard Walker, Glenda by Brian Storrie, Eric A. Gillaspn and Jill Sible of Virginia Polytechnic Institute and State University and updated by Cindy Klevickis of James Madison University and Greg M. Kelly of the University of Western Ontario, contains complete workedout solutions to all the end-of-chapter problems in the textbook. NEW: eBook (ISBN: 1,-4292-0955-0)New to the sixth edition, this customizable eBook fully integrates the complete contents of the text and its interactive media in a format that features a variety of helpful study tools, including fulltext searching,note-taking, bookmarking, highlighting, and more. Easily accessibleon any Internet-connectedcomputer via a standard rWebbrowser, the eBook enablesstudents to take an active approach to their learning in an intuitive, easy-

to learn

For Instructors Companion Web Site www.whfreeman.com/lodish6e All the student resources,plus: r All figures and tables from the book in ipeg and layered PowerPoint formats, which instructors can edit or project section by section, allowing students to follow underlying concepts.Optimized for lecture-hall presentation' including enhancedcolors, enlarged labels, and boldface rype. r Test Bank in editable Microsoft'Word format now featuringnetu and reuisedquestionsfor every chapter.The test bank is written by Brian Storrie of the University of Arkansas for Medical Sciencesand Eric A. Wong, Richard Walker, Glenda GillaspS and Jill Sible of Virginia Polytechnic Institute and StateUniversity and revisedby Cindy Klevickis of JamesMadison University and Greg M. Kelly of the University of Ontario. r Additional Analyze the Data problems are available in PDF format. r NEW: Lecture-ready Personal ResponseSystem "clicker" questions are available as Microsoft'Word files and Microsoft PowerPoint slides. Instructor's Resource CD-ROM (ISBN: 1'-4292-0126-6) includesall the instructor's resourcesfrom the Web site' including all the illustrations from the text, animations, videos, test bank files, clicker questions, and the solutions manual files. 'l'-4292-0477-X) conOverhead tansparency Set (ISBN: for optimized text' the from key illustrations tains 250 presentation. lecture-hall

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PREFACE

'

ACKNOWLEDGMENTS In updating, revising and rewriting this book, we were given 'We invaluable help by many colleagues. thank the following people who generouslygave of their time and expertiseby making contributions to specific chapters in their areas of interest, providing us with detailed information about their courses, or by reading and commenting on one or more chapters: Steven Ackerman, (Jniuersity of Massacbusetts,Boston Richard AdIe4 IJniuersity of Michigan, Dearborn Karen Aguirre, Coastal Carolina lJniuersity Jeff Bachant, Uniuersity of California, Riuerside Kenneth Balazovich, Uniuersity of Michigan Ben A. Barres, Stanford Uniuersity Karen K. Bernd, Dauidson College Sanford Bernstein, San Diego State (Jniuersity Doug Black, Howard Hughes Medical Institute and (Jniuersity of California, Los Angeles Richard L. Blanton, North Carolina State (Jniuersny Justin Blau, New York [Jniuersity Steven Block, Stanford IJniuersity Jonathan E. Boyson, Uniuersity of Vermont Janet Braam. Rice Uniuersity Roger Bradleg Montana State Uniuersity IilTilliam S. Bradshaq Brigham Young (Jniuersity Gregory G. Brown, McGill (Jniuersity \Tilliam J. Brown, Cornell IJniuersity Max M. Burger, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland David Burgess,Boston College Robin K. Cameron, McMaster [Jniuersity \7. Zacheus Cande, (Jniuersity of California, Berkeley Steven A. Carr, Broad lnstitute of Haruard (Jniuersity and Massacbusetts Institute of Technology Alice Y. Cheung, IJniuersity of Massacbusetts, Amberst Dennis O. Clegg, Uniuersity of California, Santa Barbara Paul Clifton, Utah State IJniuersity Randy \7. Cohen, California State [Jniuersity, Northridge Richard Dickerson, (Jniuersity of California, Los Angeles Patrick J. DiMario, Louisiana State Uniuersity Santosh R. D'Mello, [Jniuersity of Texas, Dallas Chris Doe, HHMI and [Jniuersity of Oregon Robert S. Dotson, Tulane [Jniuersity 'Sfilliam Dowhan, IJniuersity of Texas-Houston Medical School Gerald B. Downes, (Jniuersity of Massachusetts,Amherst Erastus C. Dudley, Huntingdon College Susan Dutcher, V/ashington (Jniuersity School of Medicine Matt Elrod-Erickson, Middle TennesseeState Uniuersity Susan Ely, Cornell Uniuersity Charles P. Emerson Jr., Boston Biomedical ResearchInstitute Irene M. Evans, Rochester Institute of Technology James G. Evans,.Whitehead Institute Bio Imaging Center, Massachusetts Institute of Technology Marilyn Gist Farquhar, Uniuersity of California, San Diego

X

PREFACE

Xavier Fernandez-Busquets, Bioengineering Instituteof Catalonia, Uniuersitatde Barcelona,Spain TerrenceG. Frey,San Diego State Uniuersity Margaret T. Fuller,Stanford UniuersitySchoolof Medicine KendraJ. Golden, WbitmanCollege David S. Goldfarb,RochesterUniuersity Martha J. Grossel,ConnecticutCollege LawrenceI. Grossman,WayneStateUniuersitySchoolof Medicine Michael Grunstein, Uniuersityof California, Los Angeles, Schoolof Medicine Barry M. Gumbiner, Uniuersityof Virginia 'Wei Guo, Uniuersityof Pennsyluarua Leah Haimo, Uniuersityof California, Riuerside Heidi E. Hamm, Vanderbilt(JniuersityMedicalSchool Craig M. Hart, Louisiana State Uniuersity Merill B. Hille, Uniuersityof Washington Jerry E. Honts, Drake Uniuersity H. Robert Horvitz, Massachusetts Institute of Technology Richard Hynes, Massachusetts lnstitute of Technologyand Howard HughesMedical Inshtute Harry Itagaki,Kenyon College ElizabethR. Jamieson,Smitb College Marie A. Janicke,State Uniuersityof New York, Buffalo Bradley'W.Jones,Uniuersityof Mississippi Mark Kainz, ColgateUniuersity Naohiro Kato, Louisiana State Uniuersity Amy E. Keating, Massachusetts lnstitute of Technology CharlesH. Keith, Uniuersityof Georgia Thomas C. S. Keller lll, Florida State Uniuersity 'V/estern Greg M. Kelly, Uniuersityof Ontario StephenKendall, California State Uniuersity,Fullerton FelipeKierszenbaum, MichiganStateUniuersity Cindy Klevickis,JamesMadison lJniuersity Brian Kobilka, StanfordUniuersityMedicalSchool. Martina Koniger, WellesleyUniuersfiy CatherineKoo, Caldwell College Keith G. Kozminski, Uniuersityof Virginia StevenI(. IJHernault, Emory Uruuerstty Douglas Lauffenburger, Massacbusetts Institute of Tecbnology RobertJ. Lefkowitz, HHMI and Duke UniuersityMedical School R. L. Levine,McGill Uniuersity FangJu Ltn, CoastalCarolina [Jniuersity ElizabethLord, Uniuersityof California, Riuerside Liqun Luo, StanfordUniuersity Grant MacGregor, Uniuersityof California, Iruine Jennifer O. Manilay, IJniuersityof California, Merced Barry Margul ies,Towson Uniuer stty C. William McCurdy, Uniuersityof California, Dauis, and LawrenceBerkeleyNational Laboratory Dennis \il/. McGee, State Uniuersityof New York, Binghamton JamesMcGrath, RochesterSchoolof Medicine David D. McKemy, Uniuersityof SouthernCalifornia Roderick MacKinnon, Rockefeller(Jniuersity JamesA. McNew, Rice Uniuersity

X

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CONTENTS IN BRIEF

Part I 1. 2. 3. Part ll 4. 5. 6. 7. 8. Part lll 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Chemical and MolecularFoundations1 L i f e B e g i n sw i th C e l l s 1 C h e mi caFl o u n d a ti o n s3 1 ProteinStructureand Function 63

Genetics and MolecularBiology 111 B a s i cM o l e c u l aG r e n e t i cM e c h a n i s m1s 1 1 M o l e cu l a Ge r n e ti cT e ch n i ques1G5 G e n e s,Ge n o mi cs, a n d C h rom osomes 215 Transcriptional Controlof Gene Expression269 Post-transcriptional GeneControl 323

CeflStructureand Function 371 V i su a l i zi n gF, ra cti o n a ti n g, and Cultur ingCells 371 BiomembraneStructure 409 Transmembrane Transportof lonsand small Molecures437 C e ll u l a rE n e rg e ti cs4 7 9 M o vi n gP ro te i n si n to Me mbr anes and Or ganelles533 VesicularTraffic,Secretion,and Endocytosis579 C e llS i g n a l i n gl : S i g n a T l ra nsduction and Shor t- Ter m CellularResponses 62 3 c e l l si g n a l i n gl l : si g n a l i n gPathways That contr ol GeneActivity 665 C e l lOrg a n i za ti o n a n d Mo v ementl: M icr ofilaments713 C e l lOrg a n i za ti o n a n d Mo v ementll: Micr otubules and Inter m ediate Filaments157 I n t e g ra ti n gC e l l si n to T i ssues801

Part lV CellGrowth and Development847 20. Regulatingthe EukaryoticCellCycle 847 21. 22. 23. 24. 25.

C e l lB i rth ,L i n e a g ea, n d D e ath 905 T h e Mo l e cu l a C r e l lB i o l o g yof Developm entg4g N e rveC e l l s 1 0 0 1 l m m u n o l o g y1 0 5 5 Cancer 1107

CONTENTS

PartI Chemical and MolecularFoundations

E

The Diversityand Commonality of Cells

18

C e l l sG r o w a n d D i v i d e

WITHCELLS 1 L I F EB E G I N S

CellsDie from AggravatedAssaultor an Internal Program 19

1

!!|

InvestigatingCellsand Their Parts 20

All CellsAre Prokaryoticor Eukaryotic

1

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s

4

VirusesAre the Ultimate Parasites

6

Cell BiologyRevealsthe Size,Shape,Location, 20 and Movementsof Cell Components Biochemistryand BiophysicsRevealthe Molecular Structureand Chemistryof PurifiedCell Constituents 21

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

6

of DamagedGenes 22 GeneticsRevealsthe Consequences

EvenSingleCellsCan HaveSex

7

We Developfrom a SingleCell

8

GenomicsRevealsDifferencesin the Structure of EntireGenomes and Expression

23

DevelopmentalBiologyRevealsChangesin the Propertiesof Cellsas They Specialize

23

Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities

@The

Choosingthe Right ExperimentalOrganismfor the Job 25 BiologicalStudiesUse Multiple The Most Successful 27 Approaches

M o l e c u l eos f a c e l l

Small MoleculesCarryEnergy,TransmitSignals, a n d A r e L i n k e di n t o M a c r o m o l e c u l e s ProteinsGive CellsStructureand Perform Most CellularTasks

'

s|

on Evolution 28 A GenomePerspective

10

Metabolic Proteins,the GeneticCode,and Organelle StructuresAre NearlYUniversal

't1

The Genome ls Packagedinto Chromosomesand R e p l i c a t e dD u r i n gC e l lD i v i s i o n

Darwin'sldeasAbout the Evolutionof Whole Animals Are Relevantto Genes

12

Mutations May Be Good, Bad, or lndifferent

13

Many GenesControllingDevelopmentAre Remarkably 28 S i m i l a ri n H u m a n sa n d O t h e r A n i m a l s Human Medicinels Informed by Researchon Other 29 Organisms

NucleicAcidsCarryCoded Information for Making Proteinsat the Right Time and Place

E

Thework of cells

28

14

C e l l sB u i l da n d D e g r a d eN u m e r o u sM o l e c u l e s and Structures

15

Animal CellsProduceTheir Own External E n v i r o n m e nat n d G l u e s

16

CellsChangeShapeand Move

16

CellsSenseand Send Information

16

covalent Bondsand Noncovalent lnteractions

to CellsRegulateTheir Gene Expression M e e t C h a n g i n gN e e d s

17

the Structureof an Atom Determines TheElectronic of CovalentBondslt CanMake 33 Numberand Geometry

31

FOUNDATIONS 2 CHEMICAL ![

CONTENTS

.

32

E l e c t r o n sM a y B e S h a r e dE q u a l l yo r U n e q u a l l y i n C o v a l e n tB o n d s

34

L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l eC h e m i c a l Reactionswith EnergeticallyFavorableReactions 57

CovalentBondsAre Much Strongerand More StableThan NoncovalentInteractions

Hydrolysisof ATPReleases SubstantialFreeEnergy and DrivesMany CellularProcesses

lonic InteractionsAre Attractionsbetween Oppositely C h a r g e dl o n s 36 HydrogenBondsDeterminethe Water Solubility o f U n c h a r g e dM o l e c u l e s 37

ATPls GeneratedDuring Photosynthesis and Respiration 59 N A D - a n d F A DC o u p l eM a n y B i o l o g i c aO l xidation and ReductionReactions

Van der Waals InteractionsAre Causedby T r a n s i e nD t iooles

37

The HydrophobicEffectCausesNonpolar Moleculesto Adhere to One Another

38

3 P R O T E ISNT R U C T U R ED AN FUNCTION

M o l e c u l a rC o m p l e m e n t a r i tM y e d i a t e dv i a NoncovalentInteractionspermitsTight, H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s

3e

![

C h e m i c alB u i l d i n gB l o ckso f C e i l s

@

A m i n o A c i d sD i f f e r i n gO n l y i n T h e i rS i d eC h a i n s ComposeProteins

40 41

FiveDifferent NucleotidesAre Usedto Build N u c l e i cA c i d s Monosaccharides Joined by GlycosidicBonds Form Linearand Branchedpolysaccharides

44

PhospholipidsAssociateNoncovalentlyto Form the BasicBilayerStructureof Biomembranes

C h e m i c aEl q u i l i b ri u m

49

EquilibriumConstantsReflectthe Extentof a ChemicalReaction

50

![

Hierarchical Structureof proteins

SecondaryStructuresAre the Core Elements of ProteinArchitecture

66

Overall Foldingof a PolypeptideChainYields Its TertiaryStructure

67

Different Waysof Depictingthe Conformationof ProteinsConveyDifferent Typesof Information

68

StructuralMotifs Are RegularCombinationsof Secondaryand TertiaryStructures

68

S t r u c t u r aal n d F u n c t i o n aD l o m a i n sA r e M o d u l e s of TertiaryStructure

70

ProteinsAssociateinto Multimeric Structuresand M a c r o m o l e c u l aAr s s e m b l i e s

72

Membersof Protein FamiliesHavea Common EvolutionaryAncestor

72

![

BiologicalFluidsHaveCharacteristic pH Values

51

P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o Which ProteinsCan Fold

B i o c h e m i caEl n e rg e ti cs

@

SeveralFormsof EnergyAre lmportant in BiologicalSystems CellsCan TransformOne Typeof Energyinto Another The Changein Free EnergyDeterminesthe Direction of a ChemicalReaction The AG'' of a ReactionCan Be Calculated from lts K"o The Rateof a ReactionDependson the Activation EnergyNecessary to Energizethe Reactantsinto a TransitionState

xvi

.

coNTENTs

64 65

50

B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l aar n d E x t r a c e l l u l aFrl u i d s

63

The PrimaryStructureof a Protein ls lts Linear Arrangementof Amino Acids

ChemicalReactionsin CellsAre at SteadyState DissociationConstantsof Binding ReactionsReflect the Affinity of InteractingMolecules Hydrogenlons Are Releasedby Acidsand Taken Up by Bases

57

50

52 52

ProteinFolding

74 74

I n f o r m a t i o nD i r e c t i n ga P r o t e i n ' sF o l d i n gl s E n c o d e di n l t s A m i n o A c i d S e q u e n c e

74 Foldingof Proteinsin Vivo ls Promotedby Chaperones 7 5 AlternativelyFoldedProteinsAre lmplicatedin Diseases 77

54

ProteinFunction

78

S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e Functionsof Most Proteins

7g

EnzymesAre Highly Efficientand SpecificCatalysts

79

An Enzyme'sActive Site BindsSubstratesand CarriesOut Catalysis

g0

SerineProteasesDemonstrateHow an Enzyme'sActive Site Works

g1

Enzymesin a Common PathwayAre Often physically Associatedwith One Another

g4

!f, 54 55 55 56

56

EnzymesCalledMolecularMotors ConvertEnergy into Motion

!E-

!l|

r.r RegulatingProteinFunctionl: ProteinDegradation

RegulatedSynthesisand Degradationof Proteins is a FundamentalPropertyof Cells

CentrifugationCan SeparateParticlesand MoleculesThat Differ in Massor Density

86 86

The Proteasomels a ComplexMolecularMachine Usedto DegradeProteins Ubiquitin Marks CytosolicProteinsfor Degradation in Proteasomes

RegulatingProteinFunctionll: Noncovalentand Covalent Modifications NoncovalentBinding PermitsAllosteric, or Cooperative,Regulationof Proteins

Purifying,Detecting,and Proteins Characterizing

85

SeparatesMoleculeson the Basis Electrophoresis Ratio of Their Charge-to-Mass Liquid ChromatographyResolvesProteinsby Mass,Charge,or BindingAffinitY

92 92 94 96

Highly SpecificEnzymeand Antibody AssaysCan Detect IndividualProteins Toolsfor Detecting Are Indispensable Radioisotopes M o l e c u l e s Biological MassSpectrometryCan Determinethe Mass and Sequenceof Proteins

101

88

Protein PrimaryStructureCan Be Determinedby ChemicalMethods and from Gene Sequences

103

89

Protein Conformationls Determinedby Sophisticated 103 PhysicalMethods

88

NoncovalentBindingof Calciumand GTPAre Widely UsedAs AllostericSwitchesto Control ProteinActivity 90 Phosphorylationand DephosphorylationCovalently RegulateProteinActivitY

91

ProteolyticCleavagelrreversiblyActivatesor lnactivatesSome Proteins

91

Higher-OrderRegulationIncludesControl of Protein Locationand Concentration

92

g

98 99

105

Proteomics

Proteomicsls the Studyof All or a LargeSubset of Proteinsin a BiologicalSYstem AdvancedTechniquesin MassSpectrometryAre Criticalto ProteomicAnalYsis

105 106

Partll Geneticsand MolecularBiologY 4 B A S I CM O L E C U L AGRE N E T I C MECHANISMS @

structureof NucleicAcids

111 113

Organizationof GenesDiffersin Prokaryoticand EukaryoticDNA to EukaryoticPrecursormRNAsAre Processed l RNAs F o r mF u n c t i o n am the Number of AlternativeRNA Splicinglncreases from a SingleEukaryoticGene ProteinsExpressed

A NucleicAcid Strand ls a LinearPolymerwith End-to-EndDirectionality

113

@

N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y A n t i p a r a l l eS l trands

114

DNA Can Undergo ReversibleStrandSeparation

115

MessengerRNA CarriesInformation from DNA in a Three-LetterGeneticCode The FoldedStructureof IRNA Promoteslts Decoding

TorsionalStressin DNA ls Relievedby Enzymes

117

Different Typesof RNA ExhibitVarious ConformationsRelatedto Their Functions

118

@ T r a n s c r i p t i o n o f P ro te i n -C o d i ng Genesand Formationof Functional 120 MRNA A TemplateDNA Strand ls Transcribedinto a ComplementaryRNA Chain by RNA Polymerase

120

122 123 125

The Decodingof mRNAbY tRNAs 127

Functions NonstandardBasePairingOften OccursBetween C o d o n sa n d A n t i c o d o n s Amino Acids BecomeActivatedWhen Covalently L i n k e dt o t R N A s

@

StepwiseSynthesisof Proteins on Rabosomes

Machines RibosomesAre Protein-Synthesizing

CONTENTS

127 129 130 131

132 132

XVII

Methionyl-tRNA,tttRecognizes the AUG StartCodon T r a n s l a t i o nI n i t i a t i o nU s u a l l yO c c u r sa t t h e F i r s t A U G f r o m t h e 5 ' E n do f a n m R N A D u r i n gC h a i nE l o n g a t i o nE a c hl n c o m i n g Aminoacyl-tRNAMovesThrough Three RibosomalSites Translationls Terminatedby ReleaseFactors When a Stop Codon ls Reached Polysomesand RapidRibosomeRecyclingIncrease the Efficiencyof Translation

DNAReptication

!f|

133 133

E! 135

166

137

166

138

Segregationof Mutations in BreedingExperiments RevealsTheir Dominanceor Recessivity

167

ConditionalMutations Can Be Usedto Study EssentialGenesin Yeast

EO

139 140

Duplex DNA ls Unwound and Daughter StrandsAre Formedat the DNA ReplicationFork

't41 141 143

Recessive Lethal Mutations in DiploidsCan Be l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e di n Heterozygotes

171 ComplementationTestsDetermineWhether Different 't7 Recessive Mutations Are in the SameGene 1 Double Mutants Are Usefulin Assessing the Order in Which ProteinsFunction 171 GeneticSuppression and SyntheticLethalityCan RevealInteractingor Redundantproteins 173 GenesCan Be ldentified by Their Map position on the Chromosome 174

D N A R e p a i ra n d R e co mb i n a ti o n 145

!!|

p|

DNA Polymerases IntroduceCopyingErrors and Also CorrectThem

145

C h e m i c aal n d R a d i a t i o nD a m a g et o D N A C a n Leadto Mutations

145

H i g h - F i d e l i tD y N A E x c i s i o n - R e p aSiyr s t e m s Recognize a n d R e p a i rD a m a g e B a s eE x c i s i o n R e p a i r sT . G M i s m a t c h eas n d D a m a g e dB a s e s

147

MismatchExcisionRepairsOther Mismatchesand S m a l lI n s e r t i o n a s nd Deletions

147

NucleotideExcisionRepairsChemicalAdducts That Distort Normal DNA Shape Two SystemsUtilize Recombinationto Repair D o u b l e - S t r a nB d r e a k si n D N A H o m o l o g o u sR e c o m b i n a t i oC n a n R e o a i rD N A D a m a g ea n d G e n e r a t eG e n e t i cD i v e r s i t y

s f th e C e l l u l a r @ V i r u s e s : pa ra si te o GeneticSystem Most Viral Host RangesAre Narrow Viral CapsidsAre RegularArraysof One or a Few Typesof protein

147

148 149 150

154

'

CONTENTS

DNACloningand Char acter i z ati on176

RestrictionEnzymesand DNA LigasesAllow Insertionof DNA Fragmentsinto CloningVectors

176

E. coli PlasmidVectorsAre Suitablefor Cloning lsolatedDNA Fragments

178

cDNA LibrariesRepresentthe Sequences of Protein-CodingGenes

179 cDNAsPreparedby ReverseTranscriptionof Cellular mRNAsCan Be Clonedto GeneratecDNA Libraries 1 8 1 DNA LibrariesCan Be Screenedby Hybridization to an Oligonucleotidp erobe 181 YeastGenomicLibrariesCan Be Constructedwith ShuttleVectorsand Screenedby Functionar Complementation 182 Gel Electrophoresis Allows Separationof Vector DNA from Cloned Fragments 184 Cloned DNA MoleculesAre SequencedRapidly by the DideoxyChain-Termination Methoc 187 The PolymeraseChain ReactionAmplifiesa Specific DNA Sequencefrom a ComplexMixture 188

154 154

VirusesCan Be Clonedand Counted in plaqueAssays 1 5 5 LyticViral Growth CyclesLeadto the Death of Host Cells 1 5 5 Viral DNA ls Integrated into the Host-CellGenome in SomeNonlyticViral Growth Cycles 158

XVIII

GeneticAnalysisof Mutationsto ldentifyand StudyGenes

R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e sG e n e r a l l y HaveOppositeEffectson Gene Function

D N A P o l y m e r a s eR s e q u i r ea p r i m e r t o I n i t i a t e Replication

SeveralProteinsParticipatein DNA Replication D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l lfyr o m E a c hO r i g i n

5 M O L E C U L AGRE N E T T C E C H N T Q U1E6Ss

ff|

UsingClonedDNA Fragments to Study GeneExpression

191

HybridizationTechniquespermit Detection of SpecificDNA Fragmentsand mRNAs

't91

DNA MicroarraysCan Be Usedto Evaluatethe Expression of Many Genesat One Time

192

ClusterAnalysisof Multiple ExpressionExperiments ldentifies Co-regulatedGenes

193

N o n p r o t e i n - C o d i nG g e n e sE n c o d eF u n c t i o n a l RNAs

SystemsCan ProduceLarge E. coli Expression Quantitiesof Proteinsfrom ClonedGenes VectorsCan Be Designedfor PlasmidExpression U s ei n A n i m a l C e l l s

Or ganization Chr om osomal of Genesand NoncodingDNA

196

222

223

s o n t a i nM u c h G e n o m e so f M a n y O r g a n i s m C N o n f u n c t i o n aD l NA

l d e n t i f y in ga n d L o ca ti n gH u ma n Disease Ge n e s

DNAsAre Concentrated Most Simple-Sequence in SpecificChromosomalLocations

224

199

DNA FingerprintingDependson Differences DNAs in Length of SimPle-Sequence

225

D N A P o l y m o r p h i s mAsr e U s e di n L i n k a g e - M a p p i n g H u m a nM u t a t i o n s

200

SpacerDNA Occupiesa Significant Unclassified Portion of the Genome

225

LinkageStudiesCan Map DiseaseGeneswith a Resolutionof About 1 Centimorgan

201

FurtherAnalysisls Neededto Locatea DiseaseGene i n C l o n e dD N A

202

f[

Show One of Three Major Many InheritedDiseases Patternsof Inheritance

198

]f|

M a n y I n h e r i t e dD i s e a s eRs e s u l ft r o m M u l t i p l eG e n e t i c 203 Defects

ffl

Inactivatingthe Functionof S p e c i f i cGe n e si n E u ka ryo te s

Normal YeastGenesCan Be Replacedwith Mutant A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n

204 205

Transcriptionof GenesLigatedto a Regulated PromoterCan Be ControlledExperimentally SpecificGenesCan Be PermanentlyInactivatedin t h e G e r m L i n eo f M i c e

207

SomaticCell RecombinationCan InactivateGenes in SpecificTissues

208

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y l n h i b i t S o m eG e n e s RNA InterferenceCausesGene Inactivationby g RNA D e s t r o y i n gt h e C o r r e s p o n d i nm

6 G E N E SG , E N O M I C SA, N D CHROMOSOMES

][

EukaryoticGeneStructure

(Mobile)DNA Transposable Elem ents

Movement of Mobile ElementsInvolvesa D N A o r a n R N AI n t e r m e d i a t e

226

Are Presentin Prokaryotes DNA Transposons and Eukaryotes

227

BehaveLike Intracellular LTRRetrotransposons Retroviruses Transposeby a Distinct Non-LTRRetrotransposons Mechanism RNAsAre Found in Genomic Other Retrotransposed DNA M o b i l e D N A E l e m e n t sH a v eS i g n i f i c a n t l Iyn f l u e n c e d Evolution

@

215 217

Most EukaryoticGenesContain Intronsand Produce m R N A sE n c o d i n gS i n g l eP r o t e i n s

217

n nits S i m p l ea n d C o m p l e xT r a n s c r i p t i oU A r e F o u n di n E u k a r y o t i cG e n o m e s

217

P r o t e i n - C o d i nG g e n e sM a y B e S o l i t a r yo r B e l o n g t o a G e n eF a m i l y

219

HeavilyUsedGene ProductsAre Encodedby Multiple 221 Copiesof Genes

229 230 234 234

236

or ganelleDNAs

M i t o c h o n d r i aC o n t a i nM u l t i p l e m t D N A M o l e c u l e s

210

226

mtDNA ls InheritedCytoplasmically The Size,Structure,and Coding Capacityof mtDNA Vary ConsiderablyBetweenOrganisms Productsof MitochondrialGenesAre Not Exported M i t o c h o n d r i aE v o l v e df r o m a S i n g l eE n d o s y m b i o t i c Bacterium EventInvolvinga Rickettsia-like

237 237 238 240

240

MitochondrialGeneticCodesDiffer from the StandardNuclearCode

240

Mutations in MitochondrialDNA CauseSeveral G e n e t i cD i s e a s eisn H u m a n s ChloroplastsContain LargeDNAsOften Encoding M o r e T h a n a H u n d r e dP r o t e i n s

242

Anal Ys i s Genome- wide Genom ics: of GeneStructureand Expression 243 SuggestFunctionsof Newly StoredSequences ldentified Genesand Proteins

CONTENTS

243

xtx

Comparisonof RelatedSequences from Different SpeciesCan Give Cluesto Evolutionary Relationships Among proteins G e n e sC a n B e l d e n t i f i e dW i t h i n G e n o m i cD N A Sequences

244

S m a l lM o l e c u l e sR e g u l a t eE x p r e s s i oonf M a n y BacterialGenesvia DNA-BindingRepressors and Activators

273

244

TranscriptionInitiation from Some Promoters RequiresAlternativeSigmaFactors

273

Transcriptionby osa-RNAPolymerasels Controlled by ActivatorsThat Bind Farfrom the Promoter

274

Many BacterialResponses Are Controlledby Two-ComponentRegulatorySystems

275

T h e N u m b e ro f P r o t e i n - C o d i nG g e n e si n a n Organism'G s e n o m el s N o t D i r e c t l yR e l a t e d to lts BiologicalComplexity S i n g l eN u c l e o t i d eP o l y m o r p h i s masn d G e n eC o p y Number VariationAre lmportant Determinants of DifferencesBetween Individualsof a Species

246

@ St r u c t u r alOrg a n i za ti o n of EukaryoticChromosomes

][

ChromatinExistsin Extendedand CondensedForms Modificationsof HistoneTailsControl Chromatin Condensationand Function NonhistoneProteinsProvidea StructuralScaffold for Long ChromatinLoops A d d i t i o n a lN o n h i s t o n ep r o t e i n sR e g u l a t e Transcriptionand Replication

247 248

276

250

Three EukaryoticPolymerases CatalyzeFormation of Different RNAs

278

254

The LargestSubunit in RNA Polymerasell Hasan EssentialCarboxyl-Terminal Repeat

2s6

ChromosomeNumber;Size,and Shapeat Metaphase Are Species-Specific 257 During Metaphase,ChromosomesCan Be D i s t i n g u i s h ebdy B a n d i n gp a t t e r n sa n d C h r o m o s o m eP a i n t i n g 25g C h r o m o s o m eP a i n t i n ga n d D N A S e q u e n c i n g Reveal the Evolutionof Chromosomes 259 InterphasePolyteneChromosomesArise by DNA A m p l i fi c a t i o n 260 Three FunctionalElementsAre Requiredfor Replication a n d S t a b l eI n h e r i t a n c eo f C h r o m o s o m e s 261 CentromereSequences Vary Greatlyin Length 263 Addition of TelomericSequencesby Telomerase PreventsShorteningof Chromosomes

263

7 TRANSCRIPTIONALCONTROL O F G E N EE X P R E S S I O N 269 Controlof GeneExpression in Bacteria

W

271

TranscriptionInitiation by BacterialRNA polymerase RequiresAssociationwith a SigmaFactor 271 Initiation of /ac Operon TranscriptionCan Be Repressed and Activated 271

XX

O

CONTENTS

276

RegulatoryElementsin EukaryoticDNA Are Found Both Closeto and Many KilobasesAway from TranscriptionStart Sites

M o r p h o l og ya n d F u n cti o n aEl l e m ents o f E u k a r y o ti C c h ro mo so me s 257

@

overview of EukaryoticGene Controland RNAPolymerases

279 RNA Polymerasell InitiatesTranscriptionat DNA SequencesCorrespondingto the 5' Cap of mRNAs 280

E

Regulator ysequences in pr ote i nCodingGenes 282

The TATABox, Initiators,and CpG lslandsFunction as Promotersin EukaryoticDNA

282

Promoter-Proximal ElementsHelp Regulate EukaryoticGenes

282

Distant EnhancersOften Stimu late Transcription by RNA Polymerasell

284

Most EukaryoticGenesAre Regulatedby Multiple Transcription-Control Elements

s @Activator s and Repr essorof Transcription

286

Footprintingand Gel-ShiftAssaysDetect protein-DNA Interactions 286 ActivatorsAre Modular ProteinsComposed of DistinctFunctionalDomainsand promote Transcription 288 Repressors Inhibit Transcriptionand Are the FunctionalConverseof Activators

290

D N A - B i n d i n gD o m a i n sC a n B e C l a s s i f i eidn t o NumerousStructuralTypes

290

StructurallyDiverseActivation and Repression DomainsRegulateTranscription

293

TranscriptionFactorInteractionsIncrease Gene-ControlOptions

294

M u l t i p r o t e i nC o m p l e x e F s o r mo n E n h a n c e r s

295

TranscriptionInitiation by RNA Polymerasell GeneralTranscriptionFactorsPositionRNA Polymerasell at Start Sitesand Assistin Initiation

296

@

GENE 8 POST- TRANSCRIPTIONAL CONTROL

Formationof HeterochromatinSilencesGene at Telomeres,Near Centromeres, Expression and in Other Regions

299

Can Direct HistoneDeacetylationand Repressors Methylation at SpecificGenes

303

ActivatorsCan Direct HistoneAcetylationand Methylation at SpecificGenes

305

FactorsHelp Activate or Chromatin-Remodeling RepressTranscription

306

HistoneModificationsVary Greatlyin Their Stabilities 307 307

Transcriptionof Many GenesRequiresOrdered Binding 308 and Functionof Activatorsand Co-activators The YeastTwo-HybridSystemExploitsActivator Flexibility to DetectcDNAsThat EncodeInteractingProteins 310

E

312

ResponseElementsContain Inverted Nuclear-Receptor 313 or Direct Repeats Hormone Bindingto a NuclearReceptorRegulateslts 313 Activity as a TranscriptionFactor

![

RegulatedElongationand Terminationof Transcription

314

Transcriptionof the HIV Genome ls Regulatedby an Antitermination Mechanism

315

Pausingof RNA Polymerasell Promoter-Proximal Occursin Some RapidlyInducedGenes

316

@

f[

other EukaryoticTranscription Systems

TranscriptionInitiation by Pol I and Pol lll ls Analogousto That by Pol ll

315 316

of EukaryoticPre-mRNA325 Processing

The 5' Cap ls Added to NascentRNAsShortlyAfter TranscriptionInitiation

325

A DiverseSet of Proteinswith ConservedRNA-B|nding 326 DomainsAssociatewith Pre-mRNAs SplicingOccursat Short,ConservedSequencesin Pre-mRNAsvia Two TransesterificationReactions

329 330

with Pre-mRNA During Splicing,snRNAsBase-Pair Assembledfrom snRNPsand a Spliceosomes, Pre-mRNA,CarryOut SPlicing

330

C h a i nE l o n g a t i o nb y R N AP o l y m e r a slel l s C o u p l e d Factors to the Presenceof RNA-Processing

333

SRProteinsContributeto Exon Definition in Long Pre-mRNAs Group ll Introns ProvideCluesto the Self-Splicing Evolutionof snRNAs

334

3' Cleavageand Polyadenylationof Pre-mRNAs 335 Are Tightly CouPled DegradeRNAThat ls Processed NuclearExonucleases 336 Out of Pre-mRNAs

Regulationof Transcription-Factor 311 Activity

All NuclearReceptorsSharea Common Domain Structure

323

298

o f T ra n sc r iption M o l e c u l a Me r ch a n i sms 299 and Activation Repression

The Mediator ComplexFormsa MolecularBridge BetweenActivation Domainsand Pol ll

317

296

SequentialAssemblyof ProteinsFormsthe Pol ll TranscriptionPreinitiationComplexin Vitro In Vivo TranscriptionInitiation by Pol ll Requires Additional Proteins

Mitochondrialand ChloroplastDNAsAre RNA Transcribedby Organelle-Specific Polymerases

Regulation of Pre-mRNA Processing

337

AlternativeSplicingls the PrimaryMechanismfor RegulatingmRNA Processing

337

A Cascadeof RegulatedRNA SplicingControls DrosophilaSexualDifferentiation

338

and ActivatorsControl Splicing SplicingRepressors at AlternativeSites

339

of RNA Editing Alters the Sequences SomePre-mRNAs

340

]f|

Transportof mRNAAcrossthe NuclearEnveloPe

341

NuclearPore ComplexesControl lmport and Export 342 from the Nucleus Are Not Exportedfrom in Spliceosomes Pre-mRNAs 345 the Nucleus HIV Rev Protein Regulatesthe Transportof Unspliced 346 Viral mRNAs

CONTENTS

'

xxi

CytoplasmicMechanisms of posttranscriptionalControl 347

f!|

Micro RNAsRepressTranslationof SpecificmRNAs RNA InterferenceInducesDegradationof precisely ComplementarymRNAs CytoplasmicPolyadenylationpromotesTranslation of SomemRNAs

Localizationof mRNAsPermitsProductionof Proteinsat SpecificRegionsWithin the Cytoplasm

357

347

Processing of rRNAand IRNA

358

349

f[

351

Pre-rRNAGenesFunctionas NucleolarOrganizers a n d A r e S i m i l a ri n A l l E u k a r y o t e s

359

Small NucleolarRNAsAssistin Processing Pre-rRNAs

360 363

Degradationof mRNAsin the CytoplasmOccurs by SeveralMechanisms

352

ProteinSynthesisCan Be GloballyRegulated

353

Self-Splicing Group I IntronsWere the First Examplesof CatalyticRNA

356

Pre-tRNAsUndergo ExtensiveModification in the Nucleus

363

357

N u c l e a rB o d i e sA r e F u n c t i o n a l lS y p e c i a l i z eN d uclear Domains

3G4

Sequence-Specific RNA-BindingproteinsControl SpecificmRNATranslation SurveillanceMechanismspreventTranslationof lmproperlyProcessed mRNAs

Partlll CellStructureand Function 9 V I S U A L I Z IN G, F R A C T ION A T IN G, A N D C U L T U R I NC GE L L S 371 Organellesof the EukaryoticCell

lll

372

Phase-Contrast and DifferentiallnterferenceContrast MicroscopyVisualizeUnstainedLivingCells 381 Fluorescence MicroscopyCan Localizeand euantify S p e c i f i cM o l e c u l e si n L i v eC e l l s 382 l m a g i n gS u b c e l l u l aDr e t a i l sO f t e n R e q u i r e tsh a t t h e SamplesBe Fixed,Sectioned,and Stained

T h e P l a s m aM e m b r a n eH a sM a n y C o m m o nF u n c t i o n s in All Cells 372 EndosomesTake Up 5oluble Macromolecules from the Cell Exterior 372

lmmunofluorescence MicroscopyCan Detect Specific Proteinsin FixedCells

38s

Confocaland DeconvolutionMicroscopyEnable Visualizationof Three-Dimensional Objects

386

Lysosomes Are Acidic OrganellesThat Contain a Batteryof DegradativeEnzymes

Graphicsand lnformaticsHaveTransformed Modern Microscopy

373

Peroxisomes DegradeFattyAcidsand ToxicCompounds 374 The EndoplasmicReticulumls a Network of InterconnectedInternal Membranes 375 The Golgi ComplexProcesses and SortsSecreted a n d M e m b r a n ep r o t e i n s

376

P l a n tV a c u o l e sS t o r eS m a l lM o l e c u l e sa n d E n a b l ea C e l lt o E l o n g a t eR a p i d l y

Eil

ElectronMicroscopy: Methods and Applications

388

Resolutionof Transmission ElectronMicroscopyis VastlyGreaterThan That of Light Microscopy

3gg

377

T h e N u c l e u sC o n t a i n st h e D N A G e n o m e ,R N A SyntheticApparatus,and a FibrousMatrix

CryoelectronMicroscopyAllows Visualizationof ParticlesWithout Fixationor Staining

399

378

MitochondriaAre the PrincipalSitesof ATp Productionin Aerobic NonphotosyntheticCells

378

ElectronMicroscopyof Metal-CoatedSpecimens Can RevealSurfaceFeaturesof Cellsand Their Components

390

ChloroplastsContain Internal Compartmentsin Which Photosynthesis Takesplace

379

!!| g

L i g h t M i cro sco p y: vi su a l i zi n gC ell Structureand Localizingproteins W i t h i n C el l s 380

The Resolutionof the Light Microscopels About 0.2 pm

xxii

.

coNTENrs

381

Purificationof Cell Organelles

391

Disruptionof CellsReleases Their Organelles and Other Contents

391

CentrifugationCan SeparateMany Typesof Organelles

392

Organelle-Specific AntibodiesAre Usefulin P r e p a r i n gH i g h l yP u r i f i e dO r g a n e l l e s

393

l s o l a t i o nC , u l tu re a , n d Differentiation 394 of MetazoanCells Flow CytometrySeparatesDifferent CellTypes

394

C u l t u r eo f A n i m a l C e l l sR e q u i r e sN u t r i e n t - R i c h M e d i a a n d S p e c i aSl o l i dS u r f a c e s

395

P r i m a r yC e l lC u l t u r e sC a n B e U s e dt o S t u d yC e l l Differentiation

396

P r i m a r yC e l lC u l t u r e sa n d C e l lS t r a i n sH a v ea F i n i t e L i f eS p a n

396

TransformedCellsCan Grow Indefinitelyin Culture

397

SomeCell LinesUndergo Differentiationin Culture

398

H y b r i dC e l l sC a l l e dH y b r i d o m a sP r o d u c eA b u n d a n t M o n o c l o n aA l ntibodies

400

HATMedium ls CommonlvUsedto lsolate HybridCells

402

organelles 407 Cnsstc ExprRturrur9.1 separating

ET R U C T U R E 1 O B I O M E M B R A NS s[

409

B i o m e mb ra n e L s:i p i dC o mp o sition 411 a n i za ti o n a n d S t r u ctu raOrg l

P h o s p h o l i p i dSsp o n t a n e o u s lFyo r mB i l a y e r s

411

B i l a y e r sF o r ma S e a l e dC o m p a r t m e n t Phospholipid S u r r o u n d i n ga n I n t e r n a lA q u e o u sS p a c e

411

M o t i f s H e l pT a r g e tP e r i p h e r a l Lipid-Binding Proteinsto the Membrane

427

ProteinsCan Be Removedfrom Membranes by Detergentsor High-SaltSolutions

427

$f,

sphingolipids, Phospholipids, SYnthesis andCholesterol: Movement and Intracellular

429

Fatty AcidsSynthesisls Mediated by Several lmportant Enzymes

430

SmallCytosolicProteinsFacilitateMovement of FattyAcids Incorporationof Fatty Acids into Membrane Lipids TakesPlaceon OrganelleMembranes

430 431

F l i p p a s eM s o v e P h o s p h o l i p i df sr o m O n e M e m b r a n e Leafletto the OppositeLeaflet

431

Cholesterolls Synthesizedby Enzymesin the Cytosol and ERMembrane

432

Cholesterola nd Phospholi pids Are Transported BetweenOrganellesby SeveralMechanisms

433

1 1 T R A N S M E M B R A NTER A N S P O R T OF IONSAND SMALLM OLECU LES437

E

overview of Membrane Transport

l lasses B i o m e m b r a n eC s o n t a i nT h r e eP r i n c i p aC of Lipids

415

M o s t L i p i d sa n d M a n y P r o t e i n sA r e L a t e r a l l yM o b i l e in Biomembranes

416

O n l y S m a l lH y d r o p h o b i cM o l e c u l e sC r o s sM e m b r a n e s 438 b y S i m p l eD i f f u s i o n

L i p i dC o m p o s i t i o nI n f l u e n c etsh e P h y s i c a l Propertiesof Membranes

418

Membrane ProteinsMediate Transportof Most Moleculesand All lons Across Biomembranes

Lipid Compositionls Different in the Exoplasmic and CytosolicLeaflets

419

s l u s t e rw i t h S p e c i f i c C h o l e s t e r oal n d S p h i n g o l i p i dC P r o t e i n si n M e m b r a n eM i c r o d o m a i n s

420

ProteinComponents Biomembranes: 421 and BasicFunctions

@

438

439

uniport Transportof Glucose and Water

441

Most TransmembraneProteinsHave M e m b r a n e - S p a n n i nagH e l i c e s

SeveralFeaturesDistinguishUniport Transportfrom S i m p l eD i f f u s i o n GLUT1UniporterTransportsGlucoseinto Most M a m m a l i a nC e l l s s F a m i l yo f S u g a r T h e H u m a nG e n o m eE n c o d e a TransportingGLUTProtetns

M u l t i p l e B S t r a n d si n P o r i n sF o r m M e m b r a n e - S p a n n i n"gB a r r e l s "

424

TransportProteinsCan Be EnrichedWithin Artificial M e m b r a n e sa n d C e l l s

CovalentlyAttached HydrocarbonChainsAnchor Some Proteinsto Membranes

424

$[|

ProteinsInteractwith Membranesin Three Different Ways

s re A l l T r a n s m e m b r a nPer o t e i n sa n d G l y c o l i p i dA A s y m m e t r i c a l lO y r i e n t e di n t h e B i l a y e r

421

OsmoticPressureCausesWater to Move Across Membranes AquaporinsIncreasethe Water Permeabilityof Cell Membranes

CONTENTS

O

44'l 442 443 443 4M 4M

xxiii

pumpsand the ATP-powered I n t r a c el l u l al ro n i cE n vi ro n me nt 447

@

Different Classes of PumpsExhibitCharacteristic Structuraland Functionalproperties ATP-Poweredlon PumpsGenerateand M a i n t a i nl o n i cG r a d i e n t sA c r o s sC e l l u l a r Membranes MuscleRelaxationDependson Ca2*ATpases That Pump Ca" from the Cytosolinto the S a r c o p l a s mR i ce t i c u i u m CalmodulinRegulates the plasmaMembrane Ca'* PumpsThat Control CytosolicCa2+ Concentrations N a - / K - A T P a s eM a i n t a i n st h e I n t r a c e l l u l aN r a* a n d K * C o n c e n t r a t i o nisn A n i m a l C e l l s V-ClassH* ATPases Maintain the Acidity of Lysosomes and Vacuoles BacterialPermeases Are ABC proteinsThat lmport a Variety of Nutrientsfrom the Environment

N o n g a te dto n C h a n n e l a s n d th e potential RestingMembrane

SelectiveMovement of lons Createsa TransmembraneElectricpotential Difference T h e M e m b r a n eP o t e n t i a li n A n i m a l C e l l sD e p e n d s Largelyon Potassiumlon MovementsThrough O p e n R e s t i n gK + C h a n n e l s

467

Na*-LinkedCa2*Antiporter ExportsCa2* from C a r d i a cM u s c l eC e l l s 447

448

449

451 452 453

454

The Approximately50 MammalianABCTransporters PlayDiverseand lmportant Rolesin Cell and O r g a nP h y s i o l o g y 455 C e r t a i nA B CP r o t e i n s" F l i p ' ,p h o s p h o l i p i d s and Other Lipid-SolubleSubstratesfrom One Membrane Leafletto the Opposite Leaflet 456

El

BacterialSymporterStructureRevealsthe Mechanismof SubstrateBinding

SeveralCotransportersRegulate CytosolicpH

468

A PutativeCation ExchangeProtein Playsa K e y R o l ei n E v o l u t i o no f H u m a nS k i n Pigmentation

469

NumerousTransportProteinsEnablePlant Vacuolesto AccumulateMetabolitesand lons

469

ft|

Transeprrnerar Transporr

Multiple TransportProteinsAre Neededto Move Glucoseand Amino AcidsAcross Epithelia

471

SimpleRehydrationTherapyDependson the OsmoticGradientCreatedby Absorption o f G l u c o s ea n d N a +

411

ParietalCellsAcidify the StomachContentsWhile M a i n t a i n i n ga N e u t r a lC y t o s o l i cp H

472

Cusslc ExprntueruT 11.1 stumbting Upon ActiveTransport

477

12 CELLULAR ENERGETICS 458

@ 458

470

FirstStepsof Glucoseand Fatty Acid CatabolismGlycolysis : and the CitricAcid €ycle

479

480

(Stagel), CytosolicEnzymes During Glycolysis ConvertGlucoseto Pyruvate

481

460

lon ChannelsContain a SelectivityFilter Formed from ConservedTransmembraneSegments

The Rate of Glycolysis ls Adjustedto Meet the Cell'sNeed for ATP

483

461

Glucosels FermentedUnder AnaerobicConditions

PatchClampsPermit Measurementof lon M o v e m e n t sT h r o u g hS i n g l eC h a n n e l s

485

463

U n d e r A e r o b i cC o n d i t i o n s ,M i t o c h o n d r i a E f fi c i e n t l yO x i d i z eP y r u v a t ea n d G e n e r a t e ATP (Stagesll-lV)

485

464

MitochondriaAre DynamicOrganelleswith Two Structurallyand FunctionallyDistinctMembranes

485

464

In Stagell, Pyruvatels Oxidizedto CO2and HighEnergyElectronsStored in ReducedCoenzymes

487

Novel lon ChannelsCan Be Characterizedby a Combinationof Oocyte Expression and P a t c hC l a m p i n g N a - E n t r yi n t o M a m m a l i a nC e l l sH a sa N e g a t i v e Changein FreeEnergy(AG)

Cotransportby symportersand Antiporters

@|

N a * - L i n k e dS y m p o r t e r lsm p o r t A m i n o A c i d sa n d G l u c o s ei n t o A n i m a l C e l l sA g a i n s tH i g h ConcentrationGradients

xxiv

.

coNTENTs

465

466

Transportersin the Inner MitochondrialMembrane H e l p M a i n t a i nA p p r o p r i a t eC y t o s o l i a c nd Matrix Concentrationsof NAD* and NADH MitochondrialOxidation of Fatty AcidsGenerates ATP PeroxisomalOxidation of Fatty AcidsGenerates No ATP

491

@ Th e

El e ctro nT ra n sp o rtch a i n a n d Generationof the Proton-Motive 493 Force

the StepwiseElectronTransportEfficientlyReleases EnergyStored in NADH and FADH2

493

ElectronTransportin Mitochondria ls Coupledto P r o t o nP u m p i n g

493

ElectronsFlow from FADH2and NADHto 02 Through Four Multiprotein Complexes

PhotoelectronTransportfrom EnergizedReactionCenterChlorophylla Producesa Charge Separation

514

I n t e r n a lA n t e n n a a n d L i g h t - H a r v e s t i n g C o m p l e x e sI n c r e a s et h e E f f i c i e n c yo f Photosynthesis

515

494

MolecularAnalysisof Photosystems

517

ReductionPotentialsof ElectronCarriersFavor ElectronFlow from NADH to 02

499

The SinglePhotosystemof PurpleBacteria Generatesa Proton-MotiveForcebut No 02

517

ExperimentsUsing PurifiedComplexesEstablished the Stoichiometryof Proton Pumping

499

The Q CycleIncreases the Numberof Protons Translocated as ElectronsFlow Through C o m p l e xl l l

LinearElectronFlow Through Both Plant Photosystems, PSlland PSl,Generatesa Proton-MotiveForce, 519 02, and NADPH

500

A n O x y g e n - E v o l v i nC g o m p l e xl s L o c a t e do n t h e L u m i n a lS u r f a c eo f t h e P S l lR e a c t i o n Center

520

CellsUse Multiple Mechanismsto ProtectAgainst Damagefrom ReactiveOxygenSpeciesDuring PhotoelectronTransPort

521

CyclicElectronFlow Through PSIGeneratesa Proton-MotiveForcebut No NADPHor 02

522

I and ll Are RelativeActivitiesof Photosystems Regulated

523

The Proton-MotiveForcein Mitochondria ls Due Largelyto a Voltage GradientAcrossthe Inner Membrane ToxicBy-productsof ElectronTransportCan D a m a g eC e l l s

[[

Harnessingthe Proton-Motive F o r c ef o r E n e rg y-R e q u i ri n g Processes

502 502

503

[[l

The Mechanismof ATPSynthesisls Shared Among Bacteria,Mitochondria,and Chloroplasts

505

ATPSynthaseComprisesTwo Multiprotein ComplexesTermedFeand F1

505

During co2 Metabolism Photosynthesis

524

RubiscoFixesCO2in the ChloroplastStroma

525

Synthesisof SucroseUsing FixedCO2ls Completed in the Cytosol

525

506

Light and RubiscoActivaseStimulateCO2 Fixation

525

ATP-ADPExchangeAcrossthe Inner Mitochondrial Membrane ls Poweredby the Proton-Motive Force

s09

Which Competeswith Photorespiration, ls Reducedin PlantsThat Fix Photosynthesis, CO2by the C4PathwaY

527

R a t eo f M i t o c h o n d r i aO l x i d a t i o nN o r m a l l yD e p e n d s on ADP Levels

510

Brown-FatMitochondria Usethe Proton-Motive Forceto GenerateHeat

510

Rotation of the Ft 1 Subunit,Driven by Proton Movement Through F6,Powers ATPSynthesis

@

1 3 M O V I N GP R O T E I NISN T O A N D O R G A N E L L E Ss33 MEMBRANES

Ph o t o s yn th e siasn d L i g h t-A b sor bing Pigments

ThylakoidMembranesin ChloroplastsAre the Sites in Plants of Photosynthesis

511 511

Occur Three of the Four Stagesin Photosynthesis O n l y D u r i n gl l l u m i n a t i o n

511

EachPhoton of Light Hasa DefinedAmount of Energy

513

Comprisea ReactionCenterand Photosystems Complexes AssociatedLight-Harvesting

514

$[

of secretoryProteins Translocation 535 Acr ossthe ERMembr ane

A HydrophobicN-TerminalSignalSequenceTargets NascentSecretoryProteinsto the ER

536

CotranslationalTranslocationls lnitiated by Two Proteins GTP-Hydrolyzing

537

Passageof Growing PolypeptidesThrough the Transloconls Driven by EnergyReleasedDuring Translation

539

CONTENTS

.

XXV

ATPHydrolysisPowersPost-translational Translocationof SomeSecretoryProteinsin yeast

Insertionof proteinsinto the E RM e mb ra n e

$[

540

542

543 InternalStop-Transfer and Signal-AnchorSequences proteins DetermineTopologyof Single-Pass 544 M u l t i p a s sP r o t e i n sH a v eM u l t i p l e I n t e r n a l TopogenicSequences 546 A P h o s p h o l i p iA d n c h o rT e t h e r sS o m eC e l l - S u r f a c e Proteinsto the Membrane 547 The Topologyof a Membrane ProteinOften Can Be Deducedfrom lts Sequence 547

A Preformed/V-LinkedOligosaccharide ls Added to M a n y P r o t e i n si n t h e R o u g hE R O l i g o s a c c h a r i dSei d eC h a i n sM a y p r o m o t eF o l d i n g and Stabilityof Glycoproteins D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e db y P r o t e i n si n t h e E RL u m e n C h a p e r o n eas n d O t h e r E Rp r o t e i n sF a c i l i t a t eF o l d i n g and Assemblyof Proteins lmproperlyFoldedProteinsin the ERlnduce Expression of Protein-FoldingCatalysts U n a s s e m b l eodr M i s f o l d e dP r o t e i n si n t h e E RA r e Often Transportedto the Cytosolfor Degradation

550

ss2

coNTENTs

569

Largeand Small MoleculesEnter and Leavethe Nucleusvia NuclearPoreComplexes

570

lmportinsTransportProteinsContainingNuclearL o c a l i z a t i oS n i g n a l si n t o t h e N u c l e u s

571

ExportinsTransportProteinsContainingNuclear-Export S i g n a l so u t o f t h e N u c l e u s 573 573

1 4 V E S I C U L ATRR A F F I CS, E C R E T I O N , AND ENDOCYTOSIS 579 Techniques for Studyingthe SecretoryPathway

s80

Transportof a protein Through the Secretory pathway Can Be Assayedin tiving Cells

5g2

YeastMutants Define Major Stagesand Many Componentsin VesicularTransport

584

Cell-FreeTransportAssaysAllow Dissectionof I n d i v i d u aS l t e p si n V e s i c u l aTr r a n s p o r t

585

![ ss2 555

556

and Chloroplasts

.

Transportinto and out of the Nucleus

s[

552

A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c eD sirect Proteinsto the MitochondrialMatrix 558 M i t o c h o n d r i aP l r o t e i nl m p o r t R e q u i r e sO u t e r - M e m b r a n e R e c e p t o ras n d T r a n s l o c o ni sn B o t h M e m b r a n e s 559 Studieswith ChimericProteinsDemonstratelmportant Featuresof Mitochondriallmport 550 T h r e eE n e r g yI n p u t sA r e N e e d e dt o l m p o r t P r o t e i n s into Mitochondria 501 Multiple Signalsand PathwaysTarget Proteinsto SubmitochondrialCompartments 561 T a r g e t i n go f C h l o r o p l a sSt t r o m a lP r o t e i n sl s S i m i l a rt o lmport of MitochondrialMatrix Proteins 565 ProteinsAre Targetedto Thylakoidsby Mechanisms Relatedto TranslocationAcrossthe Bacterial I n n e rM e m b r a n e 565 xxvi

s68

549

Sortingof Proteinsto Mitochondria 557

E!|

P e r o x i s o m aM l e m b r a n ea n d M a t r i x P r o t e i n sA r e Incorporated by Different Pathways

Most mRNAsAre Exportedfrom the Nucleusby a R a n - l n d e p e n d e nMt e c h a n i s m

Pr o t e i nMo d i fi ca ti o n s, F o l d i n g, and Quality Control in the ER

Sortingof Peroxisomal Proteins sG7

CytosolicReceptorTargetsProteinswith an SKL Sequenceat the C-Terminusinto the Peroxisomal Matrix

SeveralTopologicalClasses of Integral Membrane ProteinsAre Synthesizedon the ER

s[

s[

@

M olecularMechanisms of Vesicular Traffic

Assembryof a protein coat DrivesVesicle F o r m a t i o na n d S e l e c t i o no f C a r g o Molecules A ConservedSet of GTPaseSwitch proteinsControls Assemblyof Different VesicleCoats T a r g e t i n gS e q u e n c e os n C a r g o p r o t e i n sM a k e S p e c i f i cM o l e c u l a rC o n t a c t sw i t h C o a t Proteins

585

5g6 5g7

588

Rab GTPases Control Dockingof Vesicleson Target Membranes PairedSetsof SNAREproteinsMediate Fusionof Vesicleswith Target Membranes

591

Dissociationof SNAREComplexesAfter Membrane F u s i o nl s D r i v e nb y A T PH y d r o l y s i s

5g1

589

@

EarlyStagesof the Secretory Pathway

592

COPIIVesiclesMediate Transportfrom the ER to the Golgi COPIVesiclesMediate RetrogradeTransportwithin t h e G o l g ia n d f r o m t h e G o l g i t o t h e E R

ss4

AnterogradeTransportThrough the Golgi Occurs by CisternalMaturation

595

@

Later stages of the secretory Pathway

597

VesiclesCoatedwith Clathrinand/or Adapter Proteins 598 Mediate SeveralTransportSteps

l: SIGNAL 1 5 C E L LS I G N A L I N G AND SHORT- T ER M TRANSDUCTION 623 RESPONSES CELLULAR [[

Signalto cellular FromExtracellular 625 Response

Signaling S i g n a l i n gC e l l sP r o d u c ea n d R e l e a s e Molecules

625

SignalingMoleculesCan Act Locallyor at a Distance

625

B i n d i n go f S i g n a l i n gM o l e c u l e sA c t i v a t e s Receptorson Target Cells

626

D y n a m i nl s R e q u i r e df o r P i n c h i n gO f f o f C l a t h r i n Vesicles

599

$[

Mannose6-PhosphateResiduesTargetSoluble Proteinsto Lysosomes

600

ReceptorProteinsBind LigandsSpecifically

627

RevealedKey Study of LysosomalStorageDiseases Componentsof the LysosomalSorting Pathway

602

The DissociationConstantls a Measureof the Affinity of a Receptorfor lts Ligand

628

ProteinAggregation in the trans-GolgiMay Function in Sorting Proteinsto RegulatedSecretoryVesicles 602

BindingAssaysAre Usedto Detect Receptorsand DetermineTheir Affinities for Ligands

628

Some ProteinsUndergo ProteolyticProcessing After Leavingthe trans-Golgi

603

SeveralPathwaysSort Membrane Proteinsto the Apical or BasolateralRegionof PolarizedCells

to a SignalingMolecule MaximalCellularResponse UsuallyDoesNot RequireActivationof All Receptors

629

604

Sensitivityof a Cellto ExternalSignalsls Determined by the Number of SurfaceReceptorsand Their 631 Affinity for Ligand

Receptor-Mediated Endocytosis 606

631 ReceptorsCan Be Purifiedby Affinity Techniques ReceptorsAre FrequentlyExpressedfrom Cloned Genes 6 3 1

!f|

CellsTake Up Lipidsfrom the Blood in the Form of Large,Well-Defined LipoproteinComplexes

608

The Acidic pH of Late EndosomesCausesMost Receptor-Ligand Complexesto Dissociate

610

[!|

DirectingMembraneProteins and CytosolicMaterialsto the Lysosome

MultivesicularEndosomesSegregateMembrane ProteinsDestinedfor the Lysosomal Membranefrom ProteinsDestinedfor LysosomalDegradation

627

606

Receptorsfor Low-DensityLipoproteinand Other LigandsContain Sorting SignalsThat Target Them for Endocytosis

The EndocyticPathwayDeliverslron to Cellswithout Complex Dissociationof the Receptor-Transferrin in Endosomes

studying cell-Surface Receptors

611

Highly ConservedComPonents Signal-Transduction of Intracellular 632 Pathways GTP-BindingProteinsAre FrequentlyUsedAs On/Off Switches are Employedin Protein Kinasesand Phosphatases Virtually All SignalingPathways Carryand Amplify Signalsfrom SecondMessengers Many Receptors

612

612

RetrovirusesBud from the PlasmaMembrane by a Process Endosomes 614 Similarto Formationof Multivesicular

14.1 Following a Protein Cnsstc ExprRturruT 621 out of thecell

s!|

633 634 634

of G ProteinGeneralElements 535 SYstems RecePtor Coupled

G Protein-CoupledReceptorsAre a Largeand Diverse 635 Familywith a Common Structureand Function G Protein-CoupledReceptorsActivate Exchangeof G T Pf o r G D Po n t h e a S u b u n i to f a T r i m e r i cG 637 Protein Different G ProteinsAre Activated by Different GPCRs 539 and ln Turn RegulateDifferent EffectorProteins

CONTENTS

.

xxvii

G Protein-Coupled Receptors T h a t R eg u l a tel o n C h a n n e l s

640

AcetylcholineReceptorsin the Heart Muscle Activate a G ProteinThat OpensK+ Channels

641

Light ActivatesG..-CoupledRhodopsins

641

Activationof RhodopsinInducesClosingof cGMP-GatedCation Channels

642

[f|

Rod CellsAdapt to Varying Levelsof Ambient Light Becauseof Opsin Phosphorylationand Binding of Arrestin

644

G Protein-GoupledReceptorsThat Activateor Inhibit Adenylyl Cyclase 646

$!|

Adenylyl Cyclasels Stimulatedand Inhibited by Different Receptor-Ligand Complexes

646

StructuralStudiesEstablishedHow G,,.GTpBinds to and ActivatesAdenylyl Cyclase

646

cAMP ActivatesProtein KinaseA by Releasing CatalyticSubunits

547

GlycogenMetabolismls Regulatedby Hormone-lnducedActivation of Protein KinaseA

648

cAMP-MediatedActivation of Protein KinaseA ProducesDiverseResponses in Different Cell Types

649

S i g n a lA m p l i f i c a t i o nC o m m o n l yO c c u r si n M a n y SignalingPathways

550

S e v e r aM l e c h a n i s mD s o w n - R e g u l a tS e ignaling from G Protein-CoupledReceptors

651

Anchoring ProteinsLocalizeEffectsof cAMp to SpecificRegionsof the Cell

ActivatePhospholipase C

PhosphorylatedDerivativesof InositolAre lmportant SecondMessengers C a l c i u ml o n R e l e a s e from the Endoplasmic Reticulumis Triggeredby lp3 The Ca2*/Calmodulin ComplexMediatesMany CellularResponses to ExternalSignals Diacylglycerol(DAG)Activatesprotein KinaseC, Which RegulatesMany Other proteins Signal-lnducedRelaxationof VascularSmooth Musclels Mediated by cGMp-Activated Protein KinaseG

Clnsstc ExprnlveruT 15.1 TheInfancy of signal Transduction-GTPStimulationof cAMP Synthesis 663

15 CELL-SIGNALIN l lG : SIGNALING PATHWAYSTHAT C O N T R O L GENE ACTIVITY 66s

sl|

IntegratingResponses of cells t o En v i ro n me n taInl fl u e n ce s

'

CONTENTS

668 668

RadioactiveTaggingWas Usedto ldentify TGFp Receptors

669

ActivatedTGFBReceptorsPhosphorylateSmad TranscriptionFactors

670

NegativeFeedbackLoopsRegulateTGFB/Smad Signaling

671

Lossof TGFBSignalingPlaysa Key Role in Cancer

671

sf|

cytokineReceptors andthe JAK/STAT Pathway

672 672

CytokineReceptorsHaveSimilarStructuresand Activate SimilarSignalingPathways

673

JAK KinasesActivate STATTranscriptionFactors

674

654

ComplementationGeneticsRevealedThat JAK and STATProteinsTransduceCytokine Signals

677

654

Signalingfrom CytokineReceptorsls Regulated by NegativeSignals

678

655

Mutant ErythropoietinReceptorThat Cannot Be TurnedOff Leadsto IncreasedNumbersof Erythrocytes

679

553

6s6

656

657

Integrationof Multiple SecondMessengersRegulates Glycogenolysis 557

XXVIII

Activation of Smads

A TGFBSignalingMoleculels Formedby Cleavage of an InactivePrecursor

ReceprorTyrosrneKrnases

679

L i g a n dB i n d i n gL e a d st o P h o s p h o r y l a t i oann d Activation of IntrinsicKinasein RTKs

680

Overexpression of HER2,a Receptor TyrosineKinase,Occursin Some Breast Cancers

680

s[ $f|

TGFFReceptors and the Direct

CytokinesInfluenceDevelopmentof Many Cell Types

G Protein-coupled Receptors That

sfl

I n s u l i na n d G l u c a g o nW o r k T o g e t h e rt o M a i n t a i na StableBlood GlucoseLevel

HedgehogSignalingRelievesRepressionof Target Genes

D o m a i n sA r e l m p o r t a n tf o r B i n d i n gS i g n a l Conserved TransductionProteinsto ActivatedReceptors Down-regulationof RTKSignalingOccursby Endocytosis and LysosomalDegradation

683

@

700

PathwaysThat InvolveSignal - l nduc ed 703 ProteinCleavage

o f R a sa n d MA P K i n ase @ Act i v a t i o n 584 Pathways

Degradationof an Inhibitor ProteinActivatesthe NF-rBTranscriptionFactors

703

Ras,a GTPaseSwitch Protein,CyclesBetweenActive and InactiveStates

68s

Ligand-ActivatedNotch ls CleavedTwice,Releasing a TranscriptionFactor

705

ReceptorTyrosineKinasesAre Linkedto Rasby Adapter Proteins

685

GeneticStudiesin Drosophilaldentified Key Proteinsin the Ras/MAP Signal-Transducing KinasePathway

685

Binding of SosProteinto InactiveRasCausesa ConformationalChangeThat ActivatesRas

587

SignalsPassfrom Activated Rasto a Cascadeof Protein Kinases

688

MAP KinaseRegulatesthe Activity of Many Transcription 690 Genes FactorsControlling Early-Response G Protein-CoupledReceptorsTransmitSignalsto MAP Kinasein YeastMating Pathways

691

ScaffoldProteinsSeparateMultiple MAP Kinase Pathwaysin EukaryoticCells

692

The Ras/MAPKinasePathwayCan InduceDiverse C e l l u l a rR e s p o n s e s

693

sf|

Phosphoinositides as signal Transducers

Phospholipase C" ls Activatedby SomeRTKsand CytokineReceptors

694

6e4

Recruitmentof Pl-3 Kinaseto Hormone-Stimulated ReceptorsLeadsto Synthesisof Phosphorylated Phosphatidyl inositols in the Plasma Accumulationof Pl 3-Phosphates Membrane Leadsto Activation of SeveralKinases 695 Activated Protein KinaseB InducesMany CellularResponses

696

The Pl-3KinasePathwayls NegativelyRegulated by PTENPhosphatase

s!|

698

Arrestin ActivatesSeveralKinaseCascades 698 GPCR-Bound Wnt SignalsTrigger Releaseof a Transcription Factorfrom CytosolicProteinComplex

RegulatedIntramembraneProteolysisof SREBP a TranscriptionFactorThat Acts to Releases Maintain Phospholipidand CholesterolLevels

1 7 C E L LO R G A N I Z A T I OANN D 713 M O V E M E N Tl : M I C R O F I L A M E N T S

@

and Actin Microfilaments Structures

715

Actin ls Ancient,Abundant, and Highly Conserved G-ActinMonomersAssembleinto Long, Helical F-ActinPolymers

717

F-ActinHasStructuraland FunctionalPolarity

718

@

Dynamicsof Actin Filaments

717

718

Actin Polymerizationin Vitro Proceedsin Three Steps 7 1 9 Actin FilamentsGrow Fasterat (+) EndsThan at 720 (-) Ends Actin FilamentTreadmillingls Acceleratedby P r o f i l i na n d C o f i l i n Providesa Reservoirof Actin for Thymosin-B4 Polymerization CappingProteinsBlockAssemblyand Disassembly a t A c t i n F i l a m e n tE n d s

Activationof GeneTranscription Cell-Surface by Seven-spanning 697 Receptors

CREBLinkscAMP and Protein KinaseA to Activation of GeneTranscription

CatalyzeCleavageof Many Matrix Metalloproteases 706 S i g n a l i n gP r o t e i n sf r o m t h e C e l lS u r f a c e InappropriateCleavageof Amyloid PrecursorProtein Can Leadto Alzheimer'sDisease

@

of Actin Filament Mechanisms AssemblY

F o r m i n sA s s e m b l eU n b r a n c h e dF i l a m e n t s The Arp2/3 ComplexNucleatesBranchedFilament Assembly lntracellularMovementsCan Be Poweredby Actin Polymerization

CONTENTS

721 722 722

723 723 724 726 xxix

ToxinsThat Perturbthe Pool of Actin Monomers Are Usefulfor StudyingActin Dynamics

726

ChemotacticGradientsInduceAltered Phosphoinositide LevelsBetweenthe Front and Backof a Cell 750

Cnsstc ExpenlvlrruT 17.1 Looking at Muscle

o r g a n i z a ti o no f A cti n -B a secellular d Structures 728

@

C r o s s - L i n k i nPgr o t e i n sO r g a n i z eA c t i n F i l a m e n t si n t o Bundlesor Networks

728

Adaptor ProteinsLink Actin Filamentsto Membranes

728

Myosins: Actin-Based Motor Proteins

fifl

Contraction

755

1 8 C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E S AND INTERMEDIATE FILAM EN T S 757 [[

731

Micr otubuleStr uctur eand Or ganization

758

732

MicrotubuleWalls Are PolarizedStructuresBuilt f r o m a B - T u b u l i nD i m e r s

758

M y o s i n sM a k e U p a L a r g eF a m i l yo f MechanochemicalMotor proteins

733

MicrotubulesAre Assembledfrom MTOCsto GenerateDiverseOrganizations

760

C o n f o r m a t i o n aCl h a n g e si n t h e M y o s i nH e a d CoupleATPHydrolysisto Movement

736

Myosin HeadsTake DiscreteStepsAlong Actin Filaments

736

MyosinV Walks Hand Over Hand Down an Actin Filament

MicrotubulesAre DynamicStructuresDue to Kinetic Differencesat Their Ends

763

737

I n d i v i d u aM l i c r o t u b u l e sE x h i b i tD y n a m i cI n s t a bIi t y

763

LocaIized A s s e m b l ya n d " S e a r c h - a n d - C a p t u r e " H e l p O r g a n i z eM i c r o t u b u l e s

766

DrugsAffecting Tubulin PolymerizationAre Useful Experimentallyand to Treat Diseases

766

M y o s i n sH a v eH e a d ,N e c k ,a n d T a i l D o m a i n sw i t h DistinctFunctions

Myosin-poweredMovements

@

M y o s i nT h i c kF i l a m e n t sa n d A c t i n T h i n F i l a m e n t s i n S k e l e t aM l u s c l eS l i d eP a s tO n e A n o t h e r D u r i n gC o n t r a c t i o n

738

ftf|

738

[f!

S k e l e t aM l u s c l el s S t r u c t u r e db y S t a b i l i z i n ga n d ScaffoldingProteins

740

Contractionof SkeletalMusclels Regulatedby Ca2* a n d A c t i n - B i n d i n gP r o t e i n s

740

Actin and Myosin ll Form ContractileBundlesin N o n m u s c l eC e l l s

741 Myosin-DependentMechanismsRegulate C o n t r a c t i o ni n S m o o t hM u s c l ea n d N o n m u s c l eC e l l s 742 Myosin-V-Bound VesiclesAre CarriedAlong Actin Filaments 743

Microtubule Dynamics

762

Regulationof MicrotubuleStructure and Dynam ics 767

MicrotubulesAre Stabilizedby Side-and End-Binding Proteins

767

M i c r o t u b u l e sA r e D i s a s s e m b l ebdy E n d B i n d i n g and SeveringProteins

768

@

Kinesinsand Dyneins:Micr otubul eBasedMotor Proteins 769

Organellesin AxonsAre TransportedAlong Microtubulesin Both Directions

769

Kinesin-1PowersAnterogradeTransportof VesiclesDown Axons Toward the (+) End of Microtubules

770

745

KinesinsForm a LargeProtein Familywith Diverse Functions

771

747

Kinesin-l ls a Highly Processive Motor

772

C e l lM i g r a t i o nI n v o l v e st h e C o o r d i n a t eR e g u l a t i o n of Cdc42,Rac,and Rho

748

Dynein Motors TransportOrganellesTowardthe (-) E n do f M i c r o t u b u l e s

774

Migrating CellsAre Steeredby Chemotactic Molecules

750

Kinesinsand DyneinsCooperatein the Transport o f O r g a n e l l eT s hroughout he Cell

715

c e l l M i g r a t i o ns: i g n a l i n ga n d Chemotaxis

@

C e l lM i g r a t i o nC o o r d i n a t e F s o r c eG e n e r a t i o nw i t h C e l lA d h e s i o na n d M e m b r a n eR e c y c l i n g The SmallGTP-BindingProteinsCdc42,Rac,and Rho Control Actin Organization

XXX

.

CONTENTS

745

fifl

Ciliaand Flagella: Microtubule777 BasedSurfaceStructures

E u k a r y o t i cC i l i aa n d F l a g e l l aC o n t a i nL o n g D o u b l e t MicrotubulesBridged by Dynein Motors

777

r e a t i n gA r e P r o d u c e db y C i l i a r ya n d F l a g e l l a B C o n t r o l l e dS l i d i n go f O u t e r D o u b l e t Microtubules

778

IntraflagellarTransportMoves Material Up and D o w n C i l i aa n d F l a g e l l a

779

Defectsin IntraflagellarTransportCauseDisease by Affecting SensoryPrimaryCilia

780

Microfilamentsand MicrotubulesCooperateto TransportMelanosomes Cdc42Coordi nates M icrotubules and M icrofi laments D u r i n g C e l lM i g r a t i o n

1 9 I N T E G R A T I NCGE L L SI N T O TISSUES

$[

797

801

cell- celland cell- Matr ixAdhes i on: 803 An Overview

C e l l - A d h e s i oM n o l e c u l e sB i n dt o O n e A n o t h e r a n d t o I n t r a c e l l u l aPr r o t e i n s

803

782

805

C e n t r o s o m eD s u p l i c a t eE a r l yi n t h e C e l lC y c l ei n Preparationfor Mitosis

The ExtracellularMatrix Participatesin Adhesion, S i g n a l i n ga, n d O t h e r F u n c t i o n s

783

807

of The Mitotic SpindleContainsThree Classes Microtubules

The Evolutionof MultifacetedAdhesionMolecules Enabledthe Evolutionof DiverseAnimal Tissues

784

M i c r o t u b u l eD y n a m i c sI n c r e a s eDs r a m a t i c a l l y in Mitosis

784

$!|

Mitosis

MitosisCan Be Dividedinto Six Phases

781

l u r i n gM i t o s i s M i c r o t u b u l e sT r e a d m i l D

785

The KinetochoreCapturesand HelpsTransport Chromosomes

786

DuplicatedChromosomesAre Aligned by Motors a n d T r e a d m i l l i n gM i c r o t u b u l e s

788

to Polesby AnaphaseA MovesChromosomes Microtubule Shortening

789

AnaphaseB SeparatesPolesby the Combined A c t i o n o f K i n e s i n sa n d D y n e i n

789

s o n t r i b u t et o S D i n d l e A d d i t i o n a lM e c h a n i s mC Formation

789

CytokinesisSplitsthe DuplicatedCell in Two

789

e h e i r M i c r o t u b u l e sa n d P l a n tC e l l sR e o r g a n i z T B u i l da N e w C e l l W a l li n M i t o s i s

790

$fl

IntermediateFilaments

791

@

Junctio ns cell- celland celI- ECM and TheirAdhesionMolecules 808

, nd t p i c a l ,L a t e r a l a E p i t h e l i aC l e l l sH a v eD i s t i n c A BasalSurfaces

808

ThreeTypesof JunctionsMediate Many Cell-Cell and Cell-ECMInteractions

809

l l d h e s i o n si n A d h e r e n s C a d h e r i n sM e d i a t eC e l l - C e A J u n c t i o n sa n d D e s m o s o m e s

810

Tight JunctionsSealOff Body Cavitiesand Restrict D i f f u s i o no f M e m b r a n eC o m p o n e n t s

814

Adhesionsin EpithelialCells 816 IntegrinsMediateCelI-ECM s llow Small G a p J u n c t i o n sC o m p o s e do f C o n n e x i n A Moleculesto PassDirectlyBetweenAdjacent Cells 817

M atr ix l: The Extr acellular The BasalLamina

820

l a m i n aP r o v i d e sa F o u n d a t i o nf o r The BasaL Assemblyof Cellsinto Tissues

820 821

IntermediateFilamentsAre Assembledfrom S u b u n i tD i m e r s

792

M a t r i x P r o t e i n ,H e l p s L a m i n i n ,a M u l t i a d h e s i v e Cross-linkComponentsof the BasalLamina

Intermediate FilamentsProteins Are Expressed Manner in a Tissue-Specific

792

Type lV Collagenls a Major Structural Sheet-Forming 821 l amina C o m p o n e n to f t h e B a s a L

I n t e r m e d i a t eF i l a m e n t sA r e D y n a m i c

795

Defectsin Laminsand KeratinsCauseManv Diseases 795

[[

coordinationand cooperation between CytoskeletalElements 796

Proteins lntermediateFilament-Associated C o n t r i b u t et o C e l l u l a rO r g a n i z a t i o n

796

Components Perlecan,a Proteoglycan,Cross-links Receptors of the BasalLaminaand Cell-Surface

@The

Mra t r i xl l : Extracellula Connectiveand Other Tissues

s r e t h e M a j o r F i b r o u sP r o t e i n s F i b r i l l a rC o l l a g e n A in the ECMof ConnectiveTissues

CONTENTS

824

825 825

XXXI

F i b r i l l a rC o l l a g e nl s S e c r e t e da n d A s s e m b l e di n t o FibrilsOutsideof the Cell

926

ConnectionsBetweenthe ECMand Cytoskeleton Are Defectivein MuscularDystrophy

835

T y p eI a n d l l C o l l a g e n A s ssociate with Nonfibrillar Collagensto Form DiverseStructures

826

l g C A M sM e d i a t eC e l l - C e l l A d h e s i oi n N e u r o n a l and Other Tissues

836

LeukocyteMovement into Tissuesls Orchestrated by a Precisely Timed Sequenceof Adhesive Interactions

837

Proteoglycans and Their ConstituentGAGsPlay DiverseRolesin the ECM

827

HyaluronanResists Compression, Facilitates Cell Migration, and GivesCartilagelts Gel-likeProperties 829 FibronectinsInterconnectCellsand Matrix, Influencing Cell Shape,Differentiation,and Movement 830

AdhesiveInteractionsin Motile a n d N o n mo ti l eC e l l s

[[

I n t e g r i n sR e l a yS i g n a l sB e t w e e nC e l l sa n d T h e i r T h r e e - D i m e n s i o nE an l vironment

p[

PtantTissues

839

T h e P l a n tC e l lW a l l l s a L a m i n a t eo f C e l l u l o s eF i b r i l s in a Matrix of Glycoproteins

833 833

Regulationof Integrin-MediatedAdhesionand S i g n a l i n gC o n t r o l sC e l l M o v e m e n t

Looseningof the Cell Wall PermitsPlant Cell Growth

840

Plasmodesmata DirectlyConnectthe Cytosolsof A d j a c e n tC e l l si n H i g h e rP l a n t s Only a Few AdhesiveMoleculesHave Been ldentified in Plants

841

PartlV CellGrowth and Development 20 R E G U L AT IN T GH E E U K A R Y OT IC C E L LC Y C L E 847 Overviewof the CellCycle and lts Control

E[

849

The Cell Cyclels an Ordered Seriesof Events L e a d i n gt o C e l lR e p l i c a t i o n

849

RegulatedProtein Phosphorylationand DegradationControl Passage Through the Cell Cycle

849

DiverseExperimentalSystemsHave Been Usedto ldentify and lsolateCell-Cycle Control proteins

851

MPF Activity

853

Maturation-PromotingFactor(MpF)Stimulates Meiotic Maturation in Oocytesand Mitosis i n S o m a t i cC e l l s

854

Mitotic CyclinWas Firstldentified in EarlvSea U r c h i nE m b r y o s

856

CyclinB Levelsand KinaseActivity of MitosisPromoting Factor(MPF)ChangeTogetherin CyclingXenopusEgg Extracts Anaphase-Promoting Complex(APC/C)Controls Degradationof Mitotic Cyclinsand Exit from Mitosis

.

CONTENTS

8s6

8s8

Cyclin-Dependent KinaseRegulation Dur ingMitosis 8s9

MPFComponentsAre ConservedBetween Lower and Higher Eukaryotes

860

Phosphorylationof the CDKSubunit Regulatesthe KinaseActivity of MPF

851

ConformationalChangesInducedby Cyclin Binding and PhosphorylationIncrease MPFActivity

862

@

Controlof Mitosisby Cyclinsand

@

![fl

MolecularMechanisms for Regulating Mitotic Events

864

Phosphorylationof NuclearLaminsand Other ProteinsPromotesEarlyMitotic Events

864

U n l i n k i n go f S i s t e rC h r o m a t i d sI n i t i a t e sA n a p h a s e

867

ChromosomeDecondensationand Reassembly of the NuclearEnvelopeDependon Dephosphorylation of MPFSubstrates

870

!!f|

Cyclin-cDK and Ubiquitin-protein Ligase Control of S phase

872

A Cyclin-Dependent Kinase(CDK)ls Criticalfor S-PhaseEntry in S. cerevisiae

872

Three G1CyclinsAssociatewith 5. cerevrsrae CDK to Form S-Phase-Promoting Factors

874

Degradationof the S-Phase Inhibitor TriggersDNA Replication Multiple CyclinsRegulatethe KinaseActivity of 5. cerevisiaeCDK During Different Cell-CyclePhases 877 Replication a t E a c hO r i g i n l s I n i t i a t e dO n l y O n c e During the Cell Cycle

!fil

877 |ffirhe

cell-cyclecontrolin Mammalian Cells

2 1 C E L LB I R T H L, I N E A G EA, N D DEATH

879

90s

Birthof cells:Stemcells, 905

N i c h e s ,a n d L i n e a g e

Stem CellsGive Riseto Both Stem Cellsand DifferentiatingCells

906

RestrictedDuring Cell FatesAre Progressively Development

907

The CompleteCell Lineageof C. elegansls Known

908

M a m m a l i a nR e s t r i c t i o n P o i n t l s A n a l o g o u st o STARTin Yeast Cells

880

Multiple CDKsand CyclinsRegulatePassage of M a m m a l i a nC e l l sT h r o u g ht h e C e l lC y c l e

881

HeterochronicMutants ProvideCluesAbout Control of Cell Lineage

909

RegulatedExpression of Two Classes of Genes R e t u r n sG eM a m m a l i a nC e l l st o t h e C e l lC y c l e

881

Cultured EmbryonicStem CellsCan Differentiateinto V a r i o u sC e l lT y p e s

911

Passage Through the RestrictionPoint Dependson Phosphorylationof the Tumor-Suppressor Rb Protein 882

Adult Stem Cellsfor Different Animal TissuesOccupy 912 S u s t a i n i n gN i c h e s

C y c l i nA l s R e q u i r e df o r D N A S y n t h e s iasn d C D K 1 for Entry into Mitosis

883

MeristemsAre Nichesfor Stem Cellsin Postnatal Plants

Two Typesof Cyclin-CDKInhibitorsContributeto Cell-Cycle Control in Mammals

883

@

!![

in cell-Cycle Checkpoints Regulation

The Presenceof UnreplicatedDNA PreventsEntry into Mitosis lmproper Assemblyof the Mitotic SpindlePrevents the Initiation of Anaphase ProperSegregationof Daughter Chromosomesls Monitored by the Mitotic Exit Network

884 888

92O

cell-Typespecificationin Yeast

921

Mati ng-TypeTranscription FactorsSpecify CellTypes

922

MCMl and a1-MCM1ComplexesActivate Gene Transcription

923

a2-MCMI and cr2-a1ComplexesRepressTranscription 923 888

PheromonesInduceMating of ct and a Cellsto G e n e r a t ea T h i r d C e l l T Y P e

923

889

Arrest of Cellswith DamagedDNA Depends Cell-Cycle 891 on Tumor Suppressors

!!|

and Differentiation Specification 924 of Muscle

EmbryonicSomitesGive Riseto Myoblasts

925

MyogenicGenesWere Firstldentified in Studieswith Cultured Fibroblasts

925

Two Classesof Regulatory FactorsAct in Concert to Guide Productionof MuscleCells

926

CohesinSubunit Recombinationand a Meiosis-Specific for the SpecializedChromosome Are Necessary 895 Segregationin Meiosis|

Differentiationof Myoblastsls Under Positiveand NegativeControl

927

Cell-CellSignalsAre Crucialfor Determinationand Migration of Myoblasts

928

SpecialPropertiesof Rec8Regulatelts Cleavagein M e i o s i sI a n d l l

896

bHLH RegulatoryProteinsFunctionin Creationof Other Tissues

929

The Monopolin ComplexCo-orientsSister Kinetochoresin Meiosis|

898

Tensionon SpindleMicrotubulesContributesto ProperSpindleAttachment

898

!![

Meiosis:A specialTypeof cell Division

Key FeaturesDistinguishMeiosisfrom Mitosis

892 892

Protein of G1Cyclinsand a Meiosis-Specific Repression 895 KinasePromote Premeiotic5 Phase

Emerging 20.1 cellBiology Cnsstc ExpenlueruT from the Sea:The Discoveryof Cyclins

903

@

Regulationof Asymmetric CellDivision

930

YeastMating-TypeSwitchingDependsupon AsymmetricCell Division

CONTENTS

930 '

xxxiii

ProteinsThat RegulateAsymmetryAre Localizedat OppositeEndsof Dividing Neuroblastsin Drosophila 9 3 1

cell Deathand tts Regulation

!@

936

ProgrammedCell Death OccursThrough Apoptosis Neurotrophinspromote Survivalof Neurons

937

A Cascadeof CaspaseProteinsFunctionsin One Apoptotic Pathway

938

Pro-ApoptoticRegulatorsPermit CaspaseActivation in the Absenceof TrophicFactors

941

SomeTrophicFactorsInduceInactivationof a Pro-ApoptoticRegulator

942

Tumor NecrosisFactorand RelatedDeath Signals PromoteCell Murder by Activating Caspases

943

937

2 2 T H EM O L E C U L ACRE L LB I O L O G Y O FD E V E L O P M E N T 949

Controlof Body Segmentation: Themesand Variationsin Insects and Vertebrates 969 Early Drosophila Development ls an Exercisein Speed 970 TranscriptionalControl Specifiesthe Embryo's Anterior and Posterior

971

TranslationInhibitorsReinforceAnterior-Posterior Patterning

973

InsectSegmentationls Controlledby a Cascade of Transcription Factors

974

VertebrateSegmentationls Controlledby Cyclical Expression of RegulatoryGenes

977

DifferencesBetweenSegmentsAre Controlledby Hox Genes

978

Hox-GeneExpressionls Maintained by a Variety of Mechanisms

982

Flower DevelopmentRequiresSpatiallyRegulated Productionof TranscriptionFactors

983

!![ H i g h l i g h tso f D e ve l o p me n t

@

DevelopmentProgresses from Egg and Sperm to an EarlyEmbryo

As the EmbryoDevelops,Cell LayersBecomeTissues and Organs 951 GenesThat RegulateDevelopmentAre at the Heart of Evolution

Neural Development

985

N e u r u l a t i o nB e g i n sF o r m a t i o no f t h e B r a i na n d SpinalCord

986

950 9s0

952

cell-TypeSpecification in Early

SignalGradientsand TranscriptionFactorsSpecifyCell Typesin the NeuralTube and Somites 987 Most Neuronsin the BrainArise in the Innermost NeuralTube and Migrate Outward

988

LateralInhibition Mediated by Notch Signaling CausesEarlyNeural Cellsto BecomeDifferent

988

Gametogenesis and Fertilization g53

@

Growth and Patterningof Limbs 990

G e r m - l i n eC e l l sA r e A l l T h a t W e I n h e r i t

953

!![

FertilizationUnifiesthe Genome

955

Hox GenesDeterminethe Right Placesfor Limbsto Grow

990

958

Limb DevelopmentDependson Integration o f M u l t i p l e E x t r a c e l l u l aSri g n a lG r a d i e n t s

991

958

Hox GenesAlso Control Fine Patterning of Limb Structures

992

So Fa[ So Good

994

Genomiclmprinting ControlsGene Activation Accordingto Maternal or PaternalChromosome Origin Too Much of a Good Thing: The X Chromosomels Regulatedby DosageCompensation

cell Diversityand patterning

!f,

in EarlyVertebrateEmbryos

CleavageLeadsto the FirstDifferentiationEvents The Genomesof Most SomaticCellsAre Complete

Cussrc ExprRlueruT 22.1 UsingLethal

961 961

SignalGradientsMay InduceDifferent Cell Fates

963

SignalAntagonistsInfluenceCell Fatesand Tissue Induction

965

A Cascadeof SignalsDistinguishes Left from Right

956

.

coNTENTs

999

960

GastrulationCreatesMultiple TissueLayers,Which BecomePolarized

xxxiv

Mutationsto Study Development

959

23 NERVE CELLS !fl

Neurons andGlia:Building Blocksof the NervousSystem

1001

lOOz

Information FlowsThrough Neuronsfrom Dendrites to Axons 1003

NXXX

.

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u o ll) u n l p u e a r n lr n r ls :su lln q o l6 0 u nuul

SpecificMutations TransformCulturedCells into Tumor Cells

1113

A Multi-hit Model of CancerInduction ls Supported by SeveralLinesof Evidence

1114

Successive OncogenicMutations Can Be Traced in Colon Cancers

1116

PatternsCan DNA MicroarrayAnalysisof Expression RevealSubtle DifferencesBetweenTumor Cells

1116

@ Th e

G e ne ti cB a si so f ca n ce r

1119

Gai n-of-FunctionM utationsConvertProto-oncogenes 1119 into Oncogenes VirusesContainOncogenesor Cancer-Causing Activate CellularProto-oncogenes

1121

Mutations in Tumor-Suppressor Loss-of-Function GenesAre Oncogenic

1123

Genes Inherited Mutations in Tumor-Suppressor lncreaseCancerRisk

1123

Aberrationsin SignalingPathwaysThat Control DevelopmentAre Associatedwith Many Cancers 1124

!fl

oncogenicMutationsin GrowthPromoting Proteins

1127

OncogenicReceptorsCan Promote Proliferationin the Absenceof External Growth Factors

1127

Viral Activators of Growth-Factor ReceptorsAct as Oncoproteins

1128

Many OncogenesEncodeConstitutivelyActive Proteins Sig nal-Transduction

1129

InappropriateProductionof NuclearTranscription FactorsCan InduceTransformation

11 3 0

M o l e c u l a rC e l lB i o l o g yl s C h a n g i n gH o w C a n c e r ls Treated

1132

MutationsCausingLossof and Gr owth- lnhibiting Controls Cell-Cycle

1134

from MutationsThat Promote UnregulatedPassage Gr to 5 PhaseAre Oncogenic

1134

MutationsAffecting ChromatinLoss-of-Function RemodelingProteinsContributeto Tumors

1135

Lossof p53 Abolishesthe DNA-DamageCheckpoint 1136 Apoptotic GenesCan Functionas Proto-oncogenes 1137 Genes or Tumor-Suppressor CheckpointsOften Leadsto Failureof Cell-Cycle T u m o rC e l l s i n Aneuploidy

1138

and CaretakerGenes Carcinogens 11 3 9 in Cancer CarcinogensInduceCancerby DamagingDNA

11 3 9

SomeCarcinogensHave Been Linkedto Specific Cancers

1139

Lossof DNA-RepairSystemsCan Leadto Cancer

1141

Contributesto TelomeraseExpression lmmortalizationof CancerCells

1143

GLOSSARY

G-1 l-1

INDEX

CONTENTS

.

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'sJaJrloJpue seqeue 'so;n1lnl1s to sarloru ur eesuef a.rl sE Sur8ueqc-lseJe^eq pue dlprder e^ou aruos '(1-1 arn8r4) sadeqs pup sezrs lo f,lBrrcr, Surzeute ue ur otuoJ sllaJ

slla) Io f1r;euourruo)pue Alrsla^lqaql lU 's11arSurdpnts sdp.^a snorrel ar{l ruo{ urpel uef, to aA\ let{,ryrpup (suorlf,unJIeJrtrrr pup sluenlrlsuoJ Jrsgq Jroql 's11ec dlrs.rallp aqt Surcnporrurdq rerdeqr anSolord srql ;o ur truls elrN'slleJ rltpJp pue 'ayr1'qrrrg aqr 1o ,{lors petJceJ Jo -ulnru oqt 3ur,teo,ndgenper8 'splaryasegt to IIE r.uoryu,ry\pJp saqreo.rddelpluorurrodxapue slq8rsureqrrJsep11r,ue,tt 'sral -deqc SurrnolloJer{r uI 'uorletuour.radxa;o a1d1spue srseqd ',(8o1orq -rua u^\o slr spq splort eseql Jo qre1 leluarudolo,r -ep pue'eruerf,s relndruor tSolorsdqd'slrlaue8 1(docsonrur iiSoyorqrplnf,oloru'sorsdqdorq'dtlsruaqcorqreqtoSol sBur.rq (qcr.r sr lSolorq r lerll erualrs a,rrlBr8elut [e] JEInJOI6W 'puodsa; o1 sdem eleudordde tsotu oqt uo aprrep daqr moq pue sllor q8no.rqrs^{olJuorler.uro;ur^/rl,oq lnoqe dlrelnrrued 'peureal eg ol sureruoJlunotup asuerururue '11115 'Joqlo qJEe aJuenlturpu€ qrnot dagr ,vroq'urEluoJ daqt sernlcnrls tEr{na 's11ac sluouodruor ar{t tnoqp Etep.ry\au;ouorsoldxaue aJe! 1o o.tr 'suado d.rntuec tsrrJ-.{tue,v\teqr sV 'aJII Jo lrun Ietueruep 'aJIIyo sartradord>lreurller{ -unt oql sr IIar eqr tpr{t SuneJrpul aqt IIe trqrqxo susrueS.rorulnlloJrun aldurrsua,lg '1ac epurs e Jo lsrsuof, sulsrue8rodueru 1nq 'salnlf,n.rlsxalduror olur pezrve8rc sllel Jo suor1lrrtro suorllq ureluor srusrueSrore1 -NIIE]qFI.U

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(b) Eukaryoticcell

( a ) P r o k a r y o t icce l l P e r i p l a s m iscp a c e a n d c e l lw a l l

Golgi vesicles

Lysosome

O u t e rm e m b r a n e

I n n e r( p l a s m a ) membrane

Nucleoid lu'btrm I

Nucleoid

N u c l e a rm e m b r a n e P l a s m am e m b r a n e Golgi vesicles Mitochondrion Peroxisome Lysosome

I n n e r( p l a s m a )m e m b r a n e

Periplasmicspace Outer membrane R o u g he n d o p l a s m i c reticulum

1-2 Prokaryotic cellshavea simplerinternal A FIGURE micrograph of a organizationthan eukaryoticcells.(a)Electron coli a commonintestinal bacteriumThe thin sectionof Escherichia withina of the bacterial DNA,is notenclosed nucleoid, consisting by two membraneE coli andsomeotherbacterra aresurrounded spaceThethincellwallis membranes separated bythe periplasmic (b)Electron mrcrograph of a plasma adjacent to the innermembrane antibodies Onlya single cell,a typeof whitebloodcellthatsecretes (theplasma membrane membrane) surrounds thecell,butthe manymembrane-limited compartments, or interior contains Thedefining of eukaryotic cellsis organelles. characteristic

whichis DNAwithina definednucleus, of thecellular segregation is membrane Theouternuclear by a doublemembrane bounded for factory a reticulum, withthe roughendoplasmic continuous process andmodifyproteins, proteinsGolgivesicles assembling to digestcellmaterials generate lysosomes energy, mitochondria and process usingoxygen, molecules them,peroxisomes recycle them to the surfaceto release carrycellmaterials vesicles secretory E M u r r a y . P a r t ( b ) fromPC G B u r d e t t a n d R D J lPart(a)courtesyofl lJltrastructure: A Functional 1993,CellandTissue Cross andK L Mercer, andCompanyl W H Freeman Perspective,

and 40 miles up in the atmosphere;they are quite adaptable! The carbon stored in bacteria is nearly as much as the carbon stored in plants. Eukaryotic cells, unlike prokaryotic cells, contain a defined membrane-bound nucleus and extensive internal membranes that enclose other compartments called organelles (Figure 1-2b). The region of the cell lying betweenthe plasma membrane and the nucleus is the cytoplasm, comprising the cytosol (aqueousphase)and the organelles.Eukaryotescompriseall membersof the plant and animal kingdoms,including

the fungi, which exist in both multicellular forms (molds) and unicellular forms (yeasts),and the protozoans (proto, primitive zoan, animal), which are exclusively unicellular. Eukaryotic cells are commonly about 10-100 pm across' generally much larger than bacteria. A typical human fibroblast, a connective tissuecell, might be about 15 p,m acrosswith a volume and dry weight some thousandsof times those of an E. coli bacterial cell. An ameba, a single-celledprotozoan, can be more than 0.5 mm long. An ostrich egg beginsas a singlecell that is evenlarger and is easilyvisible to the naked eye. THE DIVERSITA Y N D C O M M O N A L I T YO F C E L L S

All cells are thought to have evolved from a common progenitor becausethe structures and moleculesin all cells have so many similarities. In recent years, detailed analysis of the DNA sequencesfrom a variety of prokaryotic organisms has revealed two distinct types: the bacteria and the archaea. Working on rhe assumption that organisms with more similar genesevolvedfrom a common progenitor more recently than those with more dissimilar genes,researchers have developed the evolutionary lineage tree shown in Figure 1-3. According to this tree, the archaeaand the eukaryotes diverged from bacteria billions of years ago before they diverged from each other. In addition to DNA sequence distinctions that define the three groups of organisms,

Animals

Plants Fungi

Ciliates

Euglena

Microsporidia

EUKARYOTA S I i m em o l d s Diplomonads (Giardia lamblia)

EUBACTERIA E. coli

Sulfolobus ARCHAEA B. subtilus

Thermococcus

Thermotoga

Methanobacteriu m Halococcus

Flavobacteria G r e e ns u l f u r bacteria

Halobacterium Methanococcus jannaschii

Borrelia burgdorferi

P r e s u m e dc o m m o n p r o g e n i t o r of all extant organisms P r e s u m e dc o m m o n p r o g e n i t o r of archaebacteria and eukaryotes

A FIGURE 1-3 All organismsfrom simplebacteriato complex mammalsprobablyevolvedfrom a common,single-celled progenitor.Thisfamilytreedepicts theevolutionary relations among thethreemajorlineages of organisms. Thestructure of thetreewas initially ascertained frommorphological criteria: creatures that look alikewereput closetogetherMorerecently the sequences of DNA andproteins havebeenexamined asa moreinformation-rich criterion for assigning relationships Thegreater thesimilarities in thesemacromolecular sequences, the moreclosely related organisms arethoughtto be.Thetreesbasedon morphological comparisons andthefossilrecordgenerally agreewellwith thosebasedon molecular data.Althoughallorganisms in the eubacterial and archaean lineages areprokaryotes, archaea aremoresimilar to eukaryotes ("true"bacteria) thanto eubacteria in somerespects For instance, archaean andeukaryotic genomes encodehomologous histone proteins, whichassociate with DNA;in contrast, bacteria lack histonesLikewise, the RNAandproteincomponents of archaean ribosomes aremorelikethosein eukaryotes thanthosein bacteria. C H A P T E RI

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archaeacell membraneshave chemical properties that differ dramatically from those of bacteria and eukaryotes. Many archaeansgrow in unusual,often extreme,environments that may resemblethe ancient conditions that existed when life first appeared on earth. For instance, halophiles ("salt loving") require high concentrationsof salt to survive, and thermoacidophiles("heat and acid loving") grow in hot (80' C) sulfur springs,where a pH of lessthan 2 is common. Still other archaeanslive in oxygen-free milieus and generate methane(CH+) by combining water with carbon dioxide.

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s Bacteria and archaea, the most abundant single-celledorganisms,are commonly 1-2 pm in size. Despite their small size and simple architecture,they are remarkable biochemical factories,converting simple chemicalsinto complex biological molecules.Bacteria are critical to the earth's ecology, but some cause major diseases:bubonic plague (Black Death) from Yersiniapestis,strep throat from Streptomyces, tuberculosis from Mycobacterium tubercwlosis, anthrax from Bacillus anthracis, cholera from Vibrio cholerae, and food poisoning from certain types of E. coli and Salmonella. Humans are walking repositories of bacteria, as are all plants and animals.'We provide food and shelter for a staggering number of "bugs," with the greatestconcentration in our intestines.In return for the food and shelter that allow them to reproduce, bacteria help us digest our food. One common gut bacterium,E. coli, is also a favorite experimental organism. In responseto signalsfrom bacteria such as E. coli, the intestinal cells form appropriate shapesto provide a niche where bacteria can live, thus facilitating proper digestion by the combined efforts of the bacterial and the intestinal cells. Conversely,exposure to intestinal cells changes the properties of the bacteria so that they participare more effectivelyin human digestion. Such communication and responseis a common feature of cells. The normal, peacefulmutualism of humans and bacteria is sometimesviolated by one or both parties. When bacteria begin to grow where they are dangerousto us (e.g.,in the bloodstream or in a wound), the cells of our immune system fight back, neutralizingor devouring the intruders. Powerful antibiotic medicines,which selectivelypoison prokaryotic cells, provide rapid assistanceto our relatively slow-developingimmune response.Understanding the molecular biology of bacterial cells leads to an understanding of how bacteria are normally poisonedby antibiotics,how they becomeresistantto antibiotics, and what processesor structurespresent in bacterial but not human cells might be usefully targeted by new drugs. Like bacteria) protozoa are usually beneficial members of the food chain. They play key roles in the fertility of soil, controlling bacterial populations and excreting nitrogenous and phosphatecompounds,and are key playersin wastetreatment systems-both natural and man-made.These unicellular eukaryotesare also critical parts of marine ecosystems, consuming large quantities of phytoplankton and harboring photosynthetic algae, which use sunlight to produce biologically usefulenergyforms and small fuel molecules.

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FfGURE 1-4 Plasmodiumorganisms,the parasitesthat cause malaria,are single-celledprotozoanswith a remarkablelife cycle.ManyPlasmodium species areknown,andtheycaninfecta The hosts. varrety of animals, cycling betweeninsect andvertebrate fourspecies thatcausemalaria in humansundergo several dramatic (a)Diagram withintheirhumanandmosquito hosts. transformations of the lifecycleSporozoites entera humanhostwhenan infected Anopheles mosquitobitesa personfl Theymigrateto the liver, intothe wheretheydevelop intomerozoites, whicharereleased fromsporozoites, sothis bloodE. Merozortes differsubstantially "to transform" (Greek, isa metamorphosis or "many transformation merozoites invaderedbloodcells(RBCs) and shapes")Circulating producedby somePlasmodium reproduce withinthem B Proteins RBCs, causing the cellsto species moveto the surface of infected infected RBCs adhere to thewallsof bloodvessels. Thrsprevents fromcirculating to thespleen, wherecellsof the immunesystem theyharbor woulddestroy the RBCs andthe Plasmodium organisms in RBCs for a periodof time Aftergrowingandreproducing suddenly characteristic of eachPlasmodium species, the merozoites

from largenumbersof infectedcells4. lt burstforth in synchrony chillsthatare isthiseventthatbringson thefeversandshaking of malariaSomeof the released symptoms thewell-known a cycleof production creating RBCs, infectadditional merozoites intomaleand develop somemerozoites Eventually, andinfection. cells, These 5, anothermetamorphosis. femalegametocytes cannot whichcontainhalftheusualnumberof chromosomes, in bloodIo an Anopheles theyaretransferred for longunless survive gametocytes are the stomach, mosquito s In the mosquito. yetanother intospermor eggs(gametes), transformed flagellaon of longhairlike markedby development metamorphosis zygotesZ, of spermandeggsgenerates the sperm6 Fusion wallandgrowinto intothecellsof thestomach whichimplant of Rupture sporozoites for producing factories essentially oocysts, of sporozoites E; thesemigrateto the thousands an oocystreleases glands, settingthe stagefor infectionof anotherhuman salivary and of matureoocysts micrograph electron host.(b)Scanning stomach of sudace the external abut Oocysts sporozoites. emerging them that protects withina membrane wallcellsandareencased (b)courtesy of R E Sinden ] fromthe hostimmunesystem. lPart

However, some protozoa do give us grief: Entamoeba histolytica causesdysentery;Trichomonas uaginalis,vaginitis; and Trypanosoma brucei, sleeping sickness.Each year the deadliest of the protozoa, Plasmodium falciparum and related species,is the cause of more than 300 million new casesof malaria, a diseasethat kills 1.5 to 3 million people annually. These protozoans inhabit mammals and mosquitoes alternately,changing their morphology and behavior in responseto signalsin each of theseenvironments.They also recognize receptors on the surfacesof the cells they infect. The complex life cycle of Plasmodium dramatically illustrates how a singlecell can adapt to each new challenge it encounters(Figure 1-4). All of the transformations in cell type that occur during the Plasmodium Iife cycle are gov-

erned by instructions encodedin the geneticmaterial of this parasite and triggered by environmental inputs. The other group of single-celledeukaryotes, the yeasts, also have their good and bad points with respect to humans. as do their multicellular cousins, the molds. Yeasts and molds, which collectively constitute the fungi, have an important ecological role in breaking down plant and animal remains for reuse.They also make numerous antibiotics and are used in the manufacture of bread, beer,wine, and cheese.Not so pleasant are fungal diseases'which range from relatively innocuous skin infections such as jock itch and athlete's foot to life-threatening Pneumocystis carinii pneumonia, a common cause of death among AIDS patients. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

VirusesAre the Ultimate Parasites

opportunity. Viral infections can be devastatingly destructive, causing cells to break open and tissuesto fall apart. However, many methods for manipulating cells depend on using virusesto convey geneticmaterial into cells.To do this, the portion of the viral genetic material that is potentially harmful is replaced with other genetic material, including human genes.The altered viruses, or vectors, still can enter cellstoting the introduced geneswith them (Chapter 9). One day, diseasescausedby defectivegenesmay be treated by using viral vectors to introduce a normal copy of a defective gene into patients. Current research is dedicated to overcoming the considerableobstaclesto this approach, such as getting the introduced genesto work at the right placesand trmes.

Not all microscopic pathogensare cells. The other most familiar disease-causingorganisms are the viruses, which make use of the machinery inside the cellsthey infect to copy themselves.Virus-causeddiseasesare numerous and all too familiar: chickenpox, influenza, some types of pneumonia, polio, measles,rabies,hepatitis,the common cold, and many others. Smallpox, once a worldwide scourge,was eradicated by a decade-longglobal immunization effort beginning in the mid-1960s. Mral infections in plants (e.g.,dwarf mosaic virus in corn) have a major economic impact on crop production. Planting of virus-resistantvarieties, developed by traditional breeding methods and more recently by geneticengineeringtechniques,can reduce crop lossessignificantly. Most viruses have a rather limited host range, infecting certain bacteria,plants, or animals(Figure1-5). Becausevirusescannot grow or reproduce on their own, they are in this sensenot consideredto be alive. To survive. a virus must infect a host cell and take over its internal machinery to synthesizeviral proteins and in some casesreplicate the viral geneticmaterial. When newly made virusesare releasedby budding from the cell membrane or when the infected cell bursts, the cycle starts anew. Viruses are much smaller than cells,on the order of 100 nanometer (nm) in diameter; in comparison, bacterial cells are usually >1000 nm (1 nm : 10-e meters).A virus is typically composedof a protein coat that enclosesa core containing the genetic material, which carries the information for producing more viruses (Chapter 4). The coat protects a virus from the environment and allows it to stick to, or enter,specifichost cells. In some viruses, the protein coat is surrounded by an outer membrane-likeenvelope. The ability of viruses to rransport genetic material into cells and tissuesrepresentsa medical menaceand a medical

The most remarkable feature of organismsis their ability to reproduce.Biological reproduction, combined with continuing evolutionary selectionfor a highly functional body plan, is why today's horseshoecrabs look much as they did 300 million years ago, a time span during which entire mountain ranges have risen or fallen. The Teton Mountains in S7yoming,now about 14,000 feet high and still growing, did not exist a mere 10 million years ago. Yet horseshoecrabs, with a life span of about 19 years,have faithfully reproduced their ancient selvesmore than half a million times during that period. The common impression that biological structure is transient and geologicalstructure is stableis the exact opposite of the truth. Despite the limited duration of our individual lives, reproduction gives us a potential for immortality that a mountain or a rock does not have. Whereas some species have changed little over great periods of time, other organismshave changeddramatically

(a)T4 bacteriophage

(b)Tobaccomosaicvirus

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

,

50nm

,

(c) Adenovirus

A FIGURE 1-5 Virusesmust infect a host cell to grow and reproduce.Theseelectronmicrographs illustrate someof the structural variety (a)T4 bacteriophage exhibited byviruses. (bracket) attaches to a bacterial cellviaa tailstructureViruses that infect bacteria arecalledbacteriophages, (b)Tobacco or simplyphages. mosarc viruscauses a mottlingof the leaves of infected tobacco C H A P T E R1

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plantsandstuntstherrgrowth.(c)Adenovirus causes eyeand respiratory tractrnfections in humans. Thisvirushasan outer membranous envelope fromwhichlongglycoprotein spikesprotrude [Part(a) from A Levine,1991, Viruses,ScientificAmericanLibrary,p 20 Part(b) courtesyof R C Valentine Part(c) courtesyof RobleyC Williams, Universityof Californial

during the same period. The changescame in responseto pressuresfrom the environment that causedincreasedsurvival of variant individuals. Both stasis and change are possible becausethe machinery of cells does an amazingly precisejob of copying geneticmaterial, yet its rare errors introduce some variation. If environmental conditions continue to select more or less the existing form, as in the caseof horseshoecrabs, the specieswill changelittle. If a new variant has a survival advantage, perhaps because conditions have changed, it may persist and replace the old form. Populations of bacteria exposedto antibiotics, for example, changetheir properties dramatically to escapeand live. They do this becauserare mutations, changesin the genetic material that allow antibiotic resistance,keep some cells alive while the cells without those mutations die. Most populations of any single specieshave a large repertoire of genetic alterations becausethere is a low but significant error rate in copying the genes.That error rate increasesin the presenceof radiation such as sunlight or certain chemical poisons. Current genomeproiects are exploring geneticvariation among humans. "The" human genome sequencethat has been determined already is just one version among billions. Understanding variation is essentialto learn how we respond differently to certain infections or drugs and to exploring how our geneticheritage combines with our experienceand learning to make each of us unique. Underlying the reproduction of organismsis the copying of cells, something that must be precise in order to control the size, shape, and organization of animals and to prevent unwanted growth, such as cancer.The cell is a machine that can copy itself, unlike viruses,which cannot do so on their own. As we will seein Chapters 20 and 21',the cell cyclefrom a single cell copying its own contents through division into two cells-is controlled by a seriesof elegant switches and cross-checking mechanisms. Reproducing cells accurately is a matter of life and death.

EvenSingleCellsCan Have Sex If genetic material was never shared or exchanged,each individual would be the beginning of a new clone of individuals, and the members of a clone would share most of the same genetic strengths and weaknesses.Sex is a processof mingling genetic variation from two individuals, creating new individuals with a combination of properties unlike either parent and that may be beneficial for survival and reproduction. Each chromosome except the sex chromosomes is representedtwice, one copy from the father and one from the mother. Since each pair of chromosomes trades piecesduring the formation of eggs and sperm, new combinations of genesare created and inherited together in the next generation-variation is accelerated. The other benefit of having two copiesof each chromosome is that a poorly functioning gene is backed up by the other copy. The common yeast used to make bread and beer, Saccharomycescereuisiae,appearsfairly frequently in this

Budding lS. cerevisi ael

1-6 The yeast Saccharomycescerevisraereproduces A FfGURE sexuallyand asexually'(a)Twocellsthat differin matingtype' calleda andcr,canmateto forman a/a cellIl. Thea anda cellsare theycontaina singlecopyof eachyeast meaning haploid, halfthe usualnumber'Matingyieldsa diploida/ctcell chromosome, Duringvegetative two containing copiesof eachchromosome. process an asexual by mitoticbudding, growth,diploidcellsmultiply a diploidcellsundergomeiosis, conditions, E Understarvation B. Rupture to formhaploidascospores typeof celldivision, special into whichcangerminate fourhaploidspores, releases of an ascus asexually E. (b)Scanning alsocanmultiply cells4. These haploid Aftereachbudbreaks of buddingyeastcells. micrograph electron buds free,a scaris left at the buddingsite,so the numberof previous (b) AbbeyA/isuals M bacteria are cells [Part canbe counted.Theorange lncl Unlimited, book becauseit has proven to be a great experimental organism. Like many other unicellular organisms' yeasts h"u. t*o mating types that are conceptually like the male and female gametes(eggsand sperm) of higher organisms'

ubiquitous. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

.

7

We Developfrom a SingleCell In 1827, German physician Karl von Baer discovered that mammals grow from eggs that come from the mother's ovary. Fertilization of an egg by a sperm cell yields a zygote, a visually unimpressive cell 200 pm in diameter. Every human being begins as a zygote,which housesall the necessaryinstructions for building a human body containing about 100 trillion (1014)cells, an amazing feat. Development begins with the fertrlizedegg cell dividing into two, four, then eight cells, forming the very early embryo (Figure 1-7). Continued cell proliferation and then differentiation

Chapters 16 and 22. Making different kinds of cells-muscle, skin, bone, neuron, blood cells-is not enough to produce the human body. The cells must be properly arranged and organized into tissues,organs, and appendages.Our two hands have the same kinds of cells, yet their different arrangements-in a mirror image-are critical for function. In addition, many cells exhibit distinct functional and/or structural asymmetries, a property often called polarity. From such polarized cells arise asymmetric, polarized tissuessuch as the lining of the intestinesand structures such as hands and hearts. The features that make some cellspolarized and how they arise also are coveredin later chapters,including Chapter 21.

Video:EarlyEmbryonicDevelopment < FIGURE 1-7 The first few cell divisionsof a fertilized egg set the stagefor all subsequentdevelopment.A developing mouseembryois shownat (a)the two-cell,(b) four-cell, and(c)eight-cell stages. Theembryoissurrounded by supporting membranes. The corresponding stepsin human development occurduringthe firstfew daysafterfertilization. Researchers. IClaudeEdelmann/Photo I n cl

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Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities The biology of stem cells, cells that can give rise to specific cell types and tissues,has generated, great interest. 'We can 'Sfhen contrast stem cells to the simpler types of bacteria. a bacterial E. coli cell divides, both daughter cells are pretty much equivalent in content, size, and shape. In some other bacteria and in many casesof eukaryotic cell division, the two daughters differ in important ways. Although both will have the same genetic material, the cells may differ in size, shape, and contents. The cells may have different fates, that is, they may become different types of differentiated cell. A division that produces two different daughter cells is sometimes described as an asymmetric cell division. Stem-celldivisions are a specialcaseof asymmetric division. One of the two daughter cells is identical to the parent cell; the other follows a path of differentiation, such as becoming a blood cell. The parent cell, called a stem cell, can go on reproducing itself at every division, at each division also producing anorher blood cell. Mosr tissuesin our bodies form from stem cells. Blood, for example, is produced from stem cells that residein the bone marrow and continue to produce new blood cellsfor our entire lives. This is the basis of the often successfulbone marrow transplants that are used to treat cancer patients who have had their blood stem cells damaged by cancer treatments: what is being transplanted is stem cells.However, blood stem cells produce onlv more of themselvesand blood cells, not othir cell types. Thus eachtissuemust have its own stem cells,at leastduring the period of development when the tissue is formed. Stem cells for each tissue arise from even more capable stem cells that have the ability to form multiple stem cell types. The first stem cells are found in early embryos,where all the cells are capable of producing all cell types. In mammals the ultimate stem cell is the fertilized egg, which produces early embryo cells capable of forming all the tissuesof the body. This capability is illustrated by the formation of identical twins, which occur naturally when the mass of cells composing an early embryo divides into two parts, each of which develops and grows into an individual animal. This means that the cells cannor have divided up their embryo-forming duties prior to the time of embryo division. Each cell in an eight-cell-stagemouse embryo has the potential to give rise to any part of the entire animal. Cells with this capability are referred to as embryonic stem (ES)cells. As we will learn in Chapter 22, ES cells can be grown in the laboratory (cultured) and will develop into various types of differentiated cells under appropriate conditions. The ability to make and manipulate mammalian embryos in the laboratory has led to new medical opportunities as well as various social and ethical concerns.In vitro fertilization, for instance, has allowed many otherwise infertile couples to have children. One technique involves extractron of nuclei from defective sperm incapable of normally fertilizing an egg,injection of the nuclei into eggs,and implantation of the resulting fertilized eggsinto the mother.

In recent years, nuclei taken from cells of adult animals have been used to produce new animals. In this procedure' the nucleus is removed from a body cell (e.g., skin or blood cell) of a donor animal and introduced into an unfertilized mammalian egg that has beendeprived of its own nucleus.In a step that has now been done with mice, cows, sheep, mules, and some other animals, the egg with its donor nucleus is implanted into a foster mother. The ability of such a donor nucleusto direct the developmentof an entire animal shows that all the information required for life is retained in the nuclei of some adult cells. Sinceall the cells in an animal produced in this way have the genesof the single original donor cell, the new animal is a genetic clone of the donor (Figure 1-8), though the animals may differ anyway due to their distinct environments and experiences.Repeating the processcan give rise to many clones. Nuclei taken from ES cellswork especiallywell, whereasnuclei from other parts of the body at later times in life work far lesswell. The majority of embryos produced by this technique do not survive due to birth defects,so the donor nuclei may not have all the needed information or the nuclei may be damaged by the cloning process.Even those animals that are born alive have abnormalities,including acceleratedaging. The "rooting" of plants, in contrast, is a type of cloning that is readily accomplished by gardeners,farmers, and laboratory technicians. Scientific interest in the cloning of humans is very limited. Virtually all scientistsoppose it becauseof its high risk to the embryo (also, most people don't believe there is a

critical shortage of twins and triplets). Of much gteater scientific and medical interest is the ability to generatespecific cell types starting from embryonic or adult stem cells' This procedure, somatic cell nuclear transfer (SCNT)' produces cells that are grown in culture and never turned into

If the cells are produced using a donor nucleus from a patient, the properties of the cells may allow them to escaperejection by the patient's immune system,opening new possibilities for cell-transplanttherapies.

E

The Moleculesof a Cell

Molecular cell biologists explore how all the remarkable

ture and function.

Small MoleculesCarryEnergy,TransmitSignals, and Are Linkedinto Macromolecules Much of the cell's content is a watery soup flavored with small molecules (e.g., simple sugars' amino acids, vitamins) and ions (e.g.,sodium, chloride, calcium ions)' The locations conce.tirations of small molecules and ions within the "nd controlled by numerous proteins inserted in cellular are cell membranes. These pumps' transporters, and ion channels move nearly all small moleculesand ions into or out of the cell and its organelles(Chapter 11). One of the best-known small molecules is adenosine triphosphate (ATP), which storesreadily available chemical 'When ..r"rgy itt two of its chemical bonds (seeFigure 2-31)' cells"splitapart theseenergy-richbonds in AIP' the released ..r.rgy ."n b. harnessed to power an energy-requiring pro..tt such as muscle contraction or protein biosynthesis' 1-8 Fivegeneticallyidenticalclonedsheep.An early To obtain energy for making ATR cells break down food a FIGURE intofivegroupsof cellsandeachwas sheepembryowasdivided molecules.For instance, when sugar is degraded to carbon natural like the much mother, intoa surrogate implanted separately dioxide and water, the energy stored in the original chemical process of twinningAt an earlystagethecellsareableto adjustand bonds is releasedand much of it can be "captured" in ATP the cellsbecome laterin development forman entireanimal; (Chapter 72).Bacterial,plant, and animal cells can all make way andcanno longerdo so.An alternative progressively restricted ATP ty this process.In addition, plants and a few other orsingle-celled the nucleiof multiple isto replace to cloneanimals ganisms can harvest energy from sunlight to form ATP in with donornucleifromcellsof an adultsheep'Eachembryo embryos photosynthesis. ' was to the adultfromwhichthe nucleus identical will be genetically Other small moleculesact as signalsboth within and beto theseprocedures survive of embryos obtained.Lowpercentages tween cells; such signals direct numerous cellular activities on the givehealthy andthefull impactof the techniques animals, effect on our bodies of Library/Photo (Chapters iS uttd f e ;. fne powerful Photo Tompkinson/Science animalsis not yet known.lGeoff flooding of instantaneous the from comes a frightening event Incl Researchers, S F A CELL T H E M O L E C U L EO

O

9

the body with epinephrine, a small-moleculehormone that mobilizes the "fight-or-flight" response.The movements neededto fight or flee are triggered by nerve impulses that flow from the brain to our muscleswith the aid of neurotransmitters, another type of small-moleculesignal that we discussin Chapter23.

acids. Sugars,for example, are the monomers used to form polysaccharides. These macromolecules are critical structural components of plant cell walls and insect skeletons.A typical polysaccharideis a linear or branched chain of repeating identical sugar units. Such a chain carries information: the number of units. However, if the units arenot identical, then the order and type of units carry additional information. As we will seein Chapter 6, some polysaccharides exhibit the greaterinformational complexity associated with a linear code made up of different units assembledin a particular order. But this property is most typical of the two other types of biological macromolecules-proteins and nucleic acids.

ProteinsGive CellsStructureand perform Most CellularTasks The varied, intricate structures of proteins enable them to carry out numerous functions. Cells string together Z0 dif_ ferent amino acids in a linear chain to foim a protein (see Figure 2-14).Proteins commonly range in length from 100

to 1000 amino acids, but some are much shorter and others longer. We obtain amino acids either by synthesizingthem from other moleculesor by breaking down proteins that we eat. The "essential" amino acids, from a dietary standpoint, are the eight that we cannot synthesizeand must obtain from food. Beans and corn together have all eight, making their combination particularly nutritious. Once a chain of amino acids is formed, it folds into a complex shape,conferring a distinctive three-dimensionalstructure and function on each protein (Figure1-9). Some proteins are similar to one another and therefore can be consideredmembers of a protein family. A few hundred such families have been identified. Most proreins are designedto work in particular places within a cell or to be releasedinto the extracellular (extra, "outside") space.Elaborate cellular pathways ensurethat proteins are transported to their proper intracellular (intra, "within") locations or secreted (Chapters 13 and14). Proteins can serveas structural componentsof a cell. for example,by forming an internal skeleton(Chapters tO, tZ, and 18). They can be sensorsthat changeshapeas remperature, ion concentrations, or other properties of the cell change. They can import and export substancesacross the plasmamembrane(Chapter11). They can be enzymes,causing chemicalreactionsto occur much more rapidly than they would without the aid of theseprotein catalysts(Chapter 3). They can bind to a specificgene,turning it on or off (Chapter 7). They can be extracellular signals,releasedfrom one cell to communicatewith other cells, or intracellular signals, carrying information within the cell (Chapters 15 and 16). They can be motors that move other moleculesaround, burning chemicalenergy (ATP) to do so (Chapters17 and 1g).

Insulin

Glutaminesynthetase Lrturamtne synthetase

Hemoglobin

FIGURE 1-9 Proteinsvary greatly in size,shape,and function.Thesemodels of thewater-accessible surface of some representative proteins aredrawnto a commonscaleandreveal the numerous projections andcrevices on thesurface. Eachprotern hasa definedthree-dimensional shape(conformation) thatisstabilized by numerous chemrcal interactions discussed in Chapters 2 and3. The illustrated proteins include (glutamine enzymes synthetase and

10

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L | F EB E G T N W S |TH CELLS

DNA molecule

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adenylatekinase),an antibody(immunoglobuiln), a hormone (insulin), and the bloods oxygencarrier(hemoglobin)Modelsof a segmentof the nucleicacid DNA and a smallregionof the lipid bilayerthat formscellularmembranes(seeSection1.3)demonstrate the relativewidth of thesestructures comparedwith that of tvpical proteins.[Courtesy of GarethWhite]

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'g rardeq3 ur lretepur uortElsuertpuB uorldrrrsuPrl lno d.rrel leqt stuauodruor IIer eql euruexe al1 'opof, f,rteu -aE les.ra.trundlreeu aql ot Surprorre aruanbasVNUIU oqr dq parelrrp rap.ro asne;d eqt ur sprre ourrue raglaSol s>1ur1 (uorlelsuB-rl SurrnC 'uorlelsueJl pue selqtuesseatuosoqrrar{l pell€r <sserord puocaseqt lno srrJrer'uralord pue VNU qtoq yo pasodruor eurqJprurelnJelou xalduoc ,{lsnourrouaue 'aruosogrraLIteJeH 'ruseldoldceql ol se,l.oruqcrq.u 'a1nca1ou (VNUru) y51A ra8uasserurelleruse otur possarordsr rrnpord 'sl1ar 'arelduer E se ruodre>1na u1 vNd IBrlrur aqr vNC Sursnureqr VNU p otur saprloe]f,nuJo a8e>1ur1 aql sez.{1etec 'au.,(zua 'VNC 'aserorudlod y51g e8rel pepuens-alqnop V eql Jo uorsre^ (VNU) prJp Jrelf,nuoqrrpepuerts-a13urs e otur pardor sr aua8 e yo uor8er Surpor eqt 'uorldrrcsuprl pellel 'rs-rr; '(II-I aqt uI arn8lg) suretordorur VNC ur uorlerurot -ur pepol aqt ol sorrasur sessecordo.la.lesn slla] lJeluor 'opuru sr uratord Jr{t sllal qlrr{.a uI puE uel{,{\ slorluof, teqt uo8al Ktolap8at E pue urelord e ;o acuanb -es prf,p ounue aqt sarpads rcq7 uot8at Surpoc e :strud orrrl ureluoJ dluorutuoc suralord 3ur1eu Jot suorlfnJlsur d.r.rer teqr saue8eql'000'SZ-000'02 lnoqe'suerunq lsaue8pues -noqt .,taale e^eq errolf,pq tso14 '3uo1 seprloallnu 000'00I or 000S a.re dlyecrddrqlrq^\ 'souo8 eql (strun leuorlrunt elerrslP olul poPI^lPsl VNq Jo uorlrod Surreaq-uotlEturolul oql 'puerts e 3uo1esoprloalJnuJo rapro reaurl Jrll 'aluenb -esslr ur seprser VNC dq perrrpr uorteruroturlrleueg or{J. 'gfleru lou oP ]Pql sPuprls ror{rodueu sureluotroslEuorl vNC -nlos oqt ue^a 'Surraddrzot peal 'r llr.{\ puerts dretuauralduroc rel{lo eqt Surureluocuortnlos e ur raded aql Suopos taded 'alduruxerod ;o aoarde o] Paqleue pue par;rrndsI puerls auo;t reqlo aql Sursnpue.rtseuo Surlcalaprot Intesndlauarlxa sruorl -enpuq;1.i:rl prf,e f,rolf,nur.lrns 'suorlrpuoo arnle.radtuetpue tles drz dlsnoauetuods11,udaqt 'pale.r rq8rr eqr ur raqtaSor 4ceq -edes a.le spuprts .,(reluerualduol teqt Suols os sr spuerls Jr o.,rueqr;o Surqrreru,{-ruluaualduol srr{J '(91-1 arn8r4)3 e qtr.&\peqlteru sr J qrea pue 1 e ser1puerls reqlo eqt 'y ue seq pupJls ouo Jeleror{,la.:uouJnrlsuof aydrursp spr{ x[eq elqnop 'puens rqr VNC r{leg to auoqlleq lerqeq eqr tuo4 lno Sunoal 'puerts -ord stred eseq eqt qtrlr E ur puo ol pua paurol VNCI are '9 pue 'J t'V peter^argqe'seprtoalcnuluora11rp rnoC

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a)eld pue ourrl lq6tu aql le surelord6ur4ey1ro; uolleturo+ulpapo) f.r.re1sp!)v )rel)nN '(solnralou 80I x S) utlle utotord IErnltnDS lu€punqE eqr or (salncalotu000'02) utalo.rdroldoc 'dleprm -a.r sutetord alrnb eqt uo.r; sarrB,r Surpurq-urlnsur erpr 'e8e.ra,reuo urolord tuere;trp Jo eruepunqe aql 'lce1 u1 1o addr qree Jo selnrelouruorlpu e ot osolf,suleluol IIOJE snqr isurelord ruere;Jrp000'0I rnoqe suleluor IIat re^II p leq] raprsuoJ taqrrny dels auo uollelnf,lef, srqt drrec oI '(Ez0I x ZO'9) punoduor lerlueqr due 1o ayourrad salnrelotu Jo requnu eql laqunu s,orpeSorrypue lq8ra,t uratord Ielol eqt trrorl sp IIer re^II .radsalncalou utarord 60I x 6'L lnoqe 'suratord f,rlofre>lna to reqrunu letor oqt elelnllel uer e,l.r '(1ou73) 00L'TS yo rq8rorr ;o lerrddr sr enlE^ srql Surunssy relnrelorrr p seq uratord lseef e8erarreeql '3 or_0I x L er{r 'tq8ta,trs(ller e to tuer sr ureto.rdrelnller rq8rarvr ;o I€tol -nd 97,,(laleturxo.rddpro; slunoJJe uralord erur5 '3 r_0I x g'g q8ra.uplno.vi aql'1uF E0'I Jo &lsuep 11ec e Sururns IIer -sy '(s;arqqlur ro) etunlol e seg 'aprse e*l o_0I x V't p srql '(1ac re,rr1) uo (tuc rurl eqnr E ,{1q3nor'11ac SI SI00'0) 'req ardcoledeqe se r1rns'11accrlodre>1ne lerrd& e a4et s.ta1 -unu srqt eterurtseoJ 'lasrr urelureru pue alerado ol spJJu IIar e selnrelotu urarord dueur .uoq >1serq8nu a^.\txaN 'dtlulJul 3uo1e pre,&ol 1a,'nsr sutetord algrssodIo reqrunu .ro'alqetsun'luele.trnba llleuorr eqt'elqElunorsrpasr,&\rar.{lo 'seruanb -lunJ dueru aq asaql Surunsse ua,tg p1no.l.r leqt Jo '3uo1 sprre -as uralord luereJJrp alqrssod oov17ere araqt ounue 00t rnoqe sr ureto.rd..1errddl,,E Jr lng 'ecue13lsrr; re alqrssodrursrueestI is>lserperre^ eseqlurotred or papaeu surolord ruereJtrpoqt IIE ruroJ splre ouIIuE 0Z uel ,{\oH

ur pegrrtsap are aruosrldarer{r Jo sluauodruol raqto dueru aql lpue;rs VNC € otur seprtoepnu Surlury ro; alqrsuodsa_r sr eserau,(1odVNq 'leur8r.roeql ot Iefrluepr ql€e ,serrlaq elqnop 1o ;red e sr eruortno eqI .(0I-I a.rn8rgaas)pue_us dreruetualduoJ 1y\eue olur saprloopnu elquesse ol aleldual e se qrea sasnpup seuosoruorql eqr ul vNC Ielrleq PuErls -elqnop spuertso.,vrtrqt seteredas,aruosrlde.r eqt (eurqrpru Jo uorlerrlda.ruralordrllnru a8rel e ,saprlrp 11ace arurl d.ra,rg 'pec ol 'JlpLlreqlo eq.l 'asaql !tuoy41o1 IlEq peJen og UEJ'seue8eqt;o JIeq snLlrpue '(71-1 ern8r4)sauosoruorqr seq uerunq Jo 'lpH 9t IIal lpur -rou dre,te 'rulads pue s33a uondarxe egr qtrl1 .VNC 1o to luerualduror eJrluastr sasr.rduorrusrue8roue ;o auroua8eq1 '(e7-1 a-rn8r1 aes) aql yo uor8arI€rtueJaqt ur ,saurt duetu 11ac 'sat1alncalorusrqr lq13ua1ur ralaurllru Jlesrluo >lrEqPePIoJ B tnoqE alnrelotu VNC relnrrrr a13ursB ur sJprseruorterurot 's11arrrtodrelord u1 'suretord -ut cttaue83qt to IIe ro tsoru urEtJaJqtr.4apelerJosseelnfelou VNC Jeeurl a13urse surel -uor aruosoruonll qleg '(9 ;erdeq3) saruosoruorrlf,se.{\ou{ J^\ soJnlJnrls JprlrrueJor.[l olur peplot dle,rrsuelxa ,snap -nu agl ur petef,ol sr sllJJ lrodrelna ul VNC er.[]Jo rsory

u o t s r ^ t ol l o ) 6 u u n c p o l e ) r l d o up u e saurosotuotq)olur pa6e{)ed sl oruouagoql 'paroldxa Surag ere uorteln8e.r 11rrs ,saue8 1o ed& srql Jo &rnbrqn pue sursrupq)au eql qSnoql II€ ro tsoru alelnSer deu sygg llerus seteturlsaaruos dg 'sldrrcsuerlauaS;o drrpqerspue uortrnpord ,uors Surtuln8er -sardxa aue8 uo tleJJef,rt€uerp p e^eq uel selnrelour VNU 'serurtrq8u aqr sllet le rq8rr aqr ur saua8tq8u or1tsale^ IIpruS -ItrE legt ualsds IortuoJ alrsrnbxaup ruroJ ol padoldruaa.re srolreJ uotldtnsue;l lueretlrp Jo sperpunq 'susruefiro xald -ruoJ ul 'seua8pereln8er aqr Suqcalasol dlpr;rceds Surpurq -VNC u,4o str Surtnquluoc uralord auo ueql JJou qlr.^A 'saxaldruor uleloJdrtlnu sE lJo,ln uouo sJolfet uortdr.rrsuerl ']as eqt ur aueSqrea rEau lsrxo rotret teqr roJ satrsSurpurq ;r seua8Jo las E eteln8er dlateurproor uer rotf,€J uortdr.rcs 'uorssarda;pue uortelrl -uert Jo addl auo 1o serdocaldrrlnyq '11ar -re eua8 e Jo drlclJlradseqt Suunsue ;o VNC aql ur rnl -lo e dlug .seprtoaltnu IIr.^aacuanbesrJf,nsqrpe 1o sardor.ra.a1 rnoJ Jo due aq uec uortrsodr1f,Ee erurs (9tS'gV1't) saruanb -as alqrssod o^eq uec s.rred espq 0I Sururetuor ylrlq 0rt '3uo1srred aseq y51q ;o ruaru8asy ZI-9 $oqe seouanbes t.roqs azruSoJerllr,l.r'uralord Surpurg-y51q e d1lecrdd1 'VNC s,ller e ut luaserdspuesnoqlJrll Jo rno seue8,ua1e tsnl 1o suor8a.rdroleln8ar aqr o1 dlyerluare;ardpurq ot elqp ere .{eql reql dlasnard os pedeqs are srotreJ uortdrrrsue{ '(1 retdeq3) saue8 relncrtred ;o uortdursuerl Surssard 'saqrlr,rs sE tJE pue -ar ;o SurtB,r.rlJe JerJlre VNC ot purq (srolreJuorldrrrsuprl qcrq,rrr pallEt suralordSurpurq-ypq uo spuadapdtr,rrlceaua8yo lortuoJ r{tns 'speeuluorrnr }eetu ol 'go surato.rd;o arrolrada.r rragl Surldepe ,(qareqr ro uo saue8 rrPeds Suturnt dq suortrpuof, ur se8ueqc.ro sleu8rs leuJetxe 'esrol aJrApue IeuJetxeot puodsa;ue) sllJJlueru taaoerory s11ecdaupr>1,{q peenpord tou er€ teqt suralord oruos ernp -ord s1larra,tq dq,rrr. s(teqJ 'sureto-rdaleu ot pesn pup (uo paurnt lo 'a,ttlf,eare saua8esoqtJo eruosdluo ad.{rIIac r{rea

s r r r ) H r M s N r g r sl j r ' r I

t u:ravul

zl

ur tnq 'seua3ueunq to tes IInJ aql uretuor serpogrno ur sllar eL[] IIE dl.reau'aruetsur roC 'paquf,suurl aq UEJ seua8rraql eJeq.4 pue uoq,lt IoJluoc ol sde,,vreleq s{usrup8ro 11y ' u r a t o r do t u l s J l n f , 'salncalotuy51a re8rel -elou VNUIU to uouplsuerr eqt pue (seLuosoluorqr Jo drlllqers eql Jo uollrunJ Pue erntf,nrts '3uo1 sepnoepnu 'sVNU aqr aleln8a.rd11err;roads 002-02 'dr{lqers pue Surssecord VNU pue ornl Ilprus sas€f,dueru u1 -fnrts eruosoruoJqlSurpnycurtlrrrrlre eua8 slf,odseduetu 1o Suneln8e.rur alor tuet.rodurr flqelreua; e Aeld ol puno' ueaq osle ser{VNU 'flruareg razrsaqludsuratord pue repear VNUIU luarrr]'a pue asrcard dlqe>1reua.rp a{pru ol surel -ord 69 ueqt eroril qlrrr,rdn rueat leqt sureqf,VNU JnoJ seq euosoqrr agt (oldurexa roC 'aurLlleru rplnlelou e Surplrnq ro; {ro^\euer; E se enres uer y51g 'useldoldc ol snolf,nu ruoJJ uorleruro;ur Surr.ra;supJlur aloJ slr ol uortrppe uI

ureqlrpourle olurspr)eouru.re 6ur1ur1 {1;erruaqr {q ureloLd e puearuenbes slrpear]eql sourosoqu {q punoqsr}r oloqm alqrlosse 'useldoyb y11y (VNUul) eq] o1 sa^oLr; rebuesseu ernleur oql 'llo) p ul :E de15saruanbas 6urporuou oAoLUar o1pessa:ord rrloi{re>1ne sr aq1: E dals eleldua]e sespuer]s ldursuerl VNCaq],o auo pepuerls-a16urs 6ursn]drnsuert e olurseprloapnu 6ur>1ur1 VNUU-ard esereuri;od aq_La]rsuelsoq] 'uorle)ol>rloeds VNc oq] 6uo;esenou.r 'VNC ue,Louorldursuerl sur6aq asereu{1od e ]e auebpale^r])e VNU aq]o] punoqxelduoruorlpr]rur ure1o.rdr1lnu e 1o{lquasse6urnnollo3 : g da15uJeq]ole^r]lepuelorluo),{eq}seue6:r;r:edsaq11o ,lo1eln6er aql o1purqsrolreluorldursue{:It da15'ssarord suor6el delsrllnu e {q surelold;o saruenbaspr)eourue oq} o}ul pouo^uo)s! vNc u! uollewrolu!papo)aqM-! lun9lJ v ureq3prce ouil..uv I 1 I I

v l g 1 o u o r o e ro u r p o c u o N V r u U ; ou o t 6 a l6 u t P o c - u t a 1 o l 3 vNc Jo uorOorpaqrrcsuerluoN -n2q2q2qa V N C + o u o r 0 o rp a q u 3 s u e {

uollolJcsue{

d uorleAllcv

can be "painted" for easy 1-12 Chromosomes FIGURE A normalhumanhas23 pairsof morphologically identification. fromthe onememberof eachpairis inherited chromosomes; distinct motherandthe othermemberfromthefather(Left)A chromosome whenthe froma humanbodycellmidwaythroughmitosis, spread wastreatedwith Thispreparation arefullycondensed chromosomes reaqents thatalloweachof the 22 pairs staininq fluorescent-labeled

Chapter 4. The molecular design of DNA and the remarkable properties of the replisome ensure rapid, highly accurate copying. Many DNA polymerasemoleculeswork in concert, each one copying part of a chromosome.The entire genome of fruit flies, about 1.2 x 10o nucleotideslong, can be copied in three minutes! Becauseof the accuracyof DNA replication, nearly all the cells in our bodies carry the same genetic instructions, and we can inherit Mom's brown hair and Dad's blue eyes. A rather dramatic example of genecontrol involves inac'Women tivation of an entire chromosomein human females. have two X chromosomes,whereas men have one X chromosome and one Y chromosome, which has different genes than the X chromosome. Yet the genes on the X chromosome must, for the most part, be equally active in female cells(XX) and male cells(XY). To achievethis balance'one of the X chromosomesin female cells is chemically modified and condensedinto a very small mass called a Barr body' which is inactive and never transcribed. Surprisingly,we inherit a small amount of geneticmaterial entirely and uniquely from our mothers. This is the circular DNA present in mitochondria' the organelles in eukaryotic cells that synthesizeAIP using the energy released by the breakdown of nutrients. Mitochondria contain multiple copies of their own DNA genomes,which code for some of the mitochondrialproteins(Chapter6). Becauseeachhuman inherits mitochondrial DNA only from his or her mother (it comes with the egg but not the sperm), the

colorwhen to appearin a different andtheX andY chromosomes of multiplex Thistechnique microscope viewedin a fluorescence (M-FISH) iscalled sometimes in situhybridization fluorescence (RiSht) fromthe (Chapter Chromosomes 6) painting chromosome orderof size, in pairsrndescending preparation on the leftarranged of X andY chromosomes Thepresence an arraycalleda karyotype. of M R Speicher] asmale [Courtesy the sexof the individual identifies

distinctive features of a particular mitochondrial DNA can be used to trace maternal history. Chloroplasts, the organelles that carry out photosynthesisin plants' also have Ih.i, o*tt circular genomes'Both mitochondria and chloroolasts are believedto be derived from endosymbionts,bacteii" th"t took up residenceinside eukaryotic cells in a mutually beneficialpartnership.The mitochondrial and chloroplast circular DNAs appeatto have originated as bacterial genomes' which also are usually circular,though the organellegenomes have lost most of the bacterialgenes.

Mutations May Be Good, Bad,or Indifferent Mistakes occasionallydo occur spontaneouslyduring DNA replication, causing changesin the sequenceof nucleotides' Soch ch"ng.s, or mutations, also can arise from radiation that causesdamageto the nucleotidechain or from chemical poisons, such as those in cigarette smoke, that lead to errors iuring the DNA-copying process (Chapter 25)' Mutations .o-.1., various forms: a simple swap of one nucleotide for another; the deletion, insertion, or inversion of one to millions of nucleotides in the DNA of one chromosome; and translocation of a stretch of DNA from one chromosome to another. In sexually reproducing animals such as ourselves,mutations can be inherited only if they are presentin cellsthat potentially contribute to the formation of offspring' Such germJine cells include eggs'sperm' and their precursor cells' T H E M O L E C U L EOSF A C E L L

O

13

Body cells that do not contribute to offspring are called somatic cells. Mutations that occur in these cells never are inherited, although they may contribute to the onset of cancer. Plants have a less distinct division between somatic and germ-line cells, since many plant cells can function in both capacltres. Mutated genesthat encode altered proteins or that cannot be controlled properly ."u.. .rurn..ous inherited diseases.For example, sickle-celldiseaseis atributable to a single nucleotide substitution in the hemoglobin gene, which encodesthe protein that carries oxygen in red blood cells. The single amino acid changecausedby the sickle cell muta-

Sequencingof the human genome has shown that a

transcription factors typically are only 70-12 nucleotides long, a few single-nucleotidemutations might convert a nonfunctional bit of DNA into a functional protein-binding regulatory site. Much of the nonessentialDNA in both eukaryotesand prokaryotes consistsof highly repeatedsequencesthat can move from one place in the genome to another. These mobile DNA elementscan jump (transpose)inro genes,most commonly damaging but sometimes activating them. Jumping generallyoccursrarely enoughto avoid endangering the host organism. Mobile elements,which *... di.coveredfirst in plants, are responsiblefor leaf color variegation and the diverse beautiful color patterns of Indian corn kernels.By jumping in and out of ge.resthat control prgmentationas plant developmentprogresses,the mobile elemenrsgive rise to elaboratecolorid putt...rr. Mobile elements were later found in bacteria, in which they often carry and, unfortunatelS disseminategenesfor antibiotic resrstance. Now we understand that mobile elements have multi_ plied and slowly accumulatedin genomesover evolutionary time, becoming a universal property of genomesin preseni_ day_organisms. They account for an astounding45 percent of the human genome. Some of our own mobile DNA ele_ ments are copies-often highly mutated and damaged_of genomesfrom viruses that spend part of their life iycle as DNA segmentsinserted into host-cell DNA. Thus we carry in our chromosomes the genetic residues of infections ac_ quired by our ancestors.Once viewedonly as molecularpar_ asites,mobile DNA elementsare now thoueht to have ion_ tnbuted significantly to the evolution of higher organisms (Chapter6). '14

.

cHAprE1 R | L | F EB E G t Nwsl r H c E L L s

IE

TheWorkof Cetts

In essence,any cell is simply a compartment with a watery interior that is separatedfrom the external environment by a surfacemembrane (the plasma membrane) that preventsthe free flow of moleculesin and out. In addition, as we've noted, eukaryotic cells have extensive internal membranes that further subdivide the cell into various comparrmenrs, the organelles.Each compartment has contents and properties, such as specializedproteins or a certain pH, suited to its job. The plasma membrane and other cellular membranes are composedprimarily of two layers of phospholipid molecules. These bipartite moleculeshave a "water-loving" (hydrophilic) end and a "water-hating" (hydrophobic) end. The two phospholipid layersof a membrane are orienred with all the hydrophilic ends directed toward the inner and outer surfacesand the hydrophobic ends buried within the interior (Figure1-13). Smalleramounts of other lipids, such as cholesterol, and many kinds of proteins are inserted into the phospholipid framework. The lipid moleculesand someproteins can float sidewisein the plane of the membrane,giving membranes a fluid character. This fluidity allows cells to change shape and even move. However, the attachment of some membrane proteins to other molecules inside or outside the cell restricts their lateral movement. We will learn more about membranes and how molecules cross them in Chapters10 and 11. The cytosol and the internal spacesof organellesdiffer from each other and from the cell exterior in terms of aciditg ionic composition, and protein contents. For example, the composition of saltsinside the cell is often drastically different from what is outside. Becauseof these different ,,microclimates," each cell compartment has its own assigned

Cholesterol

Water-seeking h e a dg r o u p

Water

FIGURE 1-13Thewatery interiorof cellsis surroundedby the plasmamembrane,a two-layeredshell of phospholipids. Thephospholipid molecules areoriented with theirfattyacylchains (blacksquiggly lines) facinginwardandtheirwater-seeking head groups(whitespheres) facingoutward.Thusbothsidesof the membrane arelinedby headgroups, mainlycharged phosphates, adjacent to thewateryspaces tnside andoutside thecell.All biological membranes havethesamebasicphospholipid bilayer structureCholesterol (red)andvarious (notshown)are proteins embedded in the bilayer. Theinteriorspaceisactually muchlarger relative to thevolumeof the plasma membrane depicted here.

tasks in the overall work of the cell (Chapters 10, L2, and 13). The unique functions and microclimates of the various cell compartm€nts are due largely to the proteins that reside in their membranesor interior. 'We can think of the entire cell compartment as a factory dedicatedto sustainingthe well-being of the cell. Much cellular work is performed by molecular machines, some housed in the cytosol, some attached to the cytoskeleton' and some in various organelles.Here we quickly review the major tasks that cells carry out in their pursuit of the good life.

Cells needto break down worn-out or obsoleteparts into small molecules that can be discarded or recycled' This housekeepingtask is assignedlargely to lysosomes,organelles crammedwith degradativeenzymes.The interior of a lysosome has a pH of about 5.0' roughly 100 times more acidic than that of the surrounding cytosol. This aids in the breakdown of materials by lysosomal enzymes' which are specially

CellsBuild and DegradeNumerous Moleculesand Structures As chemical factories,cells produce an enormous number of complex molecules from simple chemical building blocks. All of this syntheticwork is powered by chemical energy extracted primarily from sugarsand fats or sunlight, in the case of plant cells, and stored primarily in ATP, the universal "currency" of chemical energy (Figure 1-14). In animal and plant cells, most ATP is produced by large molecular machines located in two organelles,mitochondria and chloroplasts. Similar machines for generating ATP are located in the plasma membrane of bacterial cells. Both mitochondria and chloroplasts are thought to have originated as bacteria that took up residenceinside eukaryotic cells and then became welcome collaborators (Chapter 12). Directly or indirectlS all of our food is created by plant cells using sunlight to build complex macromolecules during photosynthesis. Even underground oil suppliesare derived from the decay of plant material.

llll+ overviewAnimation:BiologicalE

Most of the structural and functional properties of cells de-

cell and most membrane proteins, however' are made on ribosomes associatedwith the endoplasmic reticulum (ER)' This organelleproduces,processes'and ships out both proteins and lipids. Prolein chains produced on the ER move to the Golgi comple*, where they are further modified before being for*ari.d to their final destinations. Proteins that travel in this way contain short sequencesof amino acids or attached sugar ch"itts (oligosaccharides)that serve as addressesfor directing them to their correct destinations. These addresseswork becausethey arerecognizedand bound by other proteins that do the sorting and shipping in various cell compartments'

lnterconversions or Light (photosynthesis) compoundswith high potentialenergY(resPiration )

Synthesisof cellularmacromolecules(DNA, RNA, proteins, polysaccharides)

Synthesisof other cellularconstituents (suchas membrane phospholipidsand certainrequired metabolites)

Cellularmovements, includingmusclecontraction,crawling movements of entire cells, and movement of chromosomesduring mitosis

A FIGURE1-14 ATP is the most common molecule used by cells to capture and transfer energy. ATPis formed from ADP and in plantsand by the inorganicphosphate(Pi)by photosynthesis

Transportof moleculesagainst a concentration

gradient

Generationof an electricpotential acrossa memDrane

Heat

for nerve 1fi..:.,;t;,",

by andfatsin mostcells.Theenergyreleased of sugars breakdown processes (hydrolysis) of PifromATPdrivesmanycellular thesplitting

THEWORK OF CELLS

15

lntermediate f ilaments

Microtubules

FIGURE 1-15 The three types of cytoskeletalfilamentshave characteristic distributionswithin cells.Threeviewsof the same cell.A cultured fibroblast wastreatedwith threedifferent antibodv preparations. Eachantibody bindsspecifically to the protein monomers formingonetypeof filamentandischemically linkedto a differently colored fluorescent dye(green, blue,or red).Visualization

A n i m a l C e l l sP r o d u c eT h e i r O w n E x t e r n a l E n v i r o n m e nat n d G l u e s The simplest multicellular animals are sinsle cells embedded in a jelly of proteins and polysaccharides."ll.d th. extracellular matrix. Cells themselvesproduce and secretethesematerials, thus creating their own immediate environment (Chapter 19). Collagen, the single most abundant protein in the animal kingdom, is a major component of the extracellular matrix in most tissues.In animals, the extracellularmatrix cushions and lubricates cells. A specialized,especially tough matrix, the basal lamina, forms a supporting layer un_ derlying sheetlikecell layers and helps pr.u"rrt the cells from rlppmg apart. The cells in animal tissuesare ..glued" togerher by cell_ adhesionmolecules(CAMs) embeddedin their surfacemem_

Microfilaments

of the stainedcellin a fluorescence microscope reveals the location of filaments boundto a particular preparation dye-antibody In this case,intermediate filaments green;microtubules, arestained blue; and microfilaments, red.All threefibersystems contributeto the shapeandmovements of cells.lcourtesy of V Small ]

animal cells (Figure 1-15). The cytoskeleton prevents the plasma membrane of animal cells from relaxing into a sphere(Chapter 10); it also functions in cell locomotion and the intracellular transport of vesicles,chromosomes, and macromolecules(Chapters 17 and 18). The cytoskeletoncan be linked through the cell surfaceto the extracellular matrix or to the cytoskeletonof other cells,thus helping to form tissues(Chapter 19). All cytoskeletal filaments are long polymers of protein subunits. Elaborate systemsregulatethe assembly dirursembly of the cytoskeleton,thereby controlling cell".rd shape.In some cells the cytoskeletonis relatively stable, but in others it changesshapecontinuously. Shrinkageof the cytoskeleton in some parts of the cell and its growrh in other parts can produce coordinated changesin shape that result in cell locomotion. For instance,a cell can send out an extensionthat attachesto a surfaceor to other cellsand then retract the cell body from the other end. As this processcontinues due to coordinated changesin the cytoskeleton,the cell moves forward. Cells can move at rates on the order of 20 pm./second.

C e l l sC h a n g eS h a p ea n d M o v e

largement but not movement of cells from one position to another.

Cells changeshapeand move becausetheir internal skeleton. the cytoskeleton,exerts forces on the rest of the cell and its

CellsSenseand SendInformation

attachments- Three types of protein filaments, organized into networks and bundles, form the cytoskeleton within

A living cell continuously monitors its surroundings and adjusts its own activities and composition accordingly. Cells also communicate by deliberatelysendingsignalsthat can be receivedand interpreted by other cells. Suchsignalsare com_ mon not only within an individual organism but also be_ tween organisms.For instance,the odor of a pear signals a food source to us and other animals; consumption of the

16

.

c H A p r E1R | L I F E B E G t Nwst r H c E L L s

pear by an animal aids in distributing the pear'sseeds.Everyone benefits! The signals employed by cells include simple small chemicals, gases, proteins, light, and mechanical movements. Cells possessnumerous receptor proteins for detecting signals and elaborate pathways for transmitting them within the cell to evoke a response.At any time, a cell may be able to senseonly some of the signalsaround it, and how a cell responds to a signal may change with time. In some cases,receivingone signal primes a cell to respond to a subsequentdifferent signal in a particular way. Both changesin the environment (e.g.,an increaseor decreasein a particular nutrient or the light level) and signals receivedfrom other cells representexternal information that cells must process.The most rapid responsesto such signals generally involve changesin the location or activity of preexisting proteins. For instance, soon after you eat a carbohydrate-rich meal, glucosepours into your bloodstream.The rise in blood glucose is sensedby B cells in the pancreas, which respond by releasingtheir stored supply of the protein hormone insulin. The circulating insulin signal causesglucose transporters in the cytoplasm of fat and muscle cells to move to the cell surface,where they begin importing glucose. Meanwhile, liver cellsalso are furiously taking in glucosevia a different glucosetransporter.In both liver and musclecells, an intracellular signalingpathway triggeredby binding of insulin to cell-surfacereceptorsactivatesa key enzymeneeded to make glycogen,a large glucosepolymer (Figure 1-16a). The net result of thesecell responsesis that your blood glucoselevel falls and extra glucoseis stored as glycogen,which your cells can use as a glucosesource when you skip a meal to cram for a test. The ability of cells to send and respond to signalsis crucial to development. Many developmentallyimportant signals are secretedproteins produced by specific cells at specific times and places in a developing organism. Often a receiving cell integratesmultiple signals in deciding how to behave,for example, to differentiate into a particular tissue type, to extend a process,to die, to send back a confirming s i g n a l( y e s ,I ' m h e r e ! ) ,o r t o m i g r a t e . The functions of about half the proteins in humans, roundworms, yeast, and severalother eukaryotic organisms have beenpredicted basedon analysesof genomic sequences (Chapter6). Suchanalyseshave revealedthat at least10-15 percent of the proteins in eukaryotesfunction as secretedextracellular signals, signal receptors, or intracellular signaltransduction proteins, which pass along a signal through a seriesof steps culminating in a particular cellular response (e.g., increased glycogen synthesis).Clearly, signaling and signal transduction are maior activities of cells.

CellsRegulateTheir Gene Expressionto Meet C h a n g i n gN e e d s In addition to modulating the activities of existing proteins, cells often respond to changing circumstancesand to signals from other cells by altering the amount or types of proteins they contain. Gene expression,the overall processof selectively reading and using genetic information, is commonly

(a)

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lncreasedtranscription of specificgenes

1-16 Externalsignalscommonlycausea changein FIGURE the activity of preexistingproteinsor in the amountsand or of a hormone typesof proteinsthat cellsproduce.(a)Binding cantriggeran receptors to itsspecific molecule othersignaling of a the activity or decreases pathway that increases intracellular in to receptors bindingof insulin protein. Forexample, preexisting of cellsleadsto activation of liverandmuscle membrane the plasma from of glycogen in thesynthesis a keyenzyme glycogen synthase, within arelocated for steroidhormones glucose(b)Thereceptors complexes hormone-receptor The surface the cell cells,noton to increased tarqetgenes,leading of specific transcription activate that bindto proteins. Manysignals production of the encoded pathways, to alsoact,by morecomplex on the cellsurface receptors geneexPression modulate

controlled at the level of transcription, the first step in the production of proteins. In this way cells can produce a pariicular mRNA only when the encoded protein is needed' thus minimizing wasted energy. Producing an mRNA is, however,only the first in a chain of regulatedeventsthat together determine whether an active protein product is produced from a particular gene. Transcriptional control of geneexpressionwas first decisively demonstratedin the responseof the gut bacterium E. coli to different sugar sources.E. coli cells prefer glucose as a sugar source,but they can survive on lactosein a pinch' These bacteria use both a DNA-binding repressor protein and a DNA-binding actiuator protein to change the rate of transcription of three genesneededto metabolize lactosedepending on the relative amounts of glucoseand lactosepresint (Chapter 4). Such dual positiveinegativecontrol of gene exoression fine-tunes the bacterial cell's enzymatic equip-..rt fo. the job at hand. Like bacterial cells, unicellular eukaryotes may be subjected to widely varying environmental conditions that require extensivechangesin cellular structuresand function' T H EW O R K O F C E L L S

17

For instance,in starvation conditions yeastcellsstop growing and form dormant spores (seeFigure 1-6). In multicellular organisms, however, the environment around most cells is relatively constant. The major purpose of gene control in us and in other complex organismsis to tailor the properties of various cell types to the benefit of the entire animal or plant. Control of gene activity in eukaryotic cells usually involves a balance between the actions of transcriptional activators and repressors.Binding of activators to specificDNA regulatory sequencescalled enhancers turns on transcription, and binding of repressorsto other regulatory sequences called silencersturns off transcription. In Chapters 7 and 8, we take a closelook at transcriptional activators and repressors and how they operate, as well as other mechanismsfor controlling gene expression.In an extreme case,expression of a particular gene could occur only in part of the brain, only during eveninghours, only during a certain stageof development, only after a large meal, and so forth. Many external signals modify the activity of transcriptional activators and repressorsthat control specific genes. For example, lipid-soluble steroid hormones, such as estrogen and testosterone,can diffuse across the plasma membrane and bind to their specific receptors located in the cytoplasm or nucleus(Figure 1-16b).Hormone binding changes

Overview Animation: Life Cycle of a Cell {lltt Nondividing cells

o

Resting c el l s

R N Aa n d protein synthesis G2

A FIGURE 1-17 Duringgrowth, eukaryoticcellscontinually progressthrough the four stagesof the cell cycle, generatingnew daughtercells.In mostproliferating cells,the four phases of the cellcycleproceedsuccessively, takingfrom 10-20 hoursdepending on celltypeanddevelopmental state Duringinterphase, whichconsists of the G,,,S,andG2phases, the cellroughly doubles itsmassReplication of DNAdurinqthe S phaseleaves the cellwith four copiesof eachtypeof chromosome Inthe mitotic(M)phase, thechromosomes are evenlypartitioned to two daughter cells,andthecytoplasm divides roughlyin halfin mostcases. Undercertainconditions suchasstarvation or whena tissuehasreached itsfinalsize,cells willstopcycling andremainin a waitingstatecalledGs Mostcells in Gscanreenter thecycleif conditions chanqe.

18

C H A P T E R1

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L t F EB E G T N W S |TH CELLS

the shape of the receptor so that it can bind to specific enhancer sequencesin the DNA, thus turning the receptor into a transcriptional activator. By this rather simple signaltransduction pathway, steroid hormones cause cells to change which genes they transcribe (Chapter 7). Since steroid hormones can circulate in the bloodstream, they can affect the properties of many or all cells in a temporally coordinated manner. Binding of many other hormones and of growth factors to receptors on the cell surface triggers different signal-transduction pathways that also lead to changesin the transcription of specific genes (Chapters 15 and 16). Although thesepathways involve multiple components and are more complicated than those transducing steroid hormone signals,the generalidea is the same.

CellsGrow and Divide As we have discussed,reproduction is at the heart of biology; rocks don't do it. The reproduction of organisms depends on the reproduction of cells. The simplest type of reproduction entails the division of a "parent" cell into two "daughter" cells.This occurs as part of the cell cycle,a series of eventsthat preparesa cell to divide followed by the actual division process, called mitosis. The eukaryotic cell cycle commonly is representedas four stages(Figure 1-17). The chromosomes and the DNA they carry are copied during the S (synthesis)phase.The replicatedchromosomesseparare during the M (mitotic) phase,with each daughter cell getting a copy of each chromosome during cell division. The M and S phasesare separatedby two gap stages,the G1 phase and G2 phase, during which mRNAs and proteins are made. In single-celledorganisms,both daughtercellsoften (though not always) resemblethe parent cell. In multicellular organisms, stem cells can give rise to two different cells, one that resembles the parent cell and one that does not. Such asymmetric cell division is critical to the generationof different cell types in the body (Chapter21). During growth the cell cycle operatescontinuously with newly formed daughter cells immediately embarking on their own path ro mitosis. Under optimal conditions bacteria can divide to form two daughter cells once every 30 minutes. At this rate, in an hour one cell becomesfour: in a day one cell becomesmore than 101a, which if dried would weigh about 25 grams. Under normal circumstances, however, growth cannot continue at this rate becausethe food supply becomeslimiting. Most eukaryotic cells take considerably longer than bacterial cells to grow and divide. Moreover, the cell cycle in adult plants and animalsnormally is highly regulated(Chapter20). This tight control prevents imbalanced, excessivegrowth of tissueswhile ensuring that worn-out or damaged cells are replaced and that additional cells are formed in responseto new circumstancesor developmental needs.For instance, the proliferation of red blood cellsincreasessubstantiallywhen a person ascendsto a higher altitude and needs more capacity to capture oxygen. Somehighly specializedcells in adult animals, such as nervecellsand striatedmusclecells,rarely divide, if at all. The fundamental defect in cancer is loss of the ability to

control the growth and division of cells.In Chapter 25, we examine the molecular and cellular eventsthat lead to inappropriate, uncontrolled proliferation of cells. Mitosis is an asexual process since the daughter cells carry the exact samegeneticinformation as the parental cell. ln sexwalreproduction, fusion of two cells produces a third cell that contains genetic information from each parental cell. Sincesuch fusions would causean ever-increasingnumber of chromosomes, sexual reproductive cycles employ a special type of cell division, called meiosis,that reducesthe number of chromosomesin preparation for fusion (seeFigure 5-3). Cells with a full set of chromosomes are called diploid cells. During meiosis, a diploid cell replicates its chromosomes as usual for mitosis but then divides twice without copying the chromosomesin between. Each of the resulting four daughter cells, which have only half the full number of chromosomes,is said to be haploid. Sexual reproduction occurs in animals and plants and even in unicellular organismssuch as yeasts(seeFigure 1-5). Animals spendconsiderabletime and energygeneratingeggs and sperm, the haploid cells, called gametes,which are used for sexualreproduction. A human femalewill produce about half a million eggsin a lifetime, all of thesecells forming before she is born; a young human male produces about 100 million sperm each day. Gametes are formed from diploid precursor germ-line cells,which in humans contain 46 chromosomes.In humans the X and Y chromosomesare called sex chromosomes becausethey determine whether an individual is male or female. In human diploid cells, the 44 remaining chromosomes,called autosomes,occur as pairs of 22 different kinds. Through meiosis,a man produces sperm that have 22 chromosomes plus either an X or a Y, and a woman produces ova (unfertilized eggs) with 22 chromosomesplus an X. Fusion of an egg and sperm (fertilization) yields a fertilized egg,the zygote,with46 chromosomes,one pair of each of the 22 kinds and a pair of Xs in femalesor an X and a Y in males(Figure1-18). Errors during meiosiscan lead to disorders resulting from an abnormal number of chromosomes.These include Down's syndrome, caused by an extra chromosome 21, and Klinefelter's syndrome, causedby an extra X chromosome.

Diploid (2n)

Haploid(1nl

(",,o )

One type of female gamete

Two types of male gamete

Diploid (2n) Female zygote

Male zygote

meiosis 1-18 Dad madeyou a boy or girl. In animals, FIGURE Themale cellsformseggsandsperm(gametes). of diploidprecursor the sexof the parentproduces two typesof spermanddetermines sex chromosomes; Y are the here, X and asshown zygoteIn humans, fromthe maleparentto a Y chromosome the zygotemustreceive (non-sex chromosomes). developintoa male A : autosomes bing" between the fingers; the cells in the webbing subsequently die in an orderly and precisepattern that leavesthe fingers and thumb free to play the piano. Nerve cells in the brain soon die if they do not make proper or useful electrical

CellsDie from AggravatedAssaultor an lnternal Program 'lfhen

cells in multicellular organismsare badly damagedor infectedwith a virus, they die. Cell death resulting from such a traumatic event is messy and often releasespotentially toxic cell constituents that can damage surrounding cells. Cells also may die when they fail to receivea life-maintaining signal or when they receive a death signal. In this type of programmed cell death, called apoptosis, a dying cell actually produces proteins necessaryfor self-destruction.Death by apoptosis avoids the releaseof potentially toxic cell constituents(Figure1-19). Programmed cell death is critical to the proper development and functioning of our bodies (Chapter 21). During fetal life, for instance,our hands initially developwith "web-

1-19ApoPtoticcellsbreakapartwithout spewing FIGURE forth cell constituentsthat might harm neighboringcells. looklikethe cellon the left Cells Whitebloodcellsnormally likethe cellon the programmed celldeath(apoptosis), undergoing The arereleased blebsthateventually surface right,formnumerous is growth Apoptosis signals. it lackscertain cellisdyingbecause cellswherethey cells,to remove virus-infected to eliminate important (likethewebbingthatdisappears asfingersdevelop), arenot needed cellsthatwouldreactwith ourown immunesystem andto destroy Inc] Unlimited, Murtil/isuals bodies[Gopal THE WORK OF CELLS

19

connectionswith other cells. Some developinglymphocytes, the immune-systemcells intended to recognizeforeign proteins and polysaccharides,have the ability to reac against our own tissues.Such self-reactivelymphocytes becomeprogrammed to die before they fully marure. If these cells are not weededout before reaching maturity, they can causeautoimmune diseases,in which our immune system destroys the very tissuesit is meant to protect.

IE

InvestigatingCellsand TheirParts

To build an integrared understanding of how the various molecular components that underlie cellular functions work together in a living cell, we must draw on various perspectives. Here, we look at how five disciplines-cell biology, biochemistry and biophysics,genetics,genomics,and developmental biology-can conrribure to our knowledge of cell structure and function. The experimental approachesof each field probe the cell's inner workings in different ways, allowing us to ask different types of questions about cells and what they do. Cell division provides a good example to illustrate the role of different perspectivesin analyzing a complex cellular process.Although we discussthe different disciplinesseparatelyfor clarity, in practice most biologists use multiple approachesin concert. This is part of the fun of

Nanometers

The goal of cell biologists is to understandhow a cell is able to control its own shape and surface properties, transport materials to the right locations, copy itself, and receiveand send signals.Cell biologists use severaltypes of microscopy to observecells, while at the same time labeling specificcell components and altering them to seewhat happens.Analysesare generally done at the micrometer scale. Actual observation of cells awaited development of the first, crude microscopes in the early 1600s. A compound

Meters

Assemblies Macromolecules

Glucose

C-C bond

Cell Biology Revealsthe Size,Shape,Location, and Movementsof Cell Components

Millimeters

Micrometers

Small molecules Atoms

cell biologg putting together genetics with microscopy or enzymology with development. The realm of biology rangesin scalemore than a billionfold (Figure 1-20). Beyond that, it's ecology and earth science at the "macro" end, chemistry and physics at the "micro" end. The visible plants and animals that surround us are measuredin meters (100-102 m). By looking closely,we can seea biologicalworld of millimeters(1 mm : 10-3 m) and even tenths of millimeters (10-a m). Setting aside oddities like chicken eggs, most cells are 1-100 micrometers (1 pm : 10-5 m) long and thus clearlyvisibleonly when magnified. To seethe structureswithin cells, we must go farther down the sizescaleto 10-100 nanometers(1 nm : 10 v m).

Hemoglobin

M u l t i c e l l u l aor r g a n i s m s

Cells

Ribosome

C. elegans

Bacterium

Mitochondrion

Newborn human

Bumblebee

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10sm

1 0 ' 8m

'1 0-7m

0.1nm

1 nm

10nm

1 0 0n m

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1 0 - 5m 10pm

FIGURE 1-20 Biologistsare interestedin objectsrangingin sizefrom smallmolecules to the tallesttrees.A sampling of biological objects aligned on a logarithmic scale(a)TheDNAdouble helixhasa diameter of about2 nm (b)Eight-cell-stage numan embryothreedaysafterfertilization, about200 rr"macross(c)A wolf

20

CHAPTER 1

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L I F EB E G I N S WITHCELLS

1 0 - am 1 0 0u m

1 0 - 3m

1 0 2m

1 0 ' 1m

1 mm

1 0m m

100mm

1 0 0m 1m

spider, about15 mm across(d)Emperor penguins areabout1 m tall. [Part(a)Will andDeniMclntyrePart(b)YorgasNikas/photo Researchers, Inc Part(c)GaryGauglerl/isuals Unlimited, Inc Part(d)HughS Rosefuisuals Unlimited. lncl

microscope,the most useful type of light microscope,has two lenses.The total magnifying power is the product of the magnificationby each lens. As better lenseswere invented, the magnifying power and the ability to distinguish closely spacedobjects, the resolution, increasedgreatly. Modern cclmpoundmicroscopesmagnify the view about a thousandfold, so that a bacterium 1 micrometer (1 p-) long looks like it's a millimeterlong. Objectsabout 0.2 pm apart can be discernedin theseinstruments. Microscopy is most powerful when particular components of the cell are stained or labeled specifically,enabling them to be easilyseenand locatedwithin the cell. A simple exampleis stainingwith dyesthat bind specificallyto DNA to visualize the chromosomes. Specific proteins can be detected by harnessingthe binding specificityof antibodies,the proteins whose normal task is to help defend animals against infection and foreign substances.In general,eachtype of antibody binds to one protein or large polysaccharideand no other (Chapter 3). Purified antibodies can be chemically linked to a fluorescent molecule, which permits their detection in a specialfluorescencemicroscope(Chapter 3). lf a cell or tissueis treated with a detergentthat partially dissolvescell membranes,fluorescentantibodiescan drift in and bind to the specific protein they recognize. When the sample is viewed in the microscope,the bound fluorescent antibodies identify the location of the target protein (see F i g u r e1 - 1 5 ) . Betrer still is pinpointing proteins in living cells with intact membranes. One way of doing this is to introduce an engineeredgenethat codesfor a hybrid protein: part of the hybrid protein is the cellular protein of interest;the other part is a protein that fluoresceswhen struck by ultraviolet light. A common fluorescentprotein used for this purpose is green fluorescent protein (GFP), a natural protein that makes some iellyfish colorful and fluorescent. GFP "tagging" could reveal,for instance,that a particular protein is first made on the endoplasmic reticulum and then is moved by the cell into the lysosomes.In this case,first the endoplasmicreticulum and later the lysosomeswould glow in the dark. Chromosomesare visible in the light microscopeonly during mitosis,when they becomehighly condensed.The extraordinary behavior of chromosomesduring mitosis first was discoveredusing the improved compound microscopes of the late 1800s.About halfway through mitosis,the replicatedchromosomesbeginto move apart. Microtubules,one of the three types of cytoskeletalfilaments, participate in this movementof chromosomesduring mitosis.Fluorescenttagging of tubulin, the protein subunitthat polymerizesto form microtubules, reveals structural details of cell division that otherwisecould not be seenand allows observationof chromosomemovement(Figure1-21). Electron miqroscopesuse a focusedbeam of electronsinstead of a beam of light. In transmission electron microscopy, specimensare cut into very thin sections and placedunder a high vacuum,precludingexaminationof living cells. The resolution of transmission electron micro-

llll|

5e6nsAnimation:Mitosis

A FIGURE1-21 During the later stages of mitosis, microtubules (red) pull the replicated chromosomes(black) toward the ends of a dividing cell. Thisplantcell is stained and with a DNA-bindingdye (ethidium)to revealchromosomes antibodiesspecificfor tubulinto reveal with f luorescent-tagged microtubulesAt this stagein mitosis,the two copiesof each replicatedchromosome(calledchromatids)haveseparatedand are of AndrewBajer ] movinqawavf rom eachother [Courtesy

scopes,about 0.1 nm, permits fine structural details to be distinguished,and their powerful magnification would make a 1-pm-long bacterialcell look like a soccerball. Most of the organelles in eukaryotic cells and the double-layered structure of the plasma membrane were first observedwith electron microscopes(Chapter 9). \fith new specializedelectron microscopy techniques,three-dimensionalmodels of organellesand large protein complexes can be constructed from multiple images.But to obtain a more detailed look at the individual macromoleculeswithin cells, we must turn to techniqueswithin the purview of biochemistry and biophysics.

s e v e a tl h e B i o c h e m i s t r ay n d B i o p h y s i c R M o l e c u l a rS t r u c t u r ea n d C h e m i s t r yo f P u r i f i e d Cell Constituents Biochemistsextract the contentsof cellsand separatethe constituents based on differencesin their chemical or physical properties,a processcalledfractionation. The attention to individual moleculesmeans operating at the nanometer scale. Of particular interest are proteins, the workhorses of many A typical fractionation schemeinvolvesuse cellular processes. of various separationtechniquesin a sequentialfashion.These separationtechniquescommonly are basedon differencesin the size of moleculesor the electric charge on their surface (Chapter 3). To purify a particular protein of interest, a purification scheme is designed so that each step yields a

I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

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FIGURE 1-22 Biochemicalpurificationof a protein from a cell extract often requiresseveralseparationtechniques.The purificationcanbe followedby gelelectrophoresis of thestarting proteinmixture andthefractions obtained fromeachpurification step Inthisprocedure, a sample isapplied to wellsin thetop of a gelatin-like slabandan electric fieldisappliedInthe presence of appropriate saltanddetergent concentrations, the proteins move throughthefibersof the geltowardtheanode,with largerproteins movingmoreslowlythroughthe gelthansmaller ones(seeFigure 3-35)Whenthegelisstained, proteins separated arevisible as distinctbandswhoseintensities areroughlyproportional to the proteinconcentration Shownhereareschematic depictions of oels for thestarting (lane1)andsamples mixture of proteins takenaiter eachof several purification stepsIn the firststep,saltf ractionation, proteinsthat precipitated with a certainamountof saltwereredissolved; electrophoresis (lane2) showsthatit of thissample contains fewerproteins thantheoriginal mixtureThesample then wassubjected in succession to threetypesof column chromatography proteins thatseparate by electrical charge, size,or bindingaffinityfor a particular (seeFigure smallmolecule 3-37).The finalpreparation isquitepure,ascanbe seenfromtheappearance of justoneproteinbandin lane5 [AfterJBergetal,2o02,Biochemistry W H Freeman andCompany, p 87l preparation with fewer and fewer contaminating proteins, until finally only the protein of interestremains (Figure 1-22). The initial purification of a protein of interest from a cell extract often is a tedious, time-consumingtask. Once a small amount of purified protein is obtained, antibodies to it can be produced by methods discussedin Chapter 19. For a biochemist, antibodies are near-perfecttools for isolating larger amounts of a protein of interest for further analysis.In effect, antibodies can "pluck out" the protein they specifically recognizeand bind from a semipure sample containing numerous different proteins. An increasinglycommon alterna-

22

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L I F EB E G I N SW I T H C E L L S

tive is to engineera gene that encodesa protein of interest with a small attached protein "tag," which can be used to pull out the protein from whole cell extracts. Purification of a protein is a necessaryprelude to studies on how it catalyzesa chemical reaction or carries out other functions and how its activiry is regulated. Some enzymesare made of multiple protein chains (subunits) with one chain catalyzing a chemical reaction and other chains regulating when and where that reaction occurs. The molecular machines that perform many critical cell processesconstitute even larger assembliesof proteins. By separating the individual proteins composing such assemblies,their individual catalytic or other activitiescan be assessed. For example,purification and study of the activity of the individual proteins composing the DNA replication machine provided clues about how they work together to replicate DNA during cell division (Chapter 4). The folded, three-dimensionalstructure, or conformation, of a protein is vital to its function. To understand the relation between the function of a protein and its form, we need to know both what it does and its detailed structure. The most widely used method for determining the complex structures of proteins, DNA, and RNA is x-ray crystallography, one of the powerful tools of biophysics.Computer-assistedanalysisof the data often permits the location of every atom in a large, complex molecule to be determined. The double-helix structure of DNA, which is key to its role in herediry was first proposed basedon x-ray crystallographic studies.Throughout this book you will encounter numerous examplesof protein structuresas we zero in on how proteins work.

GeneticsRevealsthe Consequences of DamagedGenes Biochemical and crystallographic studies can tell us much about an individual protein, but they cannot prove that it is required for cell division or any other cell process.The importance of a protein is demonstratedmost firmly if a mutation that prevents its synthesis or makes it nonfunctional adverselyaffects the processunder study. !(/e define the genotype of an organism as its composition of genes;the term also is commonly used in referenceto different versionsof a singlegeneor a small number of genes of interest in an individual organism. A diploid organism generally carries two versions (alleles)of each gene, one derived from each parent. There are important exceptions, such as the geneson the X and Y chromosomesin males of some species,including our own. The phenotype is the visible outcome of a gene's action, such as blue eyes versus brown eyesor the shapesof peas.In the early days of genetics, the location and chemical identity of genes were unknown; only the observablecharacteristics,the phenotypes, could be followed. The concept that genesare like "beads" on a long "string," the chromosome,was p,roposedearly in the 1900s basedon geneticwork with the fruit fly Drosophila. In the classicalgeneticsapproach, mutants are isolated that lack the ability to do something a normal organism can do. Often large genetic "screens" are done to look for many different mutant individuals (e.g., fruit flies, yeast cells) that are

unable to complete a certain process,such as cell division or muscle formation. In experimental organismsor cultured cells, mutations usually are produced by treatment with a mutagen, a chemical or physical agent that promotes mutations in a largely random fashion.But how can we isolateand maintain mutant organismsor cellsthat are defectivein some process, such as cell division, that is necessaryfor survival?One way is to look for organisms with a temperature-sensitivemutation. Thesemutants are able to grow at one temperature,the permissiue temperature, but not at anothe5 usually higher temperature, the nonpermissiuetemperature.Normal cells can grow at either temperature. In most cases,a temperature-sensitivemutant produces an altered protein that works at the permissive temperaturebut unfolds and is nonfunctional at the nonpermissive temperature. Temperature-sensitive screensare readily done with viruses,bacteria, yeast,roundworms, and fruit flies. By analyzing the effectsof numerous different temperaturesensitivemutations that altered cell division, geneticistsdiscoveredall the genesnecessaryfor cell division without knowing anything, initially, about which proteins they encode or how theseproteinsparticipatein the process.The greatpower of geneticsis to reveal the existenceand relevanceof proteins without prior knowledge of their biochemicalidentity or molecular function. Eventually these "mutation-defined" genes were isolatedand replicated(cloned)with recombinantDNA techniquesdiscussedin Chapter 5. \fith the isolatedgenesin hand, the encodedproteins could be produced in the test tube or in engineeredbacteria or cultured cells. Then the biochemistscould investigatewhether the proteins associatewith other proteins or DNA or catalyze particular chemical reactions during cell division (Chapter20). The analysis of genome sequencesfrom various organisms during the past decadehas identified many previously unknown DNA regions that are likely to encode proteins (i.e., protein-coding genes).The generalfunction of the protein encoded by a sequence-identifiedgene may be deduced by analogy with known proteins of similar sequence.Rather than randomly isolating mutations in novel genes, several techniquesare now available for inactivating specific genes by engineering mutations into them or destroying their mRNA with interfering RNA molecules(Chapter 5). The effects of such deliberategene-specificinactivation procedures provide information about the role of the encoded proteins in living organisms. This application of genetic techniques starts with a gene/proteinsequenceand ends up with a mutant phenotype; traditional genetics starts with a mutant phenotype and ends up with a gene/proteinsequence.

GenomicsRevealsDifferencesin the Structure and Expressionof EntireGenomes Biochemistry and geneticsgenerally focus on one gene and '$flhile powerful, these tradiits encoded protein at a time. tional approachesdo not give a comprehensiveview of the structure and activity of an organism'sgenome,its entire set of genes.The field of genomicsdoes just that, encompassing the molecular characterizationof whole genomesand the determination of global patterns of geneexpression.The recent

completion of the genome sequencesfor more than 100 speciesof bacteria and severaleukaryotesnow permits comparisons of entire genomes from different species.The results provide overwhelming evidenceof the molecular unity of life and the evolutionary processesthat made us what we are (seeSection 1.5). Genomics-basedmethods for comparing thousands of piecesof DNA from different individuals all at the same time are proving useful in tracing the history and migrations of plants and animals and in following the inheritance of diseasesin human families. DNA microarrays can simultaneously detect all the mRNAs presentin a cell, thereby indicating which genesare being transcribed. Such global patterns of gene expression clearly show that liver cells transcribe a quite different set of genesthan do white blood cellsor skin cells. Changesin gene expressionalso can be monitored during a diseaseprocess' in responseto drugs or other external signals'and during development. For instance,the identification of all the mRNAs presentin cultured fibroblasts before, during' and after they divide has given us an overall view of transcriptional changesthat occur during cell division (Figure 1-23). Cancet diagnosis is being transformed becausepreviously indistinguishablecancer cells have distinct gene expressionpatterns and prognoses (Chapter 25). Similar studies with different organisms and cell types are revealing what is universal about the genesinvolved in cell division and what is specific to particular organisms.To find out which genesare directly regulatedby a transcription factor, chromatin containing the protein of interest can be purified with an antibody and the associated DNA analyzed on microarrays, a procedure called chromatin immunopreclpltatlon. The entire complement of proteins in a cell, its proteome' is controlled in part by changesin genetranscription.The regulated synthesis,processing,localization' and degradationof specific proteins also play roles in determining the proteome of a particular cell. Learning how proteins bind to other proteins, often in large, multiprotein complexes, is providing a comprehensiveview of the molecular machines important for cell functioning. The field of proteomics will advance dramatically once high-throughput x-ray crystallography, currently under development,permits researchersto rapidly determine the structuresof hundredsor thousandsof proteins.

DevelopmentalBiology RevealsChangesin the Propertiesof Cellsas They Specialize Another approach to viewing cells comes from studying how they changeduring development of a complex organism. Bacteria, algae, and unicellular eukaryotes (protozoans, yeasts) often, but by no means always, can work solo. The concerted actions of the trillions of cells that compose our bodies require an enormous amount of communication and division of labor. During the development of multicellular organisms, differentiation processesform hundreds of cell types, each specialized for a particular task: transmission of electric signals by neurons, transport of oxygen by red blood cells, destruction of infecting bacteria by macrophages' contraction by muscle cells, chemicalprocessingby liver cells,and so on. I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

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EXPERIMENTAL FIGURE 1-23 Microarrayanalysisof normal growing braincellsand braintumor cells.An experiment likethis isa starting pointfor learning howtumorcellsdifferfromnormal cellsRNAwasextracted fromnormalgrowingmousebraincells fromthecerebellum andfroma tumorof thecerebellum TheRNA fromthe tumorwaslabeled with a reddye,andRNAfromthe normal,non-tumorous cerebellum waslabeled with a greendye The two RNApreparations weremixedandhybridized to a microarray containing thousands of spotsof DNA Eachspotcontains the DNA sequence of onegene Unbound RNAwaswashedawayandthe microarray wasexposed to UVlight,whichcauses the dyesto fluoresce Spotsthataregreenhaveboundmostlynormalcerebellum RNA,spotsthatareredhaveboundmostlytumorRNA,andspots thatareyellowhaveboundroughly equalamounts of each.The faintlystained spotsrepresent genesfor whichthereislittleRNAin eithersampleThedataindicate whichgeneshavebeentranscribed in tumors,normalcerebellum, or both Onlypartof the datais shownhereTheentiredatasetrequires analyzing the colorsof more than25,000spots,allof whichcanbefittedontoonemicroscope slide.Precise measurements of colorintensity areactually madeby a spectrophotometer, but lookingby eyeshowsthat manygenesare morehighlyexpressed in normalor tumorcells.Someof these differences arethe consequence of the changeintotumorcells,but somemayreveal geneexpression changes thatcausethetumorsto form In addition, proteins madeexclusively in tumors,andperhaps necessary for uncontrolled growth,maybe candidate targets for discovering anti-cancer drugs [Courtesy of TalRaveh andl\y'atthew Scott, StanfordUniversitySchoolof Medicinel

Many of the differences among differentiated cells are due to production of specificsetsof proteins neededto carry out the unique functions of each cell type; that is, only a subsetof an organism'sgenesis transcribedat any given time or in any given cell. Such differential gene expressionat different times or in different cell types occurs in bacteria, fungi, plants, animals, and even viruses. Differential gene

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expression is readily apparent in an early fly embryo, in which all the cells look alike until they are stained to detect the proteins encoded by particular genes (Figure 1-24). Transcription can changewithin one cell type in responseto an external signal or in accordancewith a biological clock; some genes,for instance,undergo a dalIy cycle between low and high transcription rates. Producing different kinds of cells is not enough to make an organism, any more than collecting all the parts of a truck in one pile gives you a truck. The various cell types must be organized and assembled into all the tissues and organs. Even more remarkable, these body parts must work almost immediately after their formation and continue working during the growth process. For instance, the human heart begins to beat when it is lessthan 3 mm long, when we are mere 23-day-old embryos, and continues beating as it grows into a fist-sizemuscle. From a few hundred cells to billions, and still ticking. In the developing organism, cells grow and divide at some times and not others, they assembleand communicate, they prevent or repair errors in the developmentalprocess, and they coordinate each tissuewith others. In the adult organism,cell division largely stops in most organs.If part of an organ such as the liver is damaged or removed, cell division resumesuntil the organ is regenerated.The legend goes that Zets punished Prometheus for giving humans fire by

FIGURE 1-24 Differentialgene expressioncan be detectedin early fly embryosbefore cellsare morphologicallydifferent. An earlyDrosophila embryohasabout6000cellscoveringitssurface, mostof whichareindistinguishable by simplelightmicroscopy. lf the embryois madepermeable to antibodies with a detergent that partially dissolves membranes, theantibodies canfindandbindto the proteins theyrecognize Inthisembryowe seeantibodies tagged with a fluorescent labelboundto proteins thatarein the nuclei; each smallsphere corresponds to onenucleus. Threedifferent antibodies wereused,eachspecific proteinandeachgivinga for a different distinct green,or blue)in a fluorescence color(yellow, microscope. Theredcolorisaddedto highlight overlaps between theyellowand bluestainsThelocations proteins of thedifferent showthatthe cells arein factdifferent at thisearlystage,with particular genesturned on in specific stripes of cells. Thesegenescontrolthesubdivision of the bodyintorepeating segments, likethe blackandyellowstripes of a hornet.[Courtesy of Sean Carroll, University of Wisconsin ]

chaining him to a rock and having an eagleeat his liver. The punishment was eternal because,as the Greeks evidently knew, the liver regenerates. Developmental studies involve watching where, when, and how different kinds of cells form, discovering which signals trigger and coordinate developmentalevents,and understandingthe differential gene action that underliesdifferentiation (Chapters 16 and 21). During development we can see cells change in their normal context of other cells. Cell biology, biochemistry, cell biology, genetics, and genomics approachesare all employed in studying cells during development.

C h o o s i n gt h e R i g h t E x p e r i m e n t aOl r g a n i s m for the Job Our current understanding of the molecular functioning of cells rests on studieswith viruses,bacteria, yeast, protozoa, slime molds, plants, frogs, sea urchins, worms, insects,fish, chickens, mice, and humans. For various reasons, some organisms are more appropriate than others for answering particular questions.Becauseof the evolutionary conservation of genes,proteins, organelles,cell types, and so forth, discoveriesabout biological structuresand functions obtained with one experimental organism often apply to others. Thus researchersgenerallyconduct studieswith the organism that is most suitable for rapidly and completely answering the question being posed, knowing that the results obtained in one organismare likely to be broadly applicable.Figure1-25 summarizesthe typical experimental usesof various organisms whose genomeshave been sequencedcompletely or nearly so. The availability of the genomesequencesfor these organisms makes them particularly useful for geneticsand genomicsstudies. Bacteria have several advantagesas experimental organisms: they grow rapidlS possesselegant mechanisms for controlling gene activity, and have powerful genetics. This latter property relates to the small size of bacterial genomes,the easeof obtaining mutants, the availability of techniquesfor transferring genes into bacteria, an enormous wealth of knowledge about bacterial gene control and protein functions, and the relative simplicity of mapping genesrelative to one another in the genome. Singlecelledyeastsnot only have some of the sameadvantagesas bacteria but also possessthe cell organization,marked by the presenceof a nucleusand organelles,that is characteristic of all eukaryotes. Studiesof cellsin specializedtissuesmake use of animal and plant "models," that is, experimentalorganismswith attributes typical of many others. Nerve cells and muscle cells, for instance,traditionally were studied in mammals cells,such or in creatureswith especiallylarge or accessible as the giant neural cells of the squid and sea hare or the flight musclesof birds. More recently,muscle and nerve development have been extensively studied in fruit flies (Drosophila melanogaster),roundworms (Caenorhabditis elegans),and zebrafish(Danio rerio), in which mutants can be readily isolated. Organisms with large-celledembryos

that develop outside the mother (e.g.' frogs, sea urchins, fish, and chickens) are extremely useful for tracing the fates of cells as they form different tissues and for making extracts for biochemicalstudies.For instance,a key protein in regulating mitosis was first identified in studies with frog and sea urchin embryos and subsequentlypurified from extracts (Chapter 20lt. Using recombinant DNA techniques,researcherscan engineer specificgenesto contain mutations that inactivate or increaseproduction of their encodedproteins. Suchgenescan be introduced into the embryos of worms, flies, frogs, sea urchins, chickens,mice, a variety of plants, and other organisms,permitting the effectsof activating a geneabnormally or inhibiting a normal gene function to be assessed.This approach is being used extensivelyto produce mouse versions of human genetic diseases.Inactivating particular genes by introducing short piecesof interfering RNA is allowing quick tests of gene functions possiblein many organisms.The expansion of genomeproiectsto critically important diseaseorganisms,such as malaria, and to creaturesthat span the evolutionary tree is bringing new options for medicine and new insights into how living organisms have diversified to take advantageof every possibleecologicalniche. Mice have one enormous advantage over other experimental organisms:they are the closestto humans of any animal for which powerful geneticapproachesare feasible.Engineered mouse genes carrying mutations similar to those associatedwith a particular inherited diseasein humans can be introduced into mouse embryonic stem (ES) cells. These cells can be injected into an early embryo, which is then implanted into a pseudopregnantfemale mouse (a mouse treated with hormones to trigger physiological changes neededfor pregnancy) (Chapter 5). If the mice that develop from the injected ES cells exhibit diseasessimilar to the human disease,then the link between the diseaseand mutations in a particular geneor genesis supported. Once mouse models of a human diseaseare available, further studies on the molecular defectscausing the diseasecan be done and new treatmentscan be tested,thereby minimizing human exposure to untested treatments. Large-scalegenetic screens are being done that take advantageof newly designedmutagenic transposons.The transposons allow efficient generation of mouse mutants and rapid identification of the gene that has been hit in each one. A continuous unplanned genetic screen has been per\What we mean formed on human populations for millennia. is that all sorts of human variations have arisen and have been noticed, since they affect visible or noticeable human characteristics.Thousandsof inherited traits have beenidentified and, more recently,mapped to locations on the chromosomes.Some of these traits are inherited propensitiesto get a disease;others are eye color or other minor characteristics. Geneticvariations in virtually every aspectof cell biology can be found in human populations, allowing studiesof normal and diseasestatesand of variant cells in culture. Less-commonexperimental organisms offer possibilities for exploring unique or exotic properties of cells and for studying standard properties of cells that are exaggeratedin I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

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Podcast:Common ExperimentalOrganisms

Viruses

Bacteria

Proteinsinvolvedin DNA, RNA, protein synthesis Gene regulation Cancerand control of cell oroliferation Transportof proteinsand organellesinsidecells Infectionand immunity Possiblegene therapy approaches

Proteinsinvolvedin DNA, RNA, protein synthesis, metabolism Gene regulation Targetsfor new antibiotics Cell cycle Signaling

Yeast (Saccharo myce s cerev is i ael

Roundworm I Caenorh abd itis elegansl

Controlof cell cycle and cell division Proteinsecretionand membrane biogenesis Functionof the cytoskeleton Cell differentiation Aging Gene regulationand chromosome structure

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Developmentof the body plan C e l ll i n e a g e Formationand function of the nervous system Controlof programmedcell death Cell proliferationand cancergenes Aging Behavior Gene regulationand chromosome structure

Fruit ffy (Drosophi Ia mel anoga sterl

Zebrafish

Developmentof the body plan Generationof differentiatedcell lineages Formationof the nervoussystem, heart,and musculature Programmedcell death Geneticcontrol of behavior Cancergenesand control of cell proliferation Controlof cell polarization Effectsof drugs, alcohol,pesticides

Developmentof vertebratebody tissues Formationand function of brain and nervous syslem Birth defects Cancer

Mice, includingculturedcells

Plant (A ra b i d ops is th aIi anal

Developmentof body tissues F u n c t i o no f m a m m a l i a ni m m u n e system Formationand function of brain and nervoussystem Models of cancersand other human diseases Gene regulationand inheritance Infectiousdisease

Developmentand patterningof tissues Geneticsof cell biology Agricultural applications Physiology Gene regulation lmmunity Infectiousdisease

L I F EB E G I N SW I T H C E L L S

< FIGURE 1-25 Eachexperimentalorganismusedin cell (a) biology has advantagesfor certaintypes of studies.Viruses (b)havesmallgenomes andbacteria amenable to genetic dissection Manyinsights intogenecontrolinitially camefromstudies with these (c)hasthe cellular organismsTheyeastSaccharomyces cerevisiae organization of a eukaryote but isa relatively simplesingle-celled genetically. organism thatiseasyto growandto manipulate Inthe (d),whichhasa small nematode worm Caenorhabditis elegans numberof cellsarranged in a nearlyidentical wayin everyworm,the formation of eachindividual cellcanbe tracedThefruitfly (e),firstusedto discover Drosophila melanogaster the properties of genesthat chromosomes, hasbeenespecially valuable in identifying controlembryonic development Manyof thesegenesare evolutionarily conserved in humansThezebraf ishDaniorerio(f)is genesthatcontrol usedfor rapidgenetic screens to identify development andorganogenesis. Of theexperimental animal (g)areevolutionarily systems, mice(Musmusculus) the closestto humans andhaveprovided models for studying numerous human genetic andinfectious diseases. Themustard-family weed Arabidopsis thaliana,sometimes described asthe Drosophila of the plantkingdom, genes hasbeenusedfor genetic screens to identify involved in nearlyeveryaspect of plantlife Genome sequencing is completed for manyviruses andbacterial species, theyeast Saccharomyces cerevisiae, the roundwormC. elegans,the f ruit fly D. melanogaste4 humans,andthe plantArabidopsis thalianallis mostlycompleted for miceandin progress for zebrafish Other particularly organisms, frogs,seaurchins, chickens, andslimemolds, to be immensely continue valuable for cellbiologyresearch Increasingly, a widevariety of otherspecies for areused,especially (a)Visuals studies of evolution of cellsandmechanisms Unlimited, [Part Inc Part(b)KariLountmaa/Science Photo Library/Photo Researchers, Inc Part (c)ScimavPhoto Researchers, Inc Part(d)Photo Researchers, IncPart(e) Darwin Dale/Photo Researchers, Inc,Part(f)IngeSpencel'/isuals Inc Unlimited, Part(g)J M Labavjancanatuisuals Unlimited, Inc Part(h)Darwin Dale/Photo Researchers, Inc]

a useful fashion in a particular animal. For example, the ends of chromosomes,the telomeres,are extremely dilute in most cells.Human cells typically contain 92 telomeres(46 chromosomesX 2 endsper chromosome).In contrast,some protozoawith unusual "fragmented" chromosomescontain millions of telomeres per cell. Taking advantage of the unique properties of this well-chosen experimental organism has led to important recent discoveriesabout telomere structure.

The Most SuccessfulBiologicalStudiesUse M u l t i p l eA p p r o a c h e s We have discussedfive classesof approachesto biological problems: cell biology biochemistry and biophysics, genetics, genomics,and developmentalbiology. Each has its own types of experiments,and most biological problems require more than one approach in order to reach a satisfying understanding of mechanism. Now we will survey how these approacheshave been applied to the study of cell division to emphasizehow important it is to use multiple types of experlments.

Cell division was viewed, and indeed discovered,by some of the earliest usersof microscopes.More recently a variety of kinds of microscopy, including confocal and electron microscopy and time-lapseimaging (Chapter 9), have been used to characterizethe stepsof the cell cycle. Most biology begins with this sort of observation, defining the mysteries that must be tackled experimentally.Then manipulations begin. Antibodies were made againstproteins that play critical roles in cell division both to detect proteins and in some casesto interfere with the functions of those proteins. Key proteins were fused to fluorescentproteins, starting with the iellyfish greenfluorescentprotein (GFP),so that key proteins could be followed in living cells. Questions about when and where proteins work could be addressedand their functions defined to an extent. The apparatusof cell division, such as the mitotic spindle and other protein complexes,was purified and analyzed using the approaches of biochemistry and biophysics. Each protein had to be purified to find out whether it is part of a complex of proteins bound together as a machine, and the structuresof key proteins were determined using x-ray crystallography and other methods (Chapter 3). Previously unknown enzymatic activities were detected in extracts using assays such as measuring the attachment of phosphate groups to cell division regulatory proteins by kinases, and then the relevant kinasescould be purified. Finding a novel protein in a complex of proteins involved in cell division makes it a good bet that the protein does something important, a sort of "guilt by association," but it doesnot provide proof that the protein matters. For that one must turn to genetics. Genetics can be used to identify mutants in the newly found protein. If cell division fails in a living organism when the protein is not working, you know the protein matters. Geneticsis also a way to identify previously unknown genesand proteins sincescreenscan be done (especiallyin bacteria and yeast, but also in more complex Iab organisms)to look for all the genesthat are neededfor cell division. The newly discoveredproteins can be incorporated into a complete picture of the mechanicsof cell division machinery. Genomics provides another way to look for working parts of the cell-division machine. Since it is often true that mRNAs and proteins are produced only when they are needed, using microarrays to look for all geneswhose expressionvaries with the cell cycle is a powerful approach to identify candidatesfor cell division regulators. Having identified new genesthat are required for cell division, one must find out how thesegenes'protein products work. Simply knowing that a protein matters to cell division is not enough to understand the mechanism.Thus it is necessaryto return to biochemical and biophysical approaches to work out the molecular biology and to cell biology to monitor protein locations and movements. Finally, cell division does not happen in a vacuum; it happens in the context of the life cycle of the organism. To fully appreciatehow the regulation works and is used, it is important to use the approachesof developmental biology

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to revealwhen and where cell division normally happensand why. In theseexperimentsthe times and placesof cell division during the developmentof the organism are monitored, and then the signals that stimulate or suppresscell division are identified and studied. Errors in developmental control of cell division, revealedby studying mutants, can causeorgans and tissuesto be the wrong sizeor cancer. The samekinds of approachesthat work for cell division can be applied to many other biological challenges,such as Iearning how muscles form or how they work or how the brain functions. Often it's good to use every tool in the toolkit.

tf,|

A GenomePerspective

on Evolution Comprehensivestudies of genesand proteins from many organismsare giving us an extraordinary documentationof the history of life. Nature is a laboratory that has been conducting experimentsfor billions of years, and some of the most successfulgenomesthat emergedare still with us. 'We share with other eukaryotes thousands of individual proteins, hundredsof macromolecularmachines,and most of our organelles,all as a result of our sharedevolutionary history. New insights into molecular cell biology arising from genomicsare leadingto a fuller appreciationof rhe elegant molecular machines that arose during billions of yearsof genetictinkering and evolutionaryselectionfor the most efficient, precise designs. Due ro alternative RNA splicing,the number of proteins vastly exceedsthe number of genes,and the functions of many variant proteins and assembliesof proteins remain to be discovered. Once a more complete description of cells is in hand, we will be ready to fully investigate the rippling, flowing dynamics of living systems.

MetabolicProteins,the GeneticCode, a n d O r g a n e l l eS t r u c t u r e sA r e N e a r l yU n i v e r s a l Even organisms that look incredibly different share many biochemical properties. For instance, the enzymesthat catalyze degradation of sugars and many other simple chemical reactions in cells have similar structures and mechanisms in most living things. The genetic code whereby the nucleotide sequencesof mRNA specifiesthe amino acid sequencesof proteins can be read equally well by a bacterial cell and a human cell. Becauseof the universalnature of the genetic code, bacterial "factories" can be designedto manufacture growth factors, insulin, clotting factors, and other human proteins with therapeutic uses. The biochemical similarities among organisms also extend to the organelles found in eukaryotic cells.The basic structuresand functions of these subcellular components are largely conservedin all eukaryotes.

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Computer analysisof DNA sequencedata, now available for numerousbacterialspeciesand severaleukaryotes, can locate protein-coding geneswithin genomes.\fith the aid of the geneticcode, the amino acid sequencesof proteins can be deduced from the corresponding gene sequences.Although simple conceptually, "finding" genes and deducing the amino acid sequencesof their encoded proteins is complicated in practice becauseof the many noncodingregionsin eukaryoticDNA (Chapter5). Despite the difficulties and occasional ambiguities in analyzing DNA sequences, comparisonsof the genomesfrom a wide range of organisms provide stunning, compelling evidence for the conservation of the molecular mechanisms that build and change organisms and for the common evolut i o n a r y h i s t o r yo f a l l s p e c i e s .

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Darwin'sldeasAbout the Evolutionof Whole A n i m a l sA r e R e l e v a n t o G e n e s Darwin did not know that genesexist or how they change, but we do: the DNA replication machine makes an error, or a mutagen causesreplacement of one nucleotide with another or breakage of a chromosome. Some changesin the genomeare innocuous, some mildly harmful, some deadly; a very few are beneficial. Mutations can change the sequence of a gene in a way that modifies the activity of the encoded protein or alters when, where, and in what amounts the protein is produced in the body. Gene-sequencechangesthat are harmful will be lost from a population of organisms becausethe affected individuals cannot survive as well as their relatives. This selection process is exactly what Darwin described without knowing the underlying mechanismsthat cause organisms to vary. Thus the selectionof whole organisms for survival is really a selectionof genes,or more accurately sets of genes.A population of organismsoften contains many variants that are all roughly equally well-suited to the prevailing conditions. When conditions change-a fire, a flood, loss of preferred food supply, climate shift-variants that are better able to adapt will survive, and those less suited to the new conditions will begin to die out. In this way, rhe genetic composition of a population of organisms can change over tlme.

M a n y G e n e sC o n t r o l l i n gD e v e l o p m e n t A r e R e m a r k a b l yS i m i l a ri n H u m a n s a n d O t h e rA n i m a l s As humans, we probably have a biasedand somewhat exaggerated view of our status in the animal kingdom. Pride in our swollen forebrain and its associatedmental capabilities may blind us to the remarkably sophisticated abilities of other species:navigation by birds, the sonar systemof bats, homing by salmon, or the flight of a fIy. Despite all the evidencefor evolutionary unity at the cellular and physiological levels,everyoneexpectedthat genes

(a)

Genes

Flv

(d)

Mammal

(e)

< FIGURE 1-26 Similargenes,conservedduringevolution, processes in diverseanimals. regulatemanydevelopmental to havehada commonancestor areestimated Insects andmammals abouthalfa billionyearsago.Theysharegenesthatcontrolsimilar of the processes, suchasgrowthof heartandeyesandorganization times. conservation of functionfromancient bodyplan,indicating (a)Hoxgenesarefoundin clusters of mostor on thechromosomes proteins thatcontrolthe allanimalsHoxgenesencoderelated of activities of othergenesHoxgenesdirectthedevelopment as axisof manyanimals, alongthe head-to-tail different segments Eachgeneisactivated colors. indicated by corresponding (transcriptionally) axisand regionalongthe head-to-tail in a specific in micethe Hox there Forexample, controls the growthof tissues of vertebrae shapes genesareresponsible for thedistinctive Hoxgenesin fliescausebodypartsto formin affecting Mutations on the head. suchaslegsin lieuof antennae thewronglocations, genesprovide address andserveto direct a head-to-tail These in the rightplaces(b)Development of the rightstructures formation a genecalled eyesin fruitfliesrequires of the largecompound (c)Flieswith inactivated (namedfor the mutantphenotype). eyeless the human geneslackeyes(d)Normalhumaneyesrequire eyeless (e)Peoplelacking to eyeiess. gene,calledPax6,that corresponds anrrdia,a lackof adequatePax6functionhavethe geneticdisease encodehighlyrelatedproteins irisesin the eyesPax6andeyeless from of othergenesandaredescended theactivities that regulate (b)and(c)Andreas gene lParts Hefti, Interdepartmental the sameancestral (lEM) of BaselPart(d) of theUniversity Biocenter Microscopy Electron Unlimitedl Inc Part(e)Visuals Researchers. Fraser/Photo @Simon

that, as far as we can tell, are utterly absentfrom certain lineagesof animals. Plants, not surprisingly,exhibit many such differencesfrom animals after a billion-year separation in their evolution. Yet certain DNA-binding proteins differ between peasand cows at only two amino acids out of 1'021

regulating animal development would differ greatly from one phylum to the next. After all, insectsand seaurchins and mammals look so different. \(e must have many unique proteins to createa brain like ours . . . or must we? The fruits of research in developmental genetics during the past two decadesrevealthat insectsand mammals, which have a common ancestor about half a billion years ago, possessmany genes(Figure 1-26). Indeed, similar development-regulating a large number of these genes appear to be conserved in many and perhaps all animals. Remarkably, the developmental functions of the proteins encoded by thesegenesare also often preserved.For instance,certain proteins involved in eye development in insects are related to protein regulators of eye developmentin mammals. Samefor development of the heart, gut, lungs, and capillaries and for placementof body parts along the head-to-tail and back-to-front body axes (Chapter19). This is not to say that all genesor proteins are evolutionarily conserved. Many striking examples exist of proteins

H u m a nM e d i c i n el s I n f o r m e db y R e s e a r c h on OtherOrganisms Mutations that occur in certain genesduring the course of our lives contribute to formation of various human cancers. The normal, wild-type forms of such "cancer-causing"genes generallyencodeproteins that help regulatecell proliferation or death (Chapter 21').'Wealso can inherit from our parents mutant copies of genesthat causeall manner of genetic diseases,such as cystic fibrosis, muscular dystrophy' sickle cell anemia, and Huntington's disease.Happily we can also inherit genesthat make us robustly resist disease.A remarkable number of genesassociatedwith cancer and other human diseasesare present in evolutionarily distant animals. For example, a recent study shows that more than threequarters of the known human diseasegenes are related to genesfound in the fruit fIy Drosophila. Vith the identification of human diseasegenesin other organisms,experimental studies in experimentally tractable organisms should lead to rapid progress in understanding

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the normal functions of the disease-relatedgenes and what occurs when things go awry. ConverselS the disease states themselvesconstitute a genetic analysis with well-studied phenotypes. All the genesthat can be altered to cause a certain diseasemay encode a group of functionally related proteins. Thus clues about the normal cellular functions of pro-

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teins come from human diseasesand can be used to guide initial research into mechanism. For instance, genesinitially identified becauseof their link to cancer in humans can be studied in the context of normal development in various model organisms, providing further insight about the functions of their protein products.

CHAPTER

CHEMICAL FOUNDATIONS Polarized light microscopic imageof crystalsof ATBwhose hydrolysis isa primarysourceof energythat drivesmanycellular chemical reactions[Dr.ArthurM SiegelmaMr'isuals Unlimited ]

he life of a cell dependson thousands of chemical interactions and reactions exquisitely coordinated with one another in time and sDaceand under the influence of the cell's genetic instructions and its environment. By understandingat a molecular level theseinteractions and reactions, we can begin to answer fundamental questionsabout cellular life: How doesa cell extract critical nutrients and information from its environment? How does a cell convert the energy stored in nutrients into work (movement,synthesis of critical components)?How does a cell transform nutrients into the fundamental structuresrequired for its survival (cell wall, nucleus, nucleic acids, proteins, cytoskeleton)? How does a cell link itself to other cells to form a tissue? How do cells communicate with one another so that a complex, efficiently functioning organism can develop and thrive? One of the goals of Molecular Cell Biology is to provide answers to these and other questions about the structure and function of cells and organisms in terms of the properties of individual moleculesand ions. For example, the properties of one such molecule,water, have controlled and continue to control the evolution, structure, and function of cells. You cannot understand biology without appreciating how the properties of water control the chemistry of life. Life first arose in a watery environment. Constituting 70-80 percent by weight of most cells, water is the most abundant molecule in biological systems. It is within this aqueous milieu that small molecules and ions, which make up about 7 percent of the weight of living matter, assembleinto the larger macromoleculesand macromolecular aggregatesthat make up a cell's machinery and architecture and so the remaining mass of organisms.

These small molecules include amino acids (the building blocks of proteins), nucleotides (the building blocks of DNA and RNA), lipids (the building blocks of biomembranes), and sugars (the building blocks of starchesand cellulose). Many biomolecules(e.g.,sugars)readily dissolvein water; these molecules are called hydrophilic (water liking). Others (e.g., cholesterol)are oilS fatlike substancesthat shun water; these are said to be hydrophobic (water fearing). Still other biomolecules(e.g.,phospholipids)are a bit schizophrenic, containing both hydrophilic and hydrophobic regions; these molecule are said to be amphipathic. Phospholipids are used to build the flexible membranes that form the wall-like boundaries of cells and their internal organelles.The smooth functioning of cells, tissues' and organisms depends on all these molecules, from the smallest to the largest. Indeed, the chemistry of the simple proton (H*) can be as important to the survival of a human cell as that of each gigantic, genetic-code-carrying DNA molecule (the mass of the DNA molecule in human

OUTLINE 2.1

CovalentBondsand NoncovalentInteractions 32

2.2

of Cells BuildingBlocks Chemical

40

2.3

Equilibrium Chemical

49

2.4

Energetics Biochemical

54

31

(a) Molecularcomplementarity

(bl Chemicalbuilding blocks

ProteinA

Polymerization p&. d'

",llrill t

1

ProteinB

S m a l lm o l e c u l e subunits (dl Chemicalbond energy

{c} Chemicalequilibrium

e#

Macromolecule

"High-energy" phosphoa n h y dr i d e bonds

kr Kl

K^^_

K1

Adenosine triphosphate

k,

A FIGURE2-1 Chemistry of life: four key concepts.(a) Molecular complementarity liesat the heartof all biomolecular interactions, as when two proteinswith complementary shapesand chemical propertiescometogetherto form a tightlybound complex (b) Small moleculesserveas buildingblocksfor IargerstructuresForexample, to generatethe information-carrying macromolecule DNA,four small nucleotidebuildingblocksare covalentlylinkedinto long strings (polymers), which then wrap aroundeachother to form the double helix (c) Chemicalreactionsare reversible, and the distributionof the chemicalsbetweenstartingreagents(/eft)and the productsof the

(nght)depends reactions on the rateconstants of the forward(k1, (k,,lowerarrow)reactions upperarrow)andreverse Theratioof these,K"o,provides an informative measure of the relative amounts (d)In of products andreactants thatwill be present at equilibrium manycases, the source of energyfor chemical reactions in cellsisthe hydrolysis of the molecule ATPThisenergyisreleased whena highenergyphosphoanhydride bondlinkingthe B andy phosphates in (red)isbrokenby the addition theATPmolecule of a watermolecule,

c h r o m o s o m e1 i s 8 . 6 x 1 0 r 0 t i m e s t h a t o f a p r o t o n ! ) . T h e chemical interactions of all of these molecules,large and small, with water and with one anothet define the nature of life. Luckily, although many types of biomolecules interacr and react in numerous and complex pathways to form functional cells and organisms, a relatively small number of chemical principles are necessaryto understand cellular processes at the molecularlevel (Figure 2-l).ln this chapter we review these key principles, some of which you already know well. Sfe begin with the covalent bonds that connect atoms into a molecule and the noncovalent forcesthat stabilize groups of atoms into functional structures within and between molecules. We then consider the key properties of the basic chemical building blocks of macromoleculesand macromolecular assemblies.After reviewing those aspectsof chemical equilibrium that are most relevant to biological systems,we end the chapter with basic principles of bio-

chemical energetics,including the central role of ATP (adenosinetriphosphate) in capturing and transferring energy in cellular metabolism.

32

cHAPTER2 |

CHEMICALFOUNDATTONS

TOrmlnOAUI'311O l',.

A

CovalentBondsand

NoncovalentInteractions Strong and weak attractive forces between atoms are the "glue" that holds them together in individual moleculesand permits interactions between different biomolecules.Strong forces form a covalent bond when two atoms share one pair of electrons("single" bond) or multiple pairs of electrons ("double" bond, "triple" bond, etc.). The weak attractive forces of noncovalent interactions are equally important in determining the properties and functions of biomolecules such as proteins, nucleic acids, carbohydrates,and lipids. .We will first review covalent bonds and then discussthe four

Electrons

Covalentbond H

H

H

H

A FIGURE 2-2 Covalentbondsform by the sharingof electrons. Covalent bonds,the strongforcesthat holdatomstogetherinto molecules, formwhenatomsshareelectrons fromtherroutermost electron orbitals. Eachatomformsa definednumberandqeometrv of covalent bonds

major types of noncovalent interactions:ionic bonds, hydrogen bonds, van der Waals interactions,and the hydrophobic effect.

The ElectronicStructureof an Atom D e t e r m i n e st h e N u m b e ra n d G e o m e t r y of CovalentBondslt Can Make Hydrogen, oxygen, carbon, nitrogen,phosphorus,and sulfur are the most abundant elementsin biological molecules. Theseatoms, which rarely exist as isolatedentities,readily form covalent bonds, using electronsin the outermost electron orbitals surrounding their nuclei (Figure 2-2\. As a rule, each type of atom forms a characteristicnumber of covalent bonds with other atoms, with a well-defined geometry determined by the atom's size and by both the distribution of electrons around the nucleus and the number of electronsthat it can share.In some cases(e.g.,carbon), the number of stable covalent bonds formed is fixed; in other cases (e.g., sulfur), different numbers of stable covalent bonds are possible. All the biological building blocks are organizedaround the carbon atom, which normally forms four covalent bonds with three or four other atoms. As illustrated in Figure 2-3a for formaldehyde, carbon can bond to three atoms, all in a common plane. The carbon atom forms two typical single bonds with two atoms and a double bond (two shared electron pairs) with the third atom. In the absenceof other constraints, atoms joined by a single bond generally can rotate freely about the bond axis, whereas those connected by a double bond cannot. The rigid planarity imposed by double bonds has enormous significancefor the shapesand flexibility of biomoleculessuch as phospholipids, proteins, and nucleic acids. Carbon can also bond to four rather than three atoms. As illustrated by the methane (CHa) molecule,when carbon is bonded to four other atoms, the angle between any two bonds is 109.5' and the positions of bonded atoms define the four points of a tetrahedron(Figure2-3b). This geome-

try defines the structures of many biomolecules.A carbon (or any other) atom bonded to four dissimilar atoms or groups in a nonplanar configuration is said to be asymmetric. The tetrahedral orientation of bonds formed by an asymmetric carbon atom can be arranged in three-dimensional space in two different ways, producing molecules that are mirror imagesof eachother, a property calledchirality (from the Greek word cheir,meaning "hand") (Figure2-4). Such moleculesare called optical isomers,or stereoisomers.Many molecules in cells contain at least one asymmetric carbon atom, often called a chiral carbon atom. The different stereoisomersof a molecule usually have completely different biological activities becausethe arrangement of atoms within their structures differs, yielding their unique abilities to interact and chemically react with other molecules. Some drugs are mixtures of the stereoisomersof small moleculesin which only one stereoisomerhas the biological activity of interest.The use of a pure single stereoisomer of the chemical in place of the mixture can result in a more potent drug with reduced sideeffects.For example,one stereoisomerof the antidepressantdrug citalopram (Celexa) is

(al Formaldehyde

H

c:o H

(b) Methane H

I

H-C-H

I

H Chemical structure

Ball-and-stick model

Space-filling model

2-3 Geometryof bondswhen carbonis covalently A FIGURE linked to three or four other atoms.(a)A carbonatomcanbe (CHzO). Inthiscase,the bondedto threeatoms,asin formaldehyde participate in two singlebondsandone electrons carbon-bonding etoms , h i c ha l ll i ei n t h es a m ep l a n eU. n l i k a d o u b l eb o n dw canrotatef reelyaboutthe by a singlebond,whichusually connected by a doublebondcannot(b)Whena bondaxis,thoseconnected (CH+), the carbonatomformsfoursinglebonds,asin methane (all in the formof in space oriented case) are H in this atoms bonded indicates on the leftclearly Theletterrepresentation a tetrahedron. andthe bondingpattern of the molecule theatomiccomposition the geometric modelin the centerillustrates Theball-and-stick of the balls the diameters bonds, but and of the atoms arrangement are electrons the atomsandtheirnonbonding representing with the bondlengthsThesizesof the smallcompared unrealistically modelon the rightmore cloudsin the space-filling electron in threedimensions thestructure represent accuratelv C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

33

FIGURE 2-4 Stereoisomers. Manymolecules in cellscontain at leastoneasymmetric carbonatom Thetetrahedral orientation of bondsformedbyan asymmetric carbonatomcanbearranged in three-dimensional spacein two different ways,producing molecules that aremirrorimages, or stereoisomers, of eachother.Shownhereis thecommonstructure of an aminoacid,with itscentral asymmetric carbonandfourattached groups, including the Rgroup,discussed in Section 2 2. Aminoacidscanexistin two mirror-image forms, designated r ando. Although properties thechemical of such stereoisomers areidentical, theirbiological activities aredistinctOnly r aminoacidsarefoundin oroterns

that can participate in noncovalent interactions. Sulfur forms two covalent bonds in hydrogen sulfide (H2S)but also can accommodate six covalent bonds, as in sulfuric acid (H2SO4)and its sulfate derivatives.Nitrogen and phosphorus each have five electronsto share.In ammonia (NH3), the nitrogen atom forms three covalent bonds; the pair of electrons around the atom not involved in a covalent bond can take part in noncovalent interactions. In the ammonium ion (NH+*), nitrogen forms four covalent bonds, which have a tetrahedral geometry. Phosphoruscommonly forms five covalent bonds, as in phosphoric acid (H3POa) and its phosphate derivatives,which form the backbone of nucleic acids. Phosphategroups covalently attachedto proteins play a key role in regulating the activity of many proteins, and the central moleculein cellular energetics,ATP, contains three phosphate groups (seeSection 2.4). A summary of common covalent linkagesand functional groups (portions of molecules that confer distinctive chemical properties) is provided in Table 2-2.

ElectronsMay Be SharedEqually o r U n e q u a l l yi n C o v a l e n tB o n d s

The extent of an atom's ability to attract an electron is called its electronegatiuity. ln a bond between atoms with identical or similar electronegativities,the bonding electrons are es170 times more potent than the other. Somestereoisomershave sentially sharedequally betweenthe two atoms, as is the case very different activities. Darvon is a pain reliever,whereas its for most C-C and C-H bonds. Such bonds are called nonstereoisomer,Novrad (Daruon spelled backward), is a cough polar. In many molecules,the bonded atoms have different suppressant. One stereoisomer of ketamine is an anesthetic, electronegativities,resulting in unequal sharing of the elecwhereas the other causeshallucinations. I trons. The bond betweenthem is said to be polar. One end of a polar bond has a partial negativecharge The number of covalent bonds formed by other common (6-), and the other end has a partial positive charge ( 6+). atoms is shown in Table 2-1,.A hydrogen atom forms only In an O-H bond, for example, the greater electronegativone covalent bond. An atom of oxygen usually forms only ity of the oxygen atom relative to hydrogen results in the two covalent bonds but has two additional pairs of electrons electrons spending more time around the oxygen atom than the hydrogen. Thus the O-H bond possessesan electric dipole, a positive charge separated from an equal but opposite negative charge. The amount of 6- charge on the oxygen atom of a O-H dipole is approximately 25 percent of that of an electron, with an equivalent 6+ charge on the H atom. Becauseof its two O-H bonds that are nor AT()M AIIO USUAI. I,IUMBTB TYPICAL I)UTER TI.ICIROI'IS {]FCl)VATElrlT BI]NDS Bt]I'IIl GIOMETBY on exact opposite sides of the O atom, water molecules (HzO) are dipoles (Figure 2-5) that form electrostatic,noncovalent interactions with one another and with other molH I H ecules. These interactions play a critical role in almost o z every biochemical interaction and so are fundamental to ,ror cell biology. S 214,or5 St The polarity of the O:P double bond in H3PO4 results ,' in a resonancehybrid, a structure between the two forms N shown below in which nonbonding electrons are shown as 3or4 pairs of dots:

-T-

5

P

4

-'rP

I

-?-

H

CHAPTER2 I

CHEMICALFOUNDATIONS

I

:o

o I

H-O-P-O-H il"

o.

34

H

I

e

l*

H-O-P-O-H

I

o

FUNCTIONAL GROUPS

o -c-

o -c-o-

Carbonyl

Carboxyl

(ketone)

( c a r b o x y l i ca c i d )

o -o-P-oI

oPhosphate

oo

iltl -o-P-o-P-

o-

o-

Pyrophosphate (phosphorylated molecule)

(diphosphate)

LINKAGES

o til -c-o-cI

o -N-C-

ll

I Amide

In the resonancehybrid on the right, one of the electrons from the P:O double bond has accumulatedaround the O atom, giving it a negativecharge and leaving the P atom with a positive charge. These chargesare important in noncovalent interactions.

C o v a l e n tB o n d sA r e M u c h S t r o n g e r and More StableThan NoncovalentInteractions Covalentbonds are very stable(i.e.,consideredto be strong) becausethe energies required to break them are much greater than the thermal energy available at room temperature (25 "C) or body temperature(37'C). For example,the

ol

H

2-5 The dipolenatureof a water molecule.The A FIGURE a partialcharge(aweakerchargethantheone symbolE represents proton) in the a Because of thedifference on an electron or of H andO, eachof the polarH-O bondsin electronegativities of eachof anddirections of the dipoles waterisa dipoleThesizes the netdrstance andamountof charqe the bondsdetermine separation, or dipolemoment,of the molecule

thermal energy at25 "C is approximately 0.6 kilocalorie per mole (kcal/mol), whereas the energy required to break the carbon-carbonsingle bond (C-C) in ethane is about 140 times larger (Figure 2-6). Consequently' at room temperature (25 "C), fewer than f. in 1012ethanemoleculesis broken into a pair of 'CH3 molecules,each containing an unpaired, nonbondingelectron(calleda radical). Covalent single bonds in biological moleculeshave energies similar to the energy of the C-C bond in ethane. Because more electrons are shared between atoms in double bonds, they require more energy to break than single bonds. For instance, it takes 84 kcal/mol to break a single C-O bond but 170 kcal/mol to break a C:O double bond' The most common double bonds in biological molecules are C:O, C:N, C:C, and P:O. In contrast, the energy required to break noncovalent interactions is only 1-5 kcalimol, much less than the bond energiesof covalent bonds (seeFigure 2-5). Indeed' noncovalent interactions are weak enough that they are constantly being formed and broken at room temperature' Although these interactions are weak and have a transient existenceat physiologicaltemperatures(25-37 "C)' multiple noncovalent interactions can, as we will see' act together to produce highly stable and specific associations between different parts of alatge molecule or between different macromolecules. Below, we review the four main types of noncovalent interactions and then consider their roles in the binding of biomoleculesto one another and to other molecules. C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

35

> FIGURE 2-6 Relativeenergiesof covalent Noncovalent interactions bondsand noncovalentinteractions. Bond lonic energies aredefinedastheenergyrequired to breaka particular typeof linkageCovalent Hydrogen b o n d si ,n c l u d i nt g bonds h o s ef o r s i n g l (eC - C ) a n d double(C:C) carbon-carbon bonds.areoneto Thermal two powersof 10stronger thannoncovalent energy interactions Thelatteraresomewhat greater thanthethermalenergyof theenvironment at (25"C) Many normalroomtemperature processes biological arecoupled to the energy 0.24 released duringhydrolysis of a phosphoanhydride bondin ATP

Covalentbonds

Hydrolysisof ATP p h o s p h o a n h y d r i dbeo n d

c-c

C=C

240 kcal/mol

Increasingbond strength

lonic InteractionsAre Attractions between OppositelyChargedlons Ionic interactions result from the attraction of a positively charged ion-a cation-for a negatively charged ion-an anion. In sodium chloride (NaCl), for example, the bonding electron contributed by the sodium atom is completely transferred to the chlorine arom. (Figure 2-7a). Unlike covalent bonds, ionic interactionsdo not have fixed or specificgeometric orientations becausethe electrostatic field around an ion-its attraction for an opposite charge-is uniform in all directions. In solid NaCl, many ions pack tightly together in an alternating pattern to permit opposite charges to align and thus form a highly orderedcrystallinearray (saltcrystals)(Figure2-7b). tVhen solid saltsdissolvein water, the ions separatefrom one another and are stabilized by their interactionswith water molecules.In aqueoussolutions,simpleions of biological

(al

significance,suchas Na*, K*, Ca2*,Mg2* rand Cl-, are hydrated, surrounded by a stable shell of water moleculesheld in place by ionic interactions betweenthe central ion and the oppositely charged end of the water dipole (Figure 2-7c). Most ionic compounds dissolvereadily in water becausethe energy of hydration, the energy releasedwhen ions tightly bind water molecules,is greater than the lattice energy that stabilizesthe crystal structure. Parts or all of the aqueousbydration shell must be removed from ions when they directly interact with proteins. For example, water of hydration is lost when ions pass through protein pores in the cell membrane during nerve conduction. The relative strength of the interaction betweentwo ions, A- and C-, dependson the concentration of other ions in a solution. The higher the concentration of other ions (e.g., Na* and Cl-), the more opportunities A and C* have to

(b)

1,' -> ---.+

cl Cl

Donationof electron

.-#

s

-d q{-

+ Hro dissolving +-Crystallizing

-d

U A FIGURE 2-7 Electrostaticinteractionsof oppositelycharged dissolved in water,the ionsseparate andtheircharges, no longer ionsof salt(NaCl)in crystalsand in aqueoussolution.(a)In balanced by immediately adjacent ionsof opposite charge, are crystalline tablesalt,sodiumatomsarepositively charged stabilized ions(Na+) by interactions with polarwaterWatermolecules andthe dueto the lossof oneelectron each,whereas chloride ionsareheldtogetherby electrostatic atomsare interactions between the correspondingly (Ct I 5Ugainingoneelectron negatively charged charges on the ionandthe partialcharges on thewater'soxygenand each(b)Insolidform,ioniccompounds formneatlyordered hydrogen atomsIn aqueous arrays, solutions, all ionsaresurrounded by a or crystals, of tightlypackedionsin whichthe positive andnegatively hydration shellof watermolecules charged ionscounterbalance eachother.(c)Whenthe crvstals are

36

CHAPTER2 I

CHEMICALFOUNDATIONS

(c)

(b)

(a)

:O-H

I

H H

iuH tll O-H

I o-H

H

I :O-H

:O-H

:O-H

H

ll

H-O:

I :O-H

HH H-O

Water-water

: O-H

H-O: I

I

H HH

:o -i-rtr-

:O- CHs

Alcohol-water

I

:N-CHs H

Amine-water

H

H

o -i - o-

.H-O: I H

Peptide group-water

Ester group-water

2-8 Hydrogenbondingof water with itself and with A FIGURE in an outerelectrons Eachpairof nonbonding other compounds. atomin a hydrogen atomcanaccepta hydrogen oxygen or a nitrogen andtheaminogroupscanalsoformhydrogen bond Thehydroxyl forms bondswithwater.(a)In liquidwater,eachwatermolecule others, creating a dynamic hydrogen bondswithseveral transient

(b)Wateralsocanform molecules networkof hydrogen-bonded for the high accounting andamines, bondswith alcohols hydrogen (c)Thepeptide groupandestergroup, of thesecompounds solubility participate in commonly in manybiomolecules, whicharepresent bondswithwateror polargroupsin othermolecules. hydrogen

interact ionically with these other ions and thus the lower the energyrequired to break the interaction betweenA- and C*. As a result, increasingthe concentrationsof saltssuch as NaCl in a solution of biological moleculescan weaken and even disrupt the ionic interactions holding the biomolecules together.

O-H bonds within a singlewater molecule (Figure2-8a). The strengthof a hydrogen bond betweenwater molecules (approximately 5 kcal/mol) is much weaker than a covalent O-H bond (roughly 110 kcal/mol), although it is greater than that for many other hydrogen bonds in biological molecules(t-2 kcall mol). The extensivehydrogen bonding between water molecules accounts for many of the key properties of this compound' including its unusually high melting and boiling points and its ability to interact with (e.g.,dissolve)many other molecules. The solubility of unchargedsubstancesin an aqueousenvironment dependslargely on their ability to form hydrogen bondswith water.For instance,the hydroxyl group (-OH) in an alcohol (XCH2OH) and the amino group (-NH2) in amines (XCH2NH2) can form severalhydrogen bonds with water, enabling these moleculesto dissolvein water to high concentrations(Figure2-8b). In general,moleculeswith polar bonds that easilyform hydrogen bonds with water' as well as charged moleculesand ions that interact with the dipole in water, can readily dissolve in water; that is' they are hydrophilic (water liking). Many biological moleculescontain, in addition to hydroxyl and amino groups' peptide and ester groups, which form hydrogen bonds with water via otherwisenonbondedelectronson their carbonyl oxygens(Figure 2-8c). X-ray crystallography combined with computational analysispermits an accuratedepiction of the distribution of the outermostunbondedelectronsof atoms as well as the electrons in covalent bonds, as illustrated in Figure 2-9.

HydrogenBondsDeterminethe Water S o l u b i l i t yo f U n c h a r g e dM o l e c u l e s A hydrogen bond is the interaction of a partially positively charged hydrogen atom in a molecular dipole (e.9., water) with unpaired electronsfrom another atom, either in the same (intramolecular\or different Untermolecular)molecule.Normall5 a hydrogen atom forms a covalent bond with only one other atom. However, a hydrogen atom covalentlybonded to an electronegativedonor atom D may form an additional weak association,the hydrogen bond, with an acceptoratom A, which must have a nonbonding pair of electronsavailable for the interaction: D6--H6+

+ : 46- i-

D6--H6*:.....:: 46-

uyarolln uona The length of the covalent D-H bond is a bit longer than it would be if there were no hydrogen bond becausethe acceptor "pulls" the hydrogen away from the donor. An important feature of all hydrogen bonds is directionality. In the strongest hydrogen bonds, the donor atom, the hydrogen atom, and the acceptor atom all lie in a straight line. Nonlinear hydrogen bonds are weaker than linear ones; still, multiple nonlinear hydrogen bonds help to stabilize the three-dimensionalstructuresof many proteins. Hydrogen bonds are both longer and weaker than covalent bonds betweenthe same atoms. In water, for example, the distance between the nuclei of the hydrogen and oxygen atoms of adjacent, hydrogen-bondedmoleculesis about 0.27 nm, about twice the length of the covalent

Van der WaalsInteractions Are Causedby TransientDiPoles When any two atoms approach each other closel5 they create a weak, nonspecific attractive force called a van der Waals interaction. Thesenonspecificinteractionsresult from the momentary random fluctuations in the distribution of the electrons of any atom' which give rise to a transtent C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

37

clouds, the atoms are said to be in van der $7aalscontact. The strength of the van der rWaalsinteraction is about 1 kcal/mol. weaker than typical hydrogen bonds and only slightly higher than the averagethermal energy of moleculesat 25 "C. Thus multiple van der Waals interactions,a van der'Waalsinteraction in conjunction with other noncovalent interactions, or both are required to significantly influencethe stability of inter- and intramolecular conracrs.

The HydrophobicEffectCausesNonpolar Moleculesto Adhere to One Another Becausenonpolar molecules do not contain charged groups, possessa dipole moment, or becomehydrated,they are insoluble or almost insolublein water; that is, they are hydrophobic (water fearing).The covalentbonds betweentwo carbon atoms and between carbon and hydrogen atoms are the most common nonpolar bonds in biological systems.Hydrocarbons-molecules made up only of carbon and hydrogen-are virtually insoluble in water. Large triacylglycerols(or triglycerides),which make up A FIGURE 2-9 Distributionof bondingand outer nonbonding animal fats and vegetableoils, also are insolublein water. As we electronsin the peptidegroup.Shownhereisa peptidebond will see later, the major portion of these moleculesconsistsof linkingtwo aminoacidswithina proteincalledcrambinTheblack long hydrocarbon chains. After being shaken in water, triacyllinesrepresent thecovalent bondsbetween atomsThered(negative) glycerols form a separatephase.A familiar example is the sepaandblue(positive) linesrepresent contours of chargedetermined ration of oil from the water-basedvinegar in an oil-and-vinegar usingx-raycrystallography andcomputational methods. Thegreater salad dressing. the numberof contourlines,the higherthe charge. Thehighdensity Nonpolar moleculesor nonpolar portions of molecules of redcontourlinesbetweenatomsrepresents the covalent bonds (shared tend to aggregatein water owing to a phenomenoncalled pairs). electron Thetwo setsof redcontourlinesemanating fromtheoxygen(O)andnotfallingon a covalent the hydrophobic effect. Becausewater molecules cannot (black bond line) represent thetwo pairsof nonbonded form hydrogen bonds with nonpolar substances,they tend electrons on the oxygen that areavailable to participate in hydrogen bondingThehighdensity to form "cages" of relatiuely rigid hydrogen-bonded of bluecontourlinesnearthe hydrogen (H)bondedto nitrogen (N) pentagons and hexagons around nonpolar molecules represents a partialpositive charge, indicating thatthisH canactasa donorin hydrogen bonding. C Jelsch etal, 2000,procNat,t. [From Acad SciUSA97.3171 CourtesyofM M Teeterl

unequal distribution of electrons. If two noncovalently bonded atoms are close enough together, electrons of one atom will perturb the electronsof the other. This perturbation generatesa transient dipole in the secondatom, and the two dipoles will attract each other weakly (Figure 2-10). Similarly, a polar covalent bond in one molecule will attract an oppositely oriented dipole in another. Van der'Waals interactions, involving either transiently induced or permanent electric dipoles, occur in all types of molecules, both polar and nonpolar. In particular, van der \Waalsinteractions are responsible for the cohesion between nonpolar moleculessuch as heptane,CH3-(CH2)s-CH:, that cannot form hydrogen bonds or ionic interactionswith 'Waals other molecules.The strength of van der interactions decreasesrapidly with increasingdistance;thus thesenoncovalent bonds can form only when atoms are quite closeto one another. However, if atoms get too close together,they become repelled by the negative charges of their electrons. \fhen the van der Waals attraction between rwo aroms exactly balances the repulsion between their two electron

38

CHAPTER2 |

cHEM|CALFOUNDAT|ONS

i<---->i

k----t

Covalent radius ( 0 . 0 6n 2m )

van derWaals radius ( 0 . 1 4n m )

A FIGURE 2-10 Two oxygenmoleculesin van der Waals contact.ln thismodel,redindicates negative chargeandblue positive indicates chargeTransient dipoles in theelectron clouds of all atomsgiveriseto weakattractive forces,calledvander Waals interactions. Eachtypeof atomhasa characteristic vanderWaals radius at whichvanderWaalsinteractions withotheratomsare optimal. Because atomsrepeloneanotherif theyarecloseenough together for theirouterelectrons to overlap withoutbeingshared in a covalent bond,thevanderWaalsradiusisa measure of thesizeof theelectron cloudsurrounding an atom Thecovalent radrus indicated hereisfor thedoublebondof O:O; thesinqle-bond covalent radius of oxygenisslightly longer.

Nonpolar substance

Watersreleasedinto bulk solution H i g h l yo r d e r e d w a t e rm o l e c u l e s

\\ o o'o \.t

o

o Hydrophobic aggregation

@o Lowerentropy

Higherentropy

2-11 Schematic depictionof the hydrophobiceffect' FIGURE in molecules thatformaroundnonpolar Cagesof watermolecules in thesurrounding thanwatermolecules aremoreordered solution r esd u c et h s en u m b e r b u l kl i q u i dA g g r e g a t i o nf n o n p o l amr o l e c u l e resulting in a in highlyordered cages, involved of watermolecules compared state(nErht) moreenergetical lyf avorable higher-entropy, state(/eft) with the unaggregated

(Figure 2-1I, Ieft). This state is energeticallyunfavorable becauseit decreasesthe randomness(entropy) of the population of water molecules.(The role of entropy in chemic a l s y s t e m si s d i s c u s s e di n a l a t e r s e c t i o n . )I f n o n p o l a r moleculesin an aqueousenvironment aggregatewith their hydrophobic surfacesfacing each other, the hydrophobic s u r f a c e a r e a e x p o s e dt o w a t e r i s r e d u c e d ( F i g u r e 2 - 1 1 , right). As a consequence,lesswater is neededto form the cages surrounding the nonpolar molecules, and entropy increases(an energeticallymore favorable state)relative to the unaggregatedstate. In a sense,then, water squeezes the nonpolar moleculesinto spontaneouslyforming aggregates.Rather than constituting an attractive force such as in hydrogen bonds, the hydrophobic effect resultsfrom an a v o i d a n c e o f a n u n s t a b l e s t a t e ( e x t e n s i v ew a t e r c a g e s a r o u n d i n d i v i d u a ln o n p o l a r m o l e c u l e s ) . Nonpolar moleculescan also associate,albeit weakly' through van der Vaals interactions.The net result of the hydrophobic and van der Waals interactionsis a very powerful tendencyfor hydrophobic moleculesto interact with one another,not with water. Simply put, like dissolueslike' Polar moleculesdissolve in polar solvents such as water; nonpolar moleculesdissolvein nonpolar solventssuch as hexane.

y ediated M o l e c u l a rC o m p l e m e n t a r i t M PermitsTight, Interactions Noncovalent via H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s Both inside and outside cells, ions and molecuiesare constantly bumping into one another.The greaterthe number of copiesof any two types of moleculesper unit volume (i'e.,

the higher their concentration)' the more likely they are to encounter one another.Vhen two moleculesencounter each other, they most likely will simply bounce apart becausethe noncovalent interactions that would bind them together are weak and have a transient existenceat physiological temperatures. However, moleculesthat exhibit molecular complementarity, a lock-and-key kind of fit between their shapes, form multiple noncharges,or other physical properties,can '$7hen two such struccovalent interactions at close range. turally complementarymoleculesbump into each other' they can bind (stick) together. Figure 2-12 tllustrates how multiple, different weak bonds can bind two proteins together. Almost any other arrangement of the same groups on the two surfaceswould not allow the molecules to bind so tightly. Such multiple, specificinteractions betweencomplementary regions within a orotein molecule allow it to fold into a unique threedimensionalshape(Chapter 3) and hold the two chains of DNA togetherin a double helix (Chapter 4)' Similar interactionsunderliethe associationof groups of more than two molecules into multimolecular complexes, leading to formation of muscle fibers' to the gluelike associationsbetween cells in solid tissues,and to numerous other cellular structures. Depending on the number and strength of the noncovalent interactions betweenthe two moleculesand on their environment, their binding may be tight (strong) or loose (weak) and, as a consequence,either long lasting or transient.The higher the affinity of two moleculesfor each other, the better the molecular "fit" between them, the more noncovalent interactionscan form, and the tighter they can bind

-cH3 | -CH3 H3( ,

_cH3 H3C_

ProteinB ProteinA Stable complex

ProteinC ProteinA Less stable comPlex

andthe bindingof complementarity 2-12 Molecular FIGURE Thecomplementary proteinsvia multiplenoncovalentinteractions. of two proteinsurfaces polarity, andhydrophobicity charges, shapes, produce a whichin combination weakinteractions, permrt multiple from molecular deviations Because binding. tight and stronginteraction surface a parlicular binding, weaken substantially complementarity canbindtightlyto onlyoneor a usually of anygivenbiomolecule region of the Thecomplementarity of othermolecules verylimitednumber much more bind permits to them left on the two proteinmolecules proteins on therighi tightlythanthetwo noncomplementary INTERACTIONS T C O V A L E N TB O N D SA N D N O N C O V A L E N T

39

together.An important quantitative measureof affinity is the binding dissociationconstant K6, describedlater. As we discussin Chapter 3, nearly all the chemicalreactionsthat occur in cellsalso dependon the binding propertiesof enzymes.Theseproteins not only speedup, or cataIyze, reactions but also do so with a high degree of specificity, a reflection of their ability to bind tightly to only one or a few related molecules. The specificity of intermolecularinteractionsand reactions,which dependson molecular complementarity,is essentialfor many processes critical to life.

Covalent Bonds and Noncovalent Interactions Covalent bonds, which bind the atoms composing a olecule in a fixed orientation, consist of pairs of electrons shared by two atoms. They are stable in biological systems because the relatively high energies required to break them (50-200 kcal/mol) are much larger than the thermal kinetic energyavailableat room (25 .C) or body (37 "C) temperatures. r Many moleculesin cells contain at least one asymmetric carbon atom, which is bonded to four dissimilar atoms. Such moleculescan exist as optical isomers (mirror images),designatedn and r (seeFigure 2-4), which have different biological activities.In biological systems, nearly all sugarsare D isomers,whereasnearly all amino acids are L isomers. trons may be sharedequally or unequally in covalent Atoms that differ in electronegativityform polar cobonds in which the bonding electronsare distributed unequally. One end of a polar bond has a partial positive charge and the other end has a partial negativechaige (see Figure2-5). r Noncovalent interactions between atoms are considerably weaker than covalent bonds, with bond energlesranging from about 1-5 kcal/mol (seeFigure 2-6). r Four main types of noncovalent interactions occur in biological systems:ionic bonds, hydrogen bonds, van der \faals interactions, and interactions due to the hvdroohobic effect. r Ionic bonds result from the electrostatic attraction between the positive and negativechargesof ions. In aqueous solutions, all cations and anions are surrounded by a shell of bound water molecules(seeFigure Z-7c).Increasingthe salt (e.g.,NaCl) concentrarionof a solutioncan weakenthe relative strength of and even break the ionic bonds between biomolecules. hydrogen bond, a hydrogen atom covalently bonded electronegativeatom associateswith an accepror whose nonbonding electrons attract the hydrogen gure 2-8).

40

CHAPTER2 |

cHEMTCALFOUNDATTONS

r Weak and relatively nonspecificvan der'Sfaalsinteractions are createdwheneverany two atoms approach each other closely. They result from the attraction between transient dipoles associatedwith all molecules (seeFigure2-10). r In an aqueousenvironment, nonpolar moleculesor nonpolar portions of larger molecules are driven together by the hydrophobic effect, thereby reducing the exrent of their direct contactwith water molecules(seeFigure2-11). r Molecular complementarity is the lock-and-key fit between moleculeswhose shapes,charges,and other physical properties are complementary.Multiple noncovalent interactions can form betweencomplementary molecules,causing them to bind tightly (seeFigure2-12),but not between moleculesthat are not complementary. r The high degreeof binding specificity that results from molecular complementarity is one of the features that underlies intermolecular interactions and thus is essentialfor many processescritical to life.

E

Chemical BuildingBlocksof Cells

A common theme in biology is the construction of large molecules (macromolecules)and structures by the covalent or noncovalent associationof many similar or identical smaller molecules.The three most abundant classesof the critically important biological macromolecules-proteins, nucleic acids, and polysaccharides-are all polymers composed of multiple covalently linked building block small molecules,or monomers (Figure 2-13). Proteins are linear polymers containing 10 to severalthousand amino acids linked by peptide bonds. Nucleic acids are linear polymers containing hundreds to millions of nucleotides linked by phosphodiester bonds. Polysaccharidesare linear or branched polymers of monosaccharides(sugars)such as glucose linked by glycosidic bonds. Although the actual mechanismsby which covalent bonds betweenmonomers form are complex and will be discussedlater, the formation of a covalent bond between two monomer molecules usually involves the net loss of a hydrogen (H) from one monomer and a hydroxyl (OH) from the other monomer-or the net loss of one water-and can therefore be thought of as a debydration reaction. These bonds are stable under normal biological conditions (e.g., 37"C, neutral pH), and so thesebiopolymers are stable and can perform a wide variety of jobs in cells (store information, catalyze chemical reactions, serve as structural elements in defining cell shapeand movement, etc.). Macromolecular structures can also be assembledusing noncovalent interactions. The macromolecular two-layered (bilayer) structure of cellular membranesis built uo bv the noncovalent assemblyof many thousandsof small molecules called phospholipids (seeFigure 2-13).In this chapter, we will focus on the characteristicsof the monomeric chemical

POLYMERS

MONOMERS

ttl

H2N-C -C-OH I R

HHOHHOHHOHHO

HO

HO +

ttl

|

I R

I

l

ll,l

L ll

N-C-CrN-C-C-OH I I | R* R3 I

peptidebond

Amino acid

o tl

| ll I

| ll |

H-N-C-ciN-C-C | I lt R2 R, l"

oH

H-N-C-C-

PolYPePtide

B L

Base

?1,

HO-P-O

I

HO-P-OJs,

.OH

6Nucleic acid

Nucleotide

glYcosidicbond

Polysaccharide

Monosaccharide

I Hyoropr'irt. h e a dg r o u p J

Phospholipid

2-13 Overviewof the cell'sprincipalchemical FIGURE threemajortypesof biological buifdingblocks.(Top)The of bythe polymerization assembled are each macromolecules (monomers) type:proteins of a particular smallmolecules multiple acidsfromnucleotides 3),nucleic fromaminoacids(Chapter building blocks-amino acids,nucleotides,sugars'and phospholipids. The structure, function, and assemblyof proteins, nucleic acids, polysaccharides,and biomembranes are discussedin subsequentchapters.

A m i n o A c i d sD i f f e r i n gO n l y i n T h e i r SideChainsComposeProteins The monomeric building blocks of proteins ate 20 amino acids, which when incorporated into a protein polymer are sometimescalled residues.All amino acidshave a characteristic structureconsistingof a central alpha (cr)carbon atom (C') bonded to four different chemical groups: an amino (NHz)

(sugars)Each from monosaccharides (Chapter 4), andpolysaccharides whose reaction polymer by a linkedintothe iscovalently monomer (Boftom) In (dehydration) molecule net resultis lossof a water into a bilayer assemble noncovalently monomers phospholipid contrast, (chapter10) membranes of allcellular whichformsthebasis structure, group, a carboxylic acid or carboxyl (COOH) group (hence amino acid),a hydrogen (H) atom, and one variable Ih. *-. group, called a side chain or R group' Becausethe ct carbon i.-nali amino acids except glycine is asymmetric,thesemolecules can exist in two mirror-image forms called by convention the I (dextro) and the r- (levo) isomers (seeFigure 2-4)' The two isomerscannot be interconverted(one made identical with the other) without breaking and then re-forming a chemical bond in one of them. !7ith rare exceptions, only the I- forms of amino acids are found in proteins' To understand the three-dimensional structures and functions of proteins' discussedin detail in Chapter 3, you must be familiar with some of the distinctive properties of

B U I L D I N GB L O C K SO F C E L L S CHEMICAL

41

H Y D R O P H O BA I CM I N OA C I D S

cooI

*H3N-C-H I cHs

coo-

+ H . N - c I- H

"l

CH HsC

cooI

+H3N-c-H

CHs

cooI

cooI

+H3N-c-H

cooI

cooI

'H"N-C-H

-l

cooI

*H.N-C-H

-l

H-C - CH.

CH,

CH,

CH,

CH

C:CH

t"

lcHs

t-

HaC

t-

CHs

Lr*t OH

\/ Alanine ( A l ao r A l

Valine (Val orVl

lsoleucine ( l l eo r l l

Leucine (Leu or Ll

H Y D R O P H I LA I CM I N OA C I D S

Methionine (Met or M)

Acidic amino acids

cooI

coo-

I +H3N-c-H

I CHt

CH,

l-

NHs*

Lysine (Lys or Kl

CH,

t-

CH, lCH,

tCH, t-

Glycine (Gty or G)

.

H-C-OH

coo-

oH

cHs

Aspartate (Asp or Dl

Serine (Ser or Sl

Threonine (Thr orT)

I

'H"N-C-H

-l

CH,

IcH, )' cooGlutamate ( G l uo r E )

proline (pro or p)

amino acids, which are determined in part by their side chains. You need not memorize the detailed structure of each type of side chain to understandhow proteins work because amino acids can be classified into several broad categories basedon the size,shape,charge,hydrophobicity (a measure of water solubility), and chemical reactivity of the side chains (Figure 2-74). However, you should be familiar with the generalproperties of each category. Amino acids with nonpolar side chains are hydrophobic and so poorly soluble in water. The larger the nonpolar side chain, the more hydrophobic-less water soluble-the amino acid. The noncyclic side chains of alanine, ualine, leucine, and. isoleucine(calledaliphatic),as well as methionine,consisren_ tirely of hydrocarbons, except for the one sulfur arom in me_ thionine, and all are nonpolar. phenylalanine, tyrosine, and. 42

r"l t-i

coo+H3N-C-H

coo-

l-

NH i C: NHr+ lNHz Arginine (Argor Rl

SPECIALAMINO ACIDS

Cysteine (Cys or C)

cootl

CH,

CH,

Tryptophan (TrporW)

Polar amino acids with uncharged R groups

*H3N-C-H

I 'H"N-C-H -l

lCH,

Tyrosine (TyrorYl

+H.N-c-H

-l

coo-

Phenylalanine (Phe or Fl

c H A p r E R 2| c H E M t c A L F o u N D A T t o N s

FIGURE 2-14 The 20 commonaminoacidsusedto build proteins.Thesidechain(Rgroup;red)determines thecharacteristic properties of eachaminoacidandisthe basisfor grouping amino acidsintothreemaincategories: hydrophobic, hydrophilic, and special. Shownarethe ionized formsthatexistat the pH(=7)of the cytosolIn parentheses arethethree-letter andone-letter abbreviations for eachaminoacid.

tryptophan have large, bulky aromatic side chains. In later chapters, we will see in detail how these hydrophobic side chains under the influence of the hydrophobic effeci often pack in the interior of proteins or line the surfacesof proteins that are embeddedwithin hydrophobic regions of biomembranes. Amino acids with polar side chains are hydrophilic; the most hydrophilic of theseamino acids is the subsetwith side chains that are charged (ionized) at the pH typical of biological fluids (=7)-both inside and outside the cell (seeSectio-n 2.3). Arginine and lysine have positively charged side chains and are called basic amino acids;aspartic acid and glutamic acid have negatively charged side chains due to the carboxylic acid groups in their side chains (their charged forms are called aspartate and glutamate) and are called acidic. A fifth amino acid, histidine, has a side chain containing a ring

with two nitrogens, called imidazole, which can shift from being positively charged to uncharged depending on small changesin the acidity of its environment:

I

A c e t y ll y s i n e

C H 3 - C - N - C H 2 - C H 2 - C H 2 - C H " - C H - C-Ol O -

NH.-

o - o - Ptl o - c H 2 - c H - c o o -

ll Phosphoserine

ll O

NH"OH

pH 5.8

PH 7.8

The activities of many proteins are modulated by shifts in environmental acidity through protonation or deprotonation of histidine side chains. Asparagine and glutamine are uncharged but have polar side chains containing amide groups with extensivehydrogen-bondingcapacities'Similarl5 serine and threonine are unchargedbut have polar hydroxyl groups, which also participate in hydrogen bonds with other polar molecules. Lastl5 cysteine,glycine, and proline exhibit specialroles in proteins becauseof the unique properties of their side chains. The side chain of cysteine contains a reactive sulfhydryl group (-SH), which can oxidize to form a covato a secondcysteine: lent disulfide bond (-S-S-/

I I- H

I

I H,9-9H

3-Hydroxyproline

II H2C\ * N //CH-COOH, HC:C-CH2-CH-COOtrl NH.H3C-N-C-.N

3-Methylhistidine

H

-ooc y-Carboxyglutamate

-OOC

c H - c H' "l - c H - c o o ilH.*

of aminoacidside 2-15 €ommonmodifications A FIGURE others andnumerous residues chainsin proteins.Thesemodified groups(red)to theamino chemical of various areformedby addition chain of a polypeptide acidsidechainsduringor aftersynthesis

N-H

N-H

I

H-C-CH2-

SH+ HS-CH2q

ll

C:O

C:O

reveals that they contain upward of 100 different amino acids.Chemical modifications of the amino acids account for

ll

tl H-N

r''l ll

H-C-

ll O:C ll

C H 2- S - S -

N-H C H 2- c - H

C:O

Regionswithin a singleprotein chain (intramolecular) or in separatechains (intermolecular) sometimesare crosslinked through disulfide bonds. Disulfide bonds stabilizethe folded structure of some proteins. The smallestamino acid, glycine, has a singlehydrogen atom as its R group. Its small sizeallows it to fit into tight spaces.Unlike the other common amino acids, the side chain of proline bends around to form a ring by covalently bonding to the nitrogen atom (amino group) attached to the Co. As a result' proline is very rigid and createsa fixed kink in a protein chain, limiting how a protein can fold in the region of proline residues. Some amino acids are more abundant in proteins than others. Cysteine,tryptophan, and methionine are rare amino acids: together they constitute approximately 5 percent of the amino acids in a protein. Four amino acids-leucine, serine, lysine, and glutamic acid-are the most abundant amino acids, totaling 32 percent of all the amino acid residuesin a typical protein. However, the amino acid composition of proteins can vary widely from thesevalues. Although cellsusethe 20 amino acidsshown in Figure2-14 inthe initial synthesisof proteins' analysisof cellular proteins

and plants but less studied-perhaps becauseof the relative insta-bilityof phosphorylatedhistidine-and apparently rare in mammals.ltt. iia. chains of asparagine,serine,and threonine are sitesfor glycosylation,the attachmentof linear and branched carbohydrate chains. Many secretedproteins and membrane proteins contain glycosylated residues' Other amino acid modifications found in selectedproteins include the hydroxylation of proline and lysine residuesin collagen (Chapter 19), the methylation of histidine residuesin memb."ne rec.ptors' and the 7 carboxylation of glutamate in blood-clotting factors such as prothrombin' Acetylation, addition of an acetyl group' to the amino gro,rp oi the N-terminal residue, is the most common form acid chemical modification, affecting an estimated 6f "-ino 80 percentof all proteins:

o lllll

RO

cH3-c- r y- g- cll HH Acetylated N-terminus OF CELLS B U I L D I N GB L O C K S CHEMICAL

O

43

This modification may play an important role in controlling the life span of proteins within cells becausenonacetylateJ proteins are rapidly degraded.

PURINES NH,

O

ltl

Five Different Nucleotides A r e U s e dt o B u i l d N u c l e i cA c i d s Two types of chemically similar nucleic acids, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). are the principal genetic-information-carryingmoleculesof the cell. The monomers from which DNA and RNA polymers are built, called nucleotides,all have a common structure: a phosphategroup linked by a phosphoesterbond to a pentose (a five-carbon sugar molecule) that in turn is linked io a nitrogen- and carbon-containing ring structure commonly referred to as a base (Figure 2-16a).In RNA, the pentoseis ribose; in DNA, it is deoxyribose that at posirion 2, has a proton rather than the hydroxyl group at that site in ribose

HH Adenine (A)

(Gl Guanine

PYRIMIDINES NH,

il

tru/?--cH

r"^tl

.r'"'ii" vl H Cytosine (C)

A FIGURE 2-17 Chemical structuresof the principalbasesin nucleicacids.ln nucleic acidsandnucleotides, nitrogen 9 of purines andnitrogen1 of pyrimidines (red)arebondedto the 1, carbonof riboseor deoxyribose U isonlyin RNA,andT isonlyin DNA Both R N Aa n dD N Ac o n t a iA n , G ,a n dC .

position 1 of a pyrimidine (N1). The acidic character of

which form ionic interactions with the negatively charged phosphates. Cells and extracellular fluids in organismscontain small concentrationsof nucleosides,combinations of a baseand a sugar without a phosphate.Nucleotides are nucleosidesthat have one, two, or three phosphate groups esterified at the 5' hydroxyl. Nucleoside monophosphateshave a single esterified phosphate (seeFigure 2-16a); nucleoside diphosphates contain a pyrophosphategroup:

oo

-o-l-o-A-o-

(a) Adenine

Nhz l

(b)

'-c'

5',

N ; , 6 ; -c

\

HOCH2

daH HC.1 : 1c s,/ -'N' N

o - o - P - O - c H5',

o-

5',

HOCH2

Phosphate 2'

OH

H

Ribose Adenosine 5'-monophosphate (AMP}

2-Deoxyribose

FIGURE 2-15 Commonstructureof nucleotides. (a)Adenosine 5' -monophosphate (AMp),a nucleotide present in RNAByconvention, thecarbonatomsof the pentose sugarin nucleotides arenumbered with primes. In naturalnucleotides, the 1, carbonisjoinedby a B linkage to the base(inthiscaseadenine); boththe base(blue)and the phosphate on the 5, hydroxyl (red)extendabovethe planeof the sugarring.(b)Ribose anddeoxyribose, the pentoses in RNAand DNA,respectively 44

.

c H A p r E R 2| c H E M t c A L F o u N D A T t o N S

o-

o-

Pyrophosphate

and nucleosidetriphosphateshave a third phosphate.Table 2-3 lists the names of the nucleosidesand nucleotidesin nucleic acids and the various forms of nucleosidephosphates.The nucleosidetriphosphatesare used in the synthesisof nucleic acids,which we cover in Chapter 4. Among their other functions in the cell, GTP participares in intracellular signaling a_ndacts as an energy reservoir,particularly in protein synthesis, and ATP, discussedlater in this chapter, is the most widely usedbiologicalenergycarrier.

M o n o s a c c h a r i d eJso i n e db y G l y c o s i d i B c onds Form Linearand Branchedpolysaccharides The building blocks of the polysaccharidesare rhe simple sugars, or monosaccharides.Monosaccharides are carbohy_ drates,which are literally covalently bonded combinationsof carbon and water in a one-to-one ratio (CH2O),, where n e q u a l s3 , 4 , 5 , 6 , o r 7 . H e x o s e s( n : 6 ) a n d p e n t o s e (sn : 5 ) are the most common monosaccharides. All monosaccharides

GUANINT(G}

ADENINE(A)

nNe Jin

t,,'o^o

innNe f Iino*e

uBACrr(lJ) THYMINE(I)

cYT0srNt(q

Adenosine

Guanosine

Cytidine

Uridine

Deoxyadenosine

Deoxyguanosine

Deoxycytidine

DeoxYthYmidine

Adenylate

Guanylate

Cytidylate

UridYlate

Deoxycytidylate

Deoxythymidylate

Deoxyadenylate

DeoxyguanYlate

Nucleoside monophosphates

AMP

GMP

CMP

UMP

Nucieosidediphosphates

ADP

GDP

CDP

UDP

Nucleoside triphosphates

ATP

GTP

CTP

UTP

Deoxynucleoside mono-, di-, and triphosphates

dAMP,etc.

dGMP,etc'

dCMP, etc

dTMP' etc.

contain hydroxyl (-OH) a keto group:

oo

- c t- icl - H lll

Aldehyde

groups and either an aldehyde or

Htt,

6

(-rQ

n-J'-oH

H

- c -Lcf - c - l

1

<_

HO-J.-H

Keto HOH

Many biologically important sugarsare hexoses,including glucose,mannose,and galactose(Figure2-18). Mannose is identical with glucose except that the orientation of the groups bonded to carbon 2 is reversed.SimilarlS galactose, another hexose,differs from glucoseonly in the orientation of the groups attached to carbon 4. Interconversion of glucose and mannose or galactose requires the breaking and making of covalent bonds; such reactions are carried out by enzymescaIIedepimerases. l-Glucose (C6H12O6)is the principal externalsourceof energy for most cells in higher organisms and can exist in three different forms: a linear structure and two different hemiacetalring structures(Figure 2-18a).lf the aldehyde group on carbon 1 reactswith the hydroxyl group on carbon 5, the resulting hemiacetal,o-glucopyranose'contains a sixmember ring. In the cr anomer of o-glucopyranose,the hydroxyl group attachedto carbon 1 points "downward" from the ring as shown in Figure 2-18a; in the B anomer, this hydroxyl points "upward." In aqueoussolution the ct and B anomers readily interconvert spontaneously;at equilibrium there is about one-third ct anomer and two-thirds B, with very little of the open-chain form. Becauseenzymescan distinguish between the ct and B anomers of n-glucose, these forms have distinct bioloeical roles. Condensationof the

lo H-C-OH HOH

H- lLoH

o-Glucofuranose (rarel

o-Glucopyranose (common)

ol -cH20H o-Glucose

(b)

H,r, rO (^.

Ht,ro (-

Ho-l'?-H

H-l'-oH HO-ILH

HO-ILH

H- lLos H-lu-os

ulr,,o,-,

o-Mannose

HO-JLH H-CLoH

ul*ro*

D-Galactose

structuresof hexoses'All hexoses 2-18 Chemical FIGURE andcontainan aldehyde formula(C6H1206) havethe samechemical from aregenerated or a ketogroup.(a)Theringformsof o-glucose the 1 with at carbon aldehyde of the by reaction the linearmolecule readily are forms three 4. The carbon or 5 carbon on hydroxyl in form(right)predominates the pyranose although interconvertible, the (b)In o-mannose ando-galactose, systems. biological andOH(blue)boundto onecarbon of the H (green) configuration exist likeglucose, glucose. Thesesugars, in that atomdiffersfrom primarily asPYranoses. B U I L D I N GB L O C K SO F C E L L S CHEMICAL

.

45

hydroxyl group on carbon 4 of the linear glucose with its aldehydegroup results in the formation of o-glucofuranose. a hemiacetalcontaining a five-memberring. Although ali three forms of n-glucose exist in biological systems,the pyranose form is by far the most abundant. The pyranosering in Figure 2-18a is depictedas planar. In fact, because of the tetrahedral geometry carbon "rorrrd atoms, the most stableconformation of a pyranose ring has a nonplanar, chairlike shape.In this conformation, each bond from a ring carbon to a nonring atom (e.g.,H or O) is either nearly perpendicular to the ring, referred to as axial (a), or nearly in the plane ofthe ring, referredto as equatorial (e):

yranoses tPyranoses

bacteria, and molds produce cellulose-degradingenzymes. Cows and termites can break down cellulose becausethey harbor cellulose-degradingbacteriain their gut. The enzymes that make the glycosidic bonds linking monosaccharidesinto polysaccharidesare specific for the a or B anomer of one sugar and a particular hydroxyl group on the other. In principle, any two sugar molecules can be linked in a variety of ways becauseeachmonosaccharidehas multiple hydroxyl groups that can participate in the formation of glycosidic bonds. Furthermore, any one monosaccharide has the potential of being linked to more than two other monosaccharides,thus generating a branch point and nonlinear polymers. Glycosidic bonds are usually formed between the growing polysaccharide chain and a covalently modified form of a monosaccharide.Such modifications include a phosphate(e.g.,glucose6-phosphate)or a nucleotide (e.g.,UDP-galactose) :

c_o-Glucopyranose

into common table sugar (Figure2-19). Larger polysaccharides,containing dozensto hundredsof

polymer of the B anomer of glucose. Human digestiveenzymescan hydrolyze the c glycosidic bonds in starch but nor the B glycosidic bonds in cellulose.Many speciesof plants,

Glucose 6-phosphate

UDp-galactose

The epimeraseenzymesthat interconvert differenr monosaccharides often do so using the nucleotide sugarsrather than the unsubstitutedsugars. Many complex polysaccharidescontain modified sugars that are covalently linked to various small groups, particularly amino, sulfate, and acetyl groups. Such modifications are abundant in glycosaminoglycans,major polysaccharide components of the extracellular matrix that we describein Chapter 19.

PhospholipidsAssociateNoncovalentlyto Form the BasicBilayerStructureof Biomembranes Biomembranes are large flexible sheets that serve as the boundaries of cells and their intracellular organelles and

cHroH H

*oi

):o

il

\?'

H

HO

> FIGURE HOH 2-19 Formationof the disaccharides lactoseand Galactose sucrose.In anyglycosidic Iinkage, theanomeric carbonof onesugar CH,OH (ineithertheo or B molecule ,f.o. conformation) islinkedto a hydroxyl oxygenon anothersugar 1)a moleculeThelinkages arenamed H O OH accordingly; thuslactose contains a B(1--+4) bond,andsucrose HOH contains an ct(1-+ 2) bond Glucose

HOH Glucose

cH2oH

\T

46

CHAPTER2 |

cHEM|CALFOUNDAT|ONS

OHH Fructose

Sucrose

Fatty acid chains

Hydrophobictail PHOSPHATIDYI. FIGURE2-20 phosphatidylcholine,a typical phosphoglyceride. havinga are amphipathicphospholipids, All phosphoglycerides hydrophobictail (yellow)and a hydrophilichead(blue)in which glycerolis linkedvia a phosphategroup to an alcohol.Eitheror both

may be saturated of the fatty acylsidechainsin a phosphoglyceride acid(red),the simplestphospholipid, or unsaturatedIn phosphatidic the phosphateis not linkedto an alcohol

form the outer surfacesof some viruses.Membranes literally define what is a cell (the outer membrane and the contents within the membrane) and what is not (the extracellular space outside the membrane). Unlike the proteins, nucleic acids, and polysaccharides,membranesare assembledby the noncoualentassociationof their componentbuilding blocks. The primary building blocks of all biomembranesare phospholipids, whose physical properties are responsiblefor the formation of the sheetlike structure of membranes.The structuresand functions of membranes,which include in addition to phospholipidsa variety of other molecules(e.g'' cholesterol,glycolipids, proteins), will be describedin detail in Chapter 10. Phospholipidsconsistof two long-chain,nonpolar fatty acid groups linked (usually by an ester bond) to small, highly polar groups, including a phosphateand a short organic molecule, such as glycerol (trihydroxy propanol) ( F i g u r e2 - 2 0 ) .

Fatty acids consist of a hydrocarbon chain attachedto a carboxyl group (-COOH)' Like glucose,fatty acids are an important energy source for many cells (Chapter 12)' They differ in length, although the predominant fatty acids in cells have an even number of carbon atoms' usually 14, 16. 18. or 20. The maior fatty acids in phospholipids are listed in Table 2-4. Fatty acids often are designatedby the abbreviationCx:y, where x is the number of carbonsin the chain and y is the number of double bonds. Fatty acids containing 12 or more carbon atoms are nearly insolublein aqueous solutions becauseof their long hydrophobic hydrocarbon chains. Fatty acids with no carbon-carbon double bonds are said to be saturated;thosewith at leastone double bond are unsaturated. Unsaturated fatty acids with more than one carbon-carbon double bond are referred to as polyunsaturated. Two "essential" polyunsaturated fatty acids, linoleic acid (C18:2)and linolenicacid (C18:3),cannotbe synthesized

(IONIZEO T()RM INPARENTHESES} ABBREVIATI()N NAME OFACII) COMMON SATURATEDFATTY ACIDS Myristic (myristate)

C14:0

cH3(cH2)12cooH

Palmitic (palmitate)

C|6:0

cH3(cH2)14cooH

Stearic (stearate)

C18:0

cH3(cH2)15cooH

Oleic (oleate)

C18:1

CH3(CH2)7CH:CH(CHz)7COOH

Linoleic (linoleate)

C18:2

CH3(CH2)4CH:CHCHzCH:CH(CHz)7COOH

Arachidonic (arachidonate)

C20:4

CH3(CH2)4(CH:CHCHz):CH:CH(CH2)3COOH

UNSATURMED FATTY ACIDS

OF CELLS B U I L D I N GB L O C K S CHEMICAL

47

by mammals and must be suppliedin their diet. Mammals can synthesizeother common fatty acids. Two stereoisomeric configurations, cis and trans, are possible around each carbon-carbondouble bond:

(seeFigure 2-20) and triacylglycerols,or triglycerides,which contain three acyl groups esterfiedto glycerol:

?

H 3 C - ( C H 2 ) , - C- O - C H 2

ol H3C-(CH2)"-C-O-CH

ol Trans

A cis double bond introducesa rigid kink in the otherwise flexible straight chain of afatty acid (Figure2-21). In general, the unsaturated fatty acids in biological systems contain only cis double bonds. Saturatedfatty acids without

solid margarine sticks) and other food producrs are not natural, arising from the catalytic processused for hydrogenation. Saturated and trans fatty acids have similar physical properties, and their consumption, relative to the consumption of unsaturatedfats, is associatedwith increasedplasma cholesterollevels. Fatty acids can be covalently attached to another molecule by a type of dehydration reaction calledesterification, in which the OH from the carboxyl group of the fatty acid and a H from a hydroxyl group on the other molecule are Iost. In the combined molecule formed by this reaction, the portion derived from the fatty acid is called an acyl group, or fatty acyl group. This is illustrated by the most common form of phospholipids, phosphoglycerides,with two acyl groups attached to rwo of the hydroxyl groups of glycerol

H3C-(CH2),-C-O-CH' Triacylglycerol Fatty acyl groups also can be covalently linked to another fatty molecule,cholesterol,to form cholesterylesters. Triglycerides and cholesteryl esters are extremely waterinsoluble compounds in which fatty acids and cholesterol are either stored or transported. Tiiglycerides are the storageform of fatty acids in the fat cells of adiposetissue and are the principal components of dietary fats. Cholesteryl estersand triglycerides are transported between tissues through the bloodstreamin specializedcarrierscalledlipoproteins(Chapter 14). In phosphoglycerides,one hydroxyl group of the glycerol is esterifiedto phosphate while the other two normally are esterified to fatty acids. The simplest phospholipid, phosphatidic acid, contains only these components. In most phospholipids found in membranes,the phosphate group is also esterified to a hydroxyl group on another hydrophilic compound. In phosphatidylcholine, for example, choline is attached to the phosphate (see Figure 2-20). The negative charge on the phosphate as well as the charged or polar groups esterifiedto it can interact strongly with water. The phosphate and its associatedesterifiedgroup, the "head" group of a phospholipid, is hydrophilic, whereas the fatty acyl chains, the "tails," are hydrophobic. (Other common phosphoglycerides and associatedheadgroupsare shown in Table2-5.) Molecules such as phospholipids that have both hydrophobic and

HHHHHHHHHHHHHH

t t-t 'qt z- c, o- _ _ c _ c _ _ _ _ _ H s -c gt t-l lgl l-l lgl-t ' g c c c c c a c- \. ^ _ t t t .HtH. .Ht H. H H Ht lHt Hl tHLHt iH U T i i U Palmitate (ionized form of palmitic acid) FfGURE2-21 The effect of a double bond on the shape of fatty acids.Shownare chemicalstructuresof the ionizedform of palmiticacid,a saturatedfatty acidwith 16 C atoms,and oleicacid, an unsaturated one with 18 C atoms In saturatedfattv acids.the

48

cHAPTER2 |

cHEMTCALFOUNDATTONS

Oleate (ionized form of oleic acid) hydrocarbonchainis often linear;the cis doublebond in oleate createsa rigid kink in the hydrocarbonchain [AfterL Stryer, 1994, Biochemistry,4th ed, W H Freeman p 265l andCompany,

GBOUP PHOSPHOGIYCERIOES HEAD COMMON c H g^ , , | ,rung

Phosphatidylcholine

r Glycosidic bonds are formed betweeneither the o or the B anomer of one sugar and a hydroxyl group on another sugar, leading to formation of disaccharidesand other (seeFigure2-L9). polysaccharides

o&N\cr. Gholine H l.H

Phosphatidylethanolamine

o&N\n Ethanolamine H lrH

Phosphatidylserine

o---rN I o4o

sine (C), thymine (T), and uracil (U) (seeFigure Z-17). A, G, ! and C are in DNA, and A, G' U, and C are in RNA. r Glucoseand other hexosescan exist in three forms: an open-chain linear structure, a six-member (pyranose) ring, and a five-member(furanose)ring (seeFigure 2-18). In biological systems,the pyranose form of l-glucose predominates.

H

Serine

Phosphatidylinositol lnositol

hydrophilic regionsare calledamphipathic.In Chapter 10, we will seehow the amphipathic properties of phospholipids are responsiblefor the assemblyof phospholipidsinto sheetlike bilayer biomembranes in which the fatty acyl tails point into the center of the sheet and the head groups point outward toward the aqueousenvironment(seeFigure2-13).a

C h e m i c a lB u i l d i n g B l o c k so f C e l l s r Three major biopolymersformed by polymerizationreactions (net dehydration)of basicchemicalbuilding blocks are presentin cells:proteins,composedof amino acidslinked by peptidebonds;nucleicacids,composedof nucleotideslinked composedof by phosphodiesterbonds;and polysaccharides, (sugars) glycosidic bonds (see linked by monosaccharides fourth major chemical the Figure 2-13). Phospholipids, into biomembranes. noncovalently assemble building block, r Differences in the size, shape, charge, hydrophobicity, and reactivity of the side chains of the 20 common amino acids determine the chemical and structural properties of proteins (seeFigure2-14). r The basesin the nucleotidescomposing DNA and RNA are carbon- and nitrogen-containingrings attached to a pentose sugar. They form two groups: the purinesadenine (A) and guanine (G)-and the pyrimidines-cyto-

r Phospholipids are amphipathic molecules with a hydrophobic tail (often two fatty acyl chains) connectedby a small organic molecule (often glycerol) to a hydrophilic head (seeFigure 2-20). The long hydrocarbon chain of a fatty acid may contain no carbon-carbon double bond (saturated) or one or more double bonds (unsaturated);a cis double bond bends the chain'

E

C h e m i c aEl q u i l i b r i u m

We now shift our discussionto chemical reactions in which bonds, primarily covalent bonds in reactant chemicals' are broken and new bonds are formed to generatereaction products. At any one time, severalhundred different kinds of chemical reactions are occurring simultaneouslyin every cell, and many chemicalscan, in principle' undergo multiple chemical reactions. Both the extent to which reactions can proceed and the rate at which they take place determine the chemical composition of cells. '$7hen reactants first mix together-before any products have been formed-their rate of reaction to form products (forward reaction)is determinedin part by their initial concentrations, which determine the likelihood of reactants bumping into one another and reacting (Figure 2-22). As the reaction products accumulate' the concentration of each reactant deir."r.r and so does the forward reaction rate. Meanwhile' some of the product molecules begin to participate in the reversereaction, which re-forms the reactants (the ability of a reaction to go "backward" is called microscopicreuersibility). This reversereaction is slow at first but speedsup as the conEventually,the ratesofthe forcentrationofproduct increases. ward and reversereactionsbecomeequal, so that the concentrations of reactantsand products stop changing.The systemis then said to be in chemical equilibrium (plural: eqwilibria). At equilibrium, the ratio of products to reactants, called the equilibrium constant' is a fixed value that is independent of the rate at which the reaction occurs. The raie of a chemical reaction can be increased by a catalyst, which acceleratesthe chemical transformation (making and breaking of covalent bonds) but is not permanently changedduring a reaction (seeSection2.4)'ln this section, we discuss several aspectsof chemical equilibria; in the next section, we examine energy changes during reactions and their relationshipto equilibria.

C H E M I C A LE Q U I L I B R I U M

49

Rate."r"rr. : 1. By rearranging theseequations,we can express the equilibrium constant as the ratio of the rate consrants Rateof forward reaction (concentrationof reactantsdecreases)

K-^

1

k1 _T

t) -1\

o

C h e m i c ael o u i l i b r i u m (forwardand reverseratesare e q u a l ,n o c h a n g ei n c o n c e n t r a t i o n of reactantsand products)

; E o

Rate of reverse reaction (concentration of products increases)

When reactantsare first mixed. initial concentrationof products= 0

T i m e+ A FIGURE 2-22 Timedependence of the ratesof a chemical reaction.Theforwardandreverse ratesof a reaction dependin oart on the initialconcentrations of reactants andproducts. Thenet forwardreaction rateslowsasthe concentration of reactants decreases, whereas the netreverse reaction rateincreases asrne concentration of products increases At equilibrium, the ratesof the forwardandreverse reactions areequalandthe concentrations of reactants andproducts remainconstant

E q u i l i b r i u mC o n s t a n t sR e f l e c t h e Extent of a ChemicalReaction The equilibrium constant K.o dependson the nature of the reactants and products, the temperature, and the pressure (particularly in reacrions involving gases).Under standard physicalconditions (25'C and 1 atm pressurefor biological systems),the K.o is always the same for a given reaction, whether or not a catalystis present. For the general reaction with three reactants and three products aA + bB * cC ;

. zZ + yY + xX

(2_1)

where capital letters representparticular moleculesor atoms and lowercaseletters representthe number of each in the reaction formula, the equilibrium constant is given by

(2-2) where bracketsdenote the concentrationsof the moleculesat equilibrium. The rate of the forward reaction (left to right in Equation (2-1) is R a r e 6 o , * , ,:6 k r l A l " l B l b [ C | . where Ai is the rate constant for the forward reaction. Similarly, the rate of the reversereaction (right to left in Equation 2-1) is : k,[X]" [y lv Ratereverse [Z]" where A, is the rate constant for the reversereaction. At equilibrium the forward and reverserates are equal, so Rateso*..6/

s0

CHAPTER2 I

CHEMICALFOUNDATIONS

ChemicalReactionsin CellsAre at SteadyState Under appropriate conditions and given sufficient time, individual biochemical reactions carried out in a test tube eventually will reach equilibrium. Within cells, however, many reactions are linked in pathways in which a product of one reaction servesas a reactarftin another or is pumoed out of the cell. In this more complex situation,when the iate of formation of a substanceis equal to the rate of its consumption, the concentration of the substanceremains constant, and the systemof linked reactionsfor producing and consuming that substanceis said to be in a steady state (Figure 2-23). One consequenceof such linked reactionsis that they prevent the accumulation of excessintermediates,protecting cells from the harmful effects of intermediatesthat have the Dotential of beingtoxic at high concentrations.

DissociationConstantsof Binding Reactions Reflectthe Affinity of lnteractingMolecules The concept of equilibrium also appliesto the binding of one molecule to another. Many important cellular processesdepend on such binding "reactions," which involve the making and breaking of various noncovalentinteractionsrather than covalentbonds,as discussedabove.A common exampleis the binding of a ligand (e.g.,the hormone insulin or adrenaline)to its receptor on the surface of a cell forming a multimolecular assembly,or complex, that triggersa biological response.Another exampleis the binding of a protein to a specificsequence of basepairs in a moleculeof DNA, which frequently causes the expressionof a nearby geneto increaseor decrease(Chapter 7). lf the equilibrium constant for a binding reaction is (a)Testtube equilibriumconcentrations AAA

=

-BBB

BBB BBB

(b) Intracellularsteady-stateconcentrations n n - B B_B

BBB

_

- cC CC

A FIGURE2-23 Comparisonof reactionsat equilibrium and steady state. (a) ln the test tube, a biochemicalreaction(A -+ B) eventuallywill reachequilibrium,in which the ratesof the fon,rardand reversereactionsare equal(asindicatedby the reactionarrowsof equal length).(b) In metabolicpathwayswithin cells,the productB commonly would be consumed,in this exampleby conversionto C. A pathwayof linkedreactionsis at steadystatewhen the rateof formationof the (e g , B) equalstheir rateof consumption.As indicatedby intermediates the unequallengthof the arrows,the individualreversible reactions constitutinga metabolicpathwaydo not reachequilibrium Moreover, the concentrations of the intermedrates at steadystatecan differfrom what theywould be at eouilibrium.

CanBind MultipleLigands ffi Rodcast:Macromolecules > FIGURE 2-24 Macromolecules can havedistinctbinding M u l t i l i g a n db i n d i n gm a c r o m o l e c u l (ee . 9 . ,p r o t e i n ) (eg , a protein, sitesfor multipleligands.A largemacromolecule Bindingsite A (K6a) bindingsites(A-C)isshown;eachbinding B i n d i n gs i t e B ( K 6 s ) blue)withthreedistinct molecular to threedifferent binding complementarity siteexhibits (ligands (KdAc) partners A-C)with distinct dissociation constants L i g a n dB

known, the intracellular stability of the resultingcomplex can ( e . 9 . s, m a l l be predicted.To illustrate the generalapproach for determin- m o l e c u l e ) ing the concentrationof noncovalentlyassociatedcomplexes, we will calculatethe extent to which a protein (P) is bound to DNA (D)forming a protein-DNA complex (PD):

L i g a n dA ( e . 9 . s, m a l l p r o t e i n )

P+D.-PD Most commonly, binding reactions are describedin terms of the dissociation constant K6, which is the reciprocal of the equilibrium constant. For this binding reaction, the dissociation constant is given by

ro:

tPIIDl

TDi

L i g a n dC (e 9.,polysaccharide)

16:\ Binding s i t eC ( K 6 6 ) d

(2-4)

Typical reactionsin which a protein binds to a specificDNA sequencehavea K6 of 10-10 M, where M symbolizesmolariry or moles per liter (mol/L). To relate the magnitude of this dissociation constant to the intracellular ratio of bound to unbound DNA, let's consider the simple example of a bacterial cell hav1s L ing a volume of 1.5 x 10 and containing L moleculeof DNA and 10 moleculesof the DNA-binding protein P. In this 0 case,givena Ka of 10-1 M, 99 percentof the time this specific DNA sequencewill have a molecule of protein bound to it and 1 percent of the time it will not, even though the cell contains only 10 moleculesof the protein! Clearly P and D bind very tightly (have a high affinity), as reflectedby the low value of the dissociation constant for their binding reaction. For protein' M protein and protein-DNA binding, K6 values of <10 o -10 (nanomolar)are consideredto be tight, M (micromolar) modestlytight, and -10-' M (millimolar) relativelyweak. The large size of biological macromolecules,such as proteins, can result in the availability of multiple surfacesfor complementaryintermolecularinteractions (Figure 2-24). As a consequence,many macromoleculeshave the capacity to bind severalother moleculessimultaneously.In some cases, these binding reactionsare independent,with their own distinct Ka valuesthat are constant.In other cases,binding of a moleculeat one site on a macromoleculecan changethe threedimensionalshapeof a distant site, thus altering the binding interactionsof that distant sitewith someother molecule.This is an important mechanismby which one moleculecan alter (regulate)the activity of a secondmolecule(e.g.,a protein) by changingits capacityto interact with a third molecule.'Weexamine this regulatory mechanismin more detail in Chapter 3. RoshanKetab 021-6 69 50 639

B i o l o g i c aFl l u i d sH a v eC h a r a c t e r i s t ipcH V a l u e s The solventinsidecellsand in all extracellularfluids is water. An important characteristic of any aqueous solution is the

concentrationof positivelychargedhydrogenions (H+ ) and negatively chargedhydroxyl ions (OH-). Becausetheseions are the dissociationproducts of H2O, they are constituentsof all living systems,and they are liberatedby many reactionsthat take place between organic moleculeswithin cells.These ions also can be transported into or out of cells, as when highly acidic gastric juice is secretedby cellslining the walls of the stomach. \fhen a water moleculedissociates,one of its polar H-O bonds breaks.The resultinghydrogenion, often referredto as a proton, has a short lifetime as a free ion and quickly combines with a water molecule to form a hydronium ion (HrO*). For convenience,however'we refer to the concentration of hydrogenions in a solution, [H*], eventhough this really representsthe concentrationof hydronium ions, [H3O-]. Dissociationof H2O generatesone OH- ion along with each H*. The dissociationof water is a reversiblereaction: H2O:H*+OHAt 25 'C, [H+][OH-]_:

1O-r4 M2, so that in pure water,

l H * l: t o H - l : 1 o - ' M .

The concentration of hydrogen ions in a solution is expressedconventionally as its pH, defined as the negativelog of the hydrogen ion concentration. The pH of pure water at 25 'C is 7:

p H : - l o g [ H -:] l " s E L : b e + = - : z lnj,J

It is important to keep in mind that a 1 unit differencein pH representsa tenfold difference in the concentration of protons. On the pH scale,7.0 is consideredneutral: pH values below 7.0 indicateacidicsolutions(higher[H*]), and values above 7.0 indicate basic (alkaline) solutions (Figure 2-25)' For instance, gastric juice, which is rich in hydrochloric acid EQUILIBRIUM CHEMICAL

51

I n c r e a s i n g lbya s i c ( l o w e rH +c o n c e n t r a t i o n )

pH scale

1 4 S o d i u mh y d r o x i d(e1 N ) 13 1)

H o u s e h o lb dl e a c h A m m o n i a( 1 N )

11

1 0 1^

sea water , _.1r I n t e r i o ro f c e l l Fertilizedegg U n f e r t i l i z eedg g

8 j' 6

Urine I n t e r i o ro f t h e l y s o s o m e G r a p e f r u ijtu i c e

ric acid (HCl) or the carboxylgroup (-COOH), which tends to dissociateto form the negativelychargedcarboxylate ion (-COO ). Likewise,a baseis any molecule,ion, or chemical group that readily combineswith a H+, such as the hydroxyl ion (OH-); ammonia (NH:), which forms an ammonium ion (NH+-); or the amino group (-NH2). rWhenacid is addedto an aqueoussolution,the [H+] increases(the pH goes down). ConverselS when a base is addedto a solution,the [H+] decreases (the pH goesup). Because[H+][OH ] : 10-14M2, any increasein [H*] is coupled with a commensuratedecrease in [OH-] and vice versa. Many biologicalmoleculesconrain both acidic and basic groups.For example,in neutral solutions(pH : 7.0), many amino acidsexist predominantlyin the doubly ionizedform, in which the carboxyl group has lost a proton and the amino group has acceptedone: NH,*

t-

H-C-COOR

G a s t r i cj u i c e

where R representsthe uncharged side chain. Such a molecule, containing an equal number of positive and negative ions, is called a zwitterion. Zwitterions, having no net charge,are neutral. At extreme pH values,only one of these two ionizable groups of an amino acid will be charged. The dissociationreaction for an acid (or acid group in a I n c r e a s i n gal cy i d i c larger molecule)HA can be wrirten as HA == H+ + A . The ( gr e a t eH r +c o n c e n t r a t i o n ) equilibriumconstantfor this reaction,denotedK. (thesubscript FfGURE2-25 pH valuesof commonsolutions.ThepHof an d standsfor "acid"), is definedas K" : [H*][A ]/lHAl. Taking aqueous solution isthenegative logof thehydrogen ionconcentratron the logarithm of both sidesand rearrangingthe result yields a ThepHvalues for mostintracellular andextracellular biological fluids very usefulrelation betweenthe equilibrium constantand pH: arenear7 andarecarefully regulated to permittheproperfunctioning of cells, organelles, andcellular secretions H y d r o c h l o r i ac c i d ( 1 N )

(HCl), has a pH of about 1. ks [H+] is roughly a millionfold greaterthan that of cytoplasmwith a pH of abort 7.2. Although the cytosolof cellsnormally has a pH of about 7.2, the pH is much lower (about 4.5) in the interior of lyso, somes,one type of organellein eukaryoticcells(Chapter9). The many degradative enzymeswithin lysosomesfunction optimally in an acidic environment,whereastheir action is inhibited in the near neurral environmentof the cytoplasm. This illustratesthat mainrenanceof a specificpH is essential for proper functioning of some cellular strucrures.On the other hand, dramatic shifts in cellular pH may play an important role in controlling cellularactivity.For example,the pH of the cytoplasmof an unfertilizedeggof the seaurchin, an aquatic animal, is 6.6. Within 1 minute of fertilization, however,the pH risesto 7.2; that is, the [H+l decreases to about one-fourth its original value, a change necessaryfor subsequentgrowth and division of the egg.

Hydrogenlons Are Releasedby A c i d sa n d T a k e nU p b y B a s e s In general, an acid is any molecule, ion, or chemical group that tendsto releasea hydrogenion (H+), suchas hydrochlo52

.

c H A p r E R 2| c H E M t c A L F o u N D A T t o N S

pH: pK"+ loe#l

lr1Al

e-s)

where pK" equals-log K". From this expression,commonly known astheHendersonHasselbaLch equation,it can be seenthar the pK. of any acid is equalto the pH at which half the moleculesare dissociatedand half are neutral (undissociated). This is becausewhen [A-] : [HA], then log ([A-]/[HA]) : O, and thus pK" : pH. The Henderson-Hasselbalch equarionallows us to calculatethe degree of dissociationof an acid if both the pH of the solution and the pK, of the acid are known. Experimentally,by meas, uring the [A ] and [HA] as a function of the solution'spH, one can calculatethe pK. of the acid and thus the equilibrium constant Ka for the dissociationreacion (Figure2-26).

B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l a r a n d E x t r a c e l l u l aFr l u i d s A growing cell must maintain a consrant pH in the cytoplasm of about 7.2-7.4 despitethe metabolic production of many acids,suchas lactic acid and carbon dioxide;the latter reactswith water to form carbonicacid (H2CO3).Cellshave a reservoir of weak basesand weak acids, called buffers, which ensure that the cell's pH remains relatively constant

H2CO3:HCO3-+H* H2C03 -';i9 1 1 0 0

= =)

.oO

6P OG tsc

-o

50

0L

0

7.48 OH

2-26 lhe relationshipbetween pH, pKa,and the A FfGURE of carbonic acid of an acid.Asthe oHof a solution dissociation in the of the compound risesfrom0 to 8 5, the percentage from100 form(HzCO:) decreases or un-ionized, undissociated, from0 percentWhen percent formincreases andthatof the ionized acidhas the pH(6.4)isequalto the acidspKa,halfof the carbonic allof the acidhas ionizedWhenthe pH risesto above8, virtually form(HCOr) ionized to the bicarbonate despitesmall fluctuationsin the amountsof H* or OH- being generatedby metabolism or by the uptake or secretionof moleculesand ions by the cell. Buffers do this by "soaking up" excessH* or OH when theseions are addedto the cell or are producedby metabolism. If additional acid (or base)is added to a buffered solution whose pH is equal to the pK, of the buffer (tHAl : [A ]), the pH of the solution changes,but it changesless than it would if the buffer had not been present.This is becauseprotons releasedby the addedacid are taken up by the ionized form of the buffer (A- ); likewise, hydroxyl ions gener:atedby the addition of baseare neutralized by protons releasedby the undissociatedbuffer (HA). The capacity of a substanceto releasehydrogen ions or take them up depends partly on the extent to which the substancehas already taken up or releasedprotons, which in turn dependson the pH of the solution relative to the pK" of the substance.The ability of a buffer to minimize changesin pH, its buffering capacity,dependson the concentration of the buffer and the relationship between its pK" value and the pH, which is exequation. pressedby the Henderson-Hasselbalch The titration curve for acetic acid shown inFigure 2-27 illustratesthe effect of pH on the fraction of moleculesin the un-ionized(HA) and ionizedforms (A ). At one pH unit below the pK" of an acid, 91 percentof the moleculesare in the HA form; at one pH unit above the pK^, 9L percent are in the A form. At pH values more than one unit above or below the pK", the buffering capacity of weak acids and basesdeclines rapidly. In other words, the addition of the same number of moles of acid to a solution containing a mixture of HA and A that is at a pH near the pK" will cause lessof a pH changethan it would if the HA and A- were not present or if the pH were far from the pK. value. All biological systemscontain one or more buffers. Phosphate ions, the ionized forms of phosphoric acid, are present in considerablequantitiesin cellsand are an important factor

o

0'8 0.6 0.4 0.2 CH3COOH of dissociated Fraction AddedOH- -----)

1.0

2-27 The titration curveof the buffer aceticacid FfGURE of aceticacidto hydrogen (CH3COOH). ThepK,for the dissocration are ionsis4 75 At thispH,halftheacidmolecules andacetate thesolution scale, pHismeasured on a logarithmic Because dissociated CH3COOH at pH3.75to 9 percent CH3COOH from91 percent changes pH range in this capacity buffering maximum has at pH5 75.Theacid in maintaining, or buffering, the pH of the cytoplasm. Phosphoric acid (H3POa) has three protons that are capable of dissociating,but they do not dissociatesimultaneously.Loss of each proton can be describedby a discretedissociationreaction and pKo, as shown in Figure 2-28.The titration curve 14

P K a =1 2 ' 7 HPO42-+

12

I

PKa=7'2

H2PO4-*

o_

PKa=2'1

PO4

HPO+2-+H*

H3PO4+HrPOf+H+

Added OH- -----> A FIGURE2-28 The titration curve of phosphoric acid (H3PO4), ubiquitous a common buffer in biological systems.Thisbiologically at differentpH moleculehasthreehydrogenatomsthat dissociate acidhasthreepK" values,as notedon the values;thus phosphoric graph The shadedareasdenotethe pH ranges-withinone pH unit of the three pKuvalues-where the bufferingcapacityof phosphoric acidis high In theseregions,the additionof acid(or base)will cause smallchangesin the PH relatively EQUILIBRIUM CHEMICAL

53

for phosphoric acid shows that the pK" for the dissociationof the secondproton is 7.2. Thus at pH7.2, about 50 percentof cellula^rphosphate is H2POa- and about 50 percent is HPO4'- according to the Henderson-Hasselbalchequation. For this reason,phosphateis an excellentbuffer at pH values around 7.2, the approximate pH of the cytoplasm of cells, and at pH 7.4, the pH of human blood.

C h e m i c a lE q u i l i b r i u m r A chemical reaction is at equilibrium when the rate of the forward reaction is equal to the rate of the reversereaction (no net changein the concentration of the reactants or products). The equilibrium constant K.o of a reaction reflects the tio of products to reacrantsai equilibrium and thus is a measure of the extent of the reaction and the relative stabilities of the reactantsand products. r The K.o depends on the temperature, pressure, and chemical properties of the reactants and products but is independent of the reaction rate and of the initial concentrations of reactantsand products. For any reaction, the equilibrium constant K.o equalsthe tio of the forward rate constant to the reversi rate constant (krl&r).The rates of conversion of reactantsto products and vice versa depend on the rate constants and the concentrationsof the reactantsor products. 'Sfithin r cells, the linked reactions in metabolic pathways generallyare at steadystate,not equilibrium, at which rate of formation of the intermediatesequals their rate of consumption (seeFigure 2-23) and thus the concentrations of the intermediatesare not changing. r The dissociation constant K6 for the noncovalent binding of two molecules is a measure of the stability of the complex formed betweenthe molecules(e.g.,ligand-receptor or protein-DNA complexes). r The pH is the negativelogarithm of the concentration of hydrogen ions (-log [H+]). The pH of the cytoplasm is normally about 7.2-7.4, whereasthe interior of lysosomeshas a pH of about 4.5. r Acids releaseprotons (H+) and basesbind them. In biological molecules,the carboxyl and phosphate groups are the most common acidic groups; the amino group is the most common basicgroup. r Buffers are mixrures of a weak acid (HA) and its corresponding baseform (A ), which minimize the changein pH of a solution when acid or alkali is added. Biological systems use various buffers to maintain their pH within a very narrow range.

Biochemical Energetics

W

The production of energy,its storage,and its use are central to the economy of the cell. Energy may be defined as the ability to do work, a concept applicable to automobile 54

.

c H A p r E R 2| c H E M t c A L F o u N D A T t o N s

engines and electric power plants in our day-to-day world and to cellular enginesin the biological world. The energy associatedwith chemical bonds can be harnessedto support chemical work and the physical movements of cells.

SeveralFormsof EnergyAre lmportant i n B i o l o g i c aS l ystems There are two principal forms of energy:kinetic and potential. Kinetic energyis the energyof movement-the motion of molecules,for example.The secondform of energy,potential energy, or stored energy, is particularly important in the study of biological or chemical systems. Thermal energy, or heat, is a form of kinetic energy-the energy of the motion of molecules.For heat to do work, it must flow from a region of higher temperature-where the averagespeedof molecularmotion is greater-to one of lower temperature. Although differencesin temperature can exist betweenthe internal and external environmentsof cells,these thermal gradientsdo not usually serveas the sourceof energy for cellular activities.The thermal energy in warm-blooded animals, which have evolveda mechanismfor thermoregulation, is usedchiefly to maintain constantorganismictemperatures. This is an important function becausethe rates of many cellular activities are temperature-dependent.For example, cooling mammalian cellsfrom their normal body temperature of 37 "C to 4 "C can virtually "freeze" or stop many cellular processes(e.g.,intracellularmembranemovements). Radiant energyis the kinetic energyof photons, or waves of light, and is critical to biology. Radiant energycan be converted to thermal energy,for instancewhen light is absorbed by moleculesand the energy is converted to molecular motion. Radiant energy absorbed by moleculescan also change the electronic structure of the molecules, moving electrons into higher-energystates (orbitals), whence it can later be recoveredto perform work. For example, during photosynthesis,light energy absorbed by specializedmolecules (e.g.,chlorophyll) is subsequentlyconverted into the energy of chemical bonds (Chapter 12). Mechanical energy,a major form of kinetic energy in biology usually resultsfrom the conversionof stored chemical energy.For example, changesin the lengths of cytoskeletal filaments generate forces that push or pull on membranes and organelles(Chapters 17 and 18). Electric energy-the energy of moving electrons or other charged particles-is yet another major form of kinetic energy. Several forms of potential energy are biologically significant. Central to biology is chemicalpotential energy,the energy stored in the bonds connecting atoms in molecules.Indeed, most of the biochemicalreactionsdescribedin this book involve the making or breaking of at least one covalent chemical 'We bond. recognize this energy when chemicals undergo energy-releasingreactions. For example, the high potential energy in the covalent bonds of glucosecan be releasedby controlled enzymaticcombustion in cells (Chapter 12). This energyis harnessedby the cell to do many kinds of work. A secondbiologically important form of potential energyis the energyin a concentration gradient. \X/henthe concentration

of a substanceon one side of a barrier,such as a membrane,is different from that on the other side, a concentration gradient exists. All cells form concentration gradients between their interior and the external fluids by selectivelyexchangingnutrients, waste products, and ions with their surroundings.Also, organelleswithin cells (e.g., mitochondria, lysosomes)frequently contain different concentrationsof ions and other molecules;the concentration of protons within a lysosome, as we saw in the last section,is about 500 timesthat of the cytoplasm. A third form of potential energy in cells is an electric potential-the energy of charge separation. For instance, there is a gradient of electric charge of =200,000 volts per cm acrossthe plasma membrane of virtually all cells.We discuss how concentration gradients and the potential differenceacrosscell membranesare generatedand maintained in Chapter 11 and how they are converted to chemical potential energyin Chapter 12.

CellsCanTransformOne Type of Energyinto Another According to the first law of thermodynamics,energy is neither creatednor destroyedbut can be converted from one form to another.(In nuclear reactions,massis convertedto energy, but this is irrelevantto biological systems.)In photosynthesis, for example,the radiant energyof light is transformedinto the chemicalpotential energy of the covalent bonds betweenthe atoms in a sucroseor starch molecule.In musclesand nerves, chemical potential energy stored in covalent bonds is transinto the kinetic energyof musclecontracformed, respectively, tion and the electricenergyof nerve transmission.In all cells, potential energy,releasedby breaking certain chemicalbonds, is used to generatepotential energyin the form of concentration and electricpotential gradients.Similarly,energystoredin chemical concentration gradients or electric potential gradients is usedto synthesizechemicalbonds or to transport moleculesfrom one side of a membraneto another to generatea concentrationgradient. The latter processoccurs during the transport of nutrients such as glucoseinto certain cells and transport of many wasteproducts out of cells. Becauseall forms of energyare interconvertible,they can be expressedin the sameunits of measurement.Although the standard unit of energy is the joule, biochemistshave traditionally used an alternative unit, the calorie (1 joule : 0.239 calorie). Throughout this book, we use the kilocalorie to measureenergychanges(1 kcal : 1000 cal).

The Changein FreeEnergyDetermines the Directionof a ChemicalReaction Becausebiological systemsare generally held at constant temperatureand pressure,it is possibleto predict the direction of a chemicalreaction from the changein the free energy G, named after J. V. Gibbs, who showed that "all systems changein such a way that free energy [G] is minimized." In products, the the caseof a chemical reaction, reactants * free-energychangeAG is given by AG

:

Goroaucts -

Greactants

(a) Endergonic

1

A I

I

(\

ah

t 6

j o

c) 0)

o) (!) 0) LI

E

u

Reactants

Progressof reaction----->

Progressof reaction----->

2-29 Changesin the free energy(AG)of exergonic FIGURE the freeenergy reactions, and endergonicreactions.(a)In exergonic Consequently, islowerthanthatof the reactants of the products asthe andenergyisreleased occurspontaneously thesereactions reactions, the freeenergyof the proceed(b)In endergonic reactions do andthesereactions productsisgreaterthanthat of the reactants of energymustbe source An external notoccurspontaneously into products areto be converted suppliedif the reactants The relation of AG to the direction of any chemical reaction can be summarizedin three statements: r If AG is negative,the forward reaction will tend to occur spontaneouslyand energy usually will be releasedas the reaction takes place (exergonicreaction) (Figure2-291. r If AG is positive, the forward reaction will not occur spontaneously:energywill have to be added to the systemin order to force the reactantsto becomeproducts (endergonic reaction). r If AG is zero, both forward and reversereactions occur at equal rates and there will be no spontaneousconversion of reactantsto products (or vice versa);the systemis at equilibrium. By convention, the standard free-energychange of a reaction AGo' is the value of the change in free energy under the conditionsof 298 K (25 "C)' 1 atm pressure,pH 7.0 (as in pure water), and initial concentrationsof 1'M for all reacproJucts exceptprotons' which are kept at 10-7 M t"nts "nd (pH 7.0). Most biological reactions differ from standard conditions, particularly in the concentrations of reactants' which are normally lessthan 1 M. The free energy of a chemical system can be defined as : H - TS, where H is the bond energy'or enthalpy' of the G system; 7 is its temperature in degreesKelvin (K); and S is the entropy, a measureof its randomnessor disorder' If temperature remains constant' a reaction proceeds spontaneously only if the free-energychange AG in the following equation is negative:

AG: AH- TAS B I O C H E M I C AELN E R G E T I C S .

()-6\

55

In an exothermic reaction, the products contain less bond energy than the reactanrs, the liberated energy is usually converted to heat (the energy of molecular motion), and AH is negative.In an endothermic reaction, the products contain more bond energy than the reactants,heat is absorbed during the reaction, and AH is positive. The combined effects of the changes in the enthalpy and entropy determine if the AG for a reaction is positive or negarive. An exothermic reaction (AH < 0) in which enrropy increases(AS > 0) occurs spontaneously(AG < 0). An endothermic reaction (AH > 0) will occur spontaneouslyif AS increasesenough so that the T AS term can overcome the positive AH. Many biological reactions lead to an increase in order and thus a decreasein entropy (AS < 0). An obvious example is the reaction that links amino acids to form a protein. A solution of protein molecules has a lower entropy than does a solution of the same amino acids unlinked because the free movementof any amino acid in a protein is restricted when it is bound into a long chain. Often cells compensate for decreases in entropy by "coupling" suchsynthetic,entropylowering reactions with independent reactions that have a very highly negariveAG (seebelow). In this way cells can convert sources of energy in their environment into the building of highly organized structures and metabolic pathways that are essentialfor life. The actual changein free energy AG during a reacrion is influenced by temperature, pressure,and the initial concentrations of reactants and products and usually differs from AG''. Most biologicalreactions-like othersthat take place in aqueoussolutions-also are affected by the pH of the so'We lurion. can estimate free-energy changes for different temperaturesand initial concentrationsusing the equation AG:

AC.'-r RTln Q:

AC"'+ RTln

fproducts] Q-7) I reacrantsl

where R is the gas constanrof 1.987 call(degree.mol), T is the temperature (in degreesKelvin), and Q is the initial rctio of products to reactants. For a reaction A + B == C. in which two molecules combine to form a third, e in Equation2-7 equalstcl/lAltBl.In this case,an increasein the initial concentration of either [A] or [B] will result in a larger negative value for AG and thus drive the reaction toward more formation of C. Regardlessof the AG"' for a particular biochemicalreaction, it will proceed spontaneouslywithin cells only if AG is negative, given the intracellular concentrations of reactants and products. For example, the conversion of glyceraldehyde 3-phosphate (G3P) to dihydroxyacetonephosphate (DHAP), two inrermediatesin the breakdownof glucose, .^DHAP G3P . has a AGo' of -1840 caVmol.If the initial concentrationsof G3P and DHAP are equal, then AG : AGo' becauseRT ln 1 : 0; in this situation, the reversiblereaction G3P : DHAP will proceedspontaneouslyin the direction of DHAp formation until equilibrium is reached. However, if the initial 56

CHAPTER2 I

CHEMICALFOUNDATIONS

[DHAP] is 0.1 M and the initial [G3P] is 0.001 M, with other conditions standard,then Q in Equation 2-7 equals0.1/0.001 : 100, giving a AG of *887 caVmol.Under theseconditions, the reaction will proceed in the direction of formation of G3P. The AG for a reaction is independent of the reaction rare. Indeed, under usual physiological conditions, few if any of the biochemical reactions neededto sustain life would occur without some mechanism for increasing reaction rates. As we describe below and in more detail in Chapter 3, the rates of reactions in biological systemsare usually determinedby the activity of enzymes,the protein catalyststhat acceleratethe formation of products from reactantswithout altering the value of AG.

The Ad' of a ReactionCan Be Galculatedfrom lts K.o A chemical mixture at equilibrium is in a stable state of minimal free energy.For a system at equilibrium (AG : 0, Q : K"o), we can write AGo': -2.3RTlogK.o: -1362logK.o

Q-8)

under standard conditions (note the changeto base 10 logarithms). Thus if we determinethe concentrationsof reactants and products at equilibrium (i.e., the K.o), we can calculate the value of AGo'. For example,the K.o for the inrerconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate (G3P : DHAP) is 22.2 under standard conditions. Substitutingthis value into Equation 2-8, we can easily calculatethe AGo' for this reaction as - 1840 callmol. By rearranging Equation 2-8 and taking the antilogarithm, we obtain Keq :

10-(aG"'/2'3RT)

(2-e)

From this expression,it is clear that if AGo' is negative,the exponent will be positive and henceK.o will be greater than 1. Therefore at equilibrium rhere will be more products than reactants; in other words, the formation of products from reactants is favored. Conversely,if AG'' is positive, the exponent will be negativeand K.o will be lessthan 1.

The Rateof a ReactionDependson the Activation EnergyNecessary to Energize the Reactantsinto a TransitionState As a chemical reaction proceeds, reactants approach each otherl some bonds begin to form while others begin to break. One way to think of the state of the moleculesduring this transition is that there are strains in the electronic configurations of the atoms and their bonds. In order for the collection of atoms to move from the relatively stable state of the reactants to this intermediate state during the reaction, an introduction of energy is necessary.This is illustrated in the reaction energy diagram in Figure 2-30. Thus the collection of atoms is transiently in a higher-energysrare at some point during the course of the reaction. The state during a chemicalreaction at which the systemis at its highest energy level is called the transition state or transition-

+

II

Transitionstate (uncatalyzed)

Transitionstate (catalyzed) 6 0) 0) 0) LI

Products

Progressof reaction----> 2-30 Activationenergy of uncatalyzedand A FIGURE reactionpathway catalyzedchemicalreactions.Thishypothetical (blue)depicts proceeds A thechanges in freeenergyG asa reaction reaction willtakeplacespontaneously if thefreeenergy(G)of the (AG< 0). However, products all islessthanthatof the reactants proceed reactions throughone(shownhere)or morehighchemical isinversely states, andthe rateof a reaction energytransition proportional in to theactivation energy(AG*),whichisthe difference the reactants andthetransition stateIn a freeenergybetween (red),thefreeenergies of the reactants and reaction catalyzed products stateis areunchanged but thefreeenergyof thetransition lowered, thusincreasing thevelocity of the reaction

state intermediate.The energy neededto excite the reactants to this higher-energystate is called the activation energy of the reaction. The activation energy is usually representedby AGt, analogousto the representationof the changein Gibbs free energy (AG) already discussed.From the transition state, the collection of atoms can either releaseenergy as the reaction products are formed or releaseenergy as the atoms go "backward" and re-form the original reactants.The velocity (V) at which products are generated from reactants during the reaction under a given set of conditions (temperature, pressure,reactant concentrations)will depend on the concentration of material in the transition state, which in turn will depend on the activation energy and the characteristic rate constant (z) at which the transition state ls converted to products. The higher the activation energy, the lower the fraction of reactantsthat reach the transition state and the slower the overall rate of the reaction. The relationship between the concentration of reactants,z and V is (AG+/2 3RT) v : u lreactants]x 10 From this equation, we can seethat lowering the activation energy-that is, decreasingthe free energy of the transition stateAG+-leads to an accelerationof the overall reactionrate V. A reduction in AGt of 1.36 kcal/mol leads to a tenfold increasein the rate of the reaction, whereas a 2.72 kcal/mol reduction increasesthe rate 100-fold. Thus relatively small changesin AGt can lead to large changesin the overall rate of the reaction.

Catalystssuch as enzymes(Chapter 3) acceleratereaction rates by lowering the relative energy of the transition state and so the activation energy (seeFigure 2-30). The relative energiesof reactantsand products will determineif a reaction is thermodynamically favorable (negativeAG), whereas the activation energy will determine how rapidly products form (reaction kinetics). Thermodynamically favorable reactions will not occur if the activation energiesare too high.

L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l e s ith Energetically C h e m i c aR l e a c t i o nw FavorableReactions unfavorable(AG > 0) Many processesin cellsareenergetically include the synExamples and will not proceed spontaneously. of a substance transport nucleotides and thesis of DNA from a higher concenlower to from a plasma membrane acrossthe or endergonic, energy-requiring, out an can carry tration. Cells reaction (AGr > 0) by coupling it to an energy-releasing,or exergonic, reaction (AGz < 0) if the sum of the two reactionshas an overall net negativeAG. Suppose,for example,that the reactionA = B + X has a AG of + 5 kcaUmoland that the reactionX = Y + Zhas a L,G of -10 kcal/mol:

(1) A

B+X

AG:+Skcal/mol

( 2\ x

Y+Z

AG:-10kcal/mol

Sum: A.

^B+Y+Z

AG'' :-5kcal/mol

In the absenceof the secondreaction' there would be much more A than B at equilibrium. However, becausethe conversion of X to Y + Z is such a favorable reaction, it will pull the first process toward the formation of B and the consumption of A. Energetically unfavorable reactions in cells often are coupled to the energy-releasinghydrolysis of ATP' as we discussnext.

Hydrolysisof ATPReleasesSubstantialFree E n e r g ya n d D r i v e sM a n y C e l l u l a rP r o c e s s e s In almost all organisms, adenosinetriphosphate, or AIP, is the most important molecule for capturing' transiently storing, and subsequentlytransferring energy to perform work (e.g.,biosynthesis,mechanicalmotion). The useful energyin an ATP molecule is contained in phosphoanhydride bonds, which are covalent bonds formed from the condensationof two moleculesof phosphateby the loss of water: OO

ti o- -i-on + Ho-P-o- . * ll

o-

o-

o

llll o--P-O-?-OI I o o-

+ H2O

B I O C H E M I C AELN E R G E T I C S

57

pH. During synthesis of ATP, a large input of energy is required to force the negative charges in ADP and P1 together.ConverselSmuch energy is releasedwhen ATP is hy*zc'-a-N rtl \u Phosphoanhydride bonds drolyzed to ADP and P;. In comparison, formation of the HC__*,rC_;1 phosphoester o bond between an unchargedhydroxyl in glycto to - o - P -i ol |- lL|Pl - O _ | . P - O erol and P1requires less energy, and less energy is released cH2-owhen this bond is hydrolyzed. tll oo-o H Cells have evolved protein-mediated mechanismsfor transferringthe free energyreleasedby hydrolysis of phosphoanhydride bonds to other molecules, thereby driving HO OH reactions that would otherwise be energeticallyunfavorAdenosine triphosphate able. For example, if the AG for the reaction B + C -+ D is positive but less than the AG for hydrolysis of ATP, the reA FIGURE 2-31 Adenosinetriphosphate(ATP).Thetwo phosphoanhydride bonds(red)in ATP, whichlinkthethreephosphate action can be driven to the right by coupling it to hydrolygroups, eachhasa AG'of about-7 3 kcal/mol for hydrolysis sis of the terminal phosphoanhydride bond in ATP. In one Hydrolysis of thesebonds,especially theterminal one,rsrnesource common mechanism of such energy coupling, some of the of energythatdrivesmanyenergy-requiring reactions in biological energy stored in this phosphoanhydride bond is transferred systems. to one of the reactants by breaking the bond in ATP and forming a covalent bond between the releasedphosphate group and one of the reactants.The phosphorylatedinterAn ATP molecule has two key phosphoanhydride (also mediate generatedin this way can then react with C to calledphosphodiester)bonds (Figure 2-31). Hydrolysis of a form D * P; in a reaction that has a negativeAG: phosphoanhydride bond (-) in each of the following reactions has a highly negativeAG'' of about -7.3 kcal/mol: NH"

B+ATPTB-p+ADP Ap-p-p + H2O ----+ Ap-p + Pi + H+ (ATP) Ap-p-p+H2O(ATP)

B-p+C-+D+P;

(ADP) Ap+PPi+H+ (AMP)

The overallreaction B+C+ATP:-D+ADP+P.

Ap-p + H2O ----+ Ap + P; * H+ (ADP)

(AMP)

In thesereactions,Pi standsfor inorganic phosphate(pO+, ) and PP;for inorganic pyrophosphate,two phosphategroups linked by a phosphoanhydride bond. As the top two reactions show, the removal of a phosphate or a pyrophosphate group from ATP leaves adenosinediphosphate (ADp) or adenosinemonophosphate (AMP), respecively. A phosphoanhydride bond or other high-energy bond (commonly denoted by -) is not intrinsically different from other covalent bonds. High-energy bonds simply releaseespecially large amounts of energywhen broken by addition of water (hydrolyzed). For instance,the AG"' for hydrolysis of a phosphoanhydride bond in ATP (-7.3 kcal/mol) is more than three times the AGo' for hydrolysis of the phosphoester bond (red) in glycerol3-phosphate(-Z.2kcallmol):

ooH Ho-p-o-cHr-lH - cH2oH I

U Glycerol3-phosphate

A principal reason for this differenceis that ATp and its hydrolysis products ADP and P1are highly charged at neutral

58

CHAPTER2 I

CHEMICALFOUNDATIONS

is energeticallyfavorable (AG < 0). An alternative mechanism of energy coupling is to use the energy releasedby ATP hydrolysis to changethe conformation of the molecule to an "energy-rich" stressedstate. In turn, the energy stored as conformational stresscan be released as the molecule "relaxes" back into its unstressed conformation. If this relaxation processcan be mechanistically coupled to another reaction, the releasedenergycan be harnessedto drive important cellular processes. As with many biosynthetic reactions,transport of molecules into or out of the cell often has a positive AG and thus requires an input of energy to proceed. Such simple transport reactions do not directly involve the making or breaking of covalent bonds; thus the AGo' is 0. In the case of a substancemoving into a cell, Equation 2-7 becomes

AG: Rrt"+."9*

(2-10)

where [C1"] is the initial concentration of the substanceinside the cell and [Co",] is its concentration outside the cell. 'We can see from Equation 2-10 that AG is positive for transport of a substanceinto a cell against its concentration gradient (when [Cr"] > [C"",]); the energy to drive such "uphill" transport often is supplied by the hydrolysis of

ATP. Conversely,when a substancemoves down its concentration gradient ([C",,] > [C,"] ), AG is negative. Such "downhill" transport releasesenergythat can be coupledto an energy-requiringreaction, say, the movement of another substanceuphill acrossa membrane or the synthesisof ATP itself (seeChapters 1I and 12).

ATPls GeneratedDuring Photosynthesis and Respiration Clearlg to continue functioning, cells must constantly replenish their ATP supply. In nearly all cells,the initial energysource whose energyis ultimately transformed into the phosphoanhydride bondsof ATP and bonds in other compoundsis sunlight. In photosynthesis,plants and certain microorganismscan trap the energy in light and use it to synthesizeAIP from ADP and P1.Much of the ATP produced in photosynthesisis hydrolyzed to provide energyfor the conversionof carbon dioxide to sixcarbon sugars,a processcalledcarbon fixation: ATP ADP + P. 6co2 + 6 Hro V c 6 H 1 2 o+6 o 0 2 In animals, the free energy in sugars and other molecules derived from food is releasedin the processof respiration. All synthesisof ATP in animal cells and in nonphotosynthetic microorganismsresultsfrom the chemicaltransformation of energy-richcompounds in the diet (e.g., glucose, starch). We discussthe mechanismsof photosynthesisand cellular respiration in Chapter 12. The completeoxidation of glucoseto yield carbon dioxide, c6Flpo6 + 6 02 --->6 co2 + 6 H2o has a AG'' of -686 kcal/mol and is the reverseof photosynthetic carbon fixation. Cells employ an elaborate set of protein-mediated reactions to couple the oxidation of 1 molecule of glucoseto the synthesisof as many as 30 moleculesof ATP from 30 molecules of ADP. This oxygen-dependent (aerobic) degradation (catabolism) of glucose is the major pathway for generating ATP in all animal cells, nonphotosyntheticplant cells, and many bacterial cells. Catabolism of fatty acids can also be an important source of ATP. Light energy captured in photosynthesisis not the only source of chemical energy for all cells. Certain microorganisms that live in or around deepoceanvents,where adequate sunlight is unavailable,derive the energyfor converting ADP and Pi into ATP from the oxidation of reduced inorganic compounds.Thesereducedcompounds originate deepin the earth and are releasedat the vents.

not accompanythe formation of new chemical bonds or the releaseof energythat can be coupled to other reactions.The loss of electronsfrom an atom or a molecule is called oxidation, and the gain of electrons by an atom or a molecule is called reduction. Becauseelectrons are neither created nor destroyedin a chemical reaction, if one atom or molecule is oxidized, another must be reduced. For example' oxygen draws electronsfrom ps2+ (ferrous) ions to form Fe3* (ferric) ions, a reaction that occurs as part of the process by which carbohydrates are degraded in mitochondria. Each oxygen atom receivestwo electrons, one from each of two Fe"- ions: 2 Fe2* + Yz oz -->2 Fe3* + 02Thus Fe2*is oxidized, and 02 is reduced.Suchreactionsin which one molecule is reduced and another oxidized often are referred to as redox reactions. Oxygen is an electron acceptor in many redox reactions in cells under aerobic conditions. Many biologically important oxidation and reduction reactions involve the removal or the addition of hydrogen atoms (protons plus electrons) rather than the transfer of isolated electronson their own. The oxidation of succinate to fumarate, which also occurs in mitochondria, is an example (Figure 2-32). Protons are soluble in aqueous solutions (as H3O+), but electrons are not and must be transferred directly from one atom or molecule to another without a water-dissolvedintermediate.In this rype of oxidation reaction' electrons often are transferred to small electron-carrying molecules, sometimes referred to as coenzymes.The most common of theseelectron carriers are NAD* (nicotinamide adeninedinucleotide),which is reducedto NADH' and FAD (flavin adenine dinucleotide), which is reduced to FADH2 (Figure 2-33). The reduced forms of these coenzymescan transfer protons and electrons to other molecules, thereby reducing them.

c-oI

H-C-H | H-C-H I

c- oo

Succinate

N A D * a n d F A DC o u p l eM a n y B i o l o g i c a l Oxidation and ReductionReactions In many chemical reactions, electrons are transferred from one atom or molecule to another; this transfer may or may

o c-o

o

---\----\---2 v v ze 2H*

I c-H

c-H I

c-o o

Fumarate

to fumarate.Inthis of succinate 2-32 Conversion A FIGURE aspartof the citric in mitochondria occurs which reaction, oxidation andtwo protonsTheseare losestwo electrons acidcycle,succinate it to FADH2. to FAD,reducing transferred

B I O C H E M I C AELN E R G E T I C S

59

(b) Reduced: FADH2 Reduced: NADH

o c-NH2

+ le

I

Hll

I

Ribose I 2P

I Ribitol I 2P

Ribose I 2P

I

..1

A O e n o sn re

Adenosine

NAD++H++2e-.-

NADH

Ribitol I 2P

I

I

Adenosine

Adenosine FAD+2H- +2e-

i-

FADH2

FIGURE 2-33 Theelectron-carrying coenzymesNAD+and FAD. (a)NAD*(nicotinamide adenine dinucleotide) isreduced to NADHby the addition of two electrons andoneprotonsimultaneously In manybiological redoxreactions, a pairof hydrogen atoms(two protons andtwo electrons) areremoved froma moleculeIn some cases, oneof the protons andbothelectrons aretransferred to NAD-;the otherprotonisreleased intosolution(b)FAD(flavin

adenine dinucleotide) isreduced to FADH2 bythe additionof two electrons andtwo protons, whensuccinate asoccurs isconverted to (seeFigure fumarate 2-32)ln thistwo-stepreaction, addition of one electron together with oneprotonfirstgenerates a short-lived (notshown), semiquinone intermediate whichthenaccepts a second electron andproton.

To describeredox reactions, such as the reaction of ferrous ion (Fe2+)and oxygen (O2), it is easiestto divide them into two half-reactions:

reduce)a compound with a more positivereduction potential. In this type of reaction, the changein electricpotential AE is the sum of the reduction and oxidation potentialsfor the two half-reactions.The AE for a redox reaction is related to the changein free energyAG by the following expression:

Oxidation of Fe2n: Reduction of 02:

2 Fez* --+ 2 Fe3* -t 2 e2 e * 1/z02 -- 02-

In this case,the reducedoxygen (O2-) readily reactswith two protons to form one water molecule(HzO). The readinesswith which an atom or a molecule gains an electron is its reduction potential E. The tendencyto lose electrons,the oxidation potential, has rhe same magnitude but opposite sign as the reduction potential for the reversereaction. Reduction porentials are measuredin volts (V) from an arbitrary zero point set ar the reduction potential of the following half-reactionunder standard conditions (25 "C. 1 atm, and reactantsat 1 M):

H+ + e- IY'Z

*t,

oxidation

The value of E for a molecule or an atom under standard conditions is its standardreduction potential, E'6 A molecule or an ion with a positive E'e has a higher affinity for electronsthan the H* ion does under standardconditions. Conversely,a molecule or ion with a negative E'6 has a lower affinity for electronsthan the H* ion does under standard conditions.Like the valuesof AGo', standardreduction potentials may differ somewhat from those found under the conditions in a cell becausethe concentrations of reactants in a cell are not 1 M. In a redox reaction,electronsmove spontaneouslytoward atoms or moleculeshaving more positiuereductionporentials. In other words, a compound having a more negativereduction potential can transfer electrons spontaneouslyto (i.e.,

60

CHAPTER2 I

CHEMICALFOUNDATIONS

AG (callmol) : -n (23,064)AE (volts)

(2-11)

wheren is the number of electronstransferred.Note that a redox reactionwith a positiveAE value will have a negativeAG and thus will tend to proceedspontaneouslyfrom left to right.

BiochemicalEnergetics r The changein free energy AG is the most useful measure for predicting the direction of chemicalreactionsin biological systems.Chemical reactions tend to proceed spontaneouslyin the direction for which AG is negative.The magnitude of AG is independentof the reaction rate. r The chemicalfree-energychangeAGo' equals -2.3 RT log K.o. Thus the value of AGo' can be calculatedfrom the experimentally determinedconcentrationsof reactantsand products at equilibrium. r The rate of a reaction dependson the activation energy neededto energizereactantsto a rransition state. Catalysts such as enzymesspeedup reactions by lowering the activation energyof the transitionstate. r A chemical reaction having a positive AG can proceed if it is coupled with a reaction having a negariveAG of larger magnitude. r Many otherwise energetically unfavorable cellular processesare driven by the hydrolysis of phosphoanhydride bonds in ATP (seeFigure2-31).

r Directly or indirectlS light energycaptured by photosynthesisin plants and photosynthetic bacteria is the ultimate source of chemical energy for almost all cells. r An oxidation reaction (loss of electrons)is always coupled with a reduction reaction (gain of electrons). r Biological oxidation and reduction reactions often are coupled by electron-carryingcoenzymessuch as NADand FAD (seeFigure2-33). r Oxidation-reduction reactionswith a positive AE have a negativeAG and thus tend to proceed spontaneously.

KeyTerms acid 52

hydrophobic 3 1

a carbon atom (C*) 41

hydrophobic effect 38

amino acids41

ionic interactions 36

amphipathic 31

molecular complementarity 39

base52 buffers 52 chemicalpotential energy54

monosaccharides44

covalentbond 32

nucleotides44

dehydration r eaction 4 0 AG (free-energy change)55

oxidation 59 pH 51

disulfide bond 43

p h o s p h o a n h y d r i dbeo n d s5 7

endergonic55

phospholipid btlayers41

endothermic 55

polar 34

energy coupling 58 enthalpy (H) 55

polymer 40 redox reaction 59

entropy (S)55

reduction 59

equilibrium constant49

saturated 47

exergonic 55

steadystate50

exothermic56

stereoisomers 33

fatty acids 47

unsaturated 47

hydrogen bond 37

van der $0aals interactions37

hydrophilic 31

44 nucleosides

Review the Concepts 1,. The gecko is a reptile with an amazing ability to climb smooth surfaces,including glass.Recentdiscoveriesindicate that geckosstick to smooth surfacesvia van der'lfaals interactions between septaeon their feet and the smooth surface. How is this method of stickinessadvantageousover covalent interactions?Given that van der Sfaalsforces are among the weakest molecular interactions, how can the gecko's feet stick so effectively? 2. The K* channel is an example of a transmembraneprotein (a protein that spans the phospholipid bilayer of the plasmamembrane).What typesof amino acidsare likely to be found (a) lining the channel through which K* passes,(b) in contact with the hydrophobic core of the phospholipid bilayer

containing fatty acylgroups, (c) in the cytosolic domain of the protein, and (d) in the extracellulardomain of the protein? 3. V-M-Y-F-E-N: This is the single-letteramino acid abbreviation for a peptide. What is the net charge of this peptide can at pH 7.0? An enzyme called a protein tyrosine kinase 'Sfhat attach phosphatesto the hydroxyl groups of tyrosine. is the net charge of the peptide at pH 7.0 after it has been phosphorylated by a tyrosine kinase? \What is the likely source of phosphateutilized by the kinase for this reaction? 4. Disulfide bonds help to stabilize the three-dimensional structure of proteins. What amino acids are involved in the formation of disulfide bonds?Does the formation of a disulfide bond increaseor decreaseentropy (AS)? 5. In the 1960s, the drug thalidomide was prescribed to pregnant women to treat morning sickness. However' thalidomide caused severe limb defects in the children of some women who took the drug, and its use for morning sicknesswas discontinued.It is now known that thalidomide was administered as a mixture of two stereoisomericcomthe pounds, one of which relieved morning sicknessand 'Sfhat defects' birth for the other of which was responsible Why might two such closely related comare stereoisomers? pounds have such different physiologic effects? 6. Name the compound shown below.

HrrriS)c-)x ll :,

se C i H

;; ll ll

Hrl',t-c\fi)c-i/ o -o- P-o-o-P-o-o-?-o-9Hz I o-

I

o-

I o

5',

-o 2',

OH OH Is this nucleotide a component of DNA, RNA, or both? Name one other function of this compound. 7. The chemical basis of blood-group specificity residesin the carbohydratesdisplayedon the surfaceof red blood cells. Carbohydrateshave the potential for great structural diversity. Indeed,the structural complexity of the oligosaccharides that can be formed from four sugarsis greater than that for oligopeptidesfrom four amino acids. Sfhat propertiesof carbohydratesmake this great structural diversity possible? 8. Ammonia (NH:) is a weak basethat under acidic conditions becomesprotonated to the ammonium ion in the following reaction: NH, + H- -+ NH+NH3 freely permeates biological membranes, including those of lysosomes.The lysosomeis a subcellular organelle with a pH of about 4.5-5.0; the pH of cytoplasmis -7'0. lWhat is the effect on the pH of the fluid content of lysosomes R E V I E WT H E C O N C E P T S

61

when cells are exposedto ammonia? No/e: Protonated ammonia does not diffuse freely acrossmembranes. 9. Consider the binding reaction L + R -+ LR, where L is 'S7hen a ligand and R is its receptor. 1 x 10 3 M L is added to a solution containing5 x 10-2 M R, 90% of the L binds to form LR. rVhat is the K.o of this reaction? How will the K.o be affected by the addition of a protein that catalyzes this binding reaction?V/hat is the K6? 10. Vhat is the ionization state of phosphoric acid in the cytoplasm? Vhy is phosphoric acid such a physiologically important compound? 1 1 . T h e A G o ' f o r t h e r e a c t i o nX + Y - + X Y i s - 1 0 0 0 callmol. What is the AG at 25 'C (298 Kelvin) starting with 0.01 M each X, I and XY? Suggesttwo ways one could make this reaction energeticallyfavorable. 12. According to health experts,saturatedfatty acids,which come from animal fats, are a major factor contributing to coronary heart disease.What distinguishesa saturated fatty acid from an unsaturated fatty acid, and to what does the term saturated refer?RecentlS trans unsaturatedfatty acids, or trans fats, which raise total cholesterollevelsin the bodv. have also been implicated in heart disease.How does the cis stereoisomerdiffer from the trans configuration, and what effect does the cis configuration have on the structure of the fatty acid chain? 13. Chemicalmodifications to amino acidscontribute to the diversity and function of proteins. For instance,7-carboxylation of specific amino acids is required to make some proteins biologically active. What particular amino acid undergoes this modification, and what is the biological relevance?Warfarin, a derivative of coumarin, which is present in many plants, inhibits 7-carboxylation of this amino acid and was used in the past as a rat poison. At present, it is also used clinically in humans. What patients might be prescribedwarfarin and whv?

62

CHAPTER2 I

CHEMICALFOUNDATIONS

References Albertg R. A., and R. J. Silbey.2005. PhysicalChemistry, 4th ed. Wiley. Atkins, P.,and J. de Paula. 2005. The Elementsof Physical Chemistry,4th ed. \7. H. Freemanand Company. Berg,J. M., J. L. Tymoczko, and L. Stryer.2007. Biochemistry, 5th ed. W. H. Freemanand Company. Cantor, P. R., and C. R. Schimmel.1980. BiophysicalChemistry.V. H. Freemanand Company. Davenport,H.W. 1974. ABC of Acid-BaseChemistry,6rh ed.. University of ChicagoPress. Eisenberg,D., and D. Crothers.1,979.PhysicalChemistryuith Applications to the Life Sciences.Benjamin-Cummings. Guyton, A. C., and J. E. Hall. 2000. Textbook of Medical Physiology, 10th ed. Saunders. Hill, T. J. 1977. FreeEnergy Transductionin Biology. Academic Press. Klotz, I. M. 1978. Energy Changesin BiochemicalReactions. AcademicPress. Murray, R. K., et al. 1999.Harper'sBiocbemistry,25thed. Lange. Nicholls, D. G., and S.J. Ferguson.1992. Bioenergetics2. Academic Press. Oxtoby, D., H. Gillis, and N. Nachtrieb. 2003. Principlesof Modern Chemistry,Sth ed. Saunders. Sharon,N. 1980. Carbohydrates. Sci.Am.243(5):90-116. Tanford, C. 1980. The Hydropl;obic Effect: Formation of Micellesand Biological Membranes,2d ed. \filey. Tinoco, I., K. Sauer,and J. Wang. 200L. PhysicalChemistryPrinciplesand Applications in Biological Sciences,4thed. Prentice Hall. Van Holde, K., W. Johnson,and P.Ho. 1998. Principlesof PhysicalBiochemistry.PrenticeHall. Voet, D., and J. Voet. 2004. Biochemistry,3d,ed. \7iley. Wood, !(. B., et al. 1981,.Biochemistry:A ProblemsApproach, 2d ed. Benjamin-Cummings.

CHAPTER

STRUCTURE PROTEIN AND FUNCTION

Ribbondiagramof a betapropeller domainfrom the humansignaling (spheres) proteinKeaplTenwatermolecules areboundto eachof lvlanyproteinsarebuiltfrom multiple, the sixbladesof the propeller. X Li,C A independently stableproteindomains[FromL J Beamet Bottoms, andM Hannink,2005,ActaCrystallogr D: Biol.Crystallogr of Robert Hube[Martinsried 51(10):1335-1342I Credit.Courtesy

roteins, which are polymers of amino acids, come in many sizesand shapes.Their three-dimensionaldiversity reflects underlying structural differences: principally variations in their lengths and amino acid sequences,and in some cases,differencesalso in the number of disulfide bonds or the attachment of small moleculesor ions to their amino acid side chains. In general,the linear, unbranched polymer of amino acidscomposing any protein will fold into only one or a few closely related three-dimensional shapes-called conformations. The conformation of a protein together with the distinctive chemical properties of its amino acid side chains determines its function. As a consequence,proteins can perform a dazzling array of distinct functions inside and outside of cells that either are essentialfor life or provide selective evolutionary advantageto the cell or organism that contains them. It is, therefore, not surprising that characterizing the structuresand activities of proteins is a fundamental prerequisitefor understanding how cells work. Much of this textbook is devoted to examining how proteins act together to enablecells to live and function properly. Many proteins can be grouped into just a few broad func. Structural proteins, for example,determinethe tional classes shapesof cellsand their extracellularenvironments,and serve as guide wires or rails to direct the intracellularmovement of moleculesand organelles.They usually are formed by the assemblyof multiple protein subunitsinto very large,long strucnres. Scaffold proteins bring other proteins together into

ordered arrays to perform specific functions more efficiently than if those proteins were not assembledtogether. Enzymes are proteins that catalyze chemical reactions. Membrane transport proteins permit the flow of ions and molecules acrosscellular membranes. Regulatoryproteins act as siSnals' sensors,and switches to control the activities of cells by altering the functions of other proteins and genes.These include signaling proteins, such as hormones and cell-surfacereceptors that transmit extracellular signals to the cell interior'

OUTLIN E 3.1

HierarchicalStructureof Proteins

64

3.2

P r o t e i nF o l d i n g

74

3.3

ProteinFunction

78

3.4

RegulatingProteinFunction ProteinDegradation

3.5

R e g u l a t i n gP r o t e i nF u n c t i o nl l : Noncovalentand CovalentModifications

3.6

Purifying,Detecting,and CharacterizingProteins

3.7

Proteomics

92 105

63

Motor proteins are responsiblefor moving other proteins, organelles,cells-even whole organisms.Any one protein can be a member of more than one protein class,as is the caseof some cell-surfacesignaling receptorsthat are both enzymes and regulator proteins becausethey transmit signals from outside to inside cells by catalyztngchemical reactions. To accomplishefficientlytheir diversemissionssomeproreinsassembleinto largecomplexes,often calledmolecularmachines. How do proteinsmediateso many diversefunctions?They do this by exploiting a few simple activiries.Most fundamentally, proteins bind-to one anorher,to other macromolecules, suchas DNA, and to smallmoleculesand ions.In many cases such binding can induce a conformational changein the protein and thus influenceits activity. Binding is basedon molecular complementaritybetweena protein and its binding partner, as describedin Chapter 2. A second key activity is enzymatic catalysis.Appropriate folding of a protein will place some amino acid side chainsand carboxyl and amino groups of the backboneinto positions that permit the catalysis of covalent bond rearrangements.A third activity involves folding into a channel or pore within a membrane through which moleculesand ions flow. Although theseare especially crucial protein activities,they are not the only ones. For example,fish that live in frigid waters-rhe Antarctic borchsand Arctic cods-have antifreezeproteins in their circulatory systems to preventwater crystallizationat subzerotemperatures. A completeunderstandingof how proteins permit cells to live and thrive requiresthe identificationand characterization of all the proteins used by a cell. In a sense,molecular cell biologistswant to compile a completeprotein 'parts list' and consrructan all-inclusive"users manual" that describeshow theseprclteinswork. Compiling a comprehensive protein parts list has become feasiblein recent years with the sequencingof entire genomes-complete sets of g e n e s - o f m o r e a n d m o r e o r g a n i s m s .F r o m a c o m p u r e r analysisof genome sequences,researcherscan deducethe number of amino acidsand their sequenceof most of the encoded proteins (Chapter5). The rerrn proreomewas coined to refer to the entire protein complementof an organism.The human genome conrains 20,000-25,000 genes(only four times that of the single-cellyeast Saccharomycescereuisiae). However, it encodesabout 33.000 different protein because of variation in mRNA producrion (e.g..alteinativesplicing (Chapter8)). Even more variation is generaredby 100 types of protein modification that can produce hundreds of thousandsof distinct human proteins.By comparingprotein sequencesand structures of proteins of unknown function to those of known function, scientistscan often deduce much about their functions. In the past, characterizationof protein function by genetic, biochemical, or physiological methods often precededthe identification of particular proteins. In the moderngenomicand proteomicera,a protein is usuallyidentified prior to determining its function. In this chapter,we begin our study of how the structureof a protein givesriseto its function, a themethat recursthroughout this book (Figure 3-1). The first secrion examineshow chains of amino acid building blocks are arrangedin a threedimensional structural hierarchy.The next section discusses 64

CHAPTER 3

|

PROTEIN S T R U C T U RAEN D F U N C T I O t T

MOLECULAR STRUCTURE Primary (sequence)

S e c o n d a r y( l o c a lf o l d i n g )

Tertiary ( l o n g - r a n gf e olding)

Supramolecular (large-scale assembly)

Ouaternary (multimericstructure)

(b)

Regulation

@ FUNCTION Transport i

C a t a l y s i s4

FIGURE 3-1 Overviewof proteinstructureand function. (a)Proteins areassembled according to a hierarchy of structures A polypeptide l i 'nse asr e q u e n coef a m i n oa c i d sl i n k e db y p e p t i d e bonds(primary structure) foldsinto localhelices or sheets (secondary structure) thatpackrntolarge(longer-range) complex (tertiary three-dimensional structures structu re) Someindividual polypeptides (quaternary associate intomultichain complexes structure), whichin somecases canbeverylarge,consisting of tensto (supramolecular (b)Protein hundreds of subunits assemblies) function includes organization of thegenome, otherproteins, lipidbilayer (structure); membranes, andcytoplasm controlof proteinactivity (regulation), monitoring of theenvironment andtransmitting resultant (signaling), information flowof smallmolecules andionsacross (transport); membranes (viaenzymes); catalysis of chemical reactions (viamotorproteins) andgeneration of forcefor movement These functions andothersarisefromspecifrc bindinginteractions and conformatronal changes in thestructure of a properly foldedprotein how proteins fold into thesestructures.\(e then rurn to protein function, focusingon enzymes,the specialclassof proteinsthat catalyzechemicalreactions.Variousmechanismsthat cellsuse to control the activitiesand life spansof proteinsare coveredin the next two sections.Next comesa sectionon commonly used techniquesin the biologist'stool kit for isolating proteins and characterizingtheir properties.The chapter concludeswith a discussionof the burgeoningfield of proteomics.

ff,t

Hierarchical Structureof Proteins

A protein chain folds into a distinct three-dimensionalshape that is stabilizedby noncovalent interacrionsberween regions in the linear sequenceof amino acids.A kcy concept rn

(a) Primarystructure -Ala -Glu -Val-Thr-Asp- Pro-Gly-

(b) Secondarystructure s heli

(c) Tertiarystructure

Domain

bond formation between the amino group of one amino acid and the carboxyl group of another results in the net releaseof a water molecule (dehydration) (Figure 3-3a). The repeated amide N, cr carbon (C*), carbonyl C and oxygen atoms of each amino acid residue form the backbone of a protein molecule from which the various side-chaingroups project (Figure 3-3b, c). As a consequenceof the peptide linkage, the backbone exhibits directionality becauseall the amino groups are located on the same side of the Co atoms. Thus one end of a protein has a free (unlinked) amino group (the N-terminus), and the other end has a free carboxyl group (the C-terminus). The sequenceof a protein chain is conventionally written with its

(d) Ouaternarystructure (a)

HO tll

HO

ttl

*HsN- Cd-CR1

O- + *H3N- Co-C-

3l

ll lN t,o \7

O-

n'

HOHO

riltll

(a)Thelinear FIGURE 3-2 Fourlevelsof proteinhierarchy. peptide bondsisthe acids linked together by of amino sequence (b)Folding primary chainintolocala structure. of the polypeptide (c)Secondary represents secondary structure. helices or B sheets loopsandturnsin a single together with various structural elements polypeptide stablestructure, chainpackintoa largerindependently distinct domains; thisistertiarystructure(d) whichmayinclude polypeptides can with theirown tertiary structures Someindividual complex. a multichain intoa quaternary structure defining associate understandinghow proteins work is that function is deriued from three-dimensional structure, and three-dimensional structure, which is determined primarily by noncoualent interactions betueen regions in the linear sequenceof amino acids, is specifiedby amino acid seqwence.Indeed,principles relating biologicalstructureand function initially were formulated by the biologistsJohannvon Goethe(1'749-1,832),Ernst Haeckel (1834-1.91.9),and D'Arcy Thompson \1'860-1'948). They greatly influencedthe school of "organic" architecture pioneeredin the early rwentieth century that is epitomizedby the dicta "form follows function" (Louis Sullivan)and "form is function" (Frank Lloyd'Wright). Here, we considerthe architectureof proteins at four levelsof organization: primar5 secondary,tertiary, and quaternary (Figure 3-2).

The PrimaryStructureof a Proteinls lts Linear Arrangemeno t f AminoAcids As discussedin Chapter 2, proteins are constructed by the polymerizationof 20 differenttypesof amino acids.Individual amino acids are linked together in linear, unbranched chains by covalent amide bonds, called peptide bonds, with occasional disulfide bonds covalently linking side chains together.Peptide

PePtide bond (b)

(C-terminus)

(N-terminus) (c)

amino 3-3 Structureof a polypeptide.(a)Individual A FIGURE via reactions form which peptide bonds, by together linked are acids the R1,R2,etc.,represent thatresultin a lossof water(dehydration). polymers of ("Rgroups")of aminoacids.(b)Linear sidechains whichhave aminoacidsarecalledpolypeptides, oeptidebond-linked (C-terminus) (N-terminus) end carboxyl free and a a freeaminoend linkingthe (c)A ball-and-stick modelshowspeptidebonds(yellow) atom(blue)of oneaminoacid(aa)with thecarbonyl aminonitrogen onein thechain.TheR groups of an adjacent carbonatom(gray) (black) of theaminoacids atoms (green) cr carbon extendfromthe properties of distinct the determine largely chains side These individual oroteins EF P R O T E I N S H I E R A R C H I C ASLT R U C T U RO

65

N-terminal amino acid on the left and its C-terminal amino acid on the right, and the amino acids are numbered sequentially starting from the amino terminus (number 1). The primary structure of a protein is simply the linear arrangement, or sequence,of the amino acid residuesthat compose it. Many terms are used to denote the chains formed by the polymerization of amino acids. A short chain of amino acids linked by peptide bonds and having a defined sequenceis called an oligopeptide, or just peptide; longer chains are referred to as polypeptides. peptides generally contain fewer than 20-30 amino acid residues, whereas polypeptides are often 200-500 residueslong. The longest protein described to date is the muscle protein titin with 25,926 residues.We generally reservethe term protein for a polypeptide (or complex of polypeptides) that has a welldefined three-dimensionalstructure. It is implied that proteins and peptides are the natural products of a cell. The size of a protein or a polypeptide is reported as its massin daltons (a dalton is 1 atomic massunit) or as its molecular weight (M\7), which is a dimensionlessnumber. For example,a 10,000-MI7 protein has a mass of 10,000 daltons (Da), or 10 kilodaltons (kDa). In the penultimatesection of this chapter, we will consider different methods for measuringthe sizesand other physical characteristicsof proteins. The known and predicted proteins encoded by the yeast genome have an averagemolecular weight of 52,728 and contain, on average,466 amino acid residues.The average molecular weight of amino acids in proteins is 113, taking into account their averagerelative abundances.This value can be used to estimate the number of residuesin a protein from its molecular weight or, conversely,its molecular weight from the number of residues.

SecondaryStructuresAre the Core Elements of ProteinArchitecture The secondlevel in the hierarchy of protein strucrure is secondary structure. Secondary structures are stable spatial arrangementsof segmentsof a polypeptide chain held together by hydrogen bonds between backbone amide and carbonyl groups and often involving repeatingstructural patterns. A singlepolypeptide may contain multiple types of secondary structure in various portions of the chain, depending on its sequence.The principal secondarystructures are the alpha (c) helix, the beta (p) sheet,and a short U-shapedbeta (F) turn. Portions of the polypeptide that don't form these structures,but neverthelesshave a well-defined,stableshape, are said to have anirregular structure.The term random ioil appliesto highly flexible portions of a polypeptide chain that have no fixed three-dimensionalstructure.In an averageprotein, 50 percent of the polypeptide chain exists as ct helices and B sheets;the remainder of the molecule is in coils and turns. Thus, ct helicesand B sheetsare the major internal supportive elementsin most proteins. In this section,we exploie the shapesof secondarystructuresand the forces that iavor their formation. In later sections,we examine how linear arrays of secondary structure fold together into larger, more complex arrangementscalled tertiary structure. 66

.

c H A p r E 3R |

pRorEtN s r R u c r u RA EN DF U N c l o N

A m i n ot e r m i n u s

Carboxylterminus

A FIGURE 3-4 The ct helix, a commonsecondarystructurein proteins.Thepolypeptide (seenasa ribbon)isfoldedinto backbone a spiralthat isheldin placeby hydrogen bondsbetweenbackbone oxygenandhydrogen atomsOnlyhydrogens involved in bondingare shown.Theoutersurface of the helixiscovered bv theside-chain R groups(green)

The a Helix In a polypeptide segmentfolded into an o helix, the backbone forms a spiral structure in which the carbonyl oxygen atom of eachpeptide bond is hydrogen-bonded to the amide hydrogen atom of the amino acid four residues farther along the chain (in the direction of the C-terminus) (Figure 3-4). Within an a helix, all the backbone amino and carboxyl groups are hydrogen-bondedto one another,exceptat the very beginning and end of the helix. This periodic arrangementof bonds confersan amino-to-carboxy-terminal directionality on the helix becauseall the hydrogen bond acceptors (e.g.,the carbonyl groups) have the same orientation (pointing in the downward direction in Figure 3-4) and results in a structure in which there is a complete turn of the spiral every 3.6 residues.An ct helix 36 amino acids long has 10 turns of the helix and is 5.4 nm long (0.54 nm/turn). The stable arrangement of hydrogen-bonded amino acids in the a helix holds the bar:kbone in a straight, rodlike cylinder from which the side chains point outward. The relative hydrophobic or hydrophilic quality of a particular helix within a protein is determined entirely by the

characteristicsof the side chains, becauseall the polar amino and carboxyl groups of the peptide backboneare engagedin hydrogen bonding with one another in the helix. In watersoluble proteins, the hydrophilic helicestend to be found on the outside surfaces,where they can interact with the aqueous environment, whereas hydrophobic helices tend to be buried within the core of the folded protein. The amino acid proline is usually not found in cr helices,becausethe covalent bonding of its amino group with a carbon in the side chain preventsits participation in stabilizingthe backbonethrough normal hydrogen bonding. While the classic ct helix is the most intrinsically stable, and most common helical form in proteins, there are variations, such as more tightly or loosely twisted helices.For example, in a specializedhelix called a coiled coil (describedseveralsectionsfarther on), the helix is more tightly wound (3.5 residuesand 0.51 nm per turn). The p Sheet Another type of secondarystructure, the B sheet,consistsof laterally packed B strands.Each B strand is a short (5- to 8-residue),nearly fully extended polypeptide segment. Unlike in the ct helix (where hydrogen bonding between the amino and carboxyl groups in the backbone occursbetlveen nearly adjacentresidues),hydrogenbonding in the B sheetoccurs between backbone atoms in separate,but adjacent, B strands (Figure 3-5a). Thesedistinct B strandsmay be either within a single polypeptide chain, with short or long loops be-

(a) Topview

'lo e

"{r ...)

Amino termrnus

Carboxyl termrnus

(b) Side

A FIGURE 3-5 The p sheet,anothercommonsecondary structurein proteins.(a)Topviewof a simplethree-stranded B p strandsThestabilizing rrydrogen bonds sheetwith antiparallel lines(b)Side by greendashed areindicated the B strands between (green) projection groups aboveand R of the viewof a B sheetThe in thisview Thefixedbond belowthe planeof thesheetisobvious produce contour a pleated backbone anglesin the polypeptide

of four residues, 3-6 Structureof a p turn' Composed A FIGURE chain(=180"U-turn)The of a polypeptide thedirection B turnsreverse <0.7 nmapart, areusually of thefirstandfourthresidues C. carbons bond. a hydrogen areoftenlinkedby B turns andthoseresidues structures polypeptides into compact of long folding the facilitate

Nveenthe B strand segments'or on different polypeptide chains. Figure 3-5b shows how two or more B strands align into adiacent rows, forming a nearly two-dimensional B pleatedsheet(or simply pleated sheet), in which hydrogen bonds within the plane of the sheethold the B strandstogether as the side chains itick out aboveand below the plane. Like a helices,B strands have a directionality defined by the orientation of the peptide bond. Therefore, in a pleated sheet,adjacent p strands can be oriented in the same (parallel) or opposite (antiparallel) directions with respectto eachother' In someproteins, B sheetsform the floor of a binding pocket or a hydrophobic core; in other oroteins embedded in membranes the B sheetscurve around and form a hydrophilic central pore through which ions and small moleculesmay flow (Chapter11). p Turns Composedof four residues,B turns are locatedon the surface of a protein, forming sharp bends that reversethe direction of the polypeptide backbone, often toward the protein's interior. These short, U-shaped secondarystructures are often stabilizedby a hydrogenbond betweentheir end residues (Figure 3-6). Glycine and proline are commonly present in turns. The lack of alargeside chain in glycine and the presence of a built-in bend in proline allow the polypeptidebackboneto fold into a tight U shape.B turns help large proteins to fold into highly compact structures.There are six types of well-defined turns, their detailed structures depending on the arrangement of H-bonding interactions.A polypeptide backbonealso may contain longer bends, or loops' In contrast with tight B turns, which exhibit just a few well-defined conformations' longer loops can have many different conformations.

O v e r a l lF o l d i n go f a P o l y p e p t i d eC h a i nY i e l d sl t s TertiaryStructure Tertiary structure refers to the overall conformation of a polypeptide chain-that is, the three-dimensionalarrange-..ri oi all its amino acid residues'In contrastwith secondary EF P R O T E I N S H I E R A R C H I C ASLT R U C T U RO

67

Animation:Oil Drop Model of ProteinStructurefntt

Unfoldedprotein

Nativeprotein

FIGURE 3-7 Oil drop modelof proteinfolding.The hydrophobic residues of a polypeptide chaintendto cluster together, somewhat likean oil drop,on the inside, or core,of a foldedprotein, drivenawayfromthe aqueous surroundings bythe hydrophobic effect(Chapter 2) Charged polarside anduncharged chains appearon the protein's surface wheretheycanform stabilizing interactions with surroundinq waterandions structures, which are stabilized only by hydrogen bonds, tertrary structure is primarily stabilized by hydrophobic interactions between nonpolar side chains, together with hydrogen bonds between polar side chains and peptide bonds. These stabilizing forces compactly hold together elements of secondary structure-o helices, B strands, turns, and coils. Becausethe stabilizing interactions are weak, however, the tertiary structure of a protein is not rigidly fixed but undergoescontinual, minute fluctuations, and some segmentswithin the tertiary structure of a protein can be so very mobile they are consideredto be disordered (that is, lacking well-defined, stable,three-dimensionalstructure). This variation in structure has important consequencesfor the function and regulation of proteins. _ Chemical properties of amino acid side chains help define tertiary structure. Disulfide bonds between the side chains of cysteineresiduesin some proteins covalently link regions of proteins, thus restricting the mobility of proteins and increasingthe stability of their tertiary structures.Amino acids with charged hydrophilic polar side chains tend to be on the outer surfacesof proteins; by interacting with water, they help to make proteins soluble in aqueoussolutions and can form noncovalent interactions with other water-soluble molecules,including other proteins. In contrast, amino acids with hydrophobic nonpolar side chains are usually sequesteredaway from the water-facing surfacesof a protein, in many casesforming a water-insolublecentral core (called the oil drop model of globular proteins, becauseof the relatively hydrophobic, or 'oily', core, Figure 3-7). Uncharged hydrophilic polar side chains are found on both the surface and inner core of proteins.

do not readily dissolvein water, usually play a structural role or participate in cellular movements.Globwlar proteins are generally water-soluble, compactly folded structures, often but not exclusively spheroidal, that comprise a mixture of secondary structures (seethe structure of myoglobin, below). Integral membrane proteins are embedded within the phospholipid bilayer of the membranes that serve as the walls of cells and organelles.The three broad categoriesof proteins noted here are not mutually exclusive-some proteins are made up of combinations of two or even all three of thesecategories.

Different Ways of Depictingthe Conformation of ProteinsConveyDifferent Typesof Information The simplest way to representthree-dimensionalprotein structure is to trace the courseof the backbone atoms, sometimes only the C. atoms, with a solid line (called a Co trace, Figure 3-8a); the most complex model shows every atom (Figure 3-8b). The former shows the overall fold of the polypeptide chain without consideration of the amino acid side chains; the latter, a ball-and-stick model, details the interactions between side-chain atoms, including those that stabilize the protein's conformation and interact with other molecules, as well as the atoms of the backbone. Even though both views are useful, the elements of secondary structure are not always easily discernedin them. Another type of representationusescommon shorthand symbols for depicting secondarystructure-for example, coiled ribbons or solid cylinders for o' helices,flat ribbons or arrows for B strands,and flexible thin strandsfor B turns, coils, and loops (Figure3-8c). In variations of ribbon diagrams,ball-and-stick or space-fillingmodels of side chains can be attached to the backbone ribbon, while ribbon and cylinder models make the secondarystructuresof a protein easyto see. However, none of these three ways of representingprotein structure conveys much information about the protein surface,which is of interest becauseit is where other molecules usually bind to a protein. Computer analysiscan identify the surface atoms that are in contact with the watery environment. On this water-accessible surface,regionshaving a common chemical character (hydrophobicity or hydrophilicity) and electricalcharacter (basic or acidic) can be indicated by coloring (Figure 3-8d). Such models reveal the topography of the protein surface and the distribution of charge, both important features of binding sites, as well as clefts in the surface where small molecules bind. This view representsa protein as it is "seen" by another molecule.

S t r u c t u r aM l o t i f s A r e R e g u l a rC o m b i n a t i o n o sf Secondaryand TertiaryStructures Particular combinations of secondary and tertiary structures, called structural motifs or folds, appear often as segments within many different proteins. Structural motifs 58

o

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pRorEtN s r R u c r u RA EN DF U N c l o N

(a) Co backbonetrace

(b) Ball and stick

( c )R i b b o n s

(d) Solvent-accessible surface

contribute to the global structure of the entire protein' and any particular structural motif often performs a common function in different proteins (e.g., binding to a particular small molecule or ion). The primary sequencesresponsible for any given structural motif may be very similar to one another. In other words, a common sequencemotif can result in a common three-dimensionalstructural motif. However, it is possible for seemingly unrelated primary sequencesto result in folding into a common structural motif. Conversely, it is possiblethat a commonly occurring sequencemotif does not fold into a well-defined structural motif. Sometimes short sequencemotifs that have an unusual abundanceof a particular amino acid, e.g., proline or aspartateor glutamate, are called "domains"l however,these and other short contiguous segmentsare more appropriately called motifs than domains (which are defined below). Many proteins, including fibrous proteins and DNAregulating proteins called transcription factors (Chapter 7), assembleinto dimers or trimers by using an ct helix-based coiled coil, or heptad-repeat,structural motif. In this structural motif, ct helicesfrom two, three, or even four separate polypeptide chains coil about one another-resulting in a coil of coils, hence the name (Figure 3-9a). The individual helicesbind tightly to one another becauseeach helix has a strip of aliphatic (hydrophobic, but not aromatic) side chains (leucinevaline, etc.) running along one side of the helix that interacts with a similar strip in the adjacent helix, thus sequesteringthe hydrophobic groups away from water

3-8 Fourways to visualizeprotein < FIGURE guanine Ras, structure. a monomertc protein, isshownin allfour nucleotide-binding (GDP) always panels, diphosphate with guanosine (a) trace in blue TheC* backbone depicted istightly howthe polypeptide demonstrates (b)A ball-and-stick packedintoa smallvolume. of allatoms. the location reveals representation how B (c)A ribbonrepresentation emphasizes (red)are (lightblue)ando helices strands in the proternNotetheturnsand organized (d) pairsof helices andstrands. loopsconnecting surfacereveals A modelof the water-accessible on the lumps,bumps,andcrevices the numerous positive are charge of proteinsurfaceRegions chargeare of neqative purple;regions shaded red shaded

and stabilizing the assemblyof multiple independenthelices' These hydrophobic strips are generatedalong only one side of the helix becausethe primary sequencesof the helicesexhibit a motif of repeating segmentsof seven amino acids (heptads) in which the sides chains of the first and fourth residuesare aliphatic and the other side chains are often hydrophilic (Figure 3-9a). Becausehydrophilic side chains ext..,J fro- one side of the helix and hydrophobic side chains extend from the opposite side,the overall helical structure is amphipathic. Becauseleucine frequently appearsin the fourth poritionr and the hydrophobic side chains merge together iike the teeth of a zipper, these structural motifs are also called leucine zippers. Many other structural motifs employ a helices' A common calcium-binding motif called the EF hand uses two short helicesconnectedby a loop (Figure 3-9b). This structural motif found in more than 100 proteins is used for sensing the calcium levels in cells. The bindittg of a Ca2* ion to oxygen atoms in conservedresiduesin the loop dependson the concentration of Caz* and often induces a conformational change in the protein, altering its activity. Thus, calcium concentrationscan directly control proteins' structures and functions. Somewhat different helix-turn-helix and basic helixloop-helix (bHLH) structural motifs are used for protein binding to DNA and consequentlythe regulation of geneactivity. Yet another motif commonly found in proteins ihat bind RNA or DNA is the zinc finger, which contains three secondary structures-an ct helix and two B strands EF P R O T E I N S . H I E R A R C H I C ASLT R U C T U RO

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(a) Coiled-coilmotif

(b) EF-hand/helix-loop-helix motif

(c) Zinc-fingermotif

Ca2*

Leu (4) Asn(1)

FIGURE 3-9 Motifs of protein secondarystructure.(a)The parallel two-stranded coiled-coil motif(left)ischaracterized by two o helices woundaroundeachother.Helixpacking isstabilized by interactions betweenhydrophobic (redandblue)present sidechains at regular intervals alongeachstrand,andfoundalongtheseamof the intertwined helices. Eacho helixexhibits a characteristic heotad repeatsequence with a hydrophobic residue often,but not always, at positions 1 and4, asindicated Thecoiled-coil natureof this structural motifismoreapparent in longcoiledcoils(Rrghf drawnat different scale)(b)An EFhanda typeof helix-loop-helix motif, consists of two helices connected by a shortloopin a specific conformation commonto manyproteins, including manycalciumbindingandDNA-binding regulatory proteinsIn calcium-bindinq

with an antiparallel orienrarion-that form a fingerlike bundle held together by a zinc ion (Figure 3-9c). rWewill encounter numerous additional modfs in later discussionsof other proteins in this and other chapters.The presenceof the same structural motif in different proteins with similar functions clearly indicates that these useful combinations of secondary structures have been conserved in evolution.

S t r u c t u r aal n d F u n c t i o n aD l o m a i n sA r e M o d u l e s of TertiaryStructure Distinct regions of protein tertiary structure are often referred

70

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proteins suchascalmodulin, oxygenatomsfromfiveresidues in the acidicAlutamateandaspartate-rich loopandonewatermolecule formionrcbondswith a Ca2*ion.(c)Thezinc-finger motifispresent in manyDNA-binding proteins that helpregulate transcription. A 7n'- ionis heldbetween (blue)anda singleo a pairof B strands helix(red)by a pairof cysteine residues anda pairof histidine residues Thetwo invariant cysteine residues areusually at positions 3 and6, andthetwo invariant histidine residues areat positions 20 and24 in this25-residue motif.[See A. Lewit-Bentley andS Rety, 2000, EF-hand calcium-binding proteins, Curr. OpinStrucBiol.10:637-643; SA W o l f e , L N e k l u d o v aa, n d C O P a b o ,2 0 0 0 , D N A r e c o g n i t i o nb y C y s 2 H r sz2i n c finger proteins,Ann Rev.Biophys Biomol. Struc.29:1g3-2121

group to another molecule) or binding ability (e.g.,a DNAbinding domain or a membrane-bindingdomain). Functional domains are often identified experimentally by whittling down a protein to its smallestactive fragment with the aid of proteases,enzymesthat cleaveone or more peptide bonds in a target polypeptide.Alternatively, the DNA encoding a protein can be modified so that when the modified DNA is used to generatea protein, only a particular region, or domain, of the full-length protein is made. Thus it is possible to determine if specificportions of a protein are responsiblefor particular activities exhibited by the protein. Indeed, functional domains are often also associatedwith correspondingstructural domains. A structural dornain is a region =40 or more amino acids in length, arrangedin a stable,distinct secondaryor tertiary structure, that often can fold into its characteristic structure independently of the rest of the protein. As a consequence.distinct structural domains can be linked together-sometimes by short or long spacers-to form a large, multidomain protein. Each of the subunits in hemagglutinin, for example, contains

Globular domain

Fibrous domain

PROXIMAL

N External Viral membrane lnternal

3-10 Tertiaryand quaternarylevels < FIGURE of structure.Theprotetnpicturedhere, of (HA),isfoundon the surface hemagglutinin has molecule virus.Thislong,multimeric influenza of two eachcomposed subunits, threeidentical HA1andHA2(a)Tertiary polypeptide chains, thefolding HA subunitcomprises of each structure structure intoa compact andstrands of itshelices intotwo domains' thatis 13.5nm longanddivided isfoldedintoa domain(silver) Themembrane-distal membrane-proximal The globular conformation conformation stemlike domain(gold)hasa fibrous, of two longcthelices owingto thealignment in HAl Shortturns (cylinders) of HA2with B strands and longerloops,oftenat the surfaceof the in each andstrands the helices connect molecule, by of HAisstabilized structure chain.(b)Quaternary betweenthe longhelices lateralrnteractions of thethree (cylinders) in thefibrousdomains forminga triple(gold,blue,andgreen), subunits stalk.Eachof the distalglobular coiled-coil stranded in HA bindssialicacid(red)on thesurface domains proteins, HA of targetcellsLikemanymembrane carbohydrate linked covalently several contains (notshown). chains

t

,

I I

t

I I I

a globular domain and a fibrous domain (Figure 3-10a)' Like structural motifs (composed of secondary structures)' structural domains (composed of secondary and tertiary structures) are incorporated as modules into different proteins. The modular approach to protein architecture is particularly easyto recognizein large proteins, which tend to be mosaics of different domains that confer distinct activities and thus can perform different functions simultaneously. Structural domains frequently are also functional domains in that they can have an activity independentof the rest of the protein. In Chapter 6 we consider the mechanism by which the gene segmentsthat correspond to domains becameshuffled in the course of evolution, resulting in their appearance in many proteins. The epidermal growth factor (EGF) domain is a structural domain presentin severalproteins (Figure3-11). EGF is a small, soluble peptide hormone that binds to cells in the embryo and in skin and connectivetissue in adults, causing them to divide. It is generatedby proteolytic (breaking of peptide bond) cleavagebetween repeated EGF domains in the EGF precursor protein, which is anchored in the cell domain. EGF domains membraneby a membrane-spanning with sequencessimilar to, but not identical with, those in the EGF peptide hormone are present in other proteins and can be liberated by proteolysis.Theseproteins include tissue plasminogenactivator (TPA), a proteasethat is used to dissolve blood clots in heart attack victims; Neu protein, which takes part in embryonic differentiation; and Notch protein, a receptor protein in the plasma membrane that

functions in developmentally important signaling (Chapter 16). Besidesthe EGF domain, theseproteinshave other domains in common with other proteins. For example, TPA

EGF precursor

EGF

A FIGURE3-11 Modular nature of protein domains' Epidermal

andP Bork,1993'Curr' shapeand color.lndaptedfromI D Campbell Opin StrucBiol 3.3851 EF P R O T E I N S O H I E R A R C H I C ASLT R U C T U RO

71

the domains in all proteins. Structural domains can be recognized in proteins whose structures have been determined by x-ray crystallography or nuclear magnetic resonance (NMR) analysis or in images captured by electron microscopy. Regions of proteins that are defined by their distinctive spatial relationshipsro the rest of the protein aretopological domains. For examplc, some prorerns associatedwith iellsurfacemembranescan have a portion extending inward into the cytoplasm (cytoplasmic domain), a portion embedded within the phospholipid bilayer membrane (membranespanningdomain), and a portion extending outward into the extracellular space(extracellulardomain). Each of thesecan comprlse one or more structural motifs and structural and functional domains.

ProteinsAssociateinto MultimericStructures a n d M a c r o m o l e c u l aArs s e m b l i e s Multimeric proteins consisr of two or more polypeptide chains or subunits. A fourth level of structur;l orga-nization, quaternary structure, describes the number (stoi_ chiometry) and relative positions of the subunits in multimenc proteins. Hemagglutinin, for example,is a trimer of three identical subunits (homotrimer) held together by noncovalent bonds (Figure 3-10b). Other multimeric pro_ teins can be composed of various numbers of ideniical (homomeric) or different (hereromeric)subunits (see the discussionof hemoglobin, below). Often, the individual monomeric subunits of a multimeric protein cannot func_ tion normally unlessthey are assembledinto the multimeric protein. In some cases,assemblyinto a multimeric protein (oligomerization)permits proreins that actsequentiallyin a pathway to increasetheir efficiencyof operaiion owing to their juxtaposition in space. The highest level in the hierarchy of protein strucrure is the associationof proteinsinto macromolecularassemblies. Typicallg such structures are very large, in some casesexceeding1 MDa in mass,approaching30-300 nm in size,and containing tens to hundreds of polypeptide chains, and sometimesother biopolymers such as nucleic acids.The cap_ sid that encasesthe nucleic acidsof the viral genomeis an ex_ ample of a macromolecular assemblywith a'structural function. The bundles of cytoskeletalfilaments that support and give shape to the plasma membrane are another examole. Other macromolecular assembliesact as molecular machines, carrying out the most complex cellular processesbv integrating individual functions into one coordinared

tional components including general transcription factors, promoter-binding proteins, helicase,and other protein comp l e x e s( F i g u r e3 - 1 2 ) . R i b o s o m e sa. l s o d i s c u s s e d in Chapter 4, are complex multiprotein and multi-nucleic acid machines that synthesizeproteins. 72

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Generaltranscriptionfactors

R N Ap o l y m e r a s

DNA Promoter I

I

Y

Transcription preinitiation comprex

A FfGURE 3-12 A macromolecular machine:the transcriptioninitiationcomplex.ThecoreRNApolymerase, general transcription factors, a mediator complex containing about20 subunits, andother proteincomplexes not depicted hereassemble at a promoter in DNA Thepolymerase carries out transcription of DNA;theassociated proteins arerequired for initialbindingof polymerase to a specific promoterThemultiple components functiontoqetherasa machine

M e m b e r so f P r o t e i nF a m i l i e sH a v ea C o m m o n E v o l u t i o n a r yA n c e s t o r Studiesof myoglobin and hemoglobin, the oxygen-carrying proteins in muscle and red blood cells,respectively,provided early evidencethat a protein's function derivesfrom its threedimensional structure, which in turn is specified by amino acid sequence.X-ray crystallographic analysis showed that the three-dimensionalstructuresof myoglobin (a monomer) and the cr and B subunitsof hemoglobin (a o2g2retramer) are remarkably similar. Sequencingof myoglobin and the hemoglobin subunits revealedthat many identical or chemically similar residues are found in identical positions throughout the primary structures of both proteins. A mutation in the gene encoding the B chain that resultsin the substitution of a valine for a glutamic acid disturbs the fold, ing and function of hemoglobin and causes sickle-cell anemla. Similar comparisonsbetweenother proteins conclusively confirmed the relation between the amino acid sequence, three-dimensionalstructure, and function of proteins. Use of sequencecomparisons to deduce protein function has ex_ panded substantiallyin recent years as the genomesof more and more organismshave been sequenced. The molecular revolution in biology during the last decadesof the twentieth century also created a new scheme of biological classification based on similarities and differencesin the amino acid sequencesof proteins. proteins that have a common ancestor are referred to as homologs. The main evidencefor homology among proteins, and hence for their common ancestry,is similarity in their sequencesor

Monocot hemoglobin

Dicot hemoglobin

Annelid

Hemoglobin Protozoan Algal F un g aI Bacterial

Ancestral ygen-binding

Ps u b u n i t of hemoglobin

Myoglobin

3-13 Evolutionof the globin proteinfamily.leff: A FIGURE globinisthoughtto bethe primitive oxygen-binding monomeric and myoglobins, muscle hemoglobins, blood modern-day of ancestor that haverevealed comparisons Sequence plantleghemoglobins and of animals parallels the evolution of the globinproteins evolution of plantglobins with thedivergence occurred plantsMajorjunctions Latergene fromhemoglobin andof myoglobin fromanimalglobins

Rrght: of hemoglobin gaveriseto theo andB subunits duplication of two a andtwo B subunitsThe isa tetramer Hemoglobin and with leghemoglobin of thesesubunits similarity structural A heme is evident monomers, are which of both myoglobin, with eachglobinpolypeptide (red)noncovalently associated molecule in theseproteins[(relt) for oxygen-binding responsible isdirectly 93:5675 l AcadSciIJSA Nat'l Proc 1996, fromR C Hardison, Adapted

'We can therefore describehomologous proteins structures. as belonging to a "family" and can trace their lineage from The folded three-dimensional comparisonsof their sequences. structuresof homologous proteins are similar evenif parts of their primary structure show little evidenceof homology. Initially, proteins with relatively high sequencesimilarities (>50 percent exact matches,or "identities") and related functions or structureswere defined as an evolutionarily related family, while a swperfamilyencompassedtwo or more families in which the interfamily sequencesmatched lesswell (=30-40 percent identities)than within one family. It is generally thought that proteins with 30 percent sequenceidentity are likely to have similar three-dimensionalstructures; however, proteins with far less sequencematching can have very similar structures.Recently,revised definitions of family and superfamily have been proposed, in which a family comprisesproteins with a clear evolutionary relationship (>30 percentidentity or additional structural and functional information showing common descent but <30 percent identity), while a superfamily comprisesproteins with only a probable common evolutionary origin (e.g., lower percent sequenceidentities).Often investigatorsconsider proteins to constitute a common superfamily (have a common evolutionary origin) when they contain one or more common motifs or domains. The kinship among homologous proteins is most easily visualizedby a tree diagrambasedon sequenceanalyses.For

example, the amino acid sequencesof globins, the proteins of hemoglobin and myoglobin and their relatives from bacteria, plJnts, and animals, suggestthat they evolved from an a.r.esiral monomeric, oxygen-binding protein (Figure 3-13)' With the passageof time, the gene for this ancestralprotein slowly changed, initially diverging into lineagesleading to animal and plant globins. Subsequentchangesgave rise to myoglobin. the monomeric oxygen-storingprotein in musc1., ind to the a and B subunits of the tetrameric hemoglobin molecule (cr2B2)of the circulatory system'

HierarchicalStructure of Proteins r A protein is a linear polymer of amino.acids linked together by peptide bonds. Various, mostly noncovalent, interactions between amino acids in the linear sequence stabilizea protein's specificfolded three-dimensionalstructure, or conformation' r The cr helix, B strand and sheet' and B turn are the most prevalent elementsof protein secondarystructure' Sectndary structuresare stabilized by hydrogen bonds between atoms of the peptide backbone' r Protein tertiary structure resultsfrom hydrophobic interactions betweennonpolar side groups and hydrogen bonds between polar side groups and the polypeptide backbone' EF P R O T E I N S . H I E R A R C H I C ASLT R U C T U RO

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These interactions stabilize folding of the secondarystructure into a compact overall arrangement. r Certain combinations of secondarystructuresgive rise to different motifs, which are found in a variety of pioteins and are often associatedwith specificfunctions (seeFigure 3-9). Proteins often contain distinct domains, independently Ided regions of secondaryor tertiary structure *ith .h.racteristicstructural, functional, and topological properties ( s e eF i g u r e3 - 1 0 ) . r The incorporation of domains as modules in different proteins in the course of evolution has generateddiversity in protein structure and function. r The number and organizationof individual polypeptide subunits in multimeric proteins define their quaternary structure. r Cellscontain largemacromolecularassemblies in which all the_necessary participantsin complex cellular processes(e.g., DNA, RNA, and protein synthesis;photosynthesis;signal transduction)are integratedto form molecular machines. Homologous proteins, which have similar sequences, ructures, and functions, evolved from a common ances_ tor. They can be classifiedinto families and superfamilies.

ProteinFolding

f[

As noted above,when it comesto biologicalstructuressuch as proteins, "form follows function" and .,form is function." Thus it is essentialthat when a polypeptideis synthesizedwith its particular primary structure (sequence),it folds into the

tricaciesof translation are consideredin Chapter 4. Here, we describethe key determinantsof the proper folding of a'nas_ cent (newly formed or forming) polypeptide chain.

P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o Which ProteinsCan Fold A critical structural feature of polypeptides that limits how the chain can fold is the planar p.piid. bond. Figure 3_3 il_ lustratesthe amide group in peptide bonds in a folypeptide chain. Becausethe peptide bond itself behave,puni"lty tit. a double bond, OO-

llt Pi"\1-P2 I a-a.

<-> r--u-':fi-P,

HH

the carbonyl carbon and amide nitrogen and those atoms directly bonded to them must all lie rn a fixed plane (Fig_ 74

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A FIGURE 3-14 Rotationbetweenplanarpeptidegroupsin proteins.Rotation aboutthe Co-amino nitrogen bond(the@angle) andthe C"-carbonyl carbonbond(thery'angle)permits polypeptide backbones, in principle, to adopta verylargenumberof potential conformations However stericrestraints dueto thestructure of the polypeptide backbone andthe properties of theaminoacidside chainsdramatically restrict the potential conformations thatcanbe adoptedby anygivenprotein ure 3-14); there is no rotation possibleabout the peptide bond itself. As a consequence,the only flexibility in a polypeptide chain backbone, allowing it to adopt varying conformations (twists and turns to fold into different threedimensional shapes),is rotation of the fixed planes of peptide bonds with respectto one another about two bonJsthe C.-amino nitrogen bond (rotational angle called d) and the C"-carbonyl carbon bond (rotational angle caled r/). Yet a further constraint on the potential conformations that a polypeptide backbone chain can adopt is that onlv a limited number of $ and ry'angles porribl., b..",rr" io. ".. most d and t! angles the backbone or side chain atoms would come too closeto one another and thus the associated conformation would be highly unstable or even physically impossibleto achieve.

Information Directinga protein,sFoldingls E n c o d e di n l t s A m i n o A c i d S e q u e n c e While the consrraintsof backbonebond anglesseemvery restrictive, any polypeptide chain containing only a few residuescould, in principle,still fold inro many conformations. For example,if the @and ry'angleswere limited to only eight combinations, an a-residue-longpeptide would potentially have 8" conformations-a very large number for evena small polypeptide of only 10 residueslong (about g.6 million possibleconformations)! In general,however,any particular protein adopts only one or just a few very closely related characteristic functional conformations called the natiue state; for the vast majority of proteins, the native state is the most stably folded form of the molecule. In thermodynamic terms, the native state is usually the conformation with the lowest free energy. lfhat features of proteins limit their folding from very many conformationi to iust one? The propertiesof the sidechains (e.g.,size,hydrophobicitg ability

to form hydrogen and ionic bonds)' together with their particular sequencealong the polypeptide backbone,impose key restrictions. For example, a large side chain such as that of tryptophan might prevent (stericallyblock) one region of the chain from packing closelyagainstanother region, whereasa side chain with a positive charge such as arginine might attract a segmentof the polypeptide that has a complementary negatively charged side chain (e.g., aspartic acid). Another example we have already discussed is the effect of the aliphatic side chains in heptad repeatson the formation of coiled coils. Thus, a polypeptide's primary structure determines its secondary,tertiary, and quaternary structure. The initial evidencethat the information necessaryfor a protein to fold properly is encodedin its sequencecame from in vitro studieson the refolding of purified proteins. Various perturbations (such as thermal energy from heat' extremes of pH that alter the chargeson amino acid side chains, and chemicals, called denaturants, such as urea or guanidine hydrochloride at concentrations of 6-8 M) can disrupt the weak noncovalent interactions that stabilize the native conformation of a protein, leading to its denaturation. Treatment with the reducing agents)such as B-mercaptoethanol, that break disulfide bonds can further destabilizedisulfidecontaining proteins. Under such unfolding or denaturing conditions, entropy increaseswhen a population of uniformly folded moleculesis destabilizedand converted into a collection of many unfolded, or denatured, molecules that have many different non-native and biologically inactive conformations. As we have seen,there are very many possible non-nativeconformations(e.g.,8"-1). The spontaneousunfolding of proteins under denaturing conditions is not surprising,given the substantialincreasein entropy. What is striking, however,is that when a pure sample of a singletype of unfolded protein is shifted back to normal conditions (body temperature' normal pH levels,reduction in the concentration of denaturantsby dilution or their removal), some denatured polypeptides can spontaneously renature (refold) into their native' biologically active states. This kind of refolding experiment, as well as studies that show synthetic proteins made chemically can fold properly, showed that sufficient information must be contained in the protein's primary sequenceto direct correct refolding. Newly synthesizedproteins appear to fold into their proper conformations just as denaturedproteins do. The observedsimilarity in the folded, three-dimensional structures of proteins noted in Section3.1, prowith similar amino acid sequences, vided additional evidencethat the primary sequencealso determinesprotein folding in vivo. It appearsthat formation of secondarystructuresand structural motifs occursearly in the folding process,followed by assemblyof more compact and complex domains, which then associateinto more complex tertiary and quaternary structures(Figure 3-15).

(c)

3-15 Hypotheticalprotein-foldingpathway'Folding A FIGURE of primary hierarchy proteinfollowsthestructural of a monomeric of small (b-d)+ tertiary(e)structureFormation (a)+ secondary of morestable formation to precede motifs(c)appears structural (e) (d)andthefinaltertiary structure domains

tide. However the conditions inside a cell are not the same as those in test tubes used for in vitro refolding experi-

Foldingof Proteinsin Vivo ls Promoted by Chaperones The refolding of a denatured protein is presumed to mimic many aspectsof the folding of a newly synthesizedpolypep-

vides. lfithout such help, they would waste much energy in p R o T E t NF 6 L D I N G

o

75

FocusAnimation:lhaperone-Mediated Foldingfllt ; ibosome

U n f o l d e dp r o t e i n N u c l e o t i d e - b i n d i ndgo m a i n UT

E GrpE/BAG1

\a

< FIGURE 3-16 Chaperone-mediated protein folding.Manyproteins foldintotheirproperthreedimensional structures with theassistance of Hsp70-like proteinsThesemolecular chaperones transiently bind to a nascent polypeptide asit emerges froma ribosome or to proteins thathaveotherwise unfoldedInthe Hsp70cycle,a substrate proteinbindsin rapid unfolded equilibrium to theopenconformation of the substratebindingdomain(SBD) of Hsp7O, to whichan ATpis boundin the nucleotide-binding (s1sp domain(NBD) a; proteins (DnaJ/Hsp4O) Accessory stimulate the hydrolysis of ATPandconformational changein Hsp70,resulting in theclosed form,in whichthesubstrate islockedinro the SBD;hereproperfoldingisfacilitated (stepZ) Exchange of ATPfor the boundADp,stimulated by otheraccessory (GrpE/BAG1), proteins converts the Hsp70backto the openform(stepE), releasing the properly (step4) foldedsubstrate

ATP ADP

the synthesisof improperly folded, nonfuncrional proteins. which would have to be destroyedto prevenrtheir disrupt_ ing cell function. Cells clearly have such mechanisms,since more than 95 percent of the proteins present within cells have been shown to be in their native ionformations. The explanation for the cell's remarkableefficiencyrn Dromot_ ing the proper protein folding encodedin primary *orr.r.. is that cells make a ser of proteins, called chaperon.r, th"t facilitate protein folding. The importance of chaperonesis highlightedby the observationsthat they are evolutionarily conserved,they are found in all organismsfrom bacteriato humans, and some are highly homologous and use almost identical mechanisms to assist p.ot.in folding. Chaper_ ones, which in eukaryotes are located in every cellular compartment and organelle, bind to rhe target proteins whose folding they will assist. Two general-families of chaperonea s re recognized:

to the exposureof hydrophobic side chains that have not yet had a chanceto be buried in the inner core ofthe folded piotein. These exposed hydrophobic side chains on diffeient moleculeswill stick to one another owing to the hydrophobic effect (Chapter 2) and thus promote aggregation.'Vfh.r, a newly synthesizedmolecule begins to fold, it is at risk of

Molecular Chaperones The heat-shockprotein Hsp70 and its homologs (Hsp70 in the cytosol and mitochondrial matrix, BiP in the endoplasmicreticulum, and DnaK in bac_

r Molecular chaperones,which bind and stabilize unfolded or partly folded proteins, thereby preventing theseproteins from aggregatingand being degraded r Chaperonins,which form a small folding chamber into which an unfolded protein can be sequesteied, giving it time and an appropriate environment to fold p-p..ly One reasonthat chaperonesare neededfor intracellular pro_ tein folding is that they help prevent aggregationof unfoided proteins. Unfolded and partly folded proteins tend to aggre_ gate into large, often water insoluble masses,from which it is extremely difficult for a protein to dissociateand then fold into its proper conformation. In part this aggregationis due

76

C H A P T E R3

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p R o T E t NS T R U C T U RAEN D F U N C T T O N

causesa conformational change in the chaperone rhat re_ leasesthe target protein. Additional proteins, such as the co-chaperoneHsp40 in eukaryotes (DnaJ in bacteria), help increise efficiency of

Hsp70-mediatedfolding of many proteins by stimulating the hydrolysis of ATP by Hsp70/DnaK (seeFigure 3-15). An additional protein called GrpE in bacteria (similar activity of BAG1 in mammals) also interacts with the Hsp70/DnaK, promoting the exchange of ATP for ADP. Multiple molecular chaperonesare thought to bind all nascent polypeptide chains as they are being synthesizedon ribosomes.In bacteria, 85 percent of the proteins are releasedfrom their chaperonesand proceedto fold normally; an even higher percentageof proteins in eukaryotesfollow this pathway. Chaperonins The proper folding of a large variety of newly synthesizedproteins also requires the assistanceof another class of proteins, the chaperonins. These huge cylindrical macromolecular assembliesare formed from two rings of oligomers, which can exist in a "tight" peptidebinding state and a "relaxed" peptide-releasingstate. The eukaryotic chaperonin TriC consistsof eight subunits per ring. In the bacterial, mitochondrial, and chloroplast chaperonin, known as GroEL, each ring contains sevenidentical subunits (Figure 3-1'7a).The GroEL folding mechanism, which is better understood than TriC-mediated folding, servesas a general model (Figure 3-1'7b).A partly folded or misfolded polypeptide is inserted into the cavity of the barrel-like GroEL. where it binds to the inner wall and folds into its native conformation. In an MP-dependent step, GroEL undergoesa conformational change and releasesthe folded protein, a processassistedby a co-chaperonin, GroES, which caps the ends of GroEL. The binding of ATP and the co-chaperoninGroES to one of the rings in the tight state of GroEL causesa twofold expansion of its cavity, shifting the equilibrium toward the relaxedpeptidefolding state. There is a striking similarity between the capped-barreldesign of GroELiGroES, in which proteins are sequesteredfor folding, and the structure of the 265

proteasomethat participatesin protein degradation (discussedin Section3.4).

AlternativelyFoldedProteinsAre lmplicated in Diseases As noted earlier, each protein normally folds into a sin-

Eil gl., energeticallyfavorableconformationthat is speciRecentevidencesuggests' fied by its amino acid sequence. however, that a protein may fold into an alternative threedimensional structure as the result of mutations, inappropriate covalent modifications made after the protein is synthesized,or other as-yet-unidentifiedreasons.Such "misfolding" not only leads to a loss of the normal function of the protein but often marks it for proteolytic degradation. However, when degradation isn't complete or doesn't keep pace with misfolding, the subsequentaccumulation of the misfolded protein or its proteolytic fragments contributes to certain degenerative diseases characterizedby the presenceof insoluble protein plaques in various organs, including the liver and brain. Some neurodegenerativediseases,including Alzheimer's diseaseand Parkinson'sdiseasein humans and transmissible

ments or the soluble alternativelyfolded proteins are toxic to the cell is unclear. I

ffi viu"o:GroEl Pasecycle (b) Ribosome

Partiallyfolded or misfoldedprotein

proteinfolding.Proper 3-17 Chaperonin-mediated A FIGURE suchasthe on chaperonins depends foldingof someproteins of 14 complex isa hollow,barrel-shaped prokaryotic GroEL(a)GroEL (b) In rings in two stacked arranged subunits 60,000-MW identical in a "tight" exists of ADBGroEL of ATPor presence the absence proteins. statethatbindspartlyfoldedor misfolded conformational

Protein

Properly folded protein

to a moreopen,"relaxed"state,which Bindingof ATPshiftsGroEL is oneendof GroEL Duringthisprocess, thefoldedprotein. releases of assembly an GroES, bytheco-chaperonin blocked transiently Cell87:241' (a)fromA Roseman etal, 1996' subunits[Part 1O,OO0-MW Saibil H of l courtesy

P R O T E I NF O L D I N G

77

(a)

j r Some neurodegenerativediseasesare causedby aggrej S"t."r of proteins that are s r a b l y f o l d e d i n a n a l t e r n a r i v e conlormatlon. I

ff,l

ProteinFunction

Although proteins have many different shapesand sizesand mediate an extraordinarily diverse array of activitiesboth inside and outside of cells, most of thesediversefunctions are basedon rheabilityof proteinsro engagein a commonactiviry, the binding to themselves,other macromolecules,small mole, cules,and ions. Here we will describesomeof the key features underlyingprotein binding, and then turn to look at one group of proteins, enzymes,in greater detail. The activities of the other functional classesof proteins (structural,scaffold,transport, regulatory,motor) will be describedin other chapters.

S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e F u n c t i o n so f M o s t P r o t e i n s

-*si

-:* t2oPtt

,

100nm

,

FIGURE3-18 Alzheimer,sdiseaseis characterizedby the formation of insoluble plaques composed of amyloid protein. ( a )A t l o w r e s o l u t i o na ,n a m y l o i dp l a q u ei n t h e b r a i no f a n Alzheimers patientappearsas a tangleof filaments.(b)The reqularstructureof f rom p.aquesrsrevealedrn rhe atomicforcemicroscope. frlaments Proteolysis of the naturallyoccurringamyloidprecursorproteinyields a shortfragment,cailedp-amyloidprotern,that for unKnownreasons changesfrom an a-helicalto a B-sheetconformationThisalternative

degenerative diseases[Courtesv of K Kosk I

The molecule to which a protein binds is often calleclits ligand. In some casesligand binding causesa changein the shape of a protein. Ligand-binding-drivenconformational changesare integral to the mechanismof action of many proteins and are important in regulating protein activity. Two propertiesof a protein characterizehow it binds ligands.Specificity refersto the ability of a protein to bind one

ure of affinity (Chapter 2). The stronger the interaction be-

P r o t e i nF o l d i n g r The sequence of a proteindeterrnines its three-dimensional structure, which detern.rines its function. In short, function derivesfrom structure;structurederivesfrom sequence. Becauseprotein function derivesfrom protern srructure, wly synthesized proteinsmust fold into the correctshaoe f u n c r i o np r o p e r l y . r The planar structureof the peptidebond limits the num_ ber of conformationsa polypeptidecan have. The amino acid sequenceof a protein dictatesits foldine to a specificthree-dimensional conformarion.rhe native state. Proteins will unfold, or denature,if treated under conditionsthat disrupt rhe noncovalenrinteractionsstabi_ lizing their three-dimensional structures. olding in vivo occurswith assistance from chaoich bind ro nascentpolypeptidesemergingfrom and prevenrtheir misfoldine. 78

.

cHAprER 3

I

p R o r E l Ns r R U c r u R A E N DF U N c l o N

virus) or other foreign substances(e.g.,proteinsor polysaccha_ ridesin pollens).Different antibodiesare generatedin response to differentanrigens,and theseantibodieshavethe remarkable characteristicof binding specificallyto (..recognizing,')a por_ tion of the antigen,called an epitope,which initially induced the production of the antibody, and not to other molecules. Antibodies act as specificsensorsfor antigens,forming antibody-antigen complexesthat initiate a cascadeof proiective r e e c t i o nisn c e l l so f t h e i m m u n es y s r e m .

CDR

Light chain lnterchain disulfide bonds

"t"

Heavychain

Carbohydrate

periments discussedin subsequentchapters' \7e will see many examples of protein-ligand binding throughout this book, including hormones binding to receptors (Chapter 15), regulatory moleculesbinding to DNA (Chapt , i\. cell-adhesion molecules binding to extracellular matrix (Chapter 1'9),to name just a few. Next we will consider how the binding of one class of proteins, enzymes,to their ligands results in the catalysisof the chemical reactions essentialfor the survival and function of cells.

EnzymesAre HighlYEfficientand SpecificCatalysts Proteins that catalyze chemical reactions, the making and

3-19 Protein-ligandbindingof antibodies.(a)Ribbon A FIGURE molecule of the immunoglobulin Every antibody modelof an antibody. heavychains(lightanddarkred)and of two identical lgGclassconsists bondsThe linkedby disulfide lightchains(blue)covalently two rdentical the tvuoheavy containlng the overall structure diagram of insetshowsa and fit betweenan antibody andtwo lightchains(b)Thehand-in-glove on itstargetantrgen-inthiscase, thesiteto whichit binds(epitope) make Regions wherethetwo molecules lysozyme egg-white chicken contacts theantigenwith Theantibody contactareshownassudaces. (CDRs). Inthis regions fromallitscomplementarity-determining residues of theantigenandantibodyis complementarity view,the molecular fromtheantigensurface where"fingers"extending apparent especially surface. to "clefts"in theantibody areopposed

catalyzed by a specific efizyme. In many ways' enzymes are the cell's chemists,performing many of a cell's chemical reactions. (An additional form of catalytic macromolecule in cells is made from RNA. TheseRNAs are called ribozymes') Thousands of different types of enzymes'each of which catalyzesa single chemical reaction or set of closely related reactions,have beenidentified. Certain enzymesare found in the majority of cells becausethey catalyze the synthesisof common cellular products (e.g.,proteins, nucleic acids, and phospholipids) or take part in the production of energy (e'g'' ty th. .ottu.rsion of glucoseand oxygen into carbon dioxide and water). Other enzymesare present only in a particular

tract. or even outside the organism (e.g., toxic enzymesin the venom of poisonous snakes). Like all catalysts,enzymesincreasethe rate of a reaction but do not affect the extent of a reaction, which is deter-

AII antibodies are Y-shapedmoleculesformed from two identical heavy chains and two identical light chains (Figure 3-19a). Each arm of an antibody moleculecontains a singlelight chain linked to a heavy chain by a disulfide bond. Near the end of each arm are six highly variable loops' called complementarity-determiningregions (CDRs/, which form the antigen-binding sites.The sequencesof the six loops are highly variable among antibodies, generating unique complementary ligand-binding sites that make them specific for different epitopes (Figure 3-19b). The intimate contact between thesetwo surfaces,stabilizedby numerousnoncovalent interactions, is responsiblefor the extremely precisebinding specificity exhibited by an antibody. P R O T E I NF U N C T I O N

O

79

-__Transitionstate (uncatalyzed)

t

0) q) o o '-I

(a) Catalyticsite

(b)

Catalyticsite

Binding pocket

Transitionstate (catalyzed)

. A/l.+ ."cat

B i n d i n gp o c k e t

Products

Progressof reaction-----> A FIGURE 3-20 Effectof an enzymeon the activationenergy of a chemicalreaction.Thishypothetical reaction pathwaydepicts the changes in freeenergyG asa reaction proceeds A reaction will takeplacespontaneously onlyif the totalG of the products isless thanthatof the reactants (negative AG) However, allchemical reactions proceed throughoneor morehigh-energy transition states, andthe rateof a reaction isinversely proportional to the activation energy(AG+), whichisthedifference in freeenergybetween the reactants andthetransition state(highest pointalongthe pathway). Enzymes andothercatalysts accelerate the rateof a reactionby reducing thefreeenergyof thetransition stateandthusAG+. rates of reactions 106-1012times that of the corresponding uncatalyzedreactions under otherwise similar conditions.

An Enzyme'sActive Site BindsSubstrates a n d C a r r i e sO u t C a t a l y s i s

sitesmake up only a small fraction of the total protein, with the rest involved in folding of the polypeptide, iegulation of the active site, and interactions with other molecules.

A FIGURE 3-21 Active site of the enzymetrypsin. (a)An enzyme's activesiteiscomposed of a bindingpocket, whichbinds specifically to a substrate, anda catalytic site,whichcarnes out catalysis(b)A surfacerepresentation of the serineprotease trypsin Activesitecleftscontaining thecatalytic site(sidechains of the catalytic triadSer-195, Asp-102, andHis-57 shownasstickfigures) andthe substrate sidechainspecificity bindingpocketareclearly visible[Part(b)courtesy of PTeesdale-spittte ]

substrate-bindingsite and the substrate,which is mediated by multiple weak noncovalent interactions and is very sensitive to the shapesof substrates.Usually only one or a few substratescan fit preciselyinto a binding site. The idea that enzymesmight function by binding to rheir substratesin the manner of a key fitting into a lock was suggestedfirst by Emil Fischerin1894.Ln1913 Leonor Michaelis and Maud Leonora Menten provided crucial evidence supporting this hypothesis.They showed that the rate of an enzymatic reaction was proportional to the substrateconcentration at low substrateconcentrations, but that as the substrateconcentrations increased,the rate reached a maximal velocity V-o and became substrate concentration-independent, with the value of V-"" being directly proportional to the amount of enzymepresent in the reaction mixture (Ftgure 3-22). They deducedthat this saturation at hieh substratecon-

termediate step in the ultimately irreversible conversion of substrateto product (P) (Figure3-23): substratehas bound. In some enzymes,the catalytic and substrate-binding sites overlap; in others, the two regions are structurally as well as functionally distinct.

E + S i------^ES-+E + p and that the rate Ve of formation of product at a particular substrateconcentration [S] is given by what is now called the Mich aelis-Mentenequahon:

vo:v-"*;J!+ Km L)l

zyme. As noted above, this specificity of enzymesis a conse_ quenceof the precisemolecular complementarity betweenits 80

o

c H A p r E 3R | p R o r E t N s r R u c r u RA EN DF U N c l o N

(3-1)

where the Michaelis constant K-, a measureof the affinity of an enzyme for its substrate (seeFigure 3-22), is the substrate concentration that yields a half,maximal reaction rate

(a) c .=6

2.0 oJ

b.> t.5 ^=

. : : J /

6-

E(L

1.0

Enzyme

0.5 o:9 t

c().

Concentrationof substrateIS]

f I

etnoino no.t"t

(b)

1.0 o nR

I 0.6

High-affinity substrate L o w - a f fi n i t y s u b s t r a t e( S ' )

I

g 0.4 o

o.2 0

(tSlor tS'l) of substrate Concentration

reaction' 3-22 K^ and V."* for an enzyme-catalyzed A FfGURE of the of the dependence fromanalysis K, andVru"aredetermined Theshapeof concentration velocity on substrate initialreaction of a simpleenzymeischaracteristic kineticcurves thesehypothetical (S)isconverted into in whichonesubstrate reaction catalyzed after immediately product(P)Theinitralvelocity is measured concentration to substrate beforethe substrate additionof enzyme (a)Plotsof the initialvelocrty at two different appreciably changes concentration of enzyme concentrations [E]asa functionof substrate Michaelis rate is the yields reaction half-maximal a The that lsl [S] of theaffinityof Efor turningS intoP K., a measure constant increase a proportronal causes concentration theenzyme Quadrupling quadrupled; is V.u" velocity the maximal rate,andso in the reaction (b)Plotsof the initialvelocity versus isunaltered. theK., however, hasa Sfor whichtheenzyme witha substrate concentration substrate hasa lower S' for whichtheenzyme highaffinityandwitha substrate because affinity.Notethatthe V.u"isthe samewith bothsubstrates, substrate low-affinity for S', the Km is higher that IE]isthesame,but

(i.e.,112V-.*), and thus is analogousto the dissociationconstant K6 (Chapter 2). The smaller the value of K-, the more effectivethe enzyme is at making product from dilute solutions of substrateand the smaller the substrateconcentration neededto reach half-maximal velocity.The concentrationsof the various small moleculesin a cell vary widely, as do the Kvaluesfor the different enzymesthat act on them. A good rule of thumb is that the intracellular concentrationof a substrate is approximately the same as or somewhat greater than the K^ value of the enzymeto which it binds' The rates of reaction at substratesaturation vary enormously among enzymes.The maximum number of substrate moleculesconvertedto product at a singleenzymeactivesiteper secondis called the turnouer nwmber,which can be lessthan 1

Enzyme

3-23 Schematicmodel of an enzyme'sreaction A FIGURE (E)bind thatenzymes suggest kinetics Enzyme mechanism. (5)througha fixedandlimitednumberof sites molecules substrate tsknownasan (theactivesites). Theboundspecies on theenzymes with in equilibrium is (ES) ES complex The complex. enzyme-substrate stepin andisan intermediate andsubstrate the unboundenzyme to products(P) of substrate the conversion

substrate complexes (ES' ES', ES", etc.) generatedprior to the final releaseof the Products: E+S

.

^

--^ ES' ^ S' E S F . . . . .E

'"'E+P

The energyprofiles for such multistep reactionsinvolve multiple hills and valleys (Figure 3-24), and methods have been developedto trap the intermediatesin such reactionsto learn more about the details of how enzymescatalyzereactions'

SerineProteasesDemonstrateHow an Enzyme's Active Site Works Serineproteases,a large family of proteolytic enzymes' are used throughout the biological world-to digest meals (the FUNCTION PROTEIN

81

(a)

(b)

()

(5

Enzyme-transition statecomplex

j

6 o o

o) q) o o) LI

0) IL

EX+ E n z y m e+ substrate

Enzymesubstrate comprex

E+S E+P

Progressof reaction-----> S<-X++P A FIGURE 3-24 Free-energyreactionprofilesof uncatalyzed and multistepenzyme-catalyzed reactions.(a)Thefree-energy profileof a hypothetical reaction simpleuncatalyzed reaction converting (S)to product(p)viaa singlehigh_energy substrate transition state.(b)Manyenzymes catalyze suchreactions by dividing the process intomultiple discrete steps,in thiscasethe initial pancreatic enzymestrypsin, chymotrypsin, and elastase),to

consider how trypsin and its two evolutionarily closely related pancreatic proteases,chymotrypsin and elastase,catalyze cleavageof a peptide bond:

o

Progressof reaction-----> E+S*ES+EX+------+E+p formation of an EScomplex followedby conversion viaa single transition state(EX+) (E)andp Theacrrvatron to thefreeenzyme energyfor eachof thesestepsissignificantly lessthanthe activation energyfor the uncatalyzed reaction; thustheenzyme dramatically enhances the reaction rate. Figure 3-25a shows how a substratepolypeptide binds to the substrate-bindingsite in the active site of trypsin. There are two key binding interactions.First, the substrateand enzyme form hydrogen bonds that resemblea B sheet.Second, a key side chain of the substratethat determineswhich peptide in the substrate is to be cleaved extends into the enzyme's side-chain-specificitybinding pocket, at the bottom of which resides the negatively charged side chain of the (a)

x ,9

-:'

p(""'"tr1-P2 + HrO

p,,-C...-

+

Catalyticsite

NH3+-P2

His-57

l*o

H

Ho'

\r'r

where P1is the portion of the protein on the N-terminal sideof the peptide bond, and P2is the portion on rhe C-terminalside. S7efirst consider how serineproreasesbind specificallyto their substratesand then show in detail how catalysistakesplace.

C: O-

82

.

c H A p r E R3

|

p R o r E t Ns r R U c r u R EA N D F U N c l o N

Trypsin

Oxyanion hole

A r g i n i n es i d e c h a i n( R 3 )i n substrate

> FIGURE 3-25 Substratebindingin the activesite of typsin_ N like serineproteases. (a)Theactivesiteof trypsin(bluemolecule) H wrtha boundsubstrate (blackmolecule) Thesubstrate formsa two_ B i n d i n gs i t e stranded B sheetwith the bindingsite,andthe sidechainof an (Rr)in thesubstrate arginine is boundin the side-chain-specificitv binding p o c k e tl .t sp o s i t i v ecl h y a r g egdu a n i d i n i ugmr o u pi s stabilized by the negative chargeon the sidechainof the enzyme,s A s p - 1 8 9T h i sb i n d i n g a l i g n tsh ep e p t i d b e o n do f t h ea r g i n i n e appropriately (b) for hydrolysis catalyzed by theenzyme,s active_site c a t a l y tti rci a d( s i d ec h a i nos f S e r - 1 9 5H,i s - 5 a7 n dA s p - 1 0 2()b. )T h e a m i n oa c i d sl i n i n gt h es i d e - c h a i n - s p e c ibf i n c idt iyn g pocket d e t e r m i ni e t ss h a p ea n dc h a r g ea,n dt h u si t sb i n d i n g properties Trypsin accommodates the positively charged sidechainsof arginine and lysine; chymotrypsin, large,hydrophobic sidechainssuchas p h e n y l a l a n ianned; e l a s t a ssem , a lsl r d ec h a i nssu c ha sg l y c i naen d

alanine.[Part(a)modified from.J.J. perona andC. S. Craik,1991. J. Biol. Chen 272(48).29987-29990 I

Peptidebond to be cleaved

Side-chainspecificity binding pocket Asp-189

Chymotrypsin

Elastase

enzyme'sAsp-189. Trypsin has a marked preferencefor hydrolyzing proteins (black in Figure 3-25a) at the carboxyl side of a residue with a long positively charged side chain (arginine or lysine), becausethe side chain is stabilizedin the specificity binding pocket by the negativeAsp-189. Slight differencesin the structures of otherwise similar specificity pockets help explain the differing substratespecificities of the two related serine proteases:chymotrypsin prefers large aromatic groups (asin Phe,Tyr, Trp), and elastaseprefers the small side chains of Gly and Ala (Figure 3-25b1.The unchargedSer-189in chymotrypsinallows large, uncharged,hydrophobic side chains to bind stably in the pocket. The branchedaliphatic side chainsof valine and threoninein elastase replace glycines in the sides of the pocket in trypsin and thus prevent large side chains in substratesfrom binding, but allow stablebinding of the short alanineor glycinesidechain. In the catalytic site, all three enzymesuse the hydroxyl group on the side chain of a serine in position 195 to catalyzethe hydrolysis ofpeptide bonds in protein substrates.A catalytic triad formed by the three side chains of Ser-195, His-57, Asp-102 participates in what is essentiallya twostep reaction. Figure 3-26 shows how the catalytic triad co-

operates in breaking the peptide bond, with Asp-102 and His-57 supporting the attack of the hydroxyl oxygen of Ser-195 on the carbonyl carbon in the substrate.This attack initially forms an unstable transition state with four groups attached to this carbon (tetrahedral intermediate). Breaking of the C-N peptide bond then releasesone part of the protein (NH3-P2), while the other part remains covalently attached to the enzymevia an ester bond to the serine'soxygen, forming a relatively stable intermediate (the acyl enzyme). The subsequentreplacementof this oxygen by one from water, in a reaction involving another unstable tetrahedral intermediate, leadsto releaseof the final product (P1-COOH). The tetrahedral intermediates are partially stabilized by hydrogen bonding from the enzyme'sbackbone amino groups in what is called the oxyanion hole.The large family of serine proteasesand related enzymeswith an active-siteserine illustrates how an efficient reaction mechanism is used over and over by distinct enzymesto catalyzesimilar reactions. The serineproteasemechanismpoints out severalgeneral key featuresof enzymaticcatalysis:(1) enzymecatalytic sites are designedto stabilizethe binding of a transition state' thus lowering the activation energy and acceleratingthe overall

(c) Acyl enzyme (ES'complex)

(b) Tetrahedralintermediate (transitionstate)

( a ) E Sc o m p l e x

o H 'r'r.,..-N

D , rH; N

"c

/ Oxyanion hole

P.,/

P,,, H

T,o (f) EP complex

(d) Acyl enzyme (ES'complex)

(e) Tetrahedralintermediate (transitionstate)


,,\ _ . N

HN.

\J

A FIGURE 3-26 Mechanismof serineprotease-mediated His-57, triadof Ser-195, hydrolysisof peptidebonds.Thecatalytic proteases employs a multistep in theactive sitesof serine andAsp-102 peptidebondsin targetproteins(a)Aftera mechanism to hydrolyze polypeptide substrate bindsto the activesite(seeFigure3-23)forming the carbonyl 95 attacks oxygenof Ser-1 the hydroxyl an EScomplex, targetedpeptidebond(yellow)Movements carbonof the substrate's in the areindicated by arrows.(b)Thisattackresults of electrons in intermediate, statecalledlhe tetrahedral formationof a transition isstabilized by oxygen on thesubstrates charge whichthenegative hole.(c)Additional s oxyanion hydrogen bondsformedwiththe enzyme of the peptidebond,release resultin the breaking electronmovements

\-

,,,.\

_ HN. ,_-NH

\l

(lowpH) Inactive Active (NH2-P2), products andformationof the acyl of oneof the reaction watermolecule enzyme(ES'complex)(d)An oxygenfroma solvent (e)Thisattack enzyme the acyl of carbon the carbonyl thenattacks intermediate tetrahedral of a second in theformation results of the Ser(fl Additional resultin the breaking electronmovements of the andrelease of the EPcomplex) bond(formation 195-substrate is which (P1-COOH). of His-57, chain The side product finalreaction the side chain to bonding hydrogen by proper orientation heldin the protons anddonating bywithdrawing catalysis facilitates of Asp-102, (inset)lf the pH istoo low andthe sidechain the reaction throughout andthe in catalysis it cannotparticipate of His-57isprotonated, isinactive enzyme FUNCTION PROTEIN

83

reaction, (2) multiple side chains, togerherwith the polypeptide backbone,carefully organizedin three dimensions,work togetherto chemicallytransform substrateinto product, often by multistepreactions,and (3) acid-basecatalysismediatedby one or more amino acid side chainsis often used by enzymes, as when the imidazolegroup of His-57 in serineproteasesacts as a base to remove the hydrogen from Ser-195,shydroxyl group. As a consequence,often only a particular ionization state (protonated or nonprotonated) of one or more amrno acid side chains in the catalytic site is compatible with catalysis,and thus the enzyme'sactivity is pH-dependent. For example,the imidazoleof His-57 in serineproreases, whose pK" is =5.8, can help the Ser-195hydroxyl attack the substrateonly if it is not protonated. Thus, the activity of the proteaseis low at pH < 6.8, and the shapeof the pH activity profile in the pH range 4-8 matches the titration of the His-57 side chain, which is governed by the HendersonHasselbalchequation,with an inflection near pH 6.8 (seeFigure 3-27, right, and Chapter 2). The activity drops at higher pH values,generatinga bell-shapedcurve, becausethe proper folding of the protein is disrupted when the amrno group ar the protein's amino rerminus is deprotonated (pK" = 9); the conformation near the active site changesas a consequence. The pH sensitivity of an enzyme'sactivity can be due to changesin the ionization of catalytic groups, groups that participate directly in substratebinding, or groups that influence the conformation of the protein. Pancreaticproteasesevolved to function in the neutral or slightly basic conditions in the intestines;hence,their pH oprima are =8. proteasesand other

Lysosomalenzvme

o 0)

E N

c o) 0) G

o

2345678910 oH A FfGURE 3-27 pH dependence of enzymeactivity.lonizable (pH-titratable) groupsin theactivesitesor elsewhere in enzymes oftenmustbe eitherprotonated or deprotonated to permitproper substrate bindingor catalysis or to permitthe enzyme to adoptthe correctconformation Measurement of enzyme activity asa function of pHcanbe usedto identify the pKa'sof thesegroupsThe pancreatic serineproteases, (rightcurve), suchaschymotrypsin exhibitmaximum activity aroundpHg because of titrationof the (required activesiteHis-57 for catalysis, pKu= 6 8) andof theamino terminus of the proteln(required for properconformation, pK"= 9) Manylysosomal hydrolases haveevolved to exhibita lowerpH optimum(=45, leftcurve) to matchthe low internal pH in lysosomes in whichtheyfunction. fromp Lozano, [Adapted T.DeDiego, andJ L l b o r r a ,1 9 9 7 ,E u r J B i o c h e m2 4 8 ( 1) : 8 0 - 8 5 ,a n d W A J u d i c ee t a l , 2 0 0 4 . E u r . J B i o c h e m2 7 1 ( 5 ) : 1 0 4 6 - 1 0 5l3

84

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hydrolytic enzymesthat function in acidic conditions must employ a different catalytic mechanism.This is the casefor enzymeswithin the stomach (pH = 1) such as the protease pepsin or those within lysosomes(pH = 4.5), which play a key role in degrading macromoleculeswithin cells (seeFigure 3-27, left).Indeed, lysosomal hydrolasesthat degrade a wide variety of biomolecules(proteins, lipids, etc.) are relatively inactive at the pH in the cytosol (=7), and that helps protect a cell from self-digestionshould theseenzymesescape the confinesof the membrane-boundlysosome. One key feature of enzymatic catalysisnot seenin serine proteases,but found in many other enzymes,is a cofactor,or prosthetic (helper) group. This is a nonpolypeptide small molecule or ion (e.g., iron, zinc, copper, manganese)that is bound in the active site and plays an essentialrole in the reaction mechanism. Small organic prosthetic groups in enzymesare also caIIedcoenzymes.Someof theseare chemically modified during the reaction and thus need to be replaced or regeneratedafter each reaction; others are not. Examplesof the former include NAD* (nicotinamideadenine dinucleotide) and FAD (flavin adenine dinucleotide) (see Figure 2-33), whereas heme groups that bind oxygen in hemoglobin or transfer electrons in some cytochromes are examples of the latter (Figure 12-14). Thus, the chemistry catalyzedby enzymesis not resrrictedby the limited number of amino acids in polypeptide chains. Many vitamins-e.g., the B vitamins, thiamine (B1), riboflavin (B2), niacin (B3), and pyridoxine (86), and vitamin C-which cannot be synthesized in higher animal cells, function as or are used to generatecoenzymes.That is why supplementsof vitamins must be added to the liquid medium in which animals cells are grown in the laboratory (Chapter9). Small moleculesthat can bind to active sitesand disruot the reactionsare called enzymeinbibitors. Suchinhibitois are useful tools for studying the roles of enzymesin cells and whole organisms by allowing analysis of the consequencesof the loss of the enzyme'sactivity. Thus, inhibitors complement the use of mutations in genesfor probing an enzyme'sfunction in cells (seeChapter 5). However, interpreting results of inhibitor studies can be complicated if, as is often the case,rhe inhibitors block the activity of more than one protein. Smallmolecule inhibition of protein activity is the basis for mosr drugs (e.g., aspirin inhibits enzymescalled cyclooxygenases) and also for chemical warfare agents. Sarin and other nerve gasesreact with the active serinehydroxyl groups of both serine proteasesand a related enzym%acetylcholineesterase,which is a key enzymein regulating nerve conduction (seeChapter 23). I

Enzymesin a CommonPathwayAre Often PhysicallyAssociatedwith One Another Enzymes taking part in a common metabolic process (e.g., the degradation of glucoseto pyruvate) are generallylocated in the same cellular compartment (e.g., in the cytosol, at a membrane,or within a parricularorganelle).'Withina compartment, products from one reaction can move by diffusion to the next enzyme in the pathway. However, diffusion

(a)

Reactants

/

Reactants

Products

sized bacterium experiencesa drag force from water that stops its forward movement within a fraction of a nanometer when it stops actively swimming. To generatethe forces necessaryfor many cellular movements,cells dependon specialized enzymes commonly called molecular motors, or motor proteins. These mechanochemicalenzymes convert energyreleasedby the hydrolysis of ATP or contained within ion gradients into a mechanical force, usually generating either linear or rotary motion. From the observed activities of motor proteins' we can infer three generalproperties that they possess: r The ability to transducea source of energy'either ATP or an ion gradient, into linear or rotary movement

Products (c)

r The ability to bind and translocatealong a substrate r Net movement in a given direction \(/e will see many examples of such motors in subsequent chapters.

A FIGURE 3-28 Assemblyof enzymesinto efficient pathways reaction Inthe hypothetical multienzymecomplexes. intofinalproducts herethe initialreactants areconverted illustrated A, B,andC. (a)Whenthe actionof threeenzymes: bythe sequential withinthesame in even constrained are free solution or enzymes sequence in the reaction compartment, the intermediates cellular slow to the next,an inherently mustdiffusefromoneenzyme (b)Diffusion process. whenthe isgreatlyreduced or eliminated eitherby intomultisubunit complexes, enzymes associate individual protein(c)Theclosest or with the aidof a scaffold themselves whentheenzymes occurs of different catalytic activities integration in a single domains level,becoming arefusedat the genetic polypeptide chain

entailsrandom movement and can be a slow, relatively inefficient processfor moving moleculesbetweenwidely dispersed enzymes(Figure 3-28a). To overcomethis impediment, cells have evolved mechanismsfor bringing enzymesin a common pathway into closeproximity. In the simplest such mechanism, polypeptides with different catalytic activities cluster closely together as subunits of a multimeric enzyme or assembleon a common "scaffold" that holds them together(Figure3-28b). This arrangement allows the products of one reaction to be channeled directly to the next enzyme in the pathway. In some cases, independentproteins have been fused together at the genetic level to createa singlemultidomain, multifunctional enzyme (Figure3-28c).

E n z y m e sC a l l e dM o l e c u l a rM o t o r s C o n v e r t Energyinto Motion At the nanoscaleof cells and molecules,movement is influenced by forces that differ from those in the macroscopic world. For example, the high protein concentration (200300 mglml) of the cytoplasm preventsorganellesand vesicles from diffusing faster than 100 pm/3 hours. Even a micrometer-

Protein Function r The functions of nearly all proteins depend on their ability to bind other molecules(ligands). r The specificity of a protein for a particular ligand refers to the preferential binding of one or a few closely related ligands. r The affinity of a protein for a particular ligand refers to the strength of binding, usually expressedas the dissociation constant K6. r Ligand-binding sites on proteins and the corresponding ligands themselvesare chemically and spatially complementary. r Enzymes are catalyticproteins that acceleratethe rate of cellular reactions by lowering the activation energy and stabilizing transition-stateintermediates(seeFigure 3-20). r An enzyme active site, which is usually only a small part of the protein, comprisestwo functional parts: a substratebinding site and a catalytic site. The amino acids composing the active site are not necessarilyadjacent in the amino acid sequencebut are brought into proximity in the native conformation. r The substrate-bindingsite is responsiblefor the exquisite specificity of enzymesowing to its molecular complementarity with the substrateand the transition state. r The initial binding of substrates(S)to enzymes(E) results in the formation of an enzyme-substratecomplex (ES)' which then undergoesone or more reactions catalyzed by the catalytic groups in the active site until the products (P) are formed and diffuse away from the enzyme. r From plots of reaction rate versus substrate concentration, two characteristicparametersof an enzymecan be determined: the Michaelis constant K-, a rough measureof P R O T E I NF U N C T I O N

85

the enzyme'saffinity for converting substrateinto product, and the maximal velocity V*^*, d measureof its catalytic power (seeFigure 3-22). r The rates of enzyme-catalyzedreactions vary enormously, with the turnover numbers (number of substratemolecules converted to products at a singleactive site at substratesaturation) ranging between<1 to 6 x 10s molecules/s. r Many enzymes catalyzethe conversion of substratesto products by dividing the process into multiple discrete chemical reactions that involve multiple distinct enzyme substratecomplexes(ES',ES",etc.). r Serineproteaseshydrolyze peptide bonds in protein substratesusing as catalyticgroups the sidechainsof Ser-195, H i s - 5 7 ,a n d A s p - 1 0 2 . r Amino acids lining the specificity binding pocket in the binding site of serine proteasesdetermine the residue in a protein substrate that will be hydrolyzed and account for differencesin the specificity of trypsin, chymotrypsin, and elastase. r Enzymesoften use acid-basecatalysismediated by one or more amino acid side chains,such as the imidazolegroup of His-57 in serineproteases,to catalyzereactions. r The pH dependenceof protonation of catalytic groups (pK") is often reflected in the pH-rate profile of the enzyme's activity. The pH sensitivity of an enzyme'sactivity can be due to changesin the ionization of catalytic groups, of groups that participate directly in substrate binding, or of groups that influence the conformation of the protein. r In some enzymes,nonpolypeptide small moleculesor ions, called cofactors or prosthetic groups, can bind to the active site and play an essentialrole in enzymaric catalysis.Small organic prostheticgroups in enzymesare also called coenzymes;vitamins, which cannot be synthesized in higher animal cells, function as or are used to generate coenzymes. r Enzymesin a common pathway are located within specific cell compartments and may be further associatedas domains of a monomeric protein, subunits of a multimeric protein, or componentsof a protein complex assembledon a common scaffold(seeFigure 3-28). r Motor proteins are mechanochemicalenzymesthat convert energy releasedby ATP hydrolysis into either linear or rotary movement.

Regulating ProteinFunction l:

fll

ProteinDegradation Most processesin cells do not take place independently of one another or at a constant rare. The activities of all oroteins and other biomoleculesare regulated to integrate their functions for optimal performance for survival. For example, the catalytic activity of enzymesis regulated so that the amount of reaction product is lust sufficient to meet the 86

.

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needsof the cell. As a result, the steady-stateconcentrations of substratesand products will vary, depending on cellular conditions. Regulation of nonenzymaticproteins-the opening or closing of membrane channels or the assembly of a macromolecular complex, for example-is also essential. In general,there are three ways to regulateprotein activity. First, cells can increaseor decreasethe steady-statelevel of the protein by altering its rate of synthesis, its rate of degradation, or both. Second,cells can change the intrinsic activity, as distinct from the amounr, of the protein (e.g.,the affinity of substratebinding, the fraction of time the protein is in an active versus inactive conformation). Third. there can be a change in location or concentration within the cell of the protein itself, the target of the protein's activity (e.g., an enzyme'ssubstrate),or some other molecule required for the protein's activity (e.g., an enzyme'scofactor). All three types of regulation play essentialroles in the lives and functions of cells.

RegulatedSynthesisand Degradationof Proteinsls a FundamentalPropertyof Cells Controlof ProteinSynthesis The rateof synthesis of proteins is determined by the rate at which the DNA encoding the protein is converted to mRNA (transcription), the steady-stateamount of the active mRNA in the cell, and the rate at which the mRNA is converted into newly synthesized protein (translation). These important pathways are described in detail in Chapter 4. Control of Protein Degradation The life span of intracellular proteins varies from as short as a few minutes for mitotic cyclins, which help regulate passagethrough the mitotic stage of cell division, to as long as the age of an organism for proteins in the lens of the eye. Protein life span is controlled primarily by regulatedprotein degradation. There are two especiallyimportant rolesfor protein degradation. First, degradation removes proteins that are potentially toxic, improperly folded or assembled,or damagedincluding the products of mutated genes and proteins damaged by chemically active cell metabolites. Despite the existenceof chaperone-mediatedprotein folding, it is estimated that as many as 30 percent of newly made proteins are rapidly degraded becausethey are misfolded, their assembly into complexes is defective, or they are otherwise unsuitable. Most other proteins are degraded more slowlS about'l,J percent degradationper hour in mammalian cells. Second,the controlled destruction of otherwise normal oroteins provides a powerful mechanism for maintaining the appropriate levelsofthe proteins and their activities,and for permitting rapid changes in these levels to help the cells respond to changing conditions. Eukaryotic cells have several pathways for degrading proteins. One major pathway is degradation by enzymes within lysosomes, membrane-limited organelles whose acidic interior (pH =4.5) is filled with a host of hydrolytic enzymes. Lysosomal degradation is directed primarily toward aged or defective organelles of the cell-a process

llll+ Animation:The Proteasome 3-29 Ubiquitin-and proteasome-mediated < FIGURE (a)Computer-generated thata imagereveals proteolysis. (blue)at a 195 cap with proteasome structure cylindrical hasa Cytosolic proteins of ubiquitin-tagged target protern eachendof a 20Score Proteolysis are of the core(b)Proteins withinthe innerchamber occurs by polyubiquitination degradation for proteasomal targeted (Ub) of a ubiquitin by attachment E1isactivated Enzyme to a Ub molecule (step this transfers then E) and molecule (E3)transfers ligase o in E2(stepZ) Ubiquitin residue cysteine -NH2 of a on E2to theside-chain the boundUb molecule -NH -CUb (step Ub B) Additional in a targetprotein residue lysine protein by repeatlng target to the areadded molecules chain(step4) The steps[-El, forminga polyubiquitin cap, bythe proteasome targetis recognized polyubiquitinylated of the Ubgroups, to driveremoval whichusesATPhydrolysis proteinintothe of the unfolded andtransfer unfolding, the shortpeptide which from in the core, proteolysis chamber (a)fromw (stepE) lPart arelaterreleased fragments - U b - U b - U b n + 1 digestion of W Baumeister courtesy l etal, 1998,Cell92.357; Baumeister

E

E 1 = u b i q u i t i n - a c t i v a t i negn z y m e E 2 = u b i q u i t i n - c o n j u g a t i negn z y m e E 3 = u b i q u i t i nl i g a s e Ub = u[iqui1i6

I

s ti,iii;J;i,' t

ATPrl

)s

There are severaldistinct regulatory cap complexeswith different activities.The 195 cap has 16-18 protein subunits'6 Release Recognition of which can hydrolyze ATP (i.e., they are ATPases)to prouo U b the energy needed to unfold protein substratesand vide uu ,,n -".j0 transferthem into the inner chamber of the proteaselectively uo ub some. Genetic studiesin yeast have shown that cells cannot \ unfotding survive without functional proteasomes,thus demonstrating orrJ their importance. Furthermore, proper proteasomal activity is so important that cells will expend as much as 30 percent \ cleauage of the energy neededto synthesizea protein to degradeit in

ADP
t-/

\

Dir"hurg"

called autophagy (seeFigure 9-2)-and toward extracellular proteins taken up by the cell. Lysosomeswill be discussedat length in later chapters. Here we will focus on cytoplasmic protein degradationby proteasomes.

l s a C o m p l e xM o l e c u l a r TheProteasome MachineUsedto DegradeProteins Proteasomesare very large macromolecularmachinesconsisting of =50 protein subunitsand having a massof 2-2.4 ', 1'06 Da. They have a cylindrical, barrel-like catalytic core called the 20S protedsome(where S is a Svedbergunit basedon the sedimentationproperties of the particle and is proportional to its size).Bound to the ends of this core are one or two cap complexesthat regulate proteasomal activity. There are approximately 30,000 proteasomesin a typical mammalian cell. There are multiple forms of proteasomes.The best studied of theseis the 265 proteasome(Figure3-29a1,which has a catalyticcore approximately14.8 nm tall and 11.3 nm in diameter and a 19S cap regulatory particle at each end.

a Droteasome. The proteasomal catalytic core comprises two inner rings, with six proteolytic active sitesfacing toward the inner chamber of the =1..7-nm-diameterbarrel' and two outer rings that control substrateaccess.Proteasomescan degrade most proteins thoroughly becausethey have active sites(two each) that cleaveafter hydrophobic residues,acidic residues, and basic residues. Polypeptide substrates must enter the chamber via a regulated aperture at the center of the outer rings. In the 265 proteasome,the opening of the aperture, which is narrow and often allows the entry of only unfolded proteins, is controlled by ATPasesin the 195 cap. The short peptide products of proteasomal digestion (2-24 residues long) exit the chamber and are further degradedrapidly by cytosolic peptidases,eventually being converted to individual amino acids. Some have quipped that a proteasomeis a "cellular chamber of doom" in which proteins suffer a "death of a thousandcuts'" Inhibitors of proteasomefunction can be used therapeutically.Becauseof the global importance of proteasomes for cells, continuous, complete inhibition of proteasomeskills cells. However, partial' discontinuous p.ot.u.o-. inhibition has been introduced as an approach io .".r.., chemotherapy.To survive and groq cells normally DEGRADATION l: PROTEIN FUNCTION R E G U L A T I NPGR O T E I N

87

require the robust activity of a regulatory protein called NF^B, as well as other similar "pro-survival" proteins. In turn, NF*B can function fully and promote survival only when its inhibitor, I^B, is disengagedand degradedby proteasomes(Chapter 16). Partial inhibition of proteasomal activity by a small-moleculeinhibitor drug results in increasedlevels of I*B and, consequently,reducedNF^B activity (loss of pro-survival activity). Cells subsequentlydie by a mechanismcalled apoptosis (programmedcell death, Chapter 21). Becauseat leasrsome types of tumor cells are more sensitive to being killed by proteasome inhibitors than normal cells are, controlled administration of proteasome inhibitors (at levelsthat kill the cancer cells but not normal cells) has proved ro be an effectivetherapy for at least one type of lethal cancer,multiple myeloma. I

U b i q u i t i nM a r k sC y t o s o l i cP r o t e i n sf o r Degradationin Proteasomes If proteasomesare to rapidly degradeonly thoseproteins that are either defectiveor scheduledto be removed,they must be able to distinguish between those proteins that need to be degradedfrom most of the proteins that don't. To solve this problem, cells identify proteins that should be degraded by covalently attaching multiple copiesof a76-residuepolypeptide called ubiquitin that is highly conservedfrom yeast to humans. A complex sensingsystemhas evolved to determine which proteins are to be degraded,and then a three-step processis usedto polyubiquitinylatethe targetproteins.The 195 regulatory cap of the 265 proteasomethen recognizes the ubiquitin-labeledproreins, and unfolds and transports them into the proteasomefor degradation. The ubiquitination process(Figure 3-29b) involves: 1. Activation of wbiquitin-actiuating enzyme(E1)by the addition of a ubiquitin molecule,a reactionthar requires ATP 2. Transfer of this ubiquitin molecule to a cysteineresidue in ubiquitin- conjugating enzyme (E2) 3. Formation of an isopeptidebond betweenthe carboxyl terminus of the ubiquitin bound to E2 and the amino group of the side chain of a lysine residuein rhe targer prorein, a reaction catalyzedby ubiquitin-protein ligase (E3). Subsequent ligasereactionscovalently attach additional ubiquitins to the side chain of lysine 48 of the previously added ubiquitin to generatea linear polymer of ubiquitins, or a polyubiquitin-modifiedtargetprotein.

Specificity of Degradation Thrgetingof specificproteinsis primarily achievedthrough the substrate specificity of the E3 ligase.Thereare hundredsof E3 ligasesin mammaliancells,ensuring that the wide variety of proteins to be polyubiquitinylated can be modified when necessary. An example of the control of the activity of a key cellular protein by the ubiquitin-proteasomesystem is the regulated 88

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degradationof proteins called cyclins,which control the cell cycle (Chapter 20). Cyclins contain the internal sequence Arg-X-X-Leu-Gly-X-Ile-Gly-Asp/Asn (X can be any amino acid), which is recognizedby specificubiquitinylating enzyme complexes.At a specifictime in the cell cycle, each cyclin is phosphorylated by a cyclin kinase. This phosphorylation is thought to causea conformational change that exposesthe recognition sequenceto the ubiquitinylating enzymes,leading to polyubiquitination and proteasomaldegradation. Multifunctional Ubiquitin Tagging Someubiquitination performs cell functions other than the degradation of a t^rgetedprotein. Examplesof alternativeubiquitination schemes include (1) the covalentaddition of a singleubiquitin molecule (monoubiquitination) to a lysine on a rarget protein, (2) the addition of multiple single ubiquitins (multiubiquitination), (3) Iinking the ubiquitin to rhe N-terminus of the target protein, and (4) polyubiquitination in which the ubiquitins are linked to one another via their Lys-63residueinsteadof at the Lys-48 position. These modifications can influence the trafficking (sorting) of proteins within a cell (e.g.,internalization from the cell surface),control DNA repair and regulation of transcription,and undoubtedlyperform numerousother functions yet to be discovered.Cells also have a variery of deubiquitinylating enzymes that can remove ubiquitins from the targetproteinsand thus introducethe possibilityin somecases of reversingthe regulationcausedby the initial ubiquitination.

Regulating Protein Function l: Protein Degradation r Proteins may be regulated at the level of protein synthesis,protein degradation,or the intrinsic activity ofproteins through noncovalent or covalent interactions. r The life span of intracellular proteins is largely determined by their susceptibilityto proteolytic degradation. r Many proteins are marked for destruction with a polyubiquitin tag and then degradedwithin proteasomes,large cylindrical complexeswith multiple proreasesin their interiors (seeFigure3-29). r Variations in the nature of the covalent attachment of ubiquitin to proteins are involved in cellular functions other than proteasome-mediateddegradation, such as changesin the location or activity of proteins.

RegulatingProteinFunctionll: f[ Noncovalent and CovalentModifications The intrinsic activities of proteins are modulated by both noncovalent and covalent changesin the protein. Noncovalent modifications usually involve the binding or dissociation of a molecule and a consequentchangein the conformation of the protein. Often, in such cases,protein activation involves the releaseor rearrangementof an inhibitory subunit or domain.

Covalentmodifications include hydrolysisof the polypeptide chain or addition of a molecule to the side chain of one or more residuesor to the N- or C-terminus of the protein. Such modifications can cause a conformational change in the protein that can alter its activity (form is function). Covalent modifications can also modify the shape of a protein without changing the conformation of the polypeptide and its side chains, for example, by adding a charge or bulky group that can alter the ability of the protein to bind to other molecules.Lastly, covalent modifications can direct the protein to particular locations in a cell (e.g.,the cytoplasmic surface of the plasmamembrane). Many noncovalent and covalent modifications are reversible,thus allowing the activity of an individual protein to be enhancedor suppressedmultiple times during the lifetime of the protein. Others,suchas proteolysis,are irreversible and can be supersededonly by degradationof the modified protein and synthesis of a replacement. In the case of enzymes,these regulatory modifications alter K-, V-"*, or both. Nature has devisedmany different strategiesfor noncovalent and covalent regulation of activity. Here we discuss some common mechanisms for regulating protein function; additional examples will be describedin other chapters.

N o n c o v a l e nB t i n d i n gP e r m i t sA l l o s t e r i c , or Cooperative,Regulationof Proteins One of the most important mechanismsfor regulatingprotein function is through allosteric interactions. Broadly speaking,allostery (from the Greek "other shape") refers to any changein a protein's tertiary or quaternary structure, or in both, induced by the noncovalent binding of a ligand. lfhen a ligand binds to one site (A) in a protein and induces a conformational changeand associatedchangein activity of a different site (B), the ligand is called an allosteric effector of the protein, while site A is called an allosteric binding site, and the protein is called an allosteric protein. By definition, allostericproteins have multiple binding sitesfor either a single type of ligand or for multiple different ligands. The allosteric change in activity can be positive or negative' i.e., can induce an increaseor a decreasein protein activity. Allosteric regulation is particularly prevalent in multimeric enzymesand other proteins where conformational changes in one subunit are transmitted to an adiacent subunit. Cooperatiuity is a term often used synonymously with allostery,and usuallyrefersto the influence(positiveor negative) that the binding of a ligand at one site has on the binding of another moleculeof the sametype of ligand at a different site. Hemoglobin presentsa classicexample of positive cooperative binding in that the binding of a singleligand, oxygen, increasesthe affinity of the binding of the next oxygen molecule.Each of the four subunits in hemoglobin contains one heme molecule. The heme groups are the oxygen-binding componentsof hemoglobin(seeFigure3-13).The binding of oxygen to the heme molecule in one of the four hemoglobin subunits inducesa local conformational changewhose effect

c =5U U)

a

0

20 4 It

40 60 80 Poz(torr)

p O 2i n c a P i l l a r i e s of activemuscles

100 t

P O 2i n a l v e o l i of lungs

3-30 Hemoglobinbindsoxygen FIGURE a EXPERIMENTAL proteinhasfour hemoglobin Eachtetrameric cooperatively. allthesitesareloadedwith sites;at saturation oxygen-binding asthe measured iscommonly concentration Theoxygen oxygen. the oxygen(pOz) pO2 half at which is Pso the partialpressure it is areoccupied; concentration bindingsitesat a givenhemoglobin Thelarge reaction. to the K' for an enzymatic analogous somewhat changein theamountof oxygenboundovera smallrangeof pO2 tissues of oxygenin peripheral permits unloading efficient values percent plot saturation of of a shape The sigmoidal muscle suchas bindingIn of cooperative isindicative ligandconcentration versus a bindingcurveisa hyperbola, binding, of cooperative the absence 1995, fromL Stryer, 3-22 [Adapted in Figure to thecurves similar Company.l Freeman and W H ed, Biochemistru,4th

spreadsto the other subunits, lowering the K- (increasing the affinity) for the binding of additional oxygen molecules to the remaining hemes and yielding a sigmoidal oxygenbinding curve (Figure3-30)' Becauseof the sigmoidalshape of the oxygen-saturationcurve, it takes only a fourfold increasein oxygen concentration for the percent saturation of the oxygen binding sitesin hemoglobin to go from 10 to 90 percent- Conversely,if there were no cooperativity and the shapeof the curve was typical of that for Michaelis-Mententype binding, it would take an 81-fold increase in oxygen concentration to accomplish the same increasein loading. This cooperativity permits hemoglobin to take up oxygen very efficiently in the lungs where the oxygen concentration is high, and unload it in tissueswhere the concentration is low. Thus, cooperativity amplifies the sensitivity of a system to concentration changesin its ligands, providing in many casesselectiveevolutionary advantage. Negative cooperativity often involves the end product of a multistep biochemical pathway, which binds to and reducesthe activity of an enzymethat catalyzesan early, ratecontrolling step for that pathway. In this way excessive buildup of the product is prevented.This kind of regulation of a metabolic pathway is also called end-product inhibition or feedback inhibition.

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89

N o n c o v a l e nB t i n d i n go f C a l c i u ma n d G T pA r e Widely UsedAs AllostericSwitchesto Control ProteinActivity

( a ) C a l m o d u l i nw i t h o u t c a l c i u m

Unlike oxygen, which causesgraded allostericchangesin the activity of hemoglobin, other allosteric effectors act as switches,turning the activity of many different proteins on or off. Two important allosteric switches that we will encounter many times throughout this book are Ca2* and GTP. Ca2+/Calmodulin-Mediated Switching The concentration of Ca2* fteein the cytosol (not bounJto moleculesother than water) is kept very low (=10 7 M) by specializedmembrane transport proteins that continually pump Ca2+ out of the^cytosol.However,as we learn in Chapter 11, the cytosolic Ca'- concentrationcan increasefrom 10- to 100-fold when Ca2*-permeablechannelsin the cell surfacemembranesopen and allow extracellularCa2* ro flow into the cell. This risi in cytosolicCa2* is sensedby specializedCa2*-binding proteins, which alter cellular behavior by turning other prorelns on or off. The importanceof extracellular Ca2* for cell activity was first documentedby S. Ringer in 1883, when he discovered that isolated rat hearts suspendedin a NaCl solution made with 'hard' (Ca2*-rich) London rap warer contracted beautifully whereasthey beatpoorly and stoppedquickly if distilled water was used. Many of the Ca2*-bindingproteinsbind Ca2* using the EF hand/helix-loop-helix structural motif discussedearlier (seeFigure3-9b). The prototype EF hand prorein, calmodulin, is found in all eukaryotic cells and may exist as an individual monomenc protein or as a subunit of a multimeric protein. A dumbbell-shapedmolecule, calmodulin contains four Ca'--binding EF hands with K6's of =10-6 M. The binding of Ca2* to calmodulin causesa conformational changethal permits Ca2*/calmodulin to bind to conservedsecuencesin various target proteins, thereby swirching their activities on or off (Figure 3-31). Calmodulin and similar EF hand proteins thus function as sulitch proteins, acting in concert with changes in Ca2* levels to modulate the activity of other proteins. Switching Mediated by Guanine Nucleotide-Binding Proteins Another group of intracellular switch proteins constitutes the GTPase superfamily. As the name suggests,these proteins are enzymes, GTPases, that can hydrolyze GTp (guanosinetriphosphate) to GDp (guanosinediphosphate). They include the monomeric Ras protein (seeFigure 3-8) and the G. subunit of the trimeric G proteins, both discussedat length in Chapter 15. Both Ras and Go can bind to the plasma membrane,function in cell signaling,and play a key role in cell proliferation and differentiation. Other membersof the GTpase superfamily function in protein synthesis,the transport of proteins between the nucleus and the cytoplasm, the formation of coated vesiclesand their fusion with target membranes,and rearrangemenrsof the actin cytoskeleton.The Hsp70 chaperone proteln we encounteredearlier is an example of an ATp/ADp switch, similar in many respectsto a GTp/GDp switch.

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(bl Caz*/calmodulinbound to target peptide

A FIGURE 3-31 Conformational changesinducedby Ca2* bindingto calmodulin.Calmodulin isa widelydistributed cytosolic proteinthatcontains fourCa2*-brnding sites,onein eachof itsEF handsEachEFhandhasa helix-loop-helix motif At cytosolic Ca2+ concentrations aboveabout5 x 10-7M, bindingof Ca2*to calmodulin changes the protein's conformation fromthe dumbbellshaped, unboundform(a)to onein whichhydrophobic sidecharns becomemoreexposed to solvent. Theresulting Ca2*/calmodulin can wraparoundexposed (b),thereby helices of various targetproteins altering theiractivity.

AII the GTPase switch proteins exist in two forms, or conformations(Figure 3-32):(1) an active("on")form with bound GTP that modulates the activity of specific target proteins to which they bind and (2) an inactive ( ,.off ', ) form with bound GDP, which is generatedby the relatively slow hydrolysis of the GTP bound to the active form. The amount of time any given GTPaseswitch remains active depends on the rate of its GTPase activity. Thus the GTpase activity acts as a timer to control this switch. Cells contain a variety of proteins that can modulate the baseline (or intrinsic) rate of GTPase activity for any given GTpase switch. For example, GTPase activity can be enhanced by specific GTPase-activating proteins, called GAPs, or depressedby other proteins acting as allosteric regulators. After the switch has beenturned off (GTP hydrolysis), it can be turned back on by GTP exchange factor (GEF), which replacesthe bound GDP with a different GTp molecule from the surrounding fluid. Thus, cells can control when the switch is turned on and how long the switch remains on. 'We examine the role of various GTPase switch proteins in regulating intracellular signaling and other processesin several later chaoters.

Active ("on")

GAPs nGSs GDls

* <* *

GEFs4+ Inactive("off")

3-32 The GTPaseswitch. Conversion of the active, A FIGURE intothe inactive formby hydrolysis of GTPis GTP-bound GTPase proteins) andRGSs accelerated by GAPs(GTPase-activating (regulators of G proteinsignaling) andinhibited by GDls(guanine inhibitors) Reactivation by replacing GDPwith nucleotide dissociation (guanine factors). by GEFs nucleotide exchange GTPis promoted

P h o s p h o r y l a t i oann d D e p h o s p h o r y l a t i o n CovalentlyRegulateProteinActivity One of the most common mechanismsfor regulating protein activity is phosphorylation, the addition of phosphate groups to hydroxyl groups on serine,threonine, or tyrosine residues. Protein kinases catalyze phosphorylation, and phosphatases catalyze deph osph orylation. The counteracting activities of kinasesand phosphatasesprovide cells with a "switch" that can turn on or turn off the function of various proteins (Figure3-33). Phosphorylationchangesa protein's chargeand generallyleadsto a conformational change; these effects can significantly alter ligand binding or other featuresof the protein, leading to an increaseor decreasein lts actlvlty.

Active

o tl

R-O-P-O-

Hzo Protein phosphatase

P.

R-OH Inactive

3-33 Regulationof protein activity by the A FIGURE and kinase/phosphatase switch.Thecyclicphosphorylation mechanism for of a proteinisa commoncellular dephosphorylation proteinactivityIn thisexample, the targetproteinR is regulating (bottom) when andinactive active(top)whenphosphorylated responses to havetheopposite someproteins dephosphorylated; phosphorylation

Nearly 3 percent of all yeast proteins are protein kinases or phosphatases,indicating the importance of phosphorylation and dephosphorylation reactions even in simple cells. All classes of proteins-including structural proteins, scaffolds, enzymes, membrane channels' and signaling molecules-have members regulated by kinase/phosphatase switches.Different protein kinases and phosphatasesare specific for different target proteins and can thus regulate a variety of cellular pathways, as discussedin later chapters. Some of these enzymesact on one or a few target proteins, whereas others have many targets. The latter are useful in integrating the activities of proteins that are coordinately controlled by a single kinase/phosphatase switch. Frequently,the target of the kinase (and phosphatase)is yet another kinase or phosphatase' creating a cascade effect. There are many examples of such kinase cascades,which permit amplification of a signal and many levels of finetuning control (seeChapter 15).

ProteolyticCleavagelrreversiblyActivatesor lnactivatesSomeProteins Unlike phosphorylation, which is reversible, the activation or inactivation of protein function by proteolytic cleavageis an irreversible mechanism for regulating protein activity. For example,many polypeptidehormones,such as insulin, are synthesizedas long precursors, and prior to secretion from cells some of their peptide bonds must be hydrolyzed for them to fold properly. In some cases'a single long precursor prohormone polypeptidecan be cleavedinto several distinct active hormones. To prevent the pancreatic serine proteasesfrom inappropriately digesting proteins before they reach the small intestines, they are synthesizedas eymogens, inactive precursor proteins. Cleavageof a peptide bond near the N-terminus of trypsinogen (the zymogen of trypsin) by a highly specific protease in the small intestine generatesa new N-terminal residue (Ile-16), whose amino group can form an ionic bond with the carboxylic acid side chain of an internal aspartic acid. This causesa conformation changethat opens the substrate-bindingsite' activating the enzyme. The active trypsin can then activate trypsinogen, chymotrypsinogen, and other zymogens. Similar' but more elaborate,proteasecascades(one proteaseactivating inactive precursorsof others) that can amplify an initial signal play important roles in several systems' such as the blood-clotting cascade.The importance of carefully regulating such systemsis clear-inappropriate clotting could fatally clog the circulatory system, while insufficient clotting could lead to uncontrolledbleeding. An unusual and rare type of proteolytic processing' termed protein self-splicing,takes place in bacteria and some eukaryotes.This processis analogousto editing film: an internal segmentof a polypeptide is removed and the ends of the polypeptide are reioined (ligated). Unlike other forms of proteolytic processing, protein self-splicing is an autocatalytic process,which proceedsby itself without the participation of enzymes.The excisedpeptide appearsto eliminate itself from the protein by a mechanismsimilar to that usedin

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the processingof some RNA molecules (Chapter 8). In vertebrate cells, the processingof some proteins includes selfcleavage,but the subsequentligation step is absent. One such protein is Hedgehog, a membrane-bound signaling molecule that is critical to a number of develoomental (Chapter 15). processes

H i g h e r - O r d eRr e g u l a t i o nI n c l u d e sC o n t r o l of ProteinLocationand Concentration All the regulatory mechanismsheretofore describedaffect a protein locally at its site of action, turning its activity on or off. Normal functioning of a cell, however, also requires the segregationof proteins to particular compartments such as the mitochondria, nucleus, and lysosomes. In regard to enzymes,compartmentationnot only provides an opportunity for controlling the delivery of substrateor the exit of product, but also permits compering reactions to take place simultaneously in different parts of a cell. We describethe mechanismsthat cells use to direct various proteins to different compartmenrs in Chapters 12 and 13.

Protein Regulation ll: Noncovalent and Covalent Modifications r In allostery,the noncovalentbinding of one ligand mol, ecule, the allosteric effector, induces a conformational change that alters a protein's activity or affinity for other ligands. The allosteric effector can be identical in structure to or different from the other ligands, whose binding it affects.The allostericeffector can be a substrate.acuvaror.or inhibitor. r In multimeric proteins, such as hemoglobin, that bind multiple identicalligand molecules(e.g.,oxygen),the binding of one ligand molecule may increase or decreasethe binding affinity for subsequentligand molecules.This type of allosteryis known as cooperativiry. r Several allosteric mechanisms act as switches, turning protein activity on and off in a reversiblefashron. r Two classesof intracellular switch proteins regulatea variety of cellular processes: (1) Ca2+-bindingproteins (e.g., calmodulin) and (2) members of the GTpase superfamily (e.g.,Ras), which cycle between active GTP-bound and inactiveGDP-boundforms (seeFigure 3-32). r The phosphorylation and dephosphorylationofhydroxyl groups on serine,threonine, or tyrosine residueside chains by protein kinases and phosphatasesprovide reversible on/off regulation of numerous proteins. I Many types of covalent and noncovalent regulation are reversible, but some forms of regulation, like proteolytic cleavage,are irreversibre. r Higher-order regulation includes compartmentation of proteins and control of protein concentration.

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fp

Purifying, Detecting, and

Characterizing Proteins A protein often must be purified before its structure and the mechanism of its action can be studied in detail. However, becauseproteins vary in size, charge, and water-solubility, no single method can be used to isolate all proteins. To isolate one particular protein from the estimated 10,000 different proteins in a particular type of cell is a daunting task that requires methods both for separating proteins and for detectingthe presenceof specificproteins. Any molecule,whether protein, carbohydrate,or nucleic acid, can be separated,or resolued,from other moleculeson the basisof their differencesin one or more physical or chemical characteristics.The larger and more numerous the differences between two proteins, the easier and more efficient their separation.The two most widely usedcharacteristicsfor separatingproteins are size,defined as either length or mass, and binding affinity for specific ligands. In this section, we briefly outline several important techniques for separating proteins; these separation techniquesare also useful for the separationof nucleic acids and other biomolecules.(Specialized methods for removing membrane proteins from membranesare describedin Chapter 10 after the unique properties of theseproteins are discussed.)\Wethen consider the use of radioactive compounds for tracking biological activity. FinallS we considerseveraltechniquesfor characterizinga protein's mass,sequence,and three-dimensionalstructure.

CentrifugationCan SeparateParticlesand MoleculesThat Differ in Massor Density The first step in a typical protein purification schemeis centrifugation. The principle behind centrifugation is that two particles in suspension(cells, cell fragments, organelles,or molecules)with different massesor densitieswill settleto the bottom of a tube at different rates. Remember,mass is the weight of a sample (measuredin grams), whereas density is the ratio of its weight to volume (grams/liter).Proreinsvary greatly in mass but not in density.Unlessa protein has an attached lipid or carbohydrate, its density will not vary by more than 15 percentfrom I .37 glcm3,rhe averageprotein density.Heavier or more densemoleculessettle,or sediment. more quickly than lighter or lessdensemolecules. A centrifuge speedssedimentationby subjectingparticles in suspensionto centrifugal forces as great as 1,000,000 times the force of gravity g, which can sedimentparticles as small as 10 kDa. Modern ultracentrifugesachieve these forces by reaching speedsof 150,000 revolutions per minute (rpm) or greater.However, small particles with massesof 5 kDa or lesswill not sedimentuniformly even at such high speeds. Centrifugation is used for two basic purposes: (1) as a preparativetechniqueto separateone type of material from others and (2) as an analytical technique to measurephysical properties (e.9., molecular weight, densiry shape, and equilibrium binding constants) of macromolecules.The sedimentation

(a) Differentialcentrifugation I

Sample is poured into tube

(b) Rate-zonalcentrifugation I

Sample is layered on top of density gradient

Largerparticle Largerparticle

S m a l l e rp a r t i c l e

S m a l l e rp a r t i c l e Low density (low sucrose concentration) Centrifuge Particlessettle accordingto mass

Centrifugal+

Sucrose gradient

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High density ( h i g hs u c r o s e concentration)

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E

Stop centrifuge Collect fractions and do assay

HI

FIGURE EXPERIMENTAL 3-34 Centrifugation techniques separateparticlesthat differ in massor density.(a)In differential a cellhomogenate or othermixtureisspunlong centrifugation, (eg , cellorganelles, cells), the largerparticles enoughto sediment asa pelletat the bottomof thetube(stepE) The whichcollect (e g , soluble proteins, particles acids) remainin the nucleic smaller tube(stepB) whichcanbetransferred to another liquidsupernatant,

constant s of a protein is a measureof its sedimentationrate. The sedimentationconstant is commonly expressedin svedbergs (S), where a typical, large protein complex is about 3-5S, while a eukaryoticribosomeis 80S. The most common initial step Differential Centrifugation purification for tissuesis the separation of protein cells or in water-solubleproteins from insolublecellular material by differential centrifugation. A starting mixture, commonly a cell homogenate(mechanicallybroken cells),is poured into a tube and spun at a rotor speedand for a period of time that forces cell organellessuch as nuclei and large unbroken cells or large cell fragments to collect as a pellet at the bottom; the soluble proteinsremain in the supernatant(Figure3-34a1.The super-

massof Particles Decreasing (b)In rate-zonal a mixtureisspun(stepE) justlong centrifugation, thatdifferin massbut maybe similar molecules enoughto separate into (e.g, globular proterns, RNAmolecules) in shapeanddensity formedby a gradient commonly zoneswithina density drscrete fromthe areremoved Fractions solution. sucrose concentrated to testing(assayed). bottomof the tubeandsubjected

natant fraction then is poured off' and either it or the pellet can be subjectedto other purification methods to separatethe many differentproteinsthat they contain. On the basisof differencesin Rate-Zonal Centrifugation their masses, proteins can be separated by centrifugation through a solution of increasingdensity called a density gradient. A concentratedsucrosesolution is commonly usedto form density gradients.'Whena protein mixture is layeredon top of a sucrosegradient in a tube and subjectedto centrifugation, each protein in the mixture migrates down the tube at a rate conirolled by the factors that affectthe sedimentationconstant.All the proteins staft from a thin zone at the top of the tube and ,.pui"t. into bands, or zones (actually, disks)' of proteins of P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

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{lttt Technique Animation:SDSGel Electrophoresis [

< EXPERIMENTAL FIGURE 3-35 SDS-polyacrylamide gel (SDS-PAGE) electrophoresis separatesproteinsprimarilyon the (a)Initialtreatment basisof their masses. with SDS,a negatively charged detergent, proteins dissociates multimeric anddenatures all (steptr). Duringelectrophoresis, the polypeptide chains the SDSproteincomplexes migrate gel(stepE). throughthe polyacrylamide Smallcomplexes areableto movethroughthe poresfasterthan largeronesThusthe proteins separate intobandsaccording to their proteinbandsarevisualized sizes astheymigrate. Theseparated by staining with a dyeGtepB). (b)Example of SDS-PAGE separation of (detergent allthe proteins in a whole-cell lysate solublized cells): (/eft)the manyseparate proteins, stained appearing almostasa (right)a proteinpurified continuum; fromthe lysateby a singlestep of antibody-affinity chromatography. Theproteins werevisualized by staining with a silver-based dye lPart(b)modified fromB LiuandM Krieger, 2002,J BiolChem277(31):34125-34135 l

I Denature sample with | sodium dodecylsulfate

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Electrophoresis SeparatesMoleculeson the Basisof Their Charge-to-Mass Ratio (b)

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different masses.In this separationtechnique,called rate-zonal centrifugation, samplesare centrifuged just long enough to separatethe moleculesof interest into discretezones (Figure 3-34b). If a sample is centrifuged for too short a time, the different protein moleculeswill not separatesufficiently. If a sample is centrifuged much longer than necessary,all the proteins will end up in a pellet at the bottom of the tube. Although the sedimentation rate is strongly influenced by particle mass, rate-zonal centrifugation is seldom effective in determining precise molecular weights becausevariations in shape also affect sedimentation rate. The exact effects of shape are hard to assess,especially for proteins or other molecules,such as single-strandednucleic acid molecules,that can assumemany complex shapes.Nevertheless, rate-zonal centrifugation has proved to be the most practical method for separatingmany different types of poiy-"r, and particles.A second density-gradienttechnique,called equilibrium density-gradient centrifugation, is used mainly to separateDNA, lipoproteins that carry lipids through the crrculatory system,or organelles(seeFigure 9-26). 94

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Electrophoresisis a technique for separatingmoleculesin a mixture under the influence of an applied elecric field and is one of the most frequently usedtechniquesto study proteins and nucleic acids. Dissolved molecules in an electric field move, or migrate, at a speeddetermined by their charge-tomass (charge:mass)ratio. For example, if two molecules have the same mass and shape,the one with the greater net charge will move faster toward an electrodeof the opposite polarity. SDS-Polyacrylamide Gel Electrophoresis Becausemany proteins or nucleic acidsthat differ in sizeand shapehave nearly identical charge:massratios, electrophoresisof thesemacromoleculesin solution results in little or no separation of molecules of different lengths.However, successfulsepararionof proteins and nucleic acidscan be accomplishedby electrophoresisin various gels (semisolid suspensionsin water similar to the congealedgelatin found in desserts)rather than in a liquid solution. Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. .il(i'hena mixture of proteins is placedin a gel and an electriccurrent is applied, smallerproteins migrate faster through the gel than do larger proteins because the gel acts as a sieve,with smaller speciesable to maneuver more rapidly through the pores in the gel than larger species. The shapeof a moleculecan also influence its rate of migration (long asymmetricmoleculesmigrate more slowly than spherical onesof the samemass). Gels are cast betweena pair of glassplates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chains into a semisolidmatrix. The pore size of a gel can be varied by adjusting the concentrations of polyacrylamide and the crossIinking reagent.The rate at which a protein moves through a gel is influenced by the gel's pore size and the strength of the electric field. By suitable adjustment of theseparamerers,

proteins of widely varying sizescan be resolved(separated from one another) by polyacrylamide gel electrophoresis (PAGE). In the most powerful technique for resolving protein mixtures, proteins are exposedto the ionic detergentSDS (sodiumdodecylsulfate)beforeand during gel electrophoresis (Figure3-35). SDSdenaturesproteins,in part becauseit binds to hydrophobic side chains, destabilizing the hydrophobic interactions in the core of a protein that contribute to its stableconformation. (SDStreatmentis usually combined with heating in the presenceof reducing agents that break disulfide bonds.) As a consequence,multimeric proteins dissociateinto their subunits,and all polypeptide chains are forced into extended conformations with similar charge:massratios. SDS treatment thus eliminates the effect of differencesin shapein native structures;therefore, chain length, which correspondsto mass, is the principal determinant of the migration rate of proteins in SDSpolyacrylamide electrophoresis(SDS-PAGE).Even chains that differ in molecular weight by lessthan 10 percentcan be resolved by this technique. Moreover, the molecular weight of a protein can be estimatedby comparing the distance that it migratesthrough a gel with the distancesthat proteins of known molecular weight migrate (there is roughly a linear relationship between migration distance and the log of the molecular weight). Proteins within the

(a)

Protein mixture

Separate in first dimension by charge

E

gelscan be extractedfor further analysis(e.g.'identification by methodsdescribedbelow). of Two-Dimensional Gel Electrophoresis Electrophoresis all cellularproteinsby SDS-PAGEcan separateproteinshaving relatively large differencesin mass but cannot readily resolve proteinshaving similar masses(e.g.,a 41-kDa protein versusa 42-kDa protein). To separateproteins of similar masses'another physical characteristicmust be exploited. Most commonlg this characteristicis electric charge' which is determined by the pH and the relative number of the protein's positively and negatively charged groups, which is in turn dependenton the pKu'sof the ionizablegroups(seeChapter2). Two unrelated proteins having similar massesare unlikely to have identical net chargesbecausetheir sequences,and thus the number of acidic and basicresidues,are different. In two-dimensionalelectrophoresis,proteins are separated sequentially,first by their charges and then by their masses(Figure3-36al.In the first step'a cell or tissueextract is fully denatured by high concentrations (8 M) of urea and then layered on a gel strip that contains a continuous pH gradient. SDS cannot be used, becauseits binding changes the charge of the protein. The gradient is formed by ampholytes, a mixture of polyanionic and polycationic molecules, that are cast into the gel' with the most acidic ampholyte at one end and the most basic ampholyte at the

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s lil,i??"?l" by size FIGURE3-36 Two-dimensionalgel A EXPERIMENTAL proteins on the basis of charge and separates electrophoresis mass.(a) In this technique,proteinsarefirst separatedinto bandson focusing(step[) The the basisof their chargesby isoelectric resultinggel strip is appliedto an SD5-polyacrylamide 9el (stepZ), (step B) (b) In this mass and the proteinsare separatedinto spotsby

cells,each fromcultured gelof a proteinextract two-dimensional by canbe detected Polypeptides a singlepolypeptide spotrepresents Each as autoradiography such dyes,ashere,or by othertechniques point(pl)andmolecular by itsisoelectric polypeptide ischaracterized (b)courtesy of J Celis ] weiqht IPart

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opposite end. A charged protein will migrate through the gradient until ir reachesits isoelectricpoint (pI), the pH at which the net charge of the protein is zero. This technique, called isoelectric focusing (IEF), can resolve proteins that differ by only one charge unit. Proteins that have been separated on an IEF gel can then be separatedin a second dimension on the basis of their molecular weights. To accomplish this separation,the IEF gel strip is placed lengthwiseon one outside edge of a sheetlike (two-dimensional, or slab) polyacrylamide gel, this time saturated with SDS. When an electric field is imposed, the proteins will migrate from the IEF gel into the SDS slab gel and then separateaccording to their masses. The sequential resolution of proteins by charge and mass can achieve excellent separation of cellular proteins (Figure 3-35b). For example, two-dimensional gels have been very useful in comparing the proteomes in undifferentiated and differentiated cells or in normal and cancer cells becauseas many as 1000 proteins can be resolvedas individual spots simultaneously.Sophisticatedmethods have been developed to permit the comparison of complex patterns of proteins in two-dimensional gels from related, but distinct,samples(e.g.,tissuefrom a normal versusa mutant individual) to permir identification of differencesin rhe types or amounts of proteins in the samples (seesection on proteomics,below).

Liquid ChromatographyResolvesproteins b y M a s s ,C h a r g e o , r B i n d i n gA f f i n i t y A third common technique for separating mixtures of proteins or fragments of proteins, as well as other molecules,is basedon the principle that moleculesdissolvedin a solution can differentially interact (bind and dissociate)with a particular solid surface, depending on the physical and chemical properties of the molecule and the surface.If the solution is allowed to flow across the surface, then molecules that interact frequently with the surface will spend more time bound to the surface and thus flow past the ,,rrfac. -or. slowly than molecules that interact infrequently with it. In this technique, called liquid chromatography (LC), the sample is placed on rop of a tightly packed column of spherical beadsheld within a glassor plastic cylinder.The samplethen flows down the column, usually driven by gravitational or hydrostatic forces alone or with the assistanceof a pump, and small aliquots of fluid flowing out of the column, called fractions, are collected sequentially for subsequentanalysis for the presenceof the proteins of interest.The nature of the beads in the column determineswhether the separation of proteins dependson differencesin mass, charge, or binding affinity. Gel Filtration Chromatography proteins that differ in masscan be separatedon a column composedof porous beads made from polyacrylamide, dextran (a bacterial polysaccharide), or agarose(a seaweedderivative)-a technique called gel filtration chromatography. Although proteins flow around the spherical beads in gel filtration chromatography they spend 96

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some time within the large depressionsthat cover a bead'ssurface. Becausesmaller proteins can penetrate into thesedepressions more readily than larger proteins can, they travel through a gel filtration column more slowly than larger proteins (Figure 3-37a). (In contrast, proteins migrate throwgh the pores in an electrophoreticgel; thus smaller proteins move faster than larger ones.) The total volume of liquid required to elute (or separateand remove) a protein from a gel filtration column depends on its mass: the smaller the mass, the more time it is trapped on the beads,the greater the elution volume. By use of proteins of known mass as standards to calibrate the column, the elution volume can be usedto estimatethe massof a protein in a mixture. A protein's shapeas well as its masscan influence the elution volume. lon-Exchange Chromatography In ion-exchangechromatographg a secondtype of liquid chromatography proteins are separatedon the basis of differencesin their charges.This techniquemakesuseof speciallymodified beadswhose surfaces are coveredby amino groups or carboxyl groups and thus carry either a positive charge (NHr*) or a negativecharge(COO ) at neutral pH. The proteins in a mixture carry various net charges at any given pH. When a solution of a protein mixture flows through a column of positively charged beads,only proteins with a net negative charge (acidic proteins) adhere to the beads; neutral and positively charged (basic) proteins flow unimpeded through the column (Figure 3-37b). The acidic proteins are then eluted selectivelyfrom the column by passing a solution of increasingconcentrationsof salt (a salt gradient) through the column. At low sah concentratrons, protein moleculesand beads are attracred by their opposite charges. At higher salt concentrations, negative salt ions bind to the positively charged beads, displacing the negatively charged proteins. In a gradient of increasing salt concentration, weakly bound proteins, those with relatively low charge, are eluted first and highly charged proteins are eluted last. Similarly, a negatively charged column can be used to retain and fractionate basic (positively charged) protelns. Affinity Chromatography The ability of proteins to bind specifically to other molecules is the basis of affinity chromatography. In this technique, ligand or orher molecules that bind to the protein of interest are covalently attached to the beads used to form the column. Ligands can be enzyme substrates,inhibitors or their analogues,or other small moleculesthat bind to specificproteins. In a widely usedform of this techniqu e-anti b o dy -aff in ity, or immu n oaffinity, ch r o matography-the attached molecule is an antibody specific for the desiredprotein (Figure 3-37c). (\7e discussantibodies as tools to study proteins next). An affinity column in principle will retain only those proteins that bind the molecule attached to the beads; the remaining proteins, regardlessof their charges or masses, will pass through the column because they do not bind. However, if a retained protein is in turn bound to other molecules, forming a complex, then the entire complex is

(c)Antibody-affi nity chromatography

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proteins through column

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FTGURE 3-37 Threecommonlyusedliquid EXPERIMENTAL techniquesseparateproteinson the basisof chromatographic mass,charge,or affinity for a specificbinding partner.(a)Gel proteins thatdifferin sizeA separates chromatography filtration layered on thetop of a cylinder iscarefully mixture of proteins proteins packed travelthroughthe with porousbeadsSmaller proteins columnmoreslowlythanlargerproteinsThusdifferent in theeluateflowingout of the bottomof thecolumnat emerging in canbe collected elutionvolumes) times(different different chromatography tubes,calledfractions(b)lon-exchange separate packed with proteins thatdifferin netchargein columns separates (shown positive or a here) charge carry either a beads that special havingthe samenetchargeasthe beads charge. Proteins negative proteins having whereas andflow throughthe column, arerepelled

chargebindto the beadsmoreor lesstightly, the opposite Boundproteins-inthiscase, on theirstructures. depending a salt elutedby passing subsequently charged-are negatively As the ions the column (usually of NaClor KCI)through gradient (more bound proteins tightly the displace they bindto the beads, in orderto be released). highersaltconcentration proteins require is of proteins a mixture (c)In antibody-affinity chromatography, with beadsto whicha specific passed througha columnpacked attachedOnlyproteinwith highaffinityfor the iscovalently antibody proteins flow by thecolumn;allthe nonbinding isretained antibody the boundproteiniseluted through.Afterthe columniswashed, the with an acidicsolutionor someothersolutionthat disrupts protein flows out of then released the complexes; antigen-antibody is collected and thecolumn

retained on the column. The proteins bound to the affinity column are then eluted by adding an excessof a soluble form of the ligand or by changing the salt concentration or pH such that the binding to the moleculeon the column is

disrupted. The ability of this technique to separatepartrcular proteins dependson the selectionof appropriate binding partners that bind more tightly to the protein of interest than to other proteins. . P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

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H i g h l y S p e c i f i cE n z y m ea n d A n t i b o d yA s s a y s Can DetectIndividualProteins The purification of a protein, or any other molecule, requires a specific assay that can detect the presenceof the molecule of interest as it is separatedfrom other molecules (e.g.,in column or density-gradientfractions or gel bands or spots).An assaycapitalizeson somehighly distinctivecharacteristicof a protein: the ability to bind a particular ligand, to catalyze a particular reaction, or to be recognized by a specific antibody. An assaymust also be simple and fast to minimize errors and the possibilitythat the protein of interest becomesdenaturedor degradedwhile the assayis performed. The goal of any purification scheme is to isolate sufficient amounts of a given protein for study; thus a useful assaymust also be sensitiveenough that only a small proportion of the available material is consumed by it. Many common protein assaysrequire just 10 e to 1.0 12g of material. Chromogenic and Light-Emitting Enzyme Reactions Many assaysare tailored to detect some functional aspectof a protein.For example,enzymaticactivity assaysare basedon the ability to detect the loss of substrateor the formation of product. Some enzymatic assaysutilize chromogenic substrates, which changecolor in the courseof the reaction. (Somesubstrates are naturally chromogenic; if they are not, they can be linked to a chromogenic molecule.)Becauseof the specificiryof an enzyme for its substrate,only samples that contain the enzyme will change color in the presenceof a chromogenic substrate; the rate of the reaction provides a measure of the quantity of enzymepresent.Enzymesthat catalyzechromogenic reactionscan also be fused or chemically linked to an antibody and used to "report" the presenceor location of an antigen to which the antibody binds (seebelow). Antibody Assays As noted earlier, antibodies have the distinctive characteristicof binding tightly and specificallyto antigens.As a consequence,preparations of antibodies that recognizea protein antigen of interest can be generatedand used to detect the presenceof the protein, either in a complex mixture of other proteins (finding a needle in a haystack, as it were) or in a partially purified preparation of a particular protein. The tight binding of the antibody to its antigen, and thus the presenceof the antigen, can be visualized by labeling the antibody with an enzyme)a fluorescent molecule, or radioactive isotopes. For example, luciferase, an enzyme present in fireflies and some bacteria, can be linked to an anribody. In the presenceof ATp and the substrate luciferin, luciferase catalyzesa light-emitting reaction. In either case,after the antibody binds to the protein of in-

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naturally fluorescent protein found in jellyfish (seeFigure 9-12). Alternatively after the first antibody binds to its target protein, a second, labeled antibody is used to bind to the complex of the first antibody and its target. This combination of two antibodiespermits very high sensitivity in the detection of a targetprotein. To generatethe antibodies, the intact protein or a fragment of the protein is injected into an animal (usually a rabbit, mouse, or goat). Sometimesa short synthetic peptide of 10-15 residuesbasedon the sequenceof the protein is used as the antigen to induce antibody formation. A synthetic peptide, when coupled to a large protein carrier, can induce an animal to produce antibodies that bind to that portion (the epitope) of the full-sized, natural protein. Biosynthetically or chemically attaching the epitope to an unrelatedprotein is called epitope tagging. As we'll see throughout this book, antibodies generatedusing either peptide epitopes or intact proteins are extremely versatilereagentsfor isolating, detecting,and characterizingprotelns. Detecting Proteins in Gels Proteins embedded within a one- or two-dimensionalgel usually are not visible. The two general approachesfor detecting proteins in gels are either to Iabel or stain the proteins while they are still within the gel or to electrophoreticallytransfer the proteins to a membrane made of nitrocellulose or polyvinylidene difluoride and then detect them. Proteinswithin gels are usually stainedwith an organic dye or a silver-basedstain, both detectedwith normal visible light, or with a fluorescent dye that requires specialized detection equipment. Coomassieblue is the most commonly usedorganic dye, typically usedto detect=1000 ng of protein, with a lower limit of detectionof =4-1,0 ng. Silver stainingor fluorescencestainingare more sensitive(lower limit of =1 ng). Coomassie and other stains can also be used to visualize proteins after transfer to membranes;however.the most common method to visualize proteins in these membranes is immunoblotting, commonly called'Westernblotting. Western blotting combines the resolving power of gel electrophoresisand the specificityof antibodies.This multistep procedure is commonly used to separateproteins and then identify a specificprotein of interest. As shown in Figure 3-38, two different antibodies are used in this method, one that is specific for the desiredprotein and a secondthat binds to the first and is linked to an enzymeor other molecule that permits detectionof the first antibody (and thus the protein of interesr to which it binds). Enzymesto which the secondantibody is attachedcan either generatea visible colored product or, by a processcalled chemiluminescence,produce light that can readily be recorded by film or a sensitive detector. The two different antibodies, sometimescalled a "sandwich," are used to amplify the signalsand improve sensitivity.If an antibody is not available,but the geneencodingthe protein is available and can be used to expressthe protein, recombinant DNA methods (Chapter 5) can irrcorporatea small peptide epitope (epitope tagging) into the normal sequenceof the protein that can be detected by a commercially available antibody to that epitope.

Illl+ Technique Animation:lmmunoblotting I

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Chromogenic detection

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Incubatewith enzYmelinkedAb2 (Y); wash excess

Reactwith substrate for Ab2-linkedenzyme

FIGURE 3-38 Westernblotting A EXPERIMENTAL (immunoblotting) combinesseveraltechniquesto resolveand detect a specificprotein. StepE: Aftera proteinmixturehasbeen bands(orspots, throughan SDSgel,theseparated electrophoresed (blotted) fromthe gelonto gel)aretransferred for a two-dimensional removedStepZ: whichit isnot readily a porousmembraneJrom (Abr)specific of antibody isfloodedwith a solution Themembrane for a while.Onlythe protern andallowedto incubate for the desired thisproteinbindsthe antibody, bandcontaining membrane-bound (whoseposition cannotbe molecules forminga layerof antibody point) iswashedto remove Thenthe membrane seenat this

u n b o u nA d b ' . S t e pE : T h e m e m b r a ni sei n c u b a t ewdi t ha s e c o n d andbindsto thefirstAb1 (Ab2)thatspecifically recognizes antibody (e.9., to linked eitheran enzyme iscovalently antibody Thissecond reaction), a chromogenic can catalyze which phosphatase, alkaline can whosepresence or someothersubstance isotope, radioactive and the location Step4: Finally, with greatsensitivity be detected (e.g, by a deep-purple amountof boundAb2aredetected permitting the reaction), fromchromogenic precipitate (and the destred of the mass) therefore mobility electrophoretic (based on band aswellasitsquantity oroteinto be determined,

RadioisotopesAre lndispensableTools l olecules f o r D e t e c t i n gB i o l o g i c aM

Radioisotopes Useful in Biological Research Hundreds of biological compounds (e.g.,amino acids,nucleosides,and numerous metabolic intermediates)labeled with various radioisotopes are commercially available. These preparations vary considerably in their specific actiuity, which is the amount of radioactivity per unit of material, measured in

A sensitivemethod for tracking a protein or other biological molecule is by detecting the radioactivity emitted from radioisotopesintroduced into the molecule. At least one atom in a radiolabeled molecule is present in a radioactive form, called a radioisotope.

ISOTOPE

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dom/mmol) are available.Likewise' commercialpreparations tH-l"b.l.d nucleic acid precursorshave much ligher speoi 1aC-labeled cific activities than those of the corresponding preparations.In most experiments'the former are preferable t".uot. they allow RNA or DNA to be adequatelylabeled alter a shorter time of incorporation or require a smaller cell sample.Various phosphate-containingcompounds in which every phosphorus atom is the radioisotope phosphorus-32 P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

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a^rereadily available. Becauseof their high specific activity, 32P-labeled nucleotides are routinely ur.d to label nucleic acids in cell-freesystems. Labeled compounds in which a radioisotope replaces atoms normally present in the molecule have virtually the same chemical properties as the corresponding nonlabeled compounds. Enzymes,for instance,generally cannot distinguish betweensubstrateslabeledin this way and their nonlabeled substrates.The presenceof such radioactive atoms is indicatedwith the isotopein brackets(no hyphen) as a prefix (e.g., [3H]leucine).In .ontrurt, labeling almost all biomolecules (e.g., protein or nucleic acid) with the radioisotope iodine-125 (12sI)requires the covalent addition of 12sIto a molecule that normally does nor have iodine as part of its structure.Becausethis labelingproceduremodifies the chemical structure, the biological activity of the labeled molecule may differ somewhat from that of the nonlabeledform. The presenceof such radioactive atoms is indicated with the isotope as a prefix with a hyphen (no bracket) (e.g.,125l-trypsin). Standardmethods for labeling proteins with 12sIresult in covalent attachmentof the 125Iprimarily to the aromatic rings of tyrosine side chains (mono- and diiodotyrosine).

disintegrations per minute per small pixel of surface area. These instruments, which can be thought of as a kind of reusable electronic film, are commonly used to quantitate radioactive molecules separated by gel electrophoresisand are replacingphotographic emulsionsfor this purpose. A combination of labeling and biochemical techniques and of visual and quantitative detectionmethods is often employed in labeling experiments.For instance,to identify the major proteins synthesizedby a particular cell type, a sample of the cells is incubated with a radioactive amino acid (e.g., [35S]methionine)for a few minutes, during which time the Lbeled amino acid mixes with the cellular pool of unlabeled amino acids and some of the labeled amino acid is biosynthetically incorporated into newly synthesizedprotein. Subsequently unincorporatedradioactive amino acid is washed away from the cells. The cells are harvested,the mixture of cellular proteins is extractedfrom the cells (for example, by a detergentsolution), and then separatedby gel electrophoresis; and the gel is subjectedto autoradiography or phosphorimager analysis.The radioactive bands correspondto newly synthesizedproteins, which have incorporatei the radiolabeled amino acid. Alternatively, the proteins can be resolved by liquid chromatographS and the radioactivity in the eluted Labeling Experiments and Detection of Radiolabeled fractions can be determinedquantitatively with a counter.To Molecules Whether labeledcompoundsare detectedby audetect only one specificprotein, rather than all the proteins toradiography, a semiquantitativevisual assay or their radioacbiosyntheticallylabeled this way, a specific antibody to the tivity is measuredin an appropriate ',counter," a highly quantiprotein of interestcan be usedto precipitatethe protein away tative assaythat can determine the amount of a radiolabeled from the other proteins in the sample (immunoprecipitation). compound in a sample, depends on the nature of the experiThe precipitate is then solubilized in a detergenr,separating ment. In some experiments,both types of detection are used. the antibody from the protein, and the sampleis subjectedto In one use of autoradiographS a tissue,cell, or cell conSDS-PAGEfollowed by autoradiography.In this type of exstituent is labeledwith a radioactivecompound, unassociated periment, a fluorescent compound that is activated by the radioactivematerial is washed away, and the structure of the radiation is often infusedinto the gel so that the light emitted sample is stabilized either by chemically cross-linking the can be used to detect the presenceof the labeled protein, macromolecules('fixation') or by freezingit. The sample is either using film or a two-dimensional electronicdetector. then overlaid with a photographic emulsion sensitiveto radiPulse-chase experimentsare particularly usefulfor tracing ation. Developmentof the emulsion yields small silver grains changesin the intracellular location of proreins or the modiwhose distribution correspondsto that of the radioactivemafication of a protein or metabolit. ou.i time. In this experiterial and is usuallydetectedby microscopy.Autoradiographic mental protocol, a cell sample is exposed to a radiolabeled studiesof whole cellswere crucial in determiningthe intracelcompound that can be incorporated or otherwiseattachedto lular siteswhere various macromoleculesare synthesizedand a cellular molecule of interest-the "pulse"-for a brief pethe subsequentmovements of these macromoleculeswithin riod of time, then washedwith buffer to remove the unincorporated label, and finally incubated with an unlabeled form of the compound-the "chase" (Figure 3-39). Samplestaken periodically during the chaseperiod are assayedto determine the location or chemicalform of the radiolabel as a function of time. Often, pulse-chaseexperiments,in which the protein Quantitative measurementsof the amount of radioactivis detected by autoradiography after immunoprecipitation ity in a labeledmarerial are performed with severaldifferent and SDS-PAGE,are usedto follow the rate of synthesis,modification, and degradation of proteins by adding radioactive amino acid precursorsduring the pulse and then detectingthe amounts and characteristicsof the radioactiveprotein during the chase.One can thus observepostsyntheticmodifications of the protein that changeits electrophoreticmobility and the rate of degradation of a specificprotein. A classicuse of the pulse-chasetechniquewas in studiesto elucidatethe pathwav traversedby secretedproteins from their site of synihesisin the endoplasmicreticulum to the cell surface(Chapter 14). 100

.

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key component, which provides a measure of the relative abundancesof each of the ions in the sample.The fourth essential component is a computerized data systemthat is used to calibrate the instrument; acquire,store, and processthe resulting data; and often direct the instrument automatically to collect additional specific types of data from the sample' based on the initial observations. This type of automated feedback is used for the tandem MS (MS/MS) peptidesequencingmethods describedbelow. The two most frequently usedmethods of generatingions of proteins and protein fragmentsare (1) matrix-assistedlaser desorption/ionization(MALDI) and (2) electrospray(ES)' In MALbI (Figure 3-40) the peptide or protein sampleis mixed with a low-molecular-weight,W-absorbing organic acid (the matrix) and then dried on a metal tatget. Energy from a laser ionizes and vaporizes the sample producing singly charged molecular ions from the constituentmolecules'In ES (Figure 3-41.a),the sample of peptidesor proteins in solution is converted into a fine mist of tiny droplets by spraying through a

l"j,\'J," ;:::,'J."'Ll3,' "".' il?HT":',i"H experimentscan FIGURE 3-39 Pulse-chase A EXPERTMENTAL track the pathway of protein modificationor movement newlysynthesized within cells.(a)Tofollowthefateof a specific proteinin a cell,cellswereincubated for 0 5 hr with [3sS]methionine proteins, (thepulse) andthe radioactive to labelallnewlysynthesized intothe cellswasthenwashedaway aminoacidnot incorporated (thechase) for varying timesup to Thecellswerefurtherrncubated to weresubjected from each timeof chase 24 hours,andsamples protein(here,the lowto isolate onespecific immunoprecipitation of the immunoprecipitates receptor) SDS-PAGE lipoprotein density permitted visualization of the one followedby autoradiography (p) protein, asa smallprecursor synthesized whichis initially specific (m) of addition form by larger mature modified to a rapidly andthen from proteinwasconverted Abouthalfof the labeled carbohydrates after0.5 hourof p to m duringthe pulse,the restwasconverted to stablefor 6-8 hoursbeforeit begins chaseTheproteinremains (b) (indicated The same intensity). band by reduced be degraded in cellsin whicha mutantformof the wasperformed experiment to converted proteinismadeThemutantp formcannotbe properly thanthe normalprotein the m form,andit is morequicklydegraded 1986, J CellEloi H A Brush, andM Krieger, fromK F Kozarsky, lAdapted 1567-1s7s 102(5) I Roshanl(eab 021-66950639

arated by the mass analyzeron the basis of their mlz. The two most frequently used mass analyzersare timeof-flight (TOF) instruments and ion traps. TOF instruments e*ploit the fact that the time it takes an ion to passthrough the length of the analyzer before reaching the detector is

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MassSpectrometryCan Determinethe Mass and Sequenceof Proteins Mass spectrometry (MS) is a powerful technique for characthe tertzingproteins.MS is particularly useful in determining 'With such inmass of a protein or fragments of a protein. formation in hand, it is also possibleto determinepart of or all the protein's sequence.This method permits the very highly accuratedirect determination of the ratio of the mass (m) of a charged molecule (ion) to its charge (z), ot mlz. Techniquesare then used to deducethe absolutemass of the ion. There are four key features of all mass spectrometers. The first is an ion source,from which charge, usually in the form of protons, is transferred to the peptide or protein molecules.The formation of these ions occurs in the presenceof a high electric field that then directs the chargedmolecular ions into the second key component' the mass analyzer. The mass analyzer,which is always in a high vacuum chamber, physically separatesthe ions on the basis of their differing mass-to-charge(mlz) ratios. The mass-separated ions are subsequentlydirected to strike a detector,the third

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Time 3-40 Molecularmasscan be FIGURE a EXPERIMENTAL laserdesorption/ionization determinedby matrix-assisted massspectrometry'ln a MALDI-TOF time-of-flight (MALDI-TOF) pulses of lightfroma laserionizea proteinor massspectrometer, on a metaltarget(stepE) An that isabsorbed peptidemixture towardthe detector the ionsin the sample fieldaccelerates electric to the isproportional (steosEl andEl).Thetimeto the detector the having (mlz) ions For ratio. mass-to-charge the squarerootof timeto the ionsmovefaster(shorter the smaller samecharge, usingthetimeof flight weightiscalculated Themolecular detector). of a standard PG ROTEINS O AN , D CHARACTERIZIN P U R I F Y I N GD,E T E C T I N G

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m/z EXPERIMENTAL FIGURE 3-41 Molecularmassof proteins and peptidescan be determinedby electrosprayionization ion-trap massspectrometry.(a)Electrospray (ES)ionization proteins converts andpeptides in a solution intohighlycharged gaseous ionsby passing the solution througha needle(forming the droplets) thathasa highvoltageacross it (charging the droplets). Evaporation of thesolvent produces gaseous ionstharenrera mass spectrometer. Theionsareanalyzed by an ion-trapmassanalyzer that thendirectsionsto the detector. (b) Toppanet:Massspectrumof a mixture of threemajorandseveral minorpeptides ispresented asthe relative abundance of the ionsstriking (y axis)asa the detector functionof the mass-to-charge (m/z)ralio(x axis).Bottornpanel:ln an MS/MSinstrument (suchasthe iontrapshownin part(a)),a

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peptideioncanbe selected specific for fragmentatron intosmaller ionsthatarethenanalyzed anddetected. (also TheMS/MS spectrum calledthe product-ion spectrum) provides detailed structural information aboutthe parention,including sequence information for peptidesHere,the ionwith a mlzof 836.47wasselected, fragmented andthe m/z massspectrumof the productionswas measured Notethereis'nolongeran ionwith an m/zoI g36.47 present because it wasfragmented. Fromthevarying sizes of the productions,the understanding that peptidebondsareoftenbroken in suchexperiments, the knownmlzvalues for individual aminoacid fragments, anddatabase information, the sequence of the peptide, FIIVGYVDDTQFVR, canbe deduced. ona fiourefrom [part(a)based part(b),unpublished 5 Carr; datafromS Carr I

proportional to the square root of mlz (smaller ions move fasterthan larger oneswith the samecharge,seeFigure 3-40). In ion-trap analyzers,tunable electric fields are used to cap'trap,' ions with a specific mlz and to sequentially ture, or pass the trapped ions out of the analyzer onto the detector (seeFigure 3-4ta). By varying the electric fields, ions with a wide range of mlz values can be examined one by one, producing a massspectrum,which is a graph of mlz (x axis) versus relative abundance(y axis) (Figure 3-41'b,top panel). In tandem, or MS/MS, instruments, any given parent ion in the original mass spectrum (Figure 3-41b, top panel) can be broken into smallerions by collisionwith an inmass-selected, ert gas, and then the mlz and relative abundancesof the resulting fragment ions measured (Figure 3-41b, bottom panel), all within the samemachinein about 0.1 s per selectedparent ion. This secondround of fragmentationand analysispermits the sequencesof short peptides (<25 amino acids) to be determined, becausecollisional fragmentation occurs primarily at peptide bonds, so the differencesin massesbetweenions correspond to the in-chain massesof the individual amino acids, permitting deductionof the sequencein conjunctionwith databasesequenceinformation (Figure3-41'b,bottom panel). Mass spectrometryis highly sensitive,able to detectas little as 1 x 10-15 mol (100 attomoles)of a peptideor 10 x 10-15 mol (10 femtomoles)of a protein of 200,000 M$(. Errors in mass measurementaccuracyare dependentupon the specific mass analyzer used, but are typically =0.01 percent for peptidesand 0.05-0.1 percent for proteins. As described in the proteomicssectionthat follows, it is possibleto useMS to analyze complex mixtures of proteins as well as purified proteins. Most commonly, protein samples are digested by proteases,and the peptide digestionproducts are subjectedto analysis. An especiallypowerfully application of MS is to take a complex mixture of proteins from a biological specimen, digestit with trypsin or other proteases'partially separate the componentsusing liquid chromatography (LC), and then transferthe solution flowing out of the chromatographic column directly into an ES tandem mass spectrometer.This technique, called LC-MSIMS, permits the nearly continuous analysisof a very complex mixture of proteins' The abundancesof ions determined by mass spectrometry in any given sample are relative, not absolute' values. Therefore, if one wants to use MS to compare the amounts of a particular protein in two different samples(e.g.,from a normal versusa mutant organism), it is necessaryto have an internal standard in the sampleswhose amounts do not differ between the two samples. One then determines the amounts of the protein of interestrelative to that of the standard in each sample.This permits quantitatively accurateintersamplecomparisonsof protein levels.

from the polypeptide and identified by high-pressureliquid chromatography.The polypeptide is left one residueshorter, with a new amino acid at the N-terminus. The cycle is repeated on the ever shortening polypeptide until all the residueshave been identified. Before about 1985' biologistscommonly used the Edman

merous model organismsis expanding rapidly. As discussed in Chapter 5, the sequencesof proteins can be deducedfrom DNA sequencesthat are predictedto encodeproteins' A powerful approach for determining the primary struct,r.. o? an isolated protein combines MS and the use of sequencedatabases.First' the peptide "mass fingerprint" of the peptide mass fingerprint is the irot.i.r is obtained by MS. A of peptides that are generated weights molecular the iist of

ProteinConformationls Determined by SophisticatedPhysicalMethods In this chapter,we have emphasizedthat protein function is dependent on protein structure. Thus, to figure out how a prot.i.t works, its three-dimensional structure must be Lno*n. Determining a protein's conformation requiressophisticated physical methods and complex analysesof the data. \(e briefly describethree methods used .*p.ri-..tt"l to generatethree-dimensionalmodels of proteins' The use of x-ray crystallographyto X-ray €rystallography determine the three-dimensionalstructuresof proteins was pio-

atoms of the crystal scatterthe x-rays' which produce a drfftaction pattern of discretespots when they are interceptedby photographic film or an electronicdetector (Figure 3-42)' Suchpatextremely complex---composedof as many as 25,000 t...r, "..

ProteinPrimaryStructureCan Be Determinedby ChemicalMethods and from Gene Sequences The classicmethod for determining the amino acid sequence of a protein is Edman degradation. In this procedure, the free amino group of the N-terminal amino acid of a polypeptide is labeled, and the labeled amino acid is then cleaved

three-dimensionalelectron density map in hand' one then "fits" a molecular model of the protein to match the electron density, P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N s

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proteins. To date, the detailed three-dimensionalstructures of more than 10,000 proteins have beenestablished. Cryoelectron Microscopy Although someproteinsreadily crystallize, obtaining crystals of others-particularly large multisubunit proteins and membrane-associatedproteinsrequires a time-consuming trial-and-error effort to find just the right conditions,if they can be found at all. (Growing crystals suitablefor structural studiesis as much an art as a science.) There are several ways to determine the structures of such difficult-to-crystallize proteins. One is cryoelectron microscopy. In this technique, a protein sample is rapidly frozen in liquid helium to preserveits structure and then examined in the frozen, hydrated state in a cryoelectron microscope. pictures of the protein are taken at various anglesand recorded on film using a low dose of electronsto prevent radiation-induced damageto the structure. Sophisticatedcomputer programs analyze the imagesand reconstruct the protein's structure in three dimensions. Recent advancesin cryoelectron microscopy permit researchersto generatemolecular models that can help provide insight into how the protein functions. The use of cryoelectron microscopy and other types of electron microscopy for visualizing cell structures is discussedin Chapter 9.

a proteincan be determined.(a)Basic components of an x_ray crystallographic determination. Whena narrowbeamof x_rays strikes a crystal,partof it passes straightthroughandthe restrsscattered (diffracted) in various directions Theintensity of thediffracted waves, whichformperiodic arrangements of diffraction spots,is recorded on an x-rayfilm or with a solid-state electronic detector. (b)X-raydiffractionpatternfor a proteincrystalcollected on a solid_ statedetector. Fromcomplex analyses of patterns of spotslikethis one,the location of the atomsin a proteincanbe determined. [part

(a)adaptedfrom L Stryer,1995,Biochemistry, 4Ihed, W H Freeman and p 64; part(b)courtesy Company, of J Berger.j

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NMR Spectroscopy The three-dimensionalstrucures of small proteins containing as many as 200 amino acidscan be studied with nuclear magneric resonance (NMR) spectroscopy. In this technique, a concentratedprotein solution is placed in a magnetic field, and the effects of different radio frequencieson the nuclear spin statesof different atoms are measured.The spin state of any atom is influenced by neighboring atoms in adjacent residues,with closely spaced residues having a greater influence than distant residues. From the magnitude of the effect, the distances between residues can be calculated by a triangulation-like process; these distances are then used to generate a model of the three-dimensionalstructure of the protein. Although NMR does not requiie the crystallizarion of a protein, a definite advantage, this technique is limited to proteins smaller than about 20 kDa. However. NMR analvsis can also be applied to isolated protein domains, which can often be obtained as stable structures and tend to be small enough for this technique.

Purifying, Detecting, and Characterizingproteins r Proteins can be separated from other cell components and from one another on the basis of differencesin their physicaland chemicalproperties. r Various assaysare used to detect and quantify Droteins. Some assaysuse a light-producing reactironto generatea readily detectedsignal. Other assaysproduce an amplified colored signal with enzymesand chromogenic substrates. r Centrifugation separatesproteins on the basis of their rates of sedimentation, which are influenced by their massesand shapes.

proteinson the basisoftheir rates separates r Electrophoresis of movement in an applied electric field. SDS-polyacrylamide gel electrophoresis(PAGE) can resolve polypeptide chains differing in molecular weight by 10 percentor less(seeFigure 3-35). Two-dimensional gel electrophoresisprovides additional resolution by separating proteins first by charge (first dimension)and then by mass(seconddimension). r Liquid chromatography separatesproteins on the basis of their rates of movement through a column packed with spherical beads.Proteins differing in mass are resolved on gel filtration columns; those differing in charge, on ionexchange columns; and those differing in ligand-binding properties, on affinity columns, including antibody-based affinity chromatography(seeFigure 3-37).

investigation.This multipronged approach provided deep insight into the function and mechanismsof action of individual interacting proteins' proteins -Ho*.u.r,or relatively small numbers of proteins does studying this one-by-oneapproach to of what is picture global not efficiently provide insights into a organism' entire or tissue, happening in the proteome of a cell,

Proteomicsls the Study of All or a LargeSubset of Proteinsin a BiologicalSYstem The advent of genomics (sequencingof genomic DNA and its associatedtechnologies' such as simultaneous analysis of the levels of all mRNAs in cells and tissues)clearly showed that a

r Antibodies are powerful reagentsused to detect, quantify, and isolate proteins. 'Western blotting, is a frer Immunoblotting, also called quently usedmethod to study specificproteins that exploits the high specificity and sensitivity of protein detection by antibodies and the high-resolution separation of proteins by SDS-PAGE. r Radioisotopesplay a key role in the study of proteins and other biomolecules.They can be incorporated into molecules without changing the chemical composition of the molecule,or as add-on tags.They can be usedto help detect the synthesis,location, processing,and stability of proteins. r Autoradiography is a semiquantitativetechnique for detecting radioactively labeled molecules in cells, tissues,or electrophoreticgels. r Pulse-chaselabeling can determine the intracellular fate of proteinsand other metabolites(seeFigure 3-39). r Mass spectrometry is a very sensitiveand highly precise method of detecting, identifying, and characterizing proteins and peptides. r Three-dimensionalstructuresof proteins are obtained by x-ray crystallography,cryoelectron microscopS and NMR spectroscopy.X-ray crystallography provides the most detailedstructuresbut requiresprotein crystallization. Cryoelectron microscopy is most useful for large protein complexes, which are difficult to crystallize. Only relatively small proteins are amenableto NMR analysis.

El

Proteomics

For most of the twentieth century,the study of proteins was restrictedprimarily to the analysisof individual proteins. For example,one would study an enzymeby determiningits enzyproducts.rate of reaction,requirematic activity (substrates, ment for cofactors,pH, etc.),its structure' and its mechanism of action. In some cases,the relationshipsberweena few enzymesthat participate in a metabolic pathway might also be studied.On a broader scale,the localizationand activity of an enzymewould be examined in the context of a cell or tissue. The effectsof mutations, diseases,or drugs on the expression and activity of the enzyme might also be the subject of

studies: In a given sample (whole organism, tissue,cell' subcellur compartment), what fraction of the whole proteome is expressed(i.e.,which proteinsare present)? r Of those proteins presentin the sample,what are their relative abundances? r What are the relative amounts of the different splice forms and chemically modified forms (e.g.,phosphorylated, methylated, fatty acylated) of the proteins? comr Which proteins are presentin large multiprotein 'lfhat are plexes,anl which proteins are in each complex? interact? the functions of thesecomplexesand how do they r 'When the state (e.g.'growth rate' stageof cell cycle, differentiation, stresslevel) of a cell changes'do the proteins in the cell or secretedfrom the cell changein a characteristic

r Can such fingerprint-like changesbe used for diagnostic purposes?For example, do certain cancersor heart disease iurrr. .hur".teristic changesin blood proteins? Can the proteomic fingerprint help determine if a given cancer is re,irr".t, or sensitiveto a particular chemotherapeuticdrug? Proteomic fingerprints can also be the starting point for studiesof the meihanisms underlying the changeof state' Proteins (and other biomolecules)that show changesthat are diagnostic of a particular state are calledbiomarkers' Can changesin the proteome help define targetsfor drugs suggestmechanismsby which that drug might induce *ic side effects?If so, it might be possibleto engineer modified versionsof the drug with fewer side effects' o PROTEOMTCS

105

Theseare just a few of the questionsthat can be addressedusing proteomics.The methods used to answer thesequestions are as diverseas the questionsthemselves,and their numbers are growing rapidly.

Figure 3-43 outlines the general LC-MS/MS approach in which a complex mixture of proteins is digested with a protease, the myriad resulting peptides are fractionated by LC into multiple, less complex fractions, which are slowly but continuously injected by electrosprayionization into a tandem mass spectrometer.The fractions are then sequentially subjectedto multiple cyclesof MS/MS until sequences of many of the peptidesare determinedand used to identify from databasesthe proteins in the original biological sample. An example of the use of LC-MS/MS to identify many of the proteinsin eachorganelleis seenin Figure 3-44. Cellsfrom murine (mouse)liver tissuewere mechanicallybroken to release the organelles, and the organelles were partially separated by density-gradient centrifugarion. The locations of the organelles in the gradient were determined using immunoblotting with antibodies that recognize previously identified, organelle-specificproteins. Fractions from the gradient

AdvancedTechniquesin MassSpectrometry Are Criticalto ProteomicAnalysis Advances in proteomics technologies(e.g., mass specrrometry) are having a profound effect on the types of questionsthat can be practically studied. For many years,rwo-dimensional gel electrophoresishas allowed researchersto separate,displaS and characterizea mixture of proteins (Figure 3-36). The spots on a two-dimensional gel can be excised,the protein fragmentedby proteolysis(e.g.by trypsin digestion),and the fragments identified by MS. An alternative to this twodimensional gel method is high throughput LC-MS/MS.

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centrifugation 3-rt4 Density-gradient FIGURE < EXPERIMENTAL can be usedto identify manyof the proteinsin and LC-MS/MS

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eukaryotic cell, the yeastSaccharomycescereuisiae'Approxtmately 500 complexeshave been identified, with an average of 4.9 distinct proteins per complex' and thesein turn are involved in at least400 complex-to-complexinteractions'Such systematicproteomic studiesare providing new insights into tire organiiation of proteins within cells and how proteins work together to permit cellsto live and function'

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subcellularlevels. r Proteomicsprovides insightsinto the fundamental organizationof proteins within cells,and how this organization is influencedby the state of the cells (e.g.,differentiation into distinct cell types; responseto stress'disease,and drugs)' r A wide variety of methods are used for proteomic analyses,including two-dimensional gel electrophoresis,densitygradient centrifugation, and mass spectroscopy(MALDITOF and LC-MS/MS)' omics has helped begin to identify the proteomesof les ("organelleproteomeprofiling") and the organof individual proteins into multiprotein complexes eract In a complex network to support life and cellular function

ER/Golgi vesicles 220

Early enoosomes 76

Proteomics combined with molecular geneticsmethods are currently being usedto identify all protein complexesin a P E R S P E C T I VF EO S RT H E F U T U R E

O

107

motif 68 motor proteins 85 peptide bond 55 polypeptides 65 primary structure 66 proteasomes54 protern 66 proteome 64

quaternary str]uctvre 72 r ate-zonal centrifugation 93 secondarystructure 65 tertiary structure 67 ubiquitin 88

v^^*go

'Western blotting 98 x-ray crystallogr aphy 10 3

proteomics105

Review the Concepts

built. Becausesubunits of the complex may already be solved by crystallographSa composite,tru.tur. consistingof the x-ray-derived subunit structuresfit to the EM-deriveJ model will be generated.An example of this approach for an elec_ tron-transport "supercomplex" is describedin Chapter 12. Methods for rapid structure determination combined with identificarion of novel substratesand inhibitors will

range. Routine analysisof specimenswith such diversecon_ centrations should dramatically improve the mechanistic and diagnostic value of blood plasma proteomics.

KeyTerms cr helix 65 activation energy 79 active site 80 allostery 89 amyloid frlament 77 autoradiography100 B sheet 66 B turn 66 chaperones75 108

o

C H A P T E R 3I

conformations 63 cooperativity 89 (tomarn /u electrophoresis94 lr nomotogy /Z K- 90 ligand 78 liquid chromatography 9G molecular machine 64 P R O T E I NS T R U C T U RAEN D F U N C T I O N

7, The three-dimensional structure of a protein is deter_ mined by its primary, secondary,and tertiary structures.Define the primary, secondary, and tertiary structures. What are some of the common secondary structures? ril/hat are the forces that hold together the secondary and tertiary structures? 2. Proper folding of proteins is essentialfor biological ac_ tivity. Describethe roles of molecular chaperonesand chap_ eronins in the folding of proteins. 3. Enzymes can catalyzechemical reactions. How do en_ zymes increasethe rate of a reaction? What constitutes the active site of an enzyme?For an enzyme-catalyzedreaction, what are K- and V-""? For enzymeX, the K- for substrrte A is 0.4 mM and for substrate B is 0.01 mM. \Which sub_ strate has a higher affinity for enzyme X? 4. Motor proteins convert energy into a mechanicalforce. Describethe three generalproperties characteristicof motor proterns,

6, The function of proteins can be regulatedin a number of ways. What is cooperativitg and how does it influence pro_ tein function? Describe how protein phosphorylation and proteolytic cleavagecan modulate protein function. 7. A number oftechniques can separateproteins on the ba_ sis of their differencesin mass. Describe the use of two of thesetechniques,cenrrifugation and gel electrophoresis.The blood proteins rransferrin (M\X/ 76 kDa) and lysozyme(MW 15 kDa) can be separated by rate-zonal centrifugation or SDS-polyacrylamidegel electrophoresis.Which oi the two proteins will sediment faster during centrifugation? Which will migrate faster during electrophoresis? 8. Chromatography is an analytical method used to sepa_ rate proteins. Describethe principles for separatingproteins by gel filtration, ion-exchange,and affinity ihromatography. 9, Various methods have beendevelopedfor detectingpro_ teins. Describehow radioisotopesand autoradiography can be used for labeling and detectingproteins. Ho* doe, Ifr.rt_ ern blotting detectproteins?

10. Physical methods are often used to determine protein conformation. Describe how x-ray crystallography,cryoelectron microscopy,and NMR spectroscopycan be used to d e t e r m i n et h e s h a p eo f p r o t e i n s . 1L. Mass spectrometryis a powerful tool in proteomics.What are the four key features of a mass spectrometer?Describe briefly how MALDI and two-dimensionalpolyacrylamidegel electrophoresis(2D-PAGE)could be usedto identify a protein expressedin cancercellsbut not in normal healthycells.

mic fractions by differential centrifugation. Two-dimensional gels were run, and the stained gels are shown.below' \fhat do you conclude about the cellular localization of proteins 1-7? Control Nu c l e a r 10

4pH

4

Cytoplasmic pH 10

o

a

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Analyze the Data

a

a

Proteomicsinvolvesthe global analysisof protein expression. In one approach, all the proteins in control cells and treated cells are extracted and subsequentlyseparatedusing two-dimensional gel electrophoresis.TypicallS hundreds or thousandsof protein spots are resolvedand the steady-statelevels of each protein are compared between control and treated cells. In the following example, only a few protein spots are shown for simplicity. Proteins are separated in the first dimension on the basis of charge by isoelectricfocusing (pH a-10) and then separatedby sizeby SDS-polyacrylamide gel electrophoresis.Proteinsare detectedwith a stain such as Coomassieblue and assignednumbers for identification. a. Cellsare treatedwith a drug ("+ Drug") or left untreated ("Control"), and then proteins are extractedand separated by two-dimensional gel electrophoresis.The stained gels are shown below. What do you concludeabout the effect of the drug on the steady-statelevelsof proteins 1-7?

12 O.

+ Drug Nuclear pH 10

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Cytoplasmic pH 10

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o

a o o

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d. Summarize the overall properties of proteins 1-7, combiningthe data from parts (a), (b), and (c)' Describehow you could determinethe identity of any one of the proteins'

References General References Berg,J. M., J. L. Tymoczko, and L. Stryer.2007' Biochemistry, 6th ed. W. H. Freemanand ComPanY. Nelson, D. L., and M. M. Cox. 2005. LehningerPrinciplesof Biochemistry,4thed. !(/. H. Freemanand Company'

+ Drug 4pH10

Control 4pH10

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a

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^o t aa

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b. You suspectthat the drug may be inducinga protein kinaseand so repeatthe experimentin part (a) in the pres32P-labeled inorganicphosphate.In this experiment enceof the two-dimensional gels are exposedto x-ray film to detect 32P-labeledproteins. The x-ray films are the presenceof shown below. \fhat do you conclude from this experiment about the effect of the drug on proteins 1-7? Control pH

4

10 a

4

+ Drug pH

10

a

a c. To determinethe cellular localizationof proteins 1-7, the cellsfrom part (a) were separatedinto nuclearand cytoplas-

Entrv site into proterns,structures'genomes'and taxonomy: http://www.ncbi.nlm.nih.govlEntrezl The protein 3D structuredatabase:http://www'rcsb'org/ Structuralclassificationsof proteins: http://scop'berkeley'edu/ Sitescontaininggeneralinformation about protetns: httpy'/www.exp"tyiti; http://www.proweb'org/; http://scop'berkeley' edu/intro.html PROSITEDatabaseof protein families and domarns: te/ http://www.expasy.org/Prosr Domain organizationof proteins and large collection of multiarelPfanl ple sequencealignments:http://www.sanger'ac'uk/Softw Hierarchical Structure of Proteins and Protein Folding Branden,C., and J. Tooze. 1999. Introduction to Protein Strwcture. Garland. BrodsknJ. L., and G. Chiosis.2006'Hsp7Omolecularchaper,oies in human diseaseand identificationof small on.r' .-.tji"g molecule-lod"ulrrotr. Curr. Top. Med- Chem' 6(11'\:1215-1225' Bukau, B, J. Veissman, and A. Horwich' 2006 Molecular chaperonesand ptot.i.t quality control. Cell 125(3):443451" /' Cohen,F.E. 1999.Proteinmisfoldingand prion diseases' Mol. B iol. 293:313-320. Coulson,A. F., and J. Moult. 2002' A unifold, mesofold' and ' superfoldmodel of protein fold use.Proteins46:61'-7"1 mechaunifying a there Daggett,V, and A' R. Fersht.2003' Is ' 28(\l:18-25 Sci' Biocbem' Trends folding? foi-protein nism R E F E R E N C E S.

10 9

Dobson, C. M. 1999. Protein misfolding, evolution, and disease. TrendsBiochem. Sci.24:329-332. Gimona, M.2006. Protein linguistics-a grammar for modular protein assembly?Nat. Reu.Mol. Ceil Biol.7(Il:69_73. Gough,J. 2006. Genomic scalesub-family assignmentof pro_ tein domains.Nucl. Acids Res.34(13):3625-3633. Koonin, E. V., Y. I. Wolf, and G. p. Karev.2002. The srructure of the protein universeand genomeevolution. Nature 420:21,g_223. Lesk, A. M. 2001. Introduction to protein Architecture.Oxford. and H. S. Rye. 2005. GroEl-mediated protein folding: ,\i", V, making the impossible,possible.Crit. Reu.Biochem.Mol. Biol. 4l(4):21.1-239. Orengo, C. A., D. T. Jones,and J. M. Thornton. 1994. protein superfamiliesand domain superfoldi. Nature 372: 631_634. Patthn L. 1999. Protein Euolution. Blackwell Science. . Rochet,J.-C., and P.T. T andsbury.2000. Amyloid fibrillogene_ sis:themesand variations. Curr. Opin. Struc. Bioi. l}:60_6g. . . Voge!,C, and C. Chothia. 2006. prorein family expansionsand biological complexity. PLoS Comput. Biol. 2(5.;e4g. Yaffe,M. 8.2006. "Bits" and pieces.SclSTKE. Jun 20 (3401:pe2g. Y,oung,J. C., et aL.2004.pathways of chaperone-mediated pro_ tein folding in the cytosol. Nat. Reu.Mol. Cet[ glol. 5:7g1._797. Protein Function ^ Dressler,D. H., and H. Potter. 1997. DiscoueringEnzymes. Scientific American Libra ry. Fersht,A. 1999. Enzyme Structureand Mechanism,3d ed. '!7. H. Freemanand Company. Jeffery,C. J.2004. Molecular mechanismsfor multitasking: recentcrystal structuresof moonlighting proteins. Cwrr.Opin."Struc. Biol. l4(61:663-668. Marnett, A. B., and C. S. Craik. 2005. papa'sgot a brand new tag: advancesin identificarionof proteasesani the]. substrates. TrendsB iotechnol. 23 2\: 59-64. L. 2005. The catalytic triad of serinepeptidases.Cell _Polgar, Mol. Life Sci.62(1.9-20\:2t 6I-217 2. Radisky,E. S., et aL.2006.Insightsinto the serineprotease mechanismfrom atomic resolutionltructures of trypsin reacrion inrermediates. Proc. Nat'l Acad. Sci.USA 103(1g);6g35_6g40. Schenone,M, B. C. Furie, and B. Furie. 2004. The blood coagulationcascade.Curr. Opin. H ematol. ll(4\ :272_277. Schramm,V. L. 2005. Enzymatictransition staresand transition stateanalogues. Cun Opin. Struc.Biol. 15(6):604_613. Regulating Protein Function l: protein Degradation Glickman, M. H., and A. Ciechanover.2002Theubicuitin_ proteasomeproteolytic pathway: destructionfor the sakeof construction.Physiol. Reu. 82(2\:373-42g. Goldberg_, A.L.2003. Protein degradationand protection againstmisfolded or damagedproteinl. Nature 426:.g95_g99. Goldberg,.{. L, S.J. Elledge,and J. If. Harper. 2001. The cellu_ , r a rc h a m b e o r t d o o m . S c i .A m . 2 8 4 1 1 1 : O g _ 7 3 . Groll, M., and R. Huber. 2005. purification, crystallization,and ]^rff^f1a!fsi9 of the yeast20S proteasome.Met'h. Enzymol. 398:329-336. Kisselev,A. F., A. Callard, and A. L. Goldberg.2006. Impor_ tanceof rhe.differentproteolytic sitesof the prot;some and the efficacy ot rnhrbrrorsvarieswith the protein subsfrate. Biol. Cbem. J. 281(13):8582-8590. Rechsteiner, M., and C. p. Hilt. 2005. Mobilizing the proteolytic machine:cell biologicalrolesof proreasome acrivatorsand in_ hibitors.TrendsCell Biol. l5(l);27-J3. Zhou. P.2006. REGgamma:a shorrcutto destruction.Cel/ r24(2\:256-257. Zolk, O., C. Schenke, and A. Sarikas .2006.The ubiquitin_ proteasomesysrem:focuson the heart.Cardiouasc.Res.7Oi3\:410_12t.

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Regulating Protein Function ll: Noncovalent and Covalent Modifications Bellelli,A., et al. 2005. The allostericpropertiesof hemoglobin: insightsfrom narural and site directed-uiants. Cwrr.prot. pZo. Sci. 7(t):1745. Burack, \7. R., and A. S. Shaw-2000. Signaltransducrion:hang_ ing on a scaffold.Curr. Opin. Cell Biol. 12:271-216. _ Halling, D. B, P. Aracena-parks,and S. L. Hamilton. 2006. Reg_ uljrtion of voltage-gatedCaz+ channelsby calmodulin. Sci STKE lXn 1.7(3181:er1.. Horovitz, A., et al. 2001. Review: allosteryin chaperonins. /. Struc. Biol. 135:104-1,14. Kern, D., and E. R. Zuiderweg. 2003. The role of dynamicsin allostericregulation.Curr. Opin. Struc. Biol. 13(6):748-757. I..ane,K. T., and L. S. Beese.2006. Thematicreview series:lipid post_ translationalmodifications.Structuralbiology of protein farnesyitrans_ feraseand geranylgeranyltransferase rypeL j. Lip;d Res.4Z(41:tg1,-691. Lim, W. A.2002. The modular logic of signalingproteins:build_ ing allostericswitchesfrom simple binding domains. Curr. Opin. Struc.Biol. 12:61,-68. Martin, C., Y. Zhang.2005. The diversefunctions of histone lysinemethylation.Na/. Reu.Mol. Cell Biol.6(11):838-849. Sawyer,T. K., et al. 2005. Proreinphosphorylationand signal transductionmodulation: chemistryperspeitivesfor small-mo'iecule drug discovery.Med. Chem. l(3)2%-3I9. Xia,Z., and D. R. Storm.2005. The role of calmodulinas a sisnal integrator for synapticplasticity. Nat. Reu.Neurosci. 6(41:267-27 d. Yap, K. L., et al. 1999. Diversiry of conformational statesand changeswithin the EF-hand protein superfarnlly.proteins 37:499_507. Purifying, Detecting, and Characterizing proteins Domon, B., and R. Aebersold.2006. Mass spectrometryand protein analysis.Science3t2(5771):212-217. . Fncarnacion,S., et al. 2005. Comparativeproteomicsusing 2_D gel electrophoresisand massspectrometryas tools to dissectstimu_ lons and regulonsin bacteriawith sequencedor partially sequenced genomes.Biol. Proc. Online 7:ll 7-135. Hames, B. D. A Practical Approach. Oxiord Universiry press.A methods seriesthat describesprotein purification methods and assays. Patton, W. F.2002. Detection,technologies in proteome analysis. J. Chromatogr.B. Analyt. Technol.Biomed. Life SZl.ZZtlt.Zy:l_Zt. White, I. R., et al. 2004. A staristicalcomparisonof silver and !IP\O Ruby staining for proreomic analysis.Electrophoresis 25(17):3048-3054. Proteomics Foster,L. J., et al. 200 . A mammalian organellemap by pro_ tein correlation profiling. Cell 125(1\:187-195. Fu, Q., and J. E. Van Eyk.2006. proteomicsand heart disease: identifying biomarkers of clinical vtj\ty. Expert Reu. proteomics 3(2):237-249. _ Gavin. A. C., et a|.2006. Proreomesurveyrevealsmodularity of the yeastcell machinery.Nature 440(7084):631-636. Kislinger,T., et al. 2006. Global surveyof organ and organelle protern expre_ssion in mouse:combinedproteomicand trans&iptomic profiling. Cell 125(1):173-186. Kolker, E, R. Higdon, and J. M. Hogan. 2006. protein identifi_ cation.andexpressionanalysisusing mass spectrometry.Trends Mi crobi ol. I4\5 l:229-235 . Krogan, N. J., et al. 2006. Global landscapeof protein complexes in the yeastSaccb aromycescereuisiae. Nature-440(i0g4):637_623. Ong, S. E., and M. Mann. 2005. Mass spectrometry_based pro_ teomrcsturns quanritarive.Nar. Chem. Biol. l(5):252_262. Rifai, N., M. A. Gillette, and S. A. Carr.2006. protein bio_ T."lk.f dis.coveryand validation: the long and uncertainparh ro clinical utility. Nat. Biotecb. 24(8\971-t$.

C H A P T ER

BASICMOLECULAR GENETIC MECHANISMS Colored transmissionelectron micrograph of one ribosomal RNA transcription unit from a Xenopus oocyte. Transcription proceeds from left to right,with nascentribosomalribonucleoprotein RNA (rRNPs) growingin lengthas eachsuccessive complexes polymerase I moleculemovesalongthe DNAtemplateat the center eachrRNPis orientedeitheraboveor belowthe ln thisoreoaration so that the overallshapeis centralstrandof DNAbeingtranscribed, rRNPs of a livingcell,the nascent similarto a feather.In the nucleolus L MillerAcience Oscar likea bottlebrushlProfessor extendin alldirections, PhotoLibrary l

he extraordinary versatility of proteins as molecular machines and switches, cellular catalysts' and components of cellular structureswas describedin Chapter 3. In this chapter we consider how proteins are made' as well as other cellular processesthat are critical for the survival of an organism and its descendents.Our focus will be on the vital molecules known as nucleic acids, and how they ultimately are responsible for governing all cellular function. As introduced in Chapter 2, nucleic acids are linear polymers of four types of nucleotides(seeFigures 2-13' 2-1'6 and 2-1,7).These macromolecules( 1 ) contain in the precise sequenceof their nucleotidesthe information for determining the amino acid sequenceand hence the structure and function of all the proteins of a cell, \2) are critical functional components of the cellular macromolecular factories that select and align amino acids in the correct order as a polypeptide chain is being synthesized,and (3) catalyze a number of fundamental chemical reactions in cells, including formation of peptide bonds between amino acids during protein synthesis. Deoxyribonucleicacid (DNA)is an informational molecule that contains in the sequenceof its nucleotidesthe information required to build all the proteins of an organism, and hencethe cells and tissuesof that organism. It is ideally suited to perform this function on a molecular level. Chemically, it is extraordinarily stable under most terrestrial conditions, as exemplified by the ability to

stored in the sequenceof the four possible nucleotides that

OUTLINE 4.',|

Structureof NucleicAcids

113

4.2

Transcriptionof Protein-CodingGenes and Formationof FunctionalmRNA

120

4.3

The Decodingof mRNA bY tRNAs

127

4.4

StepwiseSynthesisof Proteinson Ribosomes 132

4.5

DNA Replication

139

4.6

DNA Repairand Recombination

145

4.7

of the Cellular Viruses:Parasites GeneticSYstem

111

make up the -3 x 10e base pairs in the human genome. Becauseof the principles of basepairing discussedin this chapter, the information is readily copied with an error rate of <1 in 10e nucleotides per geniration. The exact replication of this information in any speciesassuresits genetic continuity from generation to generation and is critical to the normal developmenrof an individual. DNA fulfills these functions so well that it is the vessel for genetic information in all forms of life known. (One exception is RNA viruses; however, these are limited to extremely short genomesbecauseof the relative instability of RNA compared with DNA, as we will see.)The discovery that virtually all forms of life use DNA to encodetheir genetic information, and also use nearly the same nucleic acid sequencecode to specifyamino acid sequence,implies that all forms of life descendedfrom a common ancesror on the basis of storageof information in nucleic acid sequence.This information is accessedand replicated by specific base pairing between bases. The information stored in DNA is arrangedin hereditary units, now known as genes,which control identifiable traits of an organism. In the processof transcription, the information stored in DNA is copied into ribonucleic acid (RNA), which has three distinct roles in protein synthesis. Portions of the DNA nucleotide sequenceare copied into

lation becausethe nucleotide sequencelanguage of DNA and RNA is translated into the amino acid sequencelan_ guageof proteins. Discovery of the structure of DNA in 1953 and subse_ quent elucidation of how DNA directs synthesisof RNA, which then directs assemblyof proteins-the so-called,cen_

decoded into a variety of proteins in the correct cells at the correct times in development. This regulation of gene expressionallows hemoglobin to be expressedonly in cells of the bone marrow (reticulocytes)destinedto develop into

1',t2

CHAPTER 4

I

circulating red blood cells (erythrocytes),and directs developing neurons to make the proper synapses(connections) with 1011other developingneurons in the human brain. The fundamental molecular generic processesof DNA replication, transcription, and translation must be carried out with extraordinary fidelity, speed,and accurate regulation in or, der for organismsas complex as prokaryotes and eukaryotes to develop normally. This is achievedby chemical processes that operate with extraordinary accuracycoupled with multiple layers of checkpoint, or surveillance,mechanismsthat test whether critical steps in these processeshave occurred correctly before the next step is initiated. The highly regulated expressionof genesnecessaryfor the developmento1 a multicellular organism requires integrating information from signalssent by distant cellsin the developingorganism, as well as from neighboring cells, and an intrinsic developmental program determined by earlier stepsin embryogenesis taken by that cell's progenitors. All this regulation is dependent on control sequencesin the DNA that function with proteins called transcription factors to coordinate the expressionof every gene. RNA sequenceswe discussin Chapter 8 that regulateRNA processingand translation also are encoded in DNA originally. Thus nucleic acids function as the "brains and central nervous system" of the cell, while proteins carry out the functions they specify. In this chapter,we first review the structuresand properties of DNA and RNA, and explore how the different characteristicsof each type of nucleic acid make them suited for their respectiveroles in the cell. In the next severalsections we discussthe basic processessummarizedin Figure 4-1: transcription of DNA into RNA precursors, processing of these precursorsto make functional RNA molecules.translation of mRNAs into proteins, and the replication of DNA. After outlining the individual roles of -RNA, IRNA, and rRNA in protein synthesis,we presenta detailed description of the components and biochemical stepsin translation. .We also consider the molecular problems involved in DNA replication and the complex cellular machinery for ensuring accuratecopying of the geneticmaterial. Along the way, we compare theseprocessesin prokaryotes and eukaryotes.The next section describeshow damageto DNA is repaired, and how regions of different DNA molecules are exchangedin the processof recombination to generatenew combinations of traits in the individual organisms of a species.The final section of the chapter presents basic information about viruses, which, in addition to being significant pathogens, are important model organisms for studying macromolecular synthesisand other cellular processes.Viruses have relatively simple structures compared with cells, and small genomesthat made them tractable for historic early studies of the basic processesof DNA replication, transcription, translation, recombination, and gene expression.Viruses continue to teach important lessonsin molecular cell biology today and have been adapted as experimental tools for introducing any desired genes into cells, tools that are currently being tested for their effectivenessin human gene rnerapy.

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4-1 Overviewof four basicmoleculargenetic A FIGURE that leadto we coverthethreeprocesses processes. In thischapter (n-B) andthe process DNA for replicating production of proteins (4) Because theyhavebeen machinery, viruses utilizehost-cell Duringtranscriptton for studying theseprocesses models important (n), thefour-base DNA geneby RNApolymerase of a protein-coding or of a proteiniscopied, the aminoacidsequence codespecifying by the RNA(pre-mRNA) messenger into a precursor transcribed, (rNTPs) monomers triphosphate polymerization of ribonucleoside to the sequences andothermodifications of noncoding Removal producea (Z), collectively processing, pre-mRNA knownasRIVA During to the cytoplasm whichistransported mRNA, functional

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Structureof NucleicAcids

DNA and RNA are chemically very similar. The primary structuresof both are linear polymerscomposedof monomers called nucleotides.Both function primarily as informational molecules,carrying information in the exact sequenceof their nucleotides.Cellular RNAs range in length from fewer than one hundred to many thousandsof nucleotides.Cellular DNA moleculescan be as long as severalhundredmillion nucleotides.These large DNA units in associationwith proteins can be stained with dyes and visualized in the light microscope as chromosomes,so named becauseof their stainability. Though chemically similar, DNA and RNA exhibit some very important differences.For example, RNA can also function as a catalytic molecule.As we'll see,it is

intothe (B), thefour-base codeof the mRNAisdecoded translation the macromolecular of proteinsRibosomes, acidlanguage 2O-amino of two subunits themRNAcode,arecomposed thattranslate machines (rRNAs) andmultiple RNAs fromribosomal in the nucleolus assembled subunits ribosomal cytoplasm, (/eft). to the Aftertransport proteins the help with protein synthesis out carry and mRNA with an associate factorsDuringDNA (tRNAs) translation andvarious RNAs of transfer to divide, (4), whichoccurs onlyin cellspreparing replication (dNTPs) arepolymerized monomers triphosphate deoxyribonucleoside molecule' DNA chromosomal each of copies identical to yieldtwo copies oneof the identical cellreceives Eachdaughter

the different and unique properties of DNA and RNA that make them each suited for their specificroles in the cell'

A N u c l e i cA c i d S t r a n dl s a L i n e a rP o l y m e r w i t h E n d - t o - E n dD i r e c t i o n a l i t Y In all organisms, DNA and RNA each comprise only four different nucleotides. Recall from Chaptet 2 that all nucleotidesconsist of an organic base linked to a five-carbon sugar that has a phosphate group attached to carbon 5' In RNA, the sugar is ribose; in DNA, deoxyribose(seeFig,r,re2-16). The nucleotides used in synthesisof DNA and RNA contain one of five different bases.The basesadenine (A) and guanine(G) arepurines,whichcontainapaftof fused rings; the basescytosine (C), tbymine (T), and uracil (U) are

ACIDS S T R U C T U ROEF N U C L E I C

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erty of the molecule. The chemical linkage berweenadjacent nucleotides,commonly called a phosphodiesterbond, actually consistsof two phosphoesterbonds, one on the 5, side of the phosphate and another on the 3' side. The linear sequenceof nucleotideslinked by phosphodiesterbonds constitutesthe primary structureof nucleic acids. Like polypeptides, polynucleotides can twist and fold into three-dimensionalconformations stabilized by noncovalent bonds. Although the primary structures of DNA and RNA are generally similar, their three-dimensionalconformations are quite different. Thesestructural differencesare critical to the different functions of the two types of nucleic acids.

N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y A n t i p a r a l l eS l trands The modern era of molecular biology began in 1953 when 'Watson and Francis H. C. Crick proposed that James D. DNA has a double-helical structure. Their proposal, based on analysis of x-ray diffraction patterns generated by Rosalind Franklin and Maurice Wilkins, and coupled with careful model building, proved correct and paved the way for our modern understandingof how DNA functions as the geneticmaterial. 3'end DNA consists of two associatedpolynucleotide strands OH H that wind together to form a double helix. The two sugarA FIGURE 4-2 Chemicaldirectionalityof a nucleicacidstrand. phosphate backbonesare on the outside of the double helix, Shownherearealternative representations of a singlestrandof DNA and the bases project into the interior. The adjoining basesin containing onlythreebases: (C), (A), cytosine adenine andguanine (G) (a)Thechemical each strand stack on top of one another in parallel planes structure showsa hydroxyl groupat the 3, endand (Figure 4-3a). The orientation of the two strands is antipara phosphate groupat the 5, end Notealsothattwo phosphoester bondslinkadjacent nucleotides; allel; that is, their 5'-+3' directions are opposite.The strands thistwo-bondlinkage commonly rs referredto asa phosphodiester bond.(b)In the ,,stick,'diagram are held in precise register by formation of base pairs be(top),thesugars areindicated asvertical linesandthe phosphodiester tween the two strands: A is paired with T through two hybondsasslanting lines;the bases aredenotedbytheirsingle_letter drogen bonds; G is paired with C through three hydrogen abbreviations Inthe simplest (bottom), representation onlythe bases bonds (Figure 4-3b). This base-paircomplementarityis a areindicatedByconvention, a polynucleotide sequence tsatways consequenceof the size,shape,and chemicalcomposition of writtenin the 5'-+3'direction (leftto righ0unless otherwise indicated the bases.The presenceof thousands of such hydrogen bonds in a DNA molecule contributes greaiy to the stability of the double helix. Hydrophobic and van der N7aalsinterpyrimidines, which contain a single ring (see Figue 2-17). actions between the stacked adjacent basepairs further staThree of thesebases-A, G, and C-are found in both DNA bilize the double-helicalstrucure. and RNA; however, T is found only in DNA, and U only in In natural DNA, A always hydrogen bonds with T and RNA. (Note that the single-letterabbreviationsfor these G with C, forming A.T and G.C basepairs, as shown in Figbasesare also commonly usedto denotethe entire nucleotides ure 4-3b. These associations,always between a larger in nucleic acid polymers.) purine and smaller pyrimidine are often called '\X/atsonA single nucleic acid strand has a backboae composedof Crick base pairs. Two polynucleotide strands, or regions repeating pentose-phosphateunits from which the purine thereof, in which all the nucleotides form such base pairs and pyrimidine basesextend as side groups. Like a poiypepare said to be complementary. However, in theory and in tide, a nucleic acid strand has an end-to-endchemical oiiensynthetic DNAs, other base pairs can form. For example, guanine(a purine) could theoreticallyform hydrogenbonds with thymine (a pyrimidine), causing only a minor distortion in the helix. The spaceavailable in the helix also would allow pairing between the two pyrimidines cytosine and thymine. Although the nonstandard G.T and C.T basepairs polynucleotide sequencesare written and read in the 5'--+3, are normally not found in DNA, G.U basepairs are quite direction (from left to right); for example,the sequenceAUG common in double-helical regions that form within otheris assumedto be (5')AUG(3,). As we will see, the 5,_+3, wise single-strandedRNA. Nonstandard base pairs do not directionality of a nucleic acid strand is an important propoccur naturally in duplex DNA becausethe DNA copying 114

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4-3 The DNAdoublehelix.(a)Space< FIGURE fillingmodelof B DNA,the mostcommonformof (lightshades) projectinward DNAin cells.Thebases (darkredand backbones fromthesugar-phosphate blue)of eachstrand,buttheiredgesareaccessible Arrowsindicate throughmajorandminorgrooves. bonds of eachstrand.Hydrogen the 5'-+3'direction betweenthe basesarein the centerof the structure arelinedby potential Themajorandminorgrooves (highlighted bonddonorsandacceptors hydrogen (b) double of DNA structure yellow). Chemical in showsthe two sugarschematic helixThisextended bondingbetween phosphate andhydrogen backbones (a)from basepairs,A T andG C. lPart theWatson-Crick (b) part R E f rom 287i755: R Wingetal, 1980,Nature i983,5ciAm 249:941 Dickerson,

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enzyme, which is described later in this chapter, does not permit them. Most DNA in cells is a right-handed helix. The x-ray diffraction pattern of DNA indicatesthat the stacked bases are regularly spaced0.34 nm apart along the helix axis. The helix makes a complete turn every 3.4 nm; thus there are about 10.1 pairs per turn. This is referredto as the B form of DNA, the normal form present in most DNA stretchesin cells. On the outside of B form DNA, the spacesbetweenthe intertwined strands form two helical grooves of different widths describedas the maior groove and the minor groove (seeFigure 4-3a). As a consequence,the atoms on the edges of each basewithin thesegroovesare accessiblefrom outside the helix, forming two types of binding surfaces.DNA-binding proteins can read the sequenceof basesin duplex DNA by contacting atoms in either the major or the minor grooves. Other structuresof DNA have beendescribedin addition to the major B form. Two of these are compared with B DNA in Figure 4-4. Under laboratory conditions in which most of the water is removed from DNA, the crystallographic structure of B DNA changesto the A form, which is wider and shorter than B DNA, with basepairs tilted rather than perpendicular to the helix axis. RNA-DNA and RNARNA helices exist in this form in cells and in vitro. Short DNA moleculescomposed of alternating purine-pyrimidine nucleotides(especiallyG's and C's) adopt an alternative lefthanded helix configuration instead of the normal righthanded helix. This structure is called Z DNA becausethe basesseemto zigzag when viewed from the side. Some evidence suggeststhat Z DNA may occur in cells, although its function is unknown.

By far, the most important modifications in the structure of standard B form DNA come about as a result of protein binding to specificDNA sequences.Although the multitude of hydrogen and hydrophobic bonds between the basesprovide stability to DNA, the double helix is flexible about its long axis. Unlike the ct helix in proteins (seeFigure 3-4)'

( a )B D N A

( b ) AD N A

( c ) ZD N A

4-4 Modelsof variousknown DNA structures.The A FIGURE whichareon the of thetwo strands, backbones sugar-phosphate (lighter areshownin redandblue;the bases in allstructures, outsrde :10.1 base inward(a)TheB formof DNAhas areoriented shades) apart' pairs nm are 0.34 base stacked turn.Adjacent pairsperhelical A formof DNAhas11 basepairsperturnand (b)Themorecompact to the helixaxis a largetilt of the basepairswith respect exhibits (c)Z DNAisa left-handed doublehelix S T R U C T U RO E F N U C L E I CA C I D S

115

\fhy did DNA evolve to be the carrier of genetic information in cellsas opposedto RNA? The hydrogen at the 2' position in the deoxyriboseof DNA makes it a far more stable molecule than RNA, which instead has a hydroxyl group at the 2' position of ribose (SeeFigure2-16).The 2'-hydroxyl groups in RNA participatein the sloq OH--catalyzedhydrolysis of phosphodiesterbonds at neutral pH (Figure4-6).The absenceof 2'-hydroxyl groups in DNA preventsthis process. Therefore, the presenceof deoxyribosein DNA makes it a more stablemolecule-a characteristiccritical to its function in the long-term storageof geneticinformation.

D N A C a nU n d e r g oR e v e r s i b l e S t r a n dS e p a r a t i o n During replication and transcription of DNA, the strands of the double helix must separateto allow the internal FIGURE 4-5 Proteininteractioncan bend DNA.Theconserved edgesof the basesto pair with the basesof the nucleotides C-terminal domainof theTATAbox-binding protein(TBp) bindsto being polymerizedinto new polynucleotidechains.In later theminorgrooveof specific DNAsequences richin A andl, untwisting sections,we describethe cellular mechanismsthat separate andsharply bending thedoublehelixTranscription of mosteukaryotic and subsequentlyreassociateDNA strandsduring replicagenesrequires participation of TBPlndapted fromD B Nikolov andS K tion and transcription. Here we discuss fundamental Burley,1997, ProcNat'lAcad SciU5A94:151 factors influencing the separation and reassociation of DNA strands.Thesepropertiesof DNA were elucidatedby in vitro expenments. thereare no hydrogenbondsparallelto the axis of the DNA The unwinding and separation of DNA strands,referred helix. This properry allows DNA to bend when complexed to as denaturation, or "melting," can be induced experiwith a DNA-binding protein (Figure4-5). Bendingof DNA mentally by increasing the temperature of a solution of is critical to the dense packing of DNA in chromatin, the DNA. As the thermal energy increases, the resulting protein-DNA complex in which nuclearDNA occursrn euincreasein molecular motion eventually breaks the hydrokaryotic cells(Chapter6). gen bonds and other forces that stabilizethe double helix;

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CHAPTER 4

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B A S I CM O L E C U L A G R E N E T TM CE C H A N T S M S

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FIGURE 4-7 G.Ccontentof DNAaffects EXPERIMENTAL at whichDNAdenatures meltingtemperature.Thetemperature with the proportion of G.Cpairs(a)Meltingof doubledincreases of ultraviolet by theabsorption stranded DNAcanbe monitored DNAunpair, the lightat 260 nm.As regions of double-stranded increases almosttwofoldThe absorption of lightby thoseregions

DNA in a double-stranded at whichhalfthe bases temperature of melting") isdenotedI' (for"temperature havedenatured sample muchlessasthe DNAchanges by single-stranded Lightabsorption (b) of the G'Ccontent function The I. is a isincreased temperature the I. the greater of the DNA;the higherthe G+C percentage,

the strandsthen separate,driven apart by the electrostatic repulsion of the negatively charged deoxyribose-phosphate backbone of each strand. Near the denaturation temperature, a small increasein temperaturecausesa rapid, near simultaneousloss of the multiple weak interactions holding the strands together along the entire length of the DNA molecules.Becausethe stackedbasepairs in duplex DNA absorb lessultraviolet (UV) light than the unstackedbases DNA, this leadsto an abrupt increasein in single-stranded the absorption of UV light, a phenomenon known as hyperchromicity (Figure 4 -7a). The mebing temperature (T-) at which DNA strands will separatedependson severalfactors.Moleculesthat contain a greater proportion of G'C pairs require higher temperatures to denature becausethe three hydrogen bonds in G'C pairs make thesebasepairs more stablethan A'T pairs, which have only two hydrogen bonds. Indeed, the percentage of G'C base pairs in a DNA sample can be estimated from its T- (Figure4-7b\.The ion concentrationalso influences the T- becausethe negatively charged phosphate groups in the two strands are shieldedby positively charged ions. When the ion concentration is low, this shielding is decreased,thus increasingthe repulsive forces between the strandsand reducingthe T-. Agentsthat destabilizehydrogen bonds, such as formamide or urea, also lower the 7-. Finalln extremesof pH denature DNA at low temperature. At low (acid) pH, the basesbecomeprotonated and thus positively charged, repelling one another. At high (alkaline) pH, the baseslose protons and become negatively charged, again repelling one another becauseof the similar charge.In cells,pH and temperatureare,for the most part, maintained. These features of DNA separation are most useful for manipulating DNA in a laboratory setting. The single-strandedDNA molecules that result from denaturation form random coils without an organized

structure. Lowering the temperature, increasing the ion concentration, or neutralizing the pH causes the two complementarystrandsto reassociateinto a perfect double helix. The extent of such renaturation is dependenton time, the DNA concentration, and the ionic concentration. Two DNA strands not related in sequencewill remain as random coils and will not renature; most importantly, they will not inhibit complementary DNA partner strands from finding each other and renaturing. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization, a powerful technique used to study the relatednessof two DNA samplesand to detect and isolate specificDNA moleculesin a mixture containing numerous different DNA sequences ( s e eF i g u r e5 - 1 6 ) .

TorsionalStressin DNA ls Relievedby Enzymes Many prokaryotic genomic DNAs and many viral DNAs are circular molecules. Circular DNA molecules also occur in mitochondria, which are present in almost all eukaryotic cells, and in chloroplasts, which are present in plants and some unicellular eukaryotes' Each of the two strands in a circular DNA molecule forms a closed structure without free ends' Localized unwinding of a circular DNA molecule, which occurs during DNA replication, induces torsional stress into the remaining portion of the molecule becausethe ends of the strands are not free to rotate. As a result, the DNA molecule twists back on itself, like a twisted rubber band, forming supercoils(Figure 4-8a). In other words, when part of the DNA helix is underwound, the remainder of the molecule becomes overwound. Bacterial and eukaryotic cells, however, contain topoisomerase .I, which can relieve any torsional stressthat developsin cellular DNA moleculesduring This enzymebinds to DNA replication or other processes. ACIDS S T R U C T U ROEF N U C L E I C

.

117

(a) Supercoiled

(b) Relaxedcircle

at random sites and breaks a phosphodiesterbond in one strand. Such a one-strand break in DNA is called a nick. The broken end then unwinds around the uncut strand, leadingto loss of supercoils(Figure4-8b). FinallS the same enzyme joins (ligates)the two ends of the broken strand. Another type of enzyme, topoisomerase11,makes breaks in both strands of a double-stranded DNA and rhen religates them. As a result, topoisomerase II can both relieve torsional stressand link together two circular DNA molecules a s i n t h e I i n k so f a c h a i n . Although eukaryotic nuclear DNA is linear,long loops of DNA are fixed in place within chromosomes (Chapter 6). Thus torsional stressand the consequentformation of supercoils also could occur during replication of nuclear DNA. As in bacterial cells, abundant topoisomeraseI in eukaryotic nuclei relieves any torsional stressin nuclear DNA that would develop in the absenceof this enzyme.

DifferentTypesof RNA Exhibit Various ConformationsRelatedto Their Functions The primary structure of RNA is generally similar to that of DNA with two exceptions: the sugar component of RNA, ribose, has a hydroxyl group at the 2, position (seeFigure 2-16b), and thymine in DNA is replaced by uracil in RNA. The presenceof thymine rarher rhan uracil in DNA is important to the long-term stability of DNA becauseof its function in DNA repair (seeSection 4.6). As noted earlier, the hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA. As a result of this labilitS RNA is cleaved into mononucleotides by alkaline solution (see Figure 4-6), whereas DNA is not. The C2 hydroxyl of RNA also provides a chemically reactive group that takes part in 118

.

cHAprER 4

I

< EXPERIMENTAT FIGURE 4-8 Topoisomerase I relievestorsionalstresson DNA.(a)Electron micrograph of SV40viralDNA.Whenthecircular DNAof the SV40virusisisolated andseoarated protein, fromitsassociated the DNAduplexis underwound andassumes thesupercoiled config(b)lf a supercoiled uration. DNAisnicked(i e., onestrandcleaved), the strands canrewind, leading to lossof a supercoil. Topoisomerase I catalyzes thisreaction andalsoreseals the broken endsAll thesupercoils in isolated SV40DNAcan be removed bythe sequential actionof thisenzyme, producing the relaxed-circle conformation For clarity, theshapes of the molecules at the bottom havebeensimplified

RNA-mediated catalysis.Like DNA, RNA is a long polynucleotide that can be double-strandedor single-stranded, linear or circular. It can also participate in a hybrid helix composed of one RNA strand and one DNA strand. As discussedabove, RNA-RNA and RNA-DNA double helices have a compact conformation like the A form of DNA (see Figure4-4b). Unlike DNA, which exists primarily as a very long double helix, most cellular RNAs are single-strandedand exhibit a vaiety of conformations (Figure 4-9). Differences in the sizesand conformations of the various types of RNA permit them to carry out specific functions in a cell. The simplest secondary structures in single-strandedRNAs are formed by pairing of complementary baseswithin a linear sequence."Hairpins" are formed by pairing of baseswithin :5-10 nucleotidesof eachother,and "stem-loops" by pairing of basesthat are separatedby >10 to severalhundred nucleotides.These simple folds can cooperate to form more complicated tertiary structures, one of which is termed a "pseudoknot." As discussedin detail later, IRNA molecules adopt a well-defined three-dimensional architecture in solution that is crucial in protein synthesis.Larger rRNA molecules also have locally well-defined three-dimensional structures, with more flexible links in between.Secondaryand tertiary structures also have been recognized in mRNA, particularly near the ends of molecules. Clearly, then, RNA moleculesare like proteins in that they have structured domains connecred by less structured, flexible stretches. The folded domains of RNA molecules not only are structurally analogous to the a helicesand B strands found in proteins, but in some casesalso have catalytic capacities.

B A s t cM o L E c u L A G R E N E T tMc E c H A N t s M S

In this chapter, we focus on the functions of mRNA, tRNA, and rRNA in gene expression.In later chapters we will encounter other RNAs, often associatedwith proteins' that participate in other cell functions.

(a) Secondarystructure

Structure of NucleicAcids Stem-loop (blTertiarystructure Ca,

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4-9 RNAsecondaryand tertiary structures.(a)StemA FIGURE andothersecondary structures canformby base loops,hairpins, pairingbetweendistantcomplementary of an RNAmolecule. segments loopbetween the base-paired thesingle-stranded In stem-loops, of nucleotrdes or eventhousands helical stemmaybe hundreds in hairpins, theshortturn maycontainasfew asfour long,whereas (b)Pseudoknots, are onetypeof RNAtertiarystructure, nucleotides. loopsthroughbasepairing formedby interaction of secondary shownformsthe core basesThestructure betweencomplementary RNAlefti Secondary-structure domainof the humantelomerase in greenandblueandsinglewith base-paired nucleotides diagram RNA of the telomerase strandedregionsin red.Middle:Sequence diagram to correspond to thesecondary-structure coredomain,colored coredomainstructure at the left.R/ghtiDiagramof the telomerase onlyanda tube bases by 2D-NMR, showingbase-paired determined to the colored to correspond backbone, for the sugar-phosphate (b),middle fromC A Theimer andright,adapted to the left [Part diagrams et al , 2005, Mol Cell 17:671]l

Such catalytic RNAs are called ribozymes. Although ribozymes usually are associatedwith proteins that stabilize the ribozyme structure, it is the RNA that acts as a catalyst. Some ribozymes can catalyzesplicing, a remarkable process in which an internal RNA sequenceis cut and removed, and the two resulting chains then ligated. This process occurs during formation of the majority of functional mRNA moleculesin multicellular eukaryotes,and also occurs in single-celled eukaryotes such as yeast, bacteria, and archaea. Remarkably, some RNAs carry out self-splicing, with the catalytic activity residing in the sequencethat is removed. The mechanismsof splicing and self-splicingare discussedin detail in Chapter 8. As noted later in this chapter, rRNA plays a catalytic role in the formation of peptide bonds during protein synthesis.

r Deoxyribonucleic acid (DNA), the geneticmaterial, carries information to specify the amino acid sequencesof proteins. It is transcribed into severaltypes of ribonucleic acid (RNA), including messengerRNA (mRNA), transfer RNA (IRNA), and ribosomal RNA (rRNA), which function in protein synthesis(seeFigure4-1). r Both DNA and RNA are long' unbranched polymers of nucleotides, which consist of a phosphorylated pentose linked to an organic base,either a purine or pyrimidine' The purines adenine (A) and guanine (G) and the pyrimine cytosine (C) are presentin both DNA and RNA. The pyrimidine thymine (T) present in DNA is replaced by the pyrimidine uracil (U) in RNA. r Adiacent nucleotides in a polynucleotide are linked by phosphodiester bonds. The entire strand has a chemical directionality with 5' and 3' ends (seeFigwe 4-2). r Natural DNA (B DNA) contains two complementary antiparallel polynucleotide strands wound together into a regular right-handed double helix with the baseson the inside and the two sugar-phosphatebackbones on the outside (seeFigure 4-3). Basepairing betweenthe strands and hydrophobic interactions between adjacent base pairs stacked perpendicular to the helix axis stabilize this natlve structure. r The bases in nucleic acids can interact via hydrogen bonds. The standard Watson-Crick base pairs are G'C, A.T (in DNA), and G'C, A'U (in RNA). Basepairing stabilizes the native three-dimensionalstructures of DNA and RNA. r Binding of protein to DNA can deform its helical structure, causing local bending or unwinding of the DNA molecule. r Heat causesthe DNA strandsto separate(denature).The melting temperature7- of DNA increaseswith the percentage of G'C basepairs. Under suitableconditions, separated complementarynucleic acid strandswill renature. r Circular DNA moleculescan be twisted on themselves' forming supercoils (see Figure 4-8). Enzymes called topoisomerasescan relieve torsional stressand remove supercoils from circular DNA molecules' Long linear DNA ian also experiencetorsional stressbecauselong loops are fixed in place within chromosomes' r Cellular RNAs are single-strandedpolynucleotides, some of which form well-defined secondary and tertiary structures (seeFigure 4-9). Some RNAs, called ribozymes' have catalytic activitY.

S T R U C T U RO E F N U C L E I CA C I D S

119

Transcription of Protein-Coding

@

Genesand Formationof Functional mRNA The simplest definition of a gene is a "unir of DNA that contains the information to specify synthesisof a single polypeptide chain or functional RNA (such as a IRNA)." The DNA moleculesof small viruses contain only a few genes,whereas the single DNA molecule in each of the chromosomesof higher animals and plants may contain severalthousandgenes.The vast maiority of genescarry rnformation to build protein molecules,and it is the RNA copies of such protein-coding genes that constitute the mRNA moleculesof cells. During synthesisof RNA, the four-base language of DNA containing A, G, C, and T is simply copied, or transcribed,into the four-baselanguageof RNA, which is identical except that U replacesT. In contrast, during protein synthesis, the four-base language of DNA and RNA is translatedinto the 20-amino acid languageof proteins. In this section,we focus on formation of functional mRNAs from protein-coding genes(seeFigure 4-1, A). A similar process yields the precursors of rRNAs and tRNAs encoded by rRNA and IRNA genes;these precursorsare then further modified to yield functional rRNAs and tRNAs. In addition, thousands of recently discovered micro RNAs (miRNAs) that function ro resulare translat i o n o f s p e c i f i ct a r g e tm R N A s a n d r r a n s c r i p t - i oonf s p e c i f i c target genesare transcribedinto precursorsby RNA polymerasesand processedinto functional miRNAs. Transcription and processingof these other types of RNA is discussed in Chapter 8. Regulation of transcription allows distinct setsof genesto be expressedin the many different types of cellsthat make up a multicellular organism.It also allows different amounrs of mRNA to be transcribedfrom different genes, resulting in differences in the amounts of the encodedproteins in a cell. Regulation of transcription is addressedin Chapter 7.

A TemplateDNA Strand ls Transcribedinto a C o m p l e m e n t a rR y N AC h a i nb y R N Ap o l y m e r a s e During transcription of DNA, one DNA strand acs as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA chain. Basesin the template DNA strand base-pair with complementary incoming rNTps, which then are joined in a polymerization reaction catalyzed by RNA polymerase.Polymerization involves a nucleophilic attack by the 3' oxygen in the growing RNA chain on the ct phosphate of the next nucleotide precursor to be added, resulting in formation of a phosphodiesterbond and release of pyrophosphate(PP;).As a consequenceof this mechanism, RNA moleculesare always synthesizedin the 5,-+3, direction (Figure 4-10a). The energeticsof the polymerization reaction strongly favor addition of ribonucleotides to the growing RNA 120

.

c H A p r E4R |

chain becausethe high-energybond betweenthe cr and B phosphate of rNTP monomers is replaced by the lower-energy phosphodiesterbond betweennucleotides.The equilibrium for the reaction is driven farther toward chain elongation by pyrophosphatase,an enzyme that catalyzescleavageof the releasedPP; into two molecules of inorganic phosphate. Like the two strands in DNA, the template DNA strand and the growing RNA strand that is base-pairedto it have opposite5'-+3' directionality. By convention, the site on the DNA at which RNA polymerasebegins transcription is numbered *1. Downstream denotes the direction in which a template DNA strand is transcribed;upstreamdenotesthe opposite direction. Nucleotide positions in the DNA sequencedownstream from a start site are indicated by a positive (+) sign; those upstream,by a negative(- ) sign. BecauseRNA is synthesized5'-+3', RNA polymerase moves down the template DNA strand in a 3'-+5' direction. The newly synthesizedRNA is complementary to the template DNA strand; therefore, it is identical with the nontemplate DNA strand, except with uracil in place of thymine (see F i g u r e4 - 1 0 b ) . Stages in Transcription To carry out transcription, RNA polymerase performs several distinct functions, as depicted in Figure 4-11. During transcription initiation, RNA polymeraserecognizesand binds to a specific site, called a promoter, in double-strandedDNA (step [). RNA polymerases require various protein factors, called general transcription factors, to help them locate promoters and initiate transcription. After binding to a promoter, RNA polymerase separates the DNA strandsin order to make the basesin the template strand available for basepairing with the basesof the ribonucleosidetriphosphatesthat it will polymerize rogether. RNA polymerasesmelt 12-t4base pairs of DNA around the transcription start site, which is located on the template strand within the promoter region (step [). This allows the template strand to enter the active site of the enzyme that catalyzesphosphodiester bond formation between ribonucleoside triphosphates that are complementary to the promoter template strand at the start site of transcription. The 12-14-base-pairregion of melted DNA in the polymeraseis known as the "transcription bubble." Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond (stepS). After several ribonucleotides have been polymerized, RNA polymerase dissociatesfrom the promoter DNA and generaltranscription factors.During the stageof strand elongation, RNA polymerasemoves along the template DNA one baseat a time, opening the double-strandedDNA in front of its direction of movement and guiding the strandstogetherso that they hybridize at the upstream end of the transcriprion bubble (Figure4-11, step@). One ribonucleotideat a time is added to the 3' end of the growing (nascent)RNA chain during strand elongation by the polymerase. The enzyme maintains a melted region of approximately 1.4 base pairs, the transcription bubble. Approximately eight nucleotidesat

B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

the 3' end of the growing RNA strand remain base-pairedto the template DNA strand in the transcription bubble. The elongation complex, comprising RNA polymerase,template DNA, and the growing (nascent)RNA strand, is extraordinarily stable. For example, RNA polymerasetranscribesthe longestknown mammalian gene,containing about 2 million basepairs, without dissociatingfrom the DNA template or releasingthe nascentRNA. SinceRNA synthesisoccurs at a rate of about 1000 nucleotidesper minute at 37 "C, the elongation complex must remain intact for more than 24 hours to assurecontinuous RNA synthesisof the pre-mRNA from this very long gene. During transcription termination, the final stagein RNA synthesis,the completed RNA molecule, or primary transcript, is releasedfrom the RNA polymerase, and the polymerase dissociatesfrom the template DNA (Figure4-11, step El). Specific sequencesin the template DNA signal the bound RNA polymeraseto terminate transcription. Once released,

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121

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Polymeraseadvances 3'+ 5'down template strand,meltingduplex DNA and addingrNTps to growingRNA. TERMINATION

!

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an RNA polymeraseis free to transcribethe samegeneagain or another gene.

Organizationof GenesDiffers in Prokaryotic a n d E u k a r y o t i cD N A

Structure of RNA Polymerases The RNA polymerases of bacteria, archaea,and eukaryotic cells are fundamentally similar in structure and function. Bacterial RNA polymerasesare composedof two relatedlarge subunits(B, and B), two copiesof a smallersubunit (cr),and one copy of a fifth subunit (o) that is not essentialfor transcription or cell viability but stabilizesrhe enzyme and assisrsin the assembly of its subunits. Archaeal and eukaryotic RNA polymerases have several additional small subunits associated with this core complex, which we describe in Chapter 7. Schematic diagrams of the transcription process generally show RNA polymerasebound to an unbent DNA molecule, as in Figure 4-11. However, X-ray crystallography and other studies of an elongating bacterial RNA poiymerase indicate that the DNA bends at rhe transcription bubble (Figwe 4-12).

Having outlined the process of transcription, we now briefly consider the large-scalearrangement of information in DNA and how this arrangement dictates the requirementsfor RNA synthesisso that information transfer goes smoothly. In recent years, sequencingof the entire genomes from several organisms has revealed not only large variations in the number of protein-codinggenesbut also differencesin their organization in prokaryotes and eukaryotes. The most common arrangementof protein-coding genes in all prokaryotes has a powerful and appealing logic: genes encoding proteins that function together, for example, the enzymesrequired to synthesizethe amino acid tryptophan, are most often found in a contiguous array in the DNA. Such an arrangement of genesin a functional group is called an operon becauseit operatesas a unit from a singlepromoter. Transcription of an operon produces a continuous strand of

122

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BASTC MOLECULAG R E N E T TM CE C H A N T S M S

mRNA that carriesthe messagefor a relatedseriesof proteins (Figure 4-13a). Each section of the mRNA representsthe unit (or gene)that encodesone of the proteins in the series. This arrangementresults in the coordinate expressionof aIl the genes in the operon. Every time an RNA polymerase molecule initiates transcription at the promoter of the operon, all the genesof the operon are transcribedand translated. In prokaryotic DNA the genesare closelypacked with very few noncoding gaps, and the DNA is transcribed directly into mRNA. BecauseDNA is not sequesteredin a nucleus in prokaryotes, ribosomes have immediate accessto the translation start sitesin the mRNA as they emergefrom the surface of the RNA polymerase. Consequently,translat i o n o f t h e m R N A b e g i n se v e n w h i l e t h e 3 ' e n d o f t h e mRNA is still being synthesizedat the active site of the

(a)

(b)

B subunit..r

-d ':' B'subunit/

r-1

IRNAZ n subunit

\o

subunit

Thisstructure corre4-12 BacterialRNApolymerase. A FIGURE phase(step4) molecule in theelongation to the polymerase sponds in the is proceeding transcriptton of Figure4-11 ln thesediagrams, DNAenters wheredownstream leftwarddirectionArrowsindicate andupstream DNAexitsat an anglefromthe downthe polymerase strandblue, streamDNA;thecodingstrandisred,the noncoding gold,B islight is subunit RNAgreenTheRNApolymerase nascent B' gray,andthe o subunitvisible fromthisangleisbrown In (a)a froman isvrewed modelof theelongation complex space-filling throughthe the bendin the DNAasit passes anglethatemphasizes polymerase is rotatedin (b)asshown,and Theelongation complex of the the structure proteins transparent to reveal aremadelargely in the that isnotvisible the polymerase bubbleinside transcription DNA to thetemplate modelNucleotides complementary space-filling RNAstrand(atthe left) The areaddedto the 3'-endof the nascent at the bottom nascent RNAexitsthe polymerase newlysynthesized a c h a n n ef ol r m e db e t w e etnh eB a n dB ' s u b u n i tTs h et r through fromthisangle[Courtesy subunitandtheothercrsubunitarevisible o f S e t hD a r s t ;s e eN K o r z h e v ae t a l , 2 0 0 0 , S c i e n c e 2 8 9 , 6 1 9 - 6 2 5a,n d N Opalkaet al , 2003, Cell'114:335-345I

RNA polymerase. This economic clustering of genes devoted to a single metabolic function does not occur in eukaryotes, even simple ones like yeasts,which can be metabolically similar to bacteria. Rather, eukaryotic genes encoding proteins that function together are most often physically separatedin the DNA; indeed such genes usually are located on different chromosomes.Each gene is transcribed from its own promoter, producing one mRNA, which generally is translated to yield a singlepolypeptide (Figure 4-13b). When researchersfirst compared the nucleotide sequences of eukaryotic mRNAs from multicellular organisms with the DNA sequencesencoding them, they were surprised to find that the uninterrupted protein-coding sequenceof a given mRNA was discontinuous in its corresponding section of DNA. They concluded that the eukaryotic gene existed in pieces of coding sequence,the exons, separated by non-protein-coding segments' the introns. This astonishingfinding implied that the long initial primary transcript-the RNA copy of the entire transcribed DNA sequence-had to be clipped apatt to remove the introns and then carefully stitched back together to produce eukaryotic mRNAs. Although introns are common in multicellular eukaryotes, they are extremely rare in bacteria and archaeaand uncommon ln many unicellular eukaryotes such as baker's yeast. However, introns are present in the DNA of viruses that infect eukaryotic cells. Indeed, the presenceof introns was first discovered in such viruses, whose DNA is transcribedby host-cellenzymes.

EukaryoticPrecursormRNAsAre Processed l RNAs t o F o r mF u n c t i o n am In prokaryotic cells, which have no nuclei, translation of an mRNA into protein can begin from the 5' end of the mRNA evenwhile the 3' end is still being synthesizedby RNA polymerase,In other words, transcription and translation occur concurrently in prokaryotes. In eukaryotic cells, however, not only is the site of RNA synthesis-the nucleusseparatedfrom the site of translation-the cytoplasm-but also the primary transcripts of protein-coding genes are precursor mRNAs (pre-mRNAs) that must undergo several

LRNA O F F U N C T I O N AM GG E N E SA N D F O R M A T I O N ON F PROTEIN.CODIN TRANSCRIPTIO

123

(a)Prokaryotes

(b) Eukaryotes Yeastchromosomes Kb

TRP|

TRP4

E. coli genome 580

V

trp operon

-

:

I

E I p I c I B l A -8kb

910 Vll

Staft site for trp mRNA synthesis

680

Xl

rranscrintion f frp mRNA

5'

fff++

trp mRNAs

Start sitesfor protein synthesis I Translation

:Proteins

v -j-

I

-a B

Proteins

1 r

Translation

2345 IIII

FIGURE 4-13 Geneorganizationin prokaryotesand eukaryotes.(a)Thetryptophan(trp)operonisa continuous segment of the E.colichromosome, containing fivegenes(blue) that encodethe enzymes necessary for the stepwise synthesis of tryptophanTheentireoperonistranscribed fromonepromoter into onelongcontinuous frp mRNA(red)Translation of thismRNAbegins at fivedifferent startsites,yielding (green)Theorderof frveproteins the genesin the bacterial genomeparallels the sequential functionof

proteins theencoded (b)Thefivegenes in thetryptophan pathway. encoding theenzymes required for tryptophan synthesis in yeast (Saccharomyces cerevisiae) arecarriedon four differentchromosomes Eachgeneistranscribed fromitsown promoter to yielda primary transcript thatisprocessed intoa functional mRNAencoding a singleprotein. Thelengths of thevarious chromosomes areqivenin (103bases) kilobases

modifications, collectively termed RNA processing,to yield a functional mRNA (seeFigure 4-I,2). This mRNA then must be exported to the cytoplasm before it can be translated into protein. Thus transcription and translation cannot occur concurrently in eukaryotic cells. All eukaryotic pre-mRNAs initially are modified at the two ends, and these modifications are retained in mRNAs. As the 5' end of a nascent RNA chain emergesfrom the surface of RNA polymerase, it is immediately acted on by several enzymes that together synthesize the 5, cap, a 7-methylguanylate that is connected to the terminal nu-

yeastsand invertebratesthan in vertebrates.Poly(A) polymeraseis part of a complex of proteins that can locate and cleavea transcript at a specific site and then add the correct number of A residues,in a processthat does not require a template. The final step in the processing of many different eukaryotic mRNA molecules is RNA splicing: the internal cleavageof a transcripr to excisethe introns, followed by ligation of the coding exons. Figure 4-15 summarizesthe basic steps in eukaryotic mRNA processing, using the B-globin gene as an example. rJTeexamine the cellular machinery for carrying out processing of mRNA, as well as IRNA and rRNA, in Chapter 8. The functional eukaryotic mRNAs produced by RNA processingretain noncoding regions,referred to as 5' and 3, wntranslated regions (UTRs), at each end. In mammalian mRNAs, the 5' UTR may be a hundred or more nucleotides long, and the 3'UTR may be severalkilobasesin length. Prokaryotic mRNAs also usually have 5' and 3' UTRs, but these are much shorter than those in eukaryotic mRNAs. generally containing fewer than 10 nucleotides.

Processingat the 3' end of a pre-mRNA involvescleavage by an endonuclease to yield a free 3,-hydroxyl group to which a string of adenylic acid residuesis added orr. ut " time by an enzyme called poly(A) polymerase. The resulting poly(A) tail contains 100-250 bases,being shorter in 124

.

c H A p r E R4

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B A s t cM o l E c u L A RG E N E I c M E C H A N t s M s

7-Methylguanylate

4-14 Structureof the 5' methylatedcap' Thedistin< FIGURE capon eukaryotic guishing features of the 5' methylated chemical to the initial of 7-methylguanylate mRNAare(1)the 5'-+5' linkage and(2)the methylgroupon the 2' of the mRNAmolecule nucleotide (base1) Boththesefeahydroxyl of the riboseof thefirstnucleotide yeasts plants; lack of higher in cells cells and all animal in turesoccur nucleotide of thesecond 1 Theribose themethylgroupon nucleotide 1976,Cell (base2) alsoismethylated in vertebrates [SeeAJ Shatkin, 9:645I

HH I

o-P:o I o - o - PI: o

l2'

A l t e r n a t i v eR N AS p l i c i n gI n c r e a s e s the Numberof ProteinsExpressed f r o m a S i n g l eE u k a r y o t i cG e n e

OH OH 5' -> 5'linkage

o I o-P:o I o

In contrast to bacterial and archaealgenes,the vast majority of genesin higher, multicellular eukaryotescontain multiple introns. As noted in Chapter 3' many proteins from higher eukaryoteshave a multidomain tertiary structure (seeFigure 3-11). Individual repeatedprotein domains often are encoded by one exon or a small number of exons that code for identical or nearly identical amino acid sequences'Such repeated exons are thought to have evolved by the accidental multiple duplication of a length of DNA lying between two sites in adiacent introns, resulting in insertion of a string of repeated exons) separatedby introns, between the original two introns. The presenceof multiple introns in many eukaryotic genespermits expressionof multiple' related proteins from a single gene by means of alternative splicing. In higher eukaryotes,alternativesplicing is an important mechanism for production of different forms of a protein' called isoforms, by different types of cells'

Base 1 1',

2'

H

o-cH3

-o-P:o

o I

,ro

o-cH3

O-P:O

I

illll}

overview Animation: Life

le of an mRNA

RNAprocessing 4-15 Overviewof RNAprocessing. > FIGURE genecontains produces mRNAin eukaryotes. TheB-globin functional exons(constituting thecodingregion,red)and threeprotein-coding (blue)Theintronsinterrupt the noncoding introns two intervening for aminoacids31 and protein-coding sequence between thecodons genes protein-coding of eukaryotic 32 and105 and106.Transcription thatencodes thefirstaminoacidand startsbeforethesequence encoding thelastaminoacid,resulting beyond thesequence extends (gray) These transcript. in noncoding regions at theendsof theprimary (UTRs) The5' cap regions areretained duringprocessing. untranslated (mtGppp) RNAtranscript, isaddedduringformation of theprimary at the poly(A) whichextendsbeyondthe poly(A)site Aftercleavage removes of multiple A residues to the3' end,splicing siteandaddition referto positions Thesmallnumbers andjoinstheexons. the introns of B-globin. acidsequence in the 147-amino

p-Globin genomrc DNA Start site for

106

147

Poly(A) site

RNA synthesis Primary 5' RNA transcnpt

Aln

p-Globin mRNA

A),

MRNA T R A N S C R I P T I OONF P R O T E I N - C O D I NGGE N E SA N D F O R M A T I O NO F F U N C T I O N A L

125

F i b r o n e c t ig ne n e

Fibroblast f i b r o n e c t i nm R N A Hepatocyte fibronectinmRNA

A FIGURE 4-16 Alternativesplicing.The=75-kbfibronectin gene (fop)contains multiple exons; splicing of frbronectin varies by cell type TheElllBandElllAexons(green) encodebindingdomains for proteins specific on thesurface of fibroblasts. Thefibronectin mRNA Fibronectin, a multidomain protein found in mammals, providesa good exampleof alternativesplicing(Figure4-16). Fibronectin is a long, adhesiveprotein secretedinto the extracellular spacethar can bind other proteins together.\7hat and where it binds dependson which domains are splicedtogether. The fibronectin gene contains numerous exons, grouped into several regions corresponding to specific domains of the protein. Fibroblasts produce fibronectin mRNAs that contain exons EIIIA r.rd EtIIn; these exons encode amino acid sequencesthat bind tightly to proteins in the fibroblast plasma membrane. Consequently,this fibronectin isoform adheresfibroblasrsto the extiacellular matrix. Alternative splicing of the fibronectin primary transcript in hepatocytes,the major type of cell in the liver, yields mRNAs that lack the EIIIA and EIIIB exons. As a result, the fibronectin secretedby hepatocyresinto the blood does not adhere tightly to fibroblasts or most other cell types, allowing it to circulate. During formation of blood clots, however, the fibrin-binding domains of hepatocyte fibronectin binds to fibrin, one of the principal constituentsof clots. The bound fibronectin then interacts with integrins on the membranesof passingplatelets,thereby expanding the clot by addition of platelets. More than 20 different isoforms of fibronectin have been identified, each encoded by a different, alternatively spliced mRNA composed of a unique combination of fibronectin gene exons. Recent sequencingof large numbers of mRNAs isolated from various tissues and comparison of their sequenceswith genomic DNA has revealeJthat nearly 60 percent of all human genesare expressedas alternativelyspliced mRNAs. Clearly, alternative RNA splicing greatly expands the number of proteins encoded by the genomesof higher, multicellular organisms.

Transcriptionof Protein-CodingGenes and Formation of Functional mRNA Transcription of DNA is carried out by RNA polymerase, hich adds one ribonucleotide ar a time to the 3, end of a growing RNA chain (seeFigure4-11). The sequenceof the template DNA strand determines the order in which ribonucleotidesare polymerized to form an RNA chain. 126

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produced in fibroblasts includes the ElllAandElllBexons, whereas theseexonsarespliced out of fibronectin mRNAin hepatocytes. In thisdiagram, introns(blacklines) arenot drawnto scale; mostof themaremuchlongerthananyof theexons. r During transcription initiation, RNA polymerase binds to a specific site in DNA (the promoter), Iocally melts the double-strandedDNA to reveal the unpaired template strand, and polymerizes the first two nucleotides complementary to the template strand. The melted region of t2-14 basepairs is known as the "transcription bubble." r During strand elongation, RNA polymerase moves down the DNA, melting the DNA aheadof the polymerase, so that the template strand can enter the active site of the enzyme,and allowing the complementary DNA strands of the region just transcribed to reanneal behind it. The transcription bubble moves with the polymeraseas the enzyme adds ribonucleotidescomplementaryto the template strand to the 3' end of the growing RNA chain. 'S7hen r RNA polymerasereachesa termination sequence in the DNA, the enzymestops rranscription, leading to releaseof the completed RNA and dissociationof the enzyme from the template DNA. r In prokaryotic DNA, severalprotein-coding genescommonly are clustered into a functional region, an operon, which is transcribed from a single promoter into one mRNA encoding multiple proteins with related functions (seeFigure 4-13a). Translation of a bacterial mRNA can begin before synthesisof the mRNA is complete. r In eukaryotic DNA, each protein-coding gene is transcribedfrom its own promoter.The initial primary transcript very often containsnoncoding regions(introns) interspersed among coding regions (exons). r Eukaryotic primary transcripts must undergo RNA processingto yield functional RNAs. During processing,the ends of nearly all primary transcripts from protein-coding genesare modified by addition of a 5' cap and 3' poly(A) tail. Transcripts from genescontaining introns undergo splicing,the removal of the introns and joining of the exons (seeFigure4-15). r The individual domains of multidomain proteins found in higher eukaryotesare often encodedby individual exons or a small number of exons. Distinct isoforms of such proteins often are expressedin specific cell types as the result of alternative splicing of exons.

BASTC M O L E C U L AG R E N E T TM CE C H A N T S M S

The Decodingof mRNAby tRNAs

!f,

Although DNA storesthe information for protein synthesis and mRNA conveysthe instructions encodedin DNA, most biological activitiesare carried out by proteins.As we saw in Chapter 3, the linear order of amino acidsin eachprotein determinesits three-dimensionalstructure and activity. For this reason,assemblyof amino acids in their correct order, as encoded in DNA, is critical to production of functional proteins and hence the proper functioning of cells and organrsms. Translation is the whole processby which the nucleotide sequenceof an mRNA is used as a template to join the amino acids in a polypeptide chain in the correct order (see Figure 4-1,,g).In eukaryoticcells,protein synthesisoccurs in the cytoplasm, where three types of RNA moleculescome together to perform different but cooperative functions (Figue 4-1.7)z 1,. MessengerRNA (mRNA) carries the geneticinformation transcribed from DNA in a linear form. The mRNA is read in setsof three-nucleotidesequences,called codons, each of which specifiesa particular amino acid. 2. Transfer RNA (IRNA) is the key to decipheringthe codons in mRNA. Each type of amino acid has its own subset of tRNAs, which bind the amino acid and carry it to the growing end of a polypeptide chain when the next codon in

aa7-tRNA7 arflvrng

Growing polypeptide chain

H

Hrru-J-R, H-9

!--o o

?=o

I

o o

tRNA4 leaving

mRNA \____YJ

gYJ

q/J

Codon Codon Codon d0t

ddZ

ElOg

M o v e m e n to f r i b o s o m e

a FIGURE 4-17 The three rolesof RNAin protein synthesis. RNA(mRNA) istranslated intoproteinbythejointaction Messenger of whichiscomposed RNA(IRNA) andthe ribosome, of transfer proteins molecules RNA(rRNA) andtwo majorribosomal numerous (notshown)Notethe basepairingbetween and IRNAanticodons of a peptidebond codonsin the mRNA.Formation complementary aa-tRNA andthe the amino-group N on the incoming between iscatalyzed C on the growingproteinchain(green) carboxy-terminal from aa : aminoacid;R : sidegroup[Adapted by oneof the rRNAs. A J F G r i f f i t h s e t a l , 1 9 9 9 ,M o d e r n G e n e t i cA n a l y s i s , WH F r e e m a na n d C o m p a n yl

the mRNA calls for it. The correct IRNA with its attached amino acid is selectedat each step becauseeach specific IRNA molecule contains a three-nucleotidesequence,an anticodon, that can base-pairwith its complementary codon in the mRNA. 3. Ribosomal RNA (rRNA) associateswith a set of proteins to form ribosomes.Thesecomplex structures,which physically move along an mRNA molecule' catalyzethe assembly of amino acids into polypeptide chains. They also bind tRNAs and various accessoryproteins necessaryfor protein synthesis.Ribosomesare composedof a large and a small subunit, each of which contains its own rRNA molecule or molecules. Thesethree types of RNA participate in the synthesisof proteins in all organisms. Indeed, development of three functionally distinct RNAs was probably the molecular key to the origin of life. In this section,we focus on the decoding of mRNA by IRNA adaptors, and how the structure of each of these RNAs relates to its specific task. How they work together with rRNA, ribosomes, and other protein factors to synthesizeproteins is detailed in the following section.Since translation is essentialfor protein synthesis,the two processes commonly are referred to interchangeably.However, the polypeptide chains resulting from translation undergo posttranslational folding and often other changes(e.g.,chemical modifications, association with other chains) that are required for production of mature' functional proteins (Chapter3).

MessengerRNACarriesInformation from DNA in a Three-LetterGeneticCode As noted above, the genetic code used by cells is a triplet code, with every three-nucleotidesequence'or codon, being "read" from a specifiedstarting point in the mRNA. Of the 54 possiblecodons in the geneticcode' 51 specify individual amino acids and three are stop codons. Table 4-1 shows that most amino acids are encoded by more than one codon. and tryptophan-have a single Only two-methionine codon; at the other extreme, leucine,serine,and arginine are each specifiedby six different codons. The different codons for a given amino acid are said to be synonymous.The code itself is termed degenerate,meaning that a particular amino acid can be specifiedby multiple codons. Synthesisof all polypeptide chains in prokaryotic and eukaryotic cells begins with the amino acid methionine. In bacteria, a specialized form of methionine is used with a formyl group linked to its amino group. In most mRNAs, the start (initiator) codon specifying this amino-terminal methionine is AUG. In a few bacterial mRNAs, GUG is used as the initiator codon, and CUG occasionallyis used as an initiator codon for methionine in eukaryotes. The three codons UAA, UGA, and UAG do not specify amino acids but, rather, constitute stop (termination) codons that mark the carboxyl terminus of polypeptide chains in almost all cells. The sequenceof codons that runs from a specific start T H E D E C O D I N GO F m R N A B Y t R N A s

127

sEcoN0 P0stTt0N

U

E

U

C

A

G

Phe

Ser

Tyr

cys

U

Phe

Ser

Tyr

cyt

C

Leu

Ser

Stop

Stop

A

Leu

Ser

Stop

Ttp

G

Leu

Pro

His

Atg

U

Leu

Pro

His

Atg

C

Leu

Pro

Gln

Arg

Leu (Met)'*

Pro

Gln

Arg

A = G

Ile

Thr

Asn

Ser

U

Ile

Thr

Asn

Ser

C

Ile

Thr

Lys

Atg

A

Met (Start)

Thr

Lys

Arg

G

Val

Ala

Asp

Glv

U

Val

Ala

Asp

Glv

C

Val

Ala

Glu

Glv

A

Val (Met)"

Ala

GIu

Glv

G

z. g I

F a e 4 @ E g

A

G

-E E o q

= o m

*AUG is the most common initiator codon; GUG usually codes for valine and CUG for leucine, but. rarely, these codons can also code for methionine to initiate a protein chain.

codon to a stop codon is called a reading frame. This precise linear array of ribonucleotides in groups of three in mRNA specifiesthe precise linear sequenceof amino acids in a polypeptide chain and also signals where synthesis of the chain starts and stops. Becausethe genetic code is a non-overlapping triplet code without divisions between codons, a particular mRNA theoretically could be translated in three different reading frames. Indeed some mRNAs have been shown to contain overlapping information that can be translatedin different reading frames, yielding different polypeptides (Figure 4-18). The vast majority of mRNAs, however,can be read in only one frame becausestop codonsencountered in the other two possiblereading frames terminatetranslation before a functional protein is produced. Very rarely,

128

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another unusual coding arrangement occurs becauseof frame-shiftins. In this case the protein-synthesizingmachinery may read four nucleotidesas one amino acid and then continue reading triplets, or it may back up one base and read all succeedingtriplets in the new frame until termination of the chain occurs. Only a few dozen such instancesare known. The meaning of each codon is the same in most known organisms-a strong argument that life on earth evolved only once. In fact, the geneticcode shown in Table 4-1 is known as the uniuersal code. However, the geneticcode has beenfound to differ for a few codons in many mitochondria, in ciliated protozoans, and in Acetabwlaria, a single-celled plant. As shown in Table 4-2, most of thesechangesinvolve reading of normal stop codons as amino acids, not an

BASTC M O L E C U L AG R E N E T TM CE C H A N T S M S

(Figure 4-19). The anticodon in the IRNA then base-pairs with a codon in mRNA so that the activatedamino acid can be added to the growing polypeptide chain (seeFigures4-17 Polypeptidel and4-18). Some 30-40 different tRNAs have been identified in Frame2 bacterialcells and as many as 50-100 in animal and plant cells.Thus the number of tRNAs in most cellsis more than 2 Polypeptide the number of amino acids used in protein synthesis(20) and also differs from the number of amino acid codons in 4-18 Multiplereadingframesin an mRNA FIGURE the genetic code (61). Consequently,many amino acids lf translation shownbeginsat two sequence. of the mRNAsequence have more than one IRNA to which they can attach (exupstream startsites(notshown), thentwo overlapping different plaining how there can be more tRNAs than amino acids); framesarepossible In thisexample, thecodonsareshifted reading in addition, many tRNAs can pair with more than one the same onebaseto the rightin the lowerframeAs a result, nucleotide sequence specifies different aminoacidsduringtranslation, codon (explaining how there can be more codons than tRNAs). in two of thethree Althoughregions of sequence thataretranslated The function of IRNA molecules,which are 70-80 nupossible framesarerare,thereareexamples in bothprokaryreading wherethe same cleotideslong, dependson their precisethree-dimensional andespecially in theirviruses, otesandeukaryotes, fromthe same isusedin two alternative mRNAs expressed sequence structures.In solution, all IRNA moleculesfold into a sim, n dt h es e q u e n ci ser e a di n o n er e a d i nfgr a m ei n region o f D N Aa ilar stem-loop arrangement that resemblesa cloverleaf o n em R N Aa n di n a n a l t e r n a t i vr eea d i nfgr a m ei n t h eo t h e rm R N A . when drawn in two dimensions (Figure 4-20a)' The four is wherethe sameshortsequence Thereareevena few instances stems are short double helicesstabilizedby Watson-Crick reading frames readin allthreepossible basepairing; three of the four stemshave loops containing sevenor eight basesat their ends,while the remaining,unloooed stem contains the free 3' and 5' ends of the chain. exchangeof one amino acid for another.Theseexceptionsto The three nucleotidescomposingthe anticodon are located the universalcode probably were later evolutionary developat the center of the middle loop' in an accessibleposition ments; that is, at no single time was the code immutably that facilitates codon-anticodon base pairing. In all tRNAs, fixed, although massivechangeswere not tolerated once a the 3' end of the unlooped amino acrdacceptor stem has the generalcode began to function early in evolution. sequenceCCA, which in most casesis added after synthesis and processingof the IRNA are complete.Severalbasesin The FoldedStructureof tRNA Promoteslts most tRNAs also are modified after transcription, creating D e c o d i n gF u n c t i o n s nonstandard nucleotidessuch as inosine, dihydrouridine, and pseudouridine. As we will see shortly, some of these Translation, or decoding, of the four-nucleotidelanguageof modified basesare known to play an important role in proDNA and mRNA into the 2O-amino acid languageof protein synthesis. Viewed in three dimensions, the folded teins requires tRNAs and enzymes called aminoacyl-tRNA IRNA molecule has an L shape with the anticodon loop synthetases.To participate in protein synthesis,a IRNA moland acceptor stem forming the ends of the two arms (Figecule must become chemically linked to a particular amino ure4-20b\. acid via a high-energy bond, forming an aminoacyl-tRNA F r a m e1

C()DON

Uf'IIVERSAL C0DE

C(]DTUNUSUAL

OCCURRENCE

UGA

Stop

Trp

My cop lasma, Spir op lasma, mitochondria of many species

CUG

Leu

Thr

Mitochondria in yeasts

UAA, UAG

Stop

Gln

A cetab ul ar ia, Tetr ah y m ena, Paramecium, etc.

UGA

Stop

cys

Euplotes

"Found in nuclear genesof the listed organisms and in mitochondrial genesas indicated. souRCE:S. Osawa et al., 1.992,Microbiol. Reu. 56:229.

OF mRNABY tRNAs THEDECODING

129

A m i n o a c i d( P h e )

High-energy ester bond

HO

til

H 2 N- C - C

-OH

r'',li,,

d

z

E Linkaqe of P;t" 1i 131114Ptre

ATP AAA AminoacyltRNA synthetase tRNA specificfor specificfor Phe Phe (tRNAPhe)

AMP + PPt AAA Aminoacyl-tRNA

FIGURE 4-19 Decodingnucleicacidsequenceinto amino acidsequence. Theprocess for translating nucleic acidsequences in mRNAintoaminoacidsequences in proteins involves two steps. Step[: An aminoacyl-tRNA synthetase f irstcouples a specific amino acid,viaa high-energy esterbond(yellow), to eitherthe 2, or 3,

AAA mRNA

hydroxyl of theterminal adenosine in the corresponding IRNA. StepE: A three-base sequence in theIRNA(theanticodon) then base-pairs with a codonin the mRNAspecifying theattached amino acid.lf an erroroccurs in eitherstep,thewrongaminoacidmaybe incorporated intoa polypeptide chainPhe: phenylalanine

NonstandardBasePairingOften Occurs BetweenCodonsand Anticodons

@ = dihYdrouridine = inosine e = ribothymidine e

If perfect Watson-Crick base pairing were demanded between codons and anticodons,cellswould have to contain at least 61 different types of tRNAs, one for each codon that specifiesan amino acid. As noted above,however,many cells contain fewer than 61 tRNAs. The explanation for the smaller number lies in the capability of a single IRNA anticodon to recognizemore than one, but not necessarilyeverg codon corresponding to a given amino acid. This broader recognition can occur becauseof nonstandard pairing between bases in the so-called uobble position: that is, the third (3') base in an mRNA codon and the corresponding first (5') basein its IRNA anticodon. The first and second basesof a codon almost always form standard Watson-Crickbasepairs with the third and secondbases,respectively,of the correspondinganticodon, but four nonstandardinteractionscan occur betweenbases in the wobble position. Particularly important is the G.U base pair, which structurally fits almost as well as the standard G.C pair. Thus, a given anticodon in IRNA with G in the first (wobble) position can base-pairwith the two

= pseudouridine @ m = methylgroup

T{/CG loop

Net result: Phe is selected by its codon

P h e - t R N A P h eb i n d s to the UUU codon

Acceptor stem

< FIGURE 4-20 Structureof tRNAs.(a)Althoughtheexact nucleotide sequence varies amongtRNAs, theyallfold intofour base-paired stemsandthreeloopsTheCCAsequence at the 3, end alsoisfoundin alltRNAsAttachment of an aminoacidto the 3, A yieldsan amrnoacyl-tRNA. Someof theA, C, G, andU residues are post-transcriptionally modified (seekey).Dihydrouridine in mosttRNAs (D)is nearlyalwayspresent in the D loop;likewise, (T) ribothymidine (V) arealmostalwayspresent andpseudouridlne in theTTITCG loop. Yeast alanine IRNA,represented here,alsocontains othermodified basesThetripletat thetip of theanticodon loopbase-pairs with the corresponding codonin mRNA(b)Three-dimensional modelof the generalized backbone of alltRNAsNotethe L shapeof the molecule. [Part(a) see R W Hollyet al , 1965, Science147i1462;part (b) from J G Arnez and D Moras, 1997, TrendsBiochem Sci 22:211 I

130

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B A S | CM O L E C U L A G R E N E T TM CE C H A N T S M S

tRNA

lf these basesare in first, or wobble, positionof a nticodon

321 12 5 ' m R N A3 '

5' mRNA 12 321

t h e nt h e t R N A m a y recognizecodons in m R N A h a v i n gt h e s e b a s e si n t h i r d p o s i t i o n

nized by the sameIRNA with the anticodon 3'-GAI-S'; the inosine in the wobble position forms nonstandard base pairs with the third basein the four codons. In the caseof in. UUR codon, a nonstandard G'U pair also forms between position 3 of the anticodon and position 1 of the codon.

Amino Acids BecomeActivatedWhen CovalentlyLinkedto tRNAs Recognition of the codon or codons specifying a given amino acid by a particular IRNA is actually the secondstep in decoding the genetic message.The first step' attachment

lf these basesare in t h i r d ,o r w o b b l e ,p o s i t i o n of codon of an mRNA then the codon may be recognizedby a tRNA havingthese basesin first position of anticodon

the 3' terminus of IRNA moleculesby an AlP-requiring reaction. In this reaction, the amino acid is linked to the tRNA by a high-energybond and is thus said to be actiuated. The energy of this bond subsequentlydrives formation of the peptide bonds linking adjacent amino acids in a growing c poiypeptide chain. The equilibrium of the aminoacylation 3', i.u.tiotr is driven further toward activation of the amino tRNA acid by hydrolysis of the high-energyphosphoanhydride 4-21 Nonstandard basepairingat the wobble A FIGURE bond in the releasedpyrophosphate (seeFigute 4-19). of an mRNA position.Thebasein thethird(orwobble)position Aminoacyl-IRNA synthetasesrecognize their cognate basepairwith the basein thefirst codonoftenformsa nonstandard tRNAs by interacting primarily with the anticodon loop (orwobble)position of a tRNAanticodonWobblepairingallowsa and acceptor stem, although interactions with other reit morethanone mRNAcodon(top);conversely, IRNAto recognize gions of a IRNA also contribute to recognition in some by morethanonekindof IRNA allowsa codonto be recognized cases.Also, specific bases in incorrect tRNAs that are (bottom), eachIRNAwill bearthesameaminoacid Note although structurally similar to a cognate IRNA will inhibit chargcan"read"(become in thewobbleposition thata IRNAwith l(inosine) ing of the incorrect IRNA. Thus, recognition of the correct pairedwith)threedifferent codons, anda IRNAwith G or U in the tRNA dependson both positive interactions and the abcanreadtwo codonsAlthoughA istheoretically wobbleposition senceof negative interactions. Still, becausesome amino possible it isalmostnever of the anticodon, in thewobbleposition acids are so similar structurally, aminoacyl-tRNA synfoundin nature thetasessometimesmake mistakes. These are corrected' however, by the enzymesthemselves,which have a proofreading activity that checks the fit in their amino correspondingcodonsthat have either pyrimidine (C or U) acid-binding pocket. If the wrong amino acid becomesatin the third position (Figure4-21).For example,the phenytached to a tRNA, the bound synthetase catalyzesremoval lalanine codons UUU and UUC (5'-+3') are both recogof the amino acid from the IRNA' This crucial function nized by the IRNA that has GAA (5'-+3') as the anticodon. : helps guarantee that a IRNA delivers the correct amino any base; In fact, any two codons of the type NNPyr (N : acid t" the protein-synthesizingmachinery. The overall pyrimidine) encode a single amino acid and are Pyr error rate for translation in E' coli is very loq approxidecoded by a single tRNA with G in the first (wobble) mately 1 per 50,000 codons, evidenceof both the fidelity position of the anticodon. of IRNA iecognition and the importance of proofreading Although adenine rarely is found in the anticodon by aminoacyl-IRNA synthetases. wobble position, many tRNAs in plants and animals conthis tain inosine (I), a deaminatedproduct of adenine, at position. Inosine can form nonstandardbasepairs with A, C, and U. A IRNA with inosine in the wobble position thus can recognizethe correspondingmRNA codons with The Decoding of mRNA bY tRNAs A , C , o r U i n t h e t h i r d ( w o b b l e )p o s i t i o n ( s e eF i g u r e4 - 2 1 ) ' r Genetic information is transcribed from DNA into mRNA For this reason,inosine-containingtRNAs are heavily emin the form of an overlapping,degeneratetriplet code' ployed in translation of the synonymouscodons that specify r Each amino acid is encoded by one or more threea single amino acid. For example, four of the six codons nucleotide sequences(codons) in mRNA. Each codon for leucine (CUA, CUC, CUU, and UUA) are all recogT H E D E C O D I N GO F m R N A B Y t R N A s

131

specifiesone amino acid, but most amino acidsare encoded by multiple codons(seeTable 4-1). r The AUG codon for methionine is the mosr common start codon, specifyingthe amino acid at the NH2-terminus of a protein chain. Three codons (UAA, UAG, UGA) function as stop codons and specify no amino acids. r A reading frame, the uninterrupted sequenceof codons in mRNA from a specific starr codon to a stop codon, is translated into the linear sequenceof amino acids in a polypeptide chain. r Decoding of the nucleotide sequencein mRNA into the amino acid sequenceof proteins depends on tRNAs and aminoacyl-IRNA synthetases. r All tRNAs have a similar three-dimensionalstructure that includes an acceptor arm for attachment of a specific amino acid and a stem-loopwith a three-baseanticodon sequence at its ends (seeFigure 4-20). The anticodon can base-pairwith its correspondingcodon in mRNA. r Becauseof nonstandard interactions, a tRNA may basepair with more than one mRNA codon; converselSa particular codon may base-pairwith multiple tRNAs. In each case,however,only the proper amino acid is insertedinto a g r o w i n gp o l y p e p t i d ec h a i n . Each of the 20 aminoacyl-tRNA synthetasesrecognrzesa ngle amino acid and covalently links it to a cognare

rRNA

IRNA, forming an aminoacyl-tRNA (seeFigure 4-19). This reaction activates the amino acid, so it can participate in peptide bond formation.

StepwiseSynthesis of proteins @ on Ribosomes The previous sectionshave introduced two of the major participants in protein synthesis-mRNA and aminoacylated IRNA. Here we first describethe third key player in protein synthesis-the rRNA-containing ribosome-before taking a detailed look at how all three components are brought together to carry out the biochemical eventsleading to formation of polypeptide chains on ribosomes. Similar to transcription, the complex processof translation can be divided into three stages-initiation, elongation, and terminationwhich we consider in order. \7e focus our description on translation in eukaryotic cells, but the mechanismof translation is fundamentally the same in all cells.

R i b o s o m eA s re Protein-Synthesizin Mga c h i n e s If the many components that participate in translating mRNA had to interact in free solution. the likelihood of simultaneouscollisions occurring would be so low that the rate of amino acid polymerization would be very slow. The

Proteins

.9

Subunits

Assembled ribosomes

Total: 31

23S (2900rNTs)

:.E

o o-

5S ( 1 2 0r N T s )

50s

Total: 21

70s

165 (1500 rNTs)

q,

o

t

o (J

o

J

lrj

( 1 9 0 0r N T s ) FfGURE4-22 Prokaryotic and eukaryotic ribosome components.In all cells,eachribosomeconsists of a largeand a small subunit.The two subunitscontainrRNAs(red)of differentlengths,as well as a differentset of proteins.All ribosomescontaintwo maior 132

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40s

80s

r R N Am o l e c u l e(s2 3 Sa n d 1 6 5r R N Ai n b a c t e r i a2;g Sa n d 1 g Sr R N Ai n vertebrates) and a 55 rRNA The largesubunitof verteorate ribosomesalsocontainsa 5 85 rRNAbase-paired to the 2gS rRNA The numberof ribonucleotides (rNTs)in eachrRNAtvpe is indicated

B A S t cM o L E c u L A G R E N E T tMc E C H A N t s M s

efficiencyof translation is greatly increasedby the binding of the mRNA and the individual aminoacyl-tRNAs to a ribosome. The ribosome, the most abundant RNA-protein complex in the cell, directselongationof a polypeptideat a rate of three to five amino acids added per second.Small proteins of 100-200 amino acids are thereforemade in a minute or less. On the other hand, it takes 2-3 hours to make the largest known protein, titin, which is found in muscle and contains about 30,000 amino acid residues.The cellular machine that accomplishesthis task must be preciseand persistent. With the aid of the electron microscope,ribosomeswere first discoveredas small, discrete,RNA-rich particles in cells that secretelarge amounts of protein. However, their role in protein synthesiswas not recognizeduntil reasonably pure ribosome preparations were obtained. In vitro radiolabeling experimentswith such preparationsshowed that radioactive amino acids were first incorporated into growing polypeptide chains that were associated with ribosomes before appearing in finished chains. Though there are differencesbetween the ribosomes of prokaryotes and eukaryotes,the great structural and functional similarities between ribosomes from all speciesreflects the common evolutionary origin of the most basic constituents of living cells. A ribosome is composedof three (in bacteria) or four (in eukaryotes)different rRNA molecules and as many as 83 proteins,organizedinto a largesubunit and a small subunit (Figure4-22).The ribosomal subunitsand the rRNA moleculesare commonly designatedin svedbergunits (S), a measureof the sedimentationrate of macromolecules a measure centrifuged under standard conditions----essentially, of size.The small ribosomal subunit contains a singlerRNA molecule, referred to as small rRNA. The large subunit contains a molecule of large rRNA and one molecule of 55 rRNA, plus an additional moleculeof 5.8SrRNA in vertebrates.The lengths of the rRNA molecules, the quantity of proteins in eachsubunit, and consequentlythe sizesof the subunitsdiffer between bacterial and eukaryotic cells. The assembledribosome is 70S in bacteriaand 80S in vertebrates. The sequencesof the small and large rRNAs from several thousand organismsare now known. Although the primary nucleotide sequencesof these rRNAs vary considerablS the sameparts of eachtype of rRNA theoreticallycan form basepaired stem-loops, which would generate a similar threedimensional structure for each rRNA in all organisms.The actual three-dimensionalstructuresof bacterial rRNAs from E. coli recently have been determined by x-ray crystallography of the 70S ribosome(Figure4-23).The multiple' much smaller ribosomal proteins for the most part are associated with the surfaceof the rRNAs. Although the number of protein molecules in ribosomes gready exceedsthe number of RNA molecules,RNA constitutes about 60 percent of the massof a ribosome.At the interfaceof the small and large ribosomal subunits,three local domains are formed' known as the A site, the P site, and the E site. As we'll seeshortly, these are the main sitesof interaction for the aminoacyl-tRNA and mRNA within the ribosome as protein synthesistakes place' During translation, a ribosome moves along an mRNA chain, interacting with various protein factors and tRNAs

and undergoing large conformational changes'Despite the complexity of the ribosome, great progresshas beenmade in deteimining the overall structure of bacterial ribosomesand in identifying various reactive sites' X-ray crystallographic studies on the T. thermophilus 705 ribosome, for instance, have not only revealed the dimensions and overall shape of the ribosomal subunits but also localized the positions of tRNAs bound to the ribosome during elongation of a growing protein chain. In addition, powerful chemical techniques r.r.h footprinting, which is describedin Chapter 7,have "r been used to identify specific nucleotide sequencesin rRNAs that bind to protein or another RNA. Some 40 yearc after the initial discovery of ribosomes' their overall structure and functioning during protein synthesisare finally becoming clear.

the AUG Recognizes Methionyl-tRNA;M"t Start Codon As noted earlier,the AUG codon for methionine functions as the start codon in the vast majority of mRNAs. A critical aspect of translation initiation is to begin protein synthesisat ihe start codon, thereby establishing the correct reading frame for the entire mRNA. Both prokaryotes and eukaryotes contain two different methionine tRNAs: tRNAlM"'can initiate protein synthesis, and IRNAM" can incorporate methionine only into a growing protein chain. The same aminoacyl-tRNA synthetase(MetRS) charges both tRNAs with met'hionine;however, only Met-tRNA,M" (i.e., activated methionine attached to tRNAiM't) can bind at the appropriate site on the small ribosomal subunit, the P site, to begin synthesisof a polypeptide chain' The regular Met-tRNAM"' and all other chargedtRNAs bind only to another ribosomal site, the A site, as described later. As mentioned earlier, in bacteria, the initiating methionine has a formyl group linked to its amino group, forming N-formylmethionine.

TranslationInitiation UsuallyOccursat the First A U G f r o m t h e 5 ' E n do f a n m R N A During the first stage of translation' the small and large ribosomal subunits assemblearound an mRNA that has an aminoacylated initiator IRNA correctly positioned at the start codon. This processis mediated by a specialset of proteins known as translation initiation factors (IFs). As each individual component ioins the complex, it is accompanied by one or more specific initiation factors. Interactions between theseinitiation factors help stabilizethe complex' Furthermore, some initiation factors are coupled to GTP, and the hydrolysis of GTP to GDP functions as a proofreading switch that allows subsequentsteps to proceed only if the

the two ribosomal subunits once the small subunit with a charged initiator IRNA (Met-tRNAiM"t) has bound to an ON RIBOSOMES O S T E P W I S sEY N T H E 5 I 5O F P R O T E I N S

133

Podcast:Structure of the Ribosome C) ZN Rotating 3-D Model of a BacterialRibosome < FfGURE4-23 Structureof E.coli 70Sribosomeas determined by x-ray crystallography.Modelof the ribosome viewedalongthe interface between the large(50S)andsmall(30S)subunitsThe165 rRNAandproteins in thesmallsubunitarecolored lightgreenand darkgreen,respectively; the23SrRNAandproteins in the largesubunit arecoloredlightpurpleanddarkpurple,respectively; andthe 55rRNA iscoloreddarkblue Thepositions of the ribosomal A, f andEsitesare indicatedNotethatthe ribosomal proteins arelocatedprimarily on the surface of the ribosome, andthe rRNAs on the inside, liningtheA, f andEsites[From B S Schuwirth etal.2005.Nature}l}:827 1

initiation codon in an mRNA. The first step of translation initiation is formation of a preinitiation complex. The preinitiation complex is formed when the 40S subunit complexed with the multisubunit eIF3 complex associateswith eIFIA and a ternary complex consisting of the MettRNAiM't and eIF2 bound to Ctp (Figure4-i4, steptr). The initiation factor eIF2 alrernates between association with GTP and GDP; it can bind Met-tRNA1M.. only when it is associatedwith GTP. Cells can regulate protein synthesis by phosphorylating a serine residue on the eIF2 bound to GDP; the phosphorylated complex is unable to exchange the bound GDP for GTP and therefore cannot bind MettRNAiM"t, thus inhibiting protein synthesis. The 5' cap of an mRNA to be translated is bound by the eIF4 cap-binding complex. The eIF4 cap-binding complex consists of several subunits with different functions; the eIF4E subunit of the eIF4 complex binds the 5/ cap structure on mRNAs (Figure 4-14).ThemRNA-eIF4 complix then as-

subunit so that it can remove short regions of RNA secondary structure in bound RNA using energy from ATp hydrol_ ysis. This multicomponent initiation complex then probably slides along, or scctns,the bound mRNA as the helicase activity of eIF4A unwinds RNA secondary structures that might otherwise interfere with scanningalong the mRNA in the 3' direction. Scanning srops when the tRNAiM.t anticodon recognizesthe start codon, which is the first AUG 134

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downstream from the 5' end in most eukaryotic mRNAs (step E). Recognition of the start codon leads to hydrolysis of the GTP associatedwith eIF2. an irreversible steD that prevents further scanning.Selectionof the initiating AUG is facilitated by specific surrounding nucleotides called the Kozak sequence,for Marilyn Kozak, who defined it: (5,) ACCAUGG (3'). The A precedingthe AUG (underlined)and the G immediately following it are the most important nucleotidesaffecting translation initiation efficiency.Once the small ribosomal subunit with its bound Met-tRNAiM". is correctly positioned at the start codon and the GTp bound by elF2 is hydrolyzed to GDP, elF1.,2,3, and 4 dissociate and the small subunit unites with the large (605) ribosomal subunit in a processcatalyzedby eIF5 and 6, completing formation of an 80S ribosome. Vith the entire complex assembled, the Met-tRNA1M"'bound to the AUG codon is situated in the P site. Recruitment of the large ribosomal subunit is accompaniedby hydrolysis of a GTP bound by eIF5, another proofreading step (step @). Coupling the ribosome-subunitjoining reaction to GTP hydrolysis allows the initiation process to continue only when the subunit interaction has occurred correctly. It also makes this an irreversiblestep, so that the ribosomal subunits do not dissociateuntil the entire mRNA is translated and protein synthesisis terminated. The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nucleotides of the 5' capped end as just described.However, somecellular mRNAs contain an internal ribosome entry site (IRES) located far downsrream of the 5' end. In addition, translation of some viral mRNAs, which lack a 5, cap, is initiated at IRES sequencesby the host-cell machinery of infectedeukaryotic cells.Someof the sametranslation initiation factors that assistin ribosome scanningfrom a 5, cap are required for locating an internal AUG start codon, but exactly how an IRES is recognizedis lessclear.Recentresultsindicate that some IRES sequencesfold into an RNA structure that binds to the E site on the ribosome (seebelow), thereby positioning a nearby internal AUG start codon in the p site. In bacteria, binding of the small ribosomal subunit to an initiation site occurs by a different mechanism that allows initiation at internal sitesin the polycistronic mRNAs transcribed from operons.In bacterial mRNAs, an :6-base sequencecomplementary to the 3' end of the small rRNA precedesthe AUG

B A s t cM o L E c u L A G R E N E T tMc E C H A N t s M s

4-24 lnitiationof translationin eukaryotes'/nset" < FIGURE the of translation, at thetermination dissociates Whena ribosome factorselF3andelF6, with initiation associate 4OSand605subunits thatcaninitiateanotherroundof formingcomplexes respectively, of the indicated addition Sequential and Steps Z: translation Il formsthe initiation complex to the40Ssubunit-elF3 components initiation of the mRNAbythe associated complexStepB: Scanning Metbound and subunit of thesmall leadsto positioning complex subuntt large of the Association Step 4: at thestartcodon. tRNAiM"t the mRNATwoiniti(605)formsan 805ribosome readyto translate proteins, elF2(step[) andelF5(step4) areGTP-binding ationfactors, precise The initiation. duringtranslation whoseboundGTPishydrolyzed yet well not is released are factors initiation timeat whichparticular discussron detailed more lAdapted for a text the See characterized

elF6

\

\,

f r o m R M e n d e za n d J D R i c h t e t2 0 0 1 , N a t u r eR e vM o l C e l lB i o l 2 : 5 2 1 l

start codon by 4-7 nucleotides.Basepairing betweenthis sequencein the mRNA, called the Shine-Dalgarnosequenceafter iis discoverers,and the small rRNA placesthe small ribosomal subunit in the proper position for initiation. Next' f-MettRNAiM" and initiation factors comparableto eIFIA' eIF2' and eIF3 associatewith the small subunit,followed by association of the large subunit to form the complete bacterial ribosomeby a mechanismsimilar to that in eukaryotes'

Preinitiation complex AUG-

(AAA)"

e l F 4( c a p - b i n d i ncgo m p l e x )+ m R N A

wret$cre mTGppp

D u r i n gC h a i nE l o n g a t i o nE a c hI n c o m i n g Aminoacyl-tRNAMovesThroughThree R i b o s o m aSl i t e s The correctly positioned ribosome-Met-tRNAiM't complex is now ready to begin the task of stepwiseaddition of amino acidsby the in-frame translation of the mRNA' As is the case with initiation, a set of special proteins, termed translation

(AAA}"

Initiationcomplex RNA u n w i n d in q , s c a n n r n g ,a n d start srte r e c o gn r tr or l

ATP

E

ADP + P; e l F 1 A ,e l F 3 ,e l F 4c o m p l e x , + P; elF2.GDP

(AAAln 3', 6 0 5 s u b u n i t - e l F 6e,l F S ' G T P g(

codon at a time along the mRNA. At the completion of translation initiation, as noted akeady,Met-tRNA1M" is bound to the P site on the assembled SOSribosome (Figure 4-25, top). This region of the ribosome is called the P site becausethe IRNA chemically linked to the growing polypeptide chain is located here' The second aminoacyl-tRNA is brought into the ribosome as a ternary complex in associationwith EFla'GTP and becomes bound to the A site, so named becauseit is where aminoacylated tRNAs bind (step E). EFlct'GTP bound to various

+ P; e l F 6 ,e | F S ' G D P l\

80S ribosome

SN R I B O S O M E S ' S T E P W I SSEY N T H E 5 IO5 F P R O T E I NO

135

FocusAnimation:proteinSynthesis flltt

Entry of next a a - t R N Aa t A site

< FIGURE 4-25 Peptidylchainelongationin eukaryotes. Once p siteis the80Sribosome with Met-tRNA,M", in the ribosome (top),a ternary assembled complex bearing thesecond aminoacid (aar)codedbythe mRNAbindsto theA site(stepO) Following a conformational changein the ribosome induced by hydrolysis of GTp (stepZ), thelargerRNAcatalyzes in EFIcTGTP peptide bondformation betweenMetiandaa2(step!) Hydrolysis of GTpin EF2.GTp causes anotherconformational changein the ribosome that results in its translocation onecodonalongthe mRNAandshiftsthe unacylated tRNAiM"t to the EsiteandtheIRNAwiththe boundpeptrde to the p site(step4) Thecyclecanbeginagainwith bjndingof a ternary complex bearing aa3to the now openA site In thesecono and subsequent elongation cycles, theIRNAat the Esiteisejected during stepI asa resultof theconformational change induced by hydrolysis of GTPin EFIa.GTP[Adapted fromK H Nierhaus etal. 2OOO. inR A Garrett et al, eds, TheRibosome: Structure, Function, Antibiotics, andCellular lnteractions, p 319l ASMPress,

E

G T Ph y d r o l y s i s ,

L

E | \ 6.oor-r, iii,'"?il1,'""", change +

Peptidebond formation

ff*l:Ii""

136

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cHAprER 4

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l'z

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EF2.GDp*pi Jl'.--

Thus, GTP hydrolysis by EFlcr is another proofreading step that allows protein synthesisto proceed only when the correct aminoacylated IRNA is bound to the A site. This ohenomenon contributes to the fidelity of protein synthesis. \fith the initiating Met-tRNAiM" at the p site and the second aminoacyl-tRNA tightly bound at the A site,the q amino group of the secondamino acid reactswith the ,,activated" (ester-linked)methionine on rhe initiator IRNA, forming a peptidebond (Figure4-25, stepB; seealsoFigure4-17).This peptidyltransferdse reaction is catalyzed by the large rRNA, which preciselyorients the interacting atoms, permitting the reaction to proceed.The catalytic ability of the large rRNA in bacteriahas beendemonstratedby carefullyremoving the vast majority of the protein from large ribosomal subunits. The nearly pure bacterial23S rRNA can catalyzea pepridyltransferasereaction betweenanalogsof aminoacylated-tRNAand peptidyl-tRNA. Further support for the catalytic role of large rRNA in protein synthesiscomesfrom crystallographicstudies showing that no proteins lie near the site of peptide bond synthesisin the crystal structureof the bacteriallarge subunit. Following peptide bond synthesis,the ribosomeis translocated along the mRNA a distanceequal to one codon. This translocation step is monitored by hydrolysis of the GTp in eukaryotic EF2.GTP. Once translocation has occurred correctly, the bound GTP is hydrolyzed, another irreversible processthat prevents the ribosome from moving along the RNA in the wrong direction or from translocatingan incorrect number of nucleotides.As a result of conformational changesin the ribosomethat accompanyproper translocation and the resulting GTP hydrolysis by EF2, tRNA;M.t, now without its activatedmethionine,is moved to the E (exit) site on the ribosome; concurrently,the secondtRNA, now covalently bound to a dipeptide(a peptidyl-rRNA), is moved to the P site (Figure4-25, stepZf). Tianslocationthus rerurnsthe ribosome conformation to a state in which the A site is ooen and able to accept another aminoacylatedIRNA complexed with EFlcr.GTP,beginninganother cycleof chain elongation. Repetition of the elongationcycle depictedin Figure 4-25 adds amino acids one at a time to the C-terminus of the

B A s t cM o L E c u L AG R E N E T tMc E c H A N I s M s

(a)

generaldomains of all tRNAs result in the movement of the tnXRr betweenthe A, P' and E sitesas the ribosome translocatesalong the mRNA one three-nucleotidecodon at a time'

Translationls Terminatedby ReleaseFactors W h e n a S t o p C o d o nl s R e a c h e d The final stageof translation, like initiation and elongation, requireshighly specificmolecular signalsthat decidethe fate of the mRNA-ribosome-peptidyl-tRNA complex' Two (b)

eukaryotic releasefactor, eRF3, is a GTP-binding protein' The eRF3'GTP actsin concertwith eRFl to promote cleavage of the peptidyl-tRNA, thus releasingthe completed protJin chain (Figure 4-27). Bacteria have two releasefactors (RF1 and RF2) that are functionally analogousto eRFl and

A FfGURE4-26 Low-resolutionmodel of E. coli 7OSribosome. images oI E colil0S (a)Toppanels microscoprc showcryoelectron showcomputerand5OSand30SsubunitsBottompanels ribosomes in the sameorientation of images of manydozens averages derived images (b)Modelof a 7OSribosome basedon thecomputer-derived are superimposed Three tRNAs studies cross-linking andon chemical polypeptide sitesThenascent andE(yellow) P(green), on theA (pink), c h a i ni sb u r i e di n a t u n n eiln t h el a r g er i b o s o msaul b u n ti th a tb e g i n s I S Gabashvili stemof theIRNAin the Psite [See closeto the acceptor of J Frank courtesy Cell1OO:537, l et al, 2OOO, growing polypeptideas directedby the mRNA sequence,until a stop codon is encountered.In subsequentcycles,the conformational changethat occurs in step f,l electsthe unacylated IRNA from the E site. As the nascent polypeptide chain becomeslonger,it threadsthrough a channelin the large ribosomal subunit, exiting at a position opposite the side that interactswith the small subunit (Figure4-26). RNAIn the absenceof the ribosome'the three-base-pair RNA hybrid between the IRNA anticodons and the mRNA codonsin the A and P siteswould not be stable;RNA-RNA duplexesbetweenseparateRNA moleculesmust be considerably longer to be stable under physiologicalconditions. However, multiple interactions between the large and small rRNAs and general domains of tRNAs (e.g., the D and TVCG loops, seeFigure 4-20) stabilizethe tRNAs in the A and P sites,while other RNA-RNA interactionssensecorrect codon-anticodonbasepairing, assuringthat the geneticcode is read properly. Then, interactions between rRNAs and the

eRFl + eRF3.GTP

t

Peptidyl-tRNA \ cleavage | \

-+

eRFl+eRF3oGDP+Pi

A FIGURE4-27 Terminationof translationin eukaryotes'When a stopcodon(UAA' proteinchainreaches bearinga nascent a ribosome probably complex, ribosomal factoreRFlentersthe UGA,UAG),release the bound of Hydrolysis eRF3'GTP with at or neartheA sitetogether chainfromtheIRNAin of the peptide bycleavage GTPisaccompanied subunits of thetRNAsandthetwo ribosomal the Psiteandrelease ON RIBOSOMES . 5 F PROTEINS S T E P W I SS E Y N T H E S IO

137

a GTP-binding factor (RF3) that is analosousto eRF3. Once again,the eRF3 GTPasemonirors the coriect recognition of a stop codon by eRF1. The peptidyl-rRNA bond of the IRNA

We can now seethat one or more GTp-binding proteins participate in each stage of translation. These proteins belong to the GTPasesuperfamily of switch proteins that cycle between a GTP-bound active form and GDp-bound inaciive form (seeFigure 3-32). Hydrolysisof the bound GTp causes a conformational change in the GTpase itself and other associatedproteins that are critical to various complex molecular processes.In translation initiation, for instance,hydrolysis of eIF2.GTP to eIF2.GDp prevenrsfurther scanning of the mRNA once the start site is encounrered and allJws binding of the large ribosomal subunit to the small subunit (seeFigure 4-24, step p). Similarly, hydrolysis of EF2.GTp to EF2.GDP during chain elongation leads to correct translocation of the ribosome along the mRNA (seeFigure 4-25, step Zl), and hydrolysisof eRF3.GTp to eRF3.GDp assurescorrect termination of translation. Since hvdrolvsis of the high-energy B-1 phosphoesterbond of GTi, is irreversible, coupling of these stepsin protein synthesisto GTp hydrolysis preventsthem from going in rhe reversedirection. One kind of mutation that can inactivate a gene in any organism is a base-pairchange that converts a codon nor_ mally encoding an amino acid into a stop codon, e.g., UAC (encodingtyrosine) -+ UAG (stop). When this occurs early in the reading frame, the resulting truncated protein usually is nonfunctional. Such mutations are called nonsense mvta_ tions becausewhen the genetic code equating each triplet codon sequencewith a single amino *"i being dlci_ ".id phered, the three stop codons were found ,rot to .rr.od. ,.ry amino acid-they did not ,,make sense.', In geneticstudieswith the bacterium E. coli, it was dis_

the protein encoded by the original gene with the nonsense mutation is produced to provide its essentialfunctions, the effect of the first mutation is said to be suppressedby the second mutation in the anticodon of the IRNA gene. This mechanism of nonsensesuppressionis a powerful tool in geneticstudiesin bacteria.For example, mutant bacterial virusescan be isolatedthat cannot grow in normal cells, but can grow in cellsexpressinga nonsense-suppressing IRNA becausethe mutant virus has a nonsensemutation in an essential gene. Such mutant viruses grown on the nonsense-suppressingcells can then be used in experimentsto analyzethe function of the mutant geneby infecting normal cellsthat do not suppressthe mutation and analyzing what step in the viral life cycle is defectivein the absenceof the mutant protein.

Polysomesand RapidRibosomeRecycling Increasethe Efficiencyof Translation

an mRNA. Simultaneoustranslation of an mRNA bv multiole ribosomesis readily observablein electronmicrographsandty sedimentation analysis,revealing mRNA attached to multiple ribosomes bearing nascentgrowing polypeptide chains. These structures, referred to as polyribosomes or polysomes, were seen to be circular in electron micrographs of some tissues. Subsequentstudieswith yeastcellsexplained the circular shape of polyribosomes and suggestedthe mechanism by which ribosomes recycleefficiently. These studies revealed that multiple copies of a cytosolic protein found in all eukaryotic cells,poly(A)-binding prroteinI (PABPI), can interacr with both an mRNA poly(A) tail and the 4G subunit of yeast eIF4. Recall that the 4E subunit of yeast eIF4 binds to the 5' end of an nRNA. As a result of thesein-

cap. The circular pathway depictedin Figure 4-Z9b,which may operate in many eukaryotic cells, would enhanceribosome re_ cycling and thus increasethe efficiency of protein synthesis.

Stepwise Synthesisof proteins on Ribosomes prokaryoric and eukaryotic ribosomes-the large .Both bonucleoproteincomplexeson which translation o..urrconsistof a small and a large subunit (seeFigure4-22).Each subunit contains numerous different proteins and one major rRNA molecule (small or large). The large subunit also contains one accessory55 rRNA in bacteriaand two acces, sory rRNAs in eukaryores(5S and 5.8S in vertebrates). 138

.

c H A p r E4R | B A S tM c oLEcuLA GRE N E TM t cE c H A N t s M s

ing GTP-binding proteins that hydrolyze their bound GTP TJGDP whe.t aitip has been completed successfully' During initiation' the ribosomal subunits assemblenear e translation start site in an mRNA molecule with the IRNA carrying the amino-terminal methionine (MettRNAiM"t) base-pairedwith the start codon (Figute 4-24)' ain elongation entails a repetitive four-step cycle: (1) binding of an incoming aminoacyl-tRNA to the A site e ribosome, (2) tight binding of the correct aminoacylIRNA to the A site accompanied by releaseof the previ-

to the E site (seeFigure4-25).

(b)

E'

leads to translocation. r Termination of translation is carried out by two types of termination factors: those that recognizestop codons and those that promote hydrolysis of peptidyl-tRNA (see Figure 4-27). Once again, correct recognition of a stop codon is monitored by a GTPase(eRF3)' The efficiency of protein synthesis is increased by the siultaneoustranslation of a singlemRNA by multiple ribo-

4-28 The circularstructureof mRNA FIGURE a EXPERIMENTAL mRNAformsa circular increasestranslationefficiency.Eukaryotic of threeproteins(a)In the presence owingto interactions structure eukaryotic protein| (PABPI), andelF4G, elF4E, of purifiedpoly(A)-binding visiblein thisatomicforcemicrograph structures, formcircular mRNAs interactlons protein-protein andprotein-mRNA Inthesestructures, the 5' and3' endsof the mRNA.(b)Model forma bridgebetween of ribosomal polysomes andrecycling on circular of proteinsynthesis translate cansimultaneously ribosomes Multipleindividual subunits. by formstabilized mRNA,shownherein circular a eukaryotic boundat the 3' and5' ends.Whena betweenproteins interactions fromthe 3' end,the anddissociates translation completes ribosome and 5' cap(m7G) nearby find the can rapidly subunits separated of A Sachs ] [Part(a)courtesy initrateanotherroundof svnthesrs. r Analogous rRNAs from many different speciesfold into quite similar three-dimensionalstructures containing numerous stem-loopsand binding sites for proteins, mRNA, and tRNAs. Much smaller ribosomal proteins are associated with the periphery of the rRNAs. r Of the two methionine tRNAs found in all cells,only one (tRNAiM't) functions in initiation of translation. r Each stage of translation-initiation, chain elongation, and termination-requires specific protein factors, includ-

!f,l

DNAReplication

Now that we haveseenhow geneticinformationencodedin

(duplex\DNA strandswould form a new double-stranded intact' In a remain molecule,and the parentalduplexwould are perstrands parental mechanism,the semiconservative with molecule a duplex and eachforms manentlyseparated, evidence Definitive it. to the daughterstrand base-paired DNA REPLICATION

139

(a) Predicted results C o n s e r v a t i vm e echanism

(b) Actual results

S e m i c o n s e r v a t i vmee c h a n r s m

Density-

Density-

Generation 0

P a r e n t asl t r a n d s s y n t h e s i z e idn 1 5 N

HH

0.7

HH

1.0 1.1 After first d o u b l i n gi n t a N

1.5 1.9 HH

/\

A

LL

/\

/\

LH

2.5

/\

3.0 4.1

After second d o u b l i n gi n t r N

0 and 1.9 mrxed 0 and4.1 mrxed HHLL

LLLL

LLHL

IGURE4-29 The Meselson_Stahl experiment be semiconservative.Thrsexperimentshowed a semiconseryative mechanism. E colicells initially weregrown in a mediumcontaining ammoniumsaltsprepared wlth "heavy"nitrogen(1sN)untilallthe cellularDNAwas labeledAfter the cellsweretransferred to a mediumcontaining the normal,,light,, isotope(raN),samples were removedperiodically from the cultures and the DNA in eachsamplewas analyzed by equilibrium density_gradient centrifugation, a procedure that separates macromolecules on the basis of theirdensity. Thistechniquecan separate (H_H), heavy_heavy light_ light(L-L),and heavy-light (H-L)duplexes into distinctbands. (a) Exp_ected compositionof daughterduplexmoleculessynthesized from 1sN-labeled DNAafterF. colicellsareshiftedto raN_containing medium if DNA replicationoccursby a conservative or semtconseryative mechanismParental heavy(H)strandsarein red;light(L)strands synthesized aftershiftto laN-containing mediumarein brue.Notethat the conservative mechanism nevergenerates H_LDNAand that the semiconservative mechanism nevergenerates H_HDNA but does generateH-LDNA duringthe secondand subsequent doublingsWith additionalreplication cycles, the lsN-labeled (H)strandsfrom the originalDNAarediluted,so that the vastbulkof the DNAwould consist that duplex DNA is replicated by a semiconservarive mech_

LHLL

L-L H-L H-H L-L H-L H-H of L-Lduplexes with eithermechanism(b)Actualbandingpatternsof DNAsubjected to equilibrium density-gradient centrifugationbefore and aftershiftinglsN-labeled E colicellsto laN-containing medium. DNA bandswerevisualized underUV lightand photographedThe traceson the left are a measureof the densityof the photographic signal,and hencethe DNAconcentratlon, alongthe lengthof the centrifugecellsfrom left to right.The numberof generations(far left) followingthe shiftto 1aN-containing mediumwas determined by countrngthe concentration of E colicellsin the cultureThisvalue corresponds to the numberof DNA replicatron cyclesthat had occurred at the time eachsamplewas taken.After one generation of growth,all the extractedDNA had the densityof H-LDNA.After '19 generations, approximately halfthe DNA hadthe densityof H-LDNA;the otherhalf had the densityof L-LDNA.With additionalgenerations, a largerand largerfractionof the extractedDNA consistedof L-Lduplexes;H_H duplexesneverappearedTheseresultsmatchthe predictedpatternfor the semiconservative replicationmechanismdepictedin (a) The bottom two centrifuge cellscontainedmixtures of H-HDNAand DNA isolated at 1 9 and 4 1 generations in orderto clearlyshowthe positions of H_H, H-1,and L-LDNA in the densitygradient.[part(b)fromM Meselson andF. W Stahl, 1958,Proc Nat'lAcadSciUSAM:6711

DNA strands. However, the vast preponderance of RNA and DNA in cells is synthesized from preexisting duplex DNA.

D N A P o l y m e r a s eRs e q u i r ea p r i m e r t o I n i t i a t eR e p l i c a t i o n Analogousto RNA, DNA is synthesized from deoxynucleoside 5'-triphosphateprecursors(dNTps). Also like RNA synthesis, DNA synthesisalwaysproceedsin the 5,+3, direction because chain growth resultsfrom formation of a phosphoesterbond 140

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cHAprER 4

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beftveenthe 3' oxygen of a growing strand and the o phosphate of a dNTP (seeFigure 4-10a). As discussedearlier, an RNA polymerasecan find an appropriate transcription start site on duplex DNA and initiate the synthesisof an RNA complementaryto the template DNA strand (seeFigure 4-11). In contrast, DNA polymerasescannot initiate chain synthesisde novo; instead, they require a short, preexisting RNA or DNA strand, called a primer, to begin chain growth. \7ith a primer base-pairedto the template strand, a DNA polymeraseadds deoxynucleotidesto the free hydroxyl group at the 3' end of the primer as directed by the sequenceof the template strand: Primer

lVhen RNA is the primer, the daughter strand that is formed is RNA at the 5' end and DNA at the 3' end.

D u p l e xD N A l s U n w o u n da n d D a u g h t e rS t r a n d s A r e F o r m e da t t h e D N A R e p l i c a t i o nF o r k In order for duplex DNA to function as a template during replication,the two intertwined strandsmust be unwound' or melted, to make the basesavailablefor basepairing with the basesof the dNTPs that are polymerizedinto the newly synthesized daughter strands. This unwinding of the parental DNA strandsis by specifichelicases,beginningat unique segments in a DNA moleculecalledreplication origins' or simply origins. The nucleotide sequencesof origins from different organisms vary gready,although they usually contain A'Trich sequences.Once helicaseshave unwound the parental DNA at an origin, a specializedRNA polymerasecalled primase forms a short RNA primer complementaryto the unwound template strands. The primer, still base-pairedto its complementary DNA strand, is then elongated by a DNA polymerase,thereby forming a new daughter strand.

cent fragments'

SeveralProteinsParticipatein DNA Replication Detailed understanding of the eukaryotic proteins that participate in DNA replication has come largely from studies witlh small viral DNAs, particularly SV40 DNA, the circular

llil+ FocusAnimation:NucleotidePolymerization by DNA Polyrngra:g DNA 4-30 leading-strandand lagging-strand > FIGURE to each areaddedby a DNApolymerase Nucleotides synthesis. (indicated by growingdaughter strandin the 5'-+3' direction f roma continuously strandissynthesized Theleading arrowheads) (red) is strand lagging end. The primer at its 5' singleRNA thatare frommultipleRNAprimers discontinuously synthesized duplexis aseachnewregionof the parental formedperiodically produces Okazaki initially of theseprimers unwoundElongation prevtous the growing approaches fragment As each fragments, areligated andthefragments primer, the primerisremoved of the entire in synthesis results eventually of thisprocess Repetition l a g g i nsgt r a n d .

5', P o i n to f j o i n i n g L a g g i n gs t r a n d Okazakifragment P a r e n t aD l NAduPlex

S h o r tR N A p r i m e r

5', Leadingstrand

DNA REPLICATION

14'l

FocusAnimation:Coordinationof Leading- and

flltt strandsynthesis

(a) SV40DNA replicationfork

E::i1,""';

31

5',

Primase

L a g g i n gs t r a n d

E

pPol E Rfc PCNA

Primer

(b}PCNA RPA

a

Doublestranded DNA

Leadingstrand

(c)RPA

FIGURE 4-31 Model of an SV40DNA replicationfork. (a)A hexamer of largeT-antigen ([), a viralprotein. functions asa helicase to unwindthe parental DNAstrands. Single-strand regions of the parental template unwoundby largeT-antigen areboundby multiple copies of the heterotrimeric proteinRpA(Z). Theleading strandissynthesized by a complex of DNApolymerase S (polS),

(E). (b)Thethreesubunits fragment of pCNA,shownin different colors, forma circular structure with a centralholethroughwhich double-stranded DNApasses. A diagram of DNAisshownjn the centerof a ribbonmodelof the pCNAtrimer.Thediaqram at the

pCNAboundto DNAin upperleftshowsthe iconrepresenting parta. (c)Thelargesubunitof RPAcontains two domains thatbind single-stranded DNA.Onthe left,thestructure determined for the two DNA-binding domains of the largesubunitboundto single_ stranded DNAisshownwith the DNAbackbone (whitebackbone with bluebases) parallel to the planeof the page Notethatthe singleDNAstrandisextended withthe bases exposed, an optimal conformation for replication by a DNApolymerase Onthe right,the viewisdownthe lengthof thesingleDNAstrand,revealing how RpA wraparoundthe DNA.Thediagram B strands at bottomcenter showsthe iconrepresenting heterotrimeric RpAboundto singlestranded DNAin part(a).[part (a)adapted fromS J Flint etal, 2000, pathogenesis, Virology: Molecular Biology, andControl, ASMpress; part(b) afterJ M Gulbis et al, 1996,Cetl87:297; andpart(c)afterA Bochkarev etal, 1997, Nature 385:176 l

Figure 4-31 depicts the multiple proteins that coordinate copying of SV40 DNA at a replication fork. The assembled proteins at a replication fork further illustrate the concept of molecular machines introduced in Chapter 3. These multi_ component complexes permit the cell to carry out an ordered sequenceof eventsthat accomplishessentialcell functions. The molecular machine that replicatesSV40 DNA con_ tains only one viral protein. All other proteins involved in 142

o

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I

B A s t cM o L E c u L A R G E N E T tM c ECHANtsMs

llll+ FocusAnimation:Bidirectional of DNA Reptication EcoRl

SV40 DNA replication are provided by the host cell. This viral protein, large T-antigen, forms a hexamer that unwinds the parental strandsat a replication fork. Primers for leading and lagging daughter-strandDNA are synthesizedby a complex of primase, which synthesizesa short RNA primer, and DNA polymerased (Pol a), which extendsthe RNA primer with deoxynucleotides,forming a mixed RNA-DNA primer' The primer is extended into daughter-strand DNA by DNA polymerase6 (Pol 6), which is lesslikely to make errors during copying of the templatestrand than is Pol ct becauseof its proofreading mechanism (see Section 4.6 below). Pol E forms a complex with R/c (replication factor C) and PCNA (proliferating cell ntclear antigen), which displaces the primase-Pol crcomplex following primer synthesis.As illustrated in Figure 4-31,b,PCNA is a homotrimeric protein that has a central hole through which the daughterduplex DNA passes, thereby preventing the PCNA-Rfc-Pol 6 complex from dissociating from the template.Pol E is the main polymeraseused by eukaryotes for elongating DNA strands during replication. After parental DNA is separatedinto single-strandedtemplatesat the replication fork, it is bound by multiple copiesof RPA (replicationprotein A), a heterotrimericprotein (Figure 4-31,c).Binding of RPA maintains the template in a uniform conformation optimal for copying by DNA polymerases. Bound RPA proteins are dislodgedfrom the parental strands by Pol cr and Pol 6 as they synthesizethe complementary strandsbase-pairedwith the parental strands. Severaleukaryotic proteins that function in DNA replication are not depictedin Figure 4-31. DNA polymerasee also contributes to the synthesisof cellular chromosomal DNA, though its exact role is uncertain. Still other specializedDNA polymerasesare involved in repair of mismatchesand damagedlesionsin DNA (seeSection4.6). A topoisomeraseassociateswith the parental DNA aheadof the helicaseto remove torsional stressintroduced by the unwinding of the parental strands. Ribonuclease H and FEN I remove the ribonucleotidesat the 5' ends of Okazaki fragments;these are replacedby deoxynucleotidesadded by DNA polymerase6 as it Okazaki extendsthe upstream Okazaki fragment. Successive fragments are coupled by DNA ligase through standard 5'-+3' phosphoesterbonds.Replicationof a linear DNA molecule presentsa specialproblem at the ends of the molecule sincethe 5'-most RNA primers of the lagging strandscannot be replacedby DNA by this mechanism.In most eukaryotes' this problem is solved by the RNA-protein complex called telomerasethat carriesits own templateas discussedin Chapter 6, Genes,Genomics,and Chromosomes.

D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l l y f r o m E a c hO r i g i n As indicatedin Figures4-30 and 4-31, both parental DNA strands that are exposedby local unwinding at a replication fork are copied into a daughter strand. In theory, DNA replication from a singleorigin could involve one replication fork

C i r c u l a vr i r a l cnromosome

o

q)

o)

E

.,*.,."0, o g;-ri.r-" o";"; r;,;*; a EXPERTME;I of microscopy bidirectionalreplicationof SV40DNA.Electron growthof DNAstrands bidirectional SV40DNAindicates replicating cells viralDNAfromSV4O-infected froman origin.Thereplicating one site recognizes which EcoRl, enzyme restriction wascut by the for a a landmark DNA.Thiswasdoneto provide in thecircular recognition the FcoRl in the 5V40genome: sequence specific asthe endsof linearDNA recognized isnoweasily sequence micrographs Electron microscopy. by electron vrsualized molecules a collection showed molecules DNA SV40 replicating of EcoRl-cut "bubbles," longerreplication with increasingly of cut molecules from eachendof the cut whosecentersarea constantdistance with Thisfindingisconsistent chaingrowthin two molecules at thecenterof a bubble, froma commonoriginlocated directions etal, G C Fareed diagrams' [See in thecorresponding asillustrated of N P Salzman l photographs courtesy J Virol10:484; 1972, that moves in one direction. Alternatively, two replication forks might assembleat a single origin and then move in opposite directions, leading to bidirectional growth of both i",rght., strands.Severaltypes of experiments,including the orr. sho*tt in Figure 4-32, provided early evidencein support of bidirectional strand growth. The general consensusis that all prokaryotic and eukaryoticlels employ a bidirectional mechanism of DNA DNA REpL1CAT;ON o

143

FocusAnimation:Coordination of Leading-and Lagging-strand flltt Synthesis Helicases

< FIGURE 4-33 Bidirectional mechanism of DNAreplication. Theleftreplication fork hereiscomparable to the replication fork diagrammed in Figure 4-31, whichalsoshowsproteins otherthan largeT-antigen lop:TwolargeT-antigen hexameric helicases first EJrn*'no'nn bindat the replication originin opposite orientations. Step[: Using energyprovided fromATPhydrolysis, the helicases movein opposite directions, unwinding the parental DNAandgenerating single-strand templates thatareboundby RPAproteinsStepE: primase_pol o complexes synthesize (red)base-paired shortprimers to eachof the parental primersynthesis separated strands. Stepg: pCNA-Rfc-pol 6 complexes E t".o,nn-.trand replace the primase-Pol crcomplexes J andextendtheshortprimers, generating (darkgreen)at eachreplication the leading strands fork Step@: Thehelicases furtherunwindthe parental strands, andRpA proteins bindto the newlyexposed single-strand regions. Stepg: I PCNA-Rfc-Pol 6 complexes extend the leading strands further Step6: extension ! | Leading-strand Primase-Pol ctcomplexes primers synthesize for lagging-strand t synthesis at eachreplication fork Stepfl: pCNA-Rfc-pol 6 complexes displace the primase-Pol o complexes andextendthe lagging-strand (lightgreen), Okazaki fragments whicheventually areligatedto the 5' endsof the leading strandsTheposition whereligation occursis represented by a circleReplication continues byfurtherunwinding of theparental strands andsynthesis of leading andlagging strands asin Steps4-Z Althoughdepicted asindividual stepsfor clarity, unwinding andsynthesis of leading andlaggingstrands occurconcunentlv I

Unlike SV40 DNA, eukaryotic chromosomal DNA moleculescontain multiple replication origins separatedby tens to hundreds of kilobases.A six-subunit protein called ORC, for origin recognition complex, binds to each origin and associateswith other proteins required to load cellular hexameric helicasescomposed of six homologous MCM proteins (for minichromosome maintenance. the genetic screen initially_used to identify the genes encoding them). Two opt.nn,"n-strand primersynrhesis posed MCM helicases separatethe parental strands at an

extension El I Leaoing-strand

+

E J

extension Z I a.nn,nn-strand

+

transcription of most genes,control of the initiation step is the primary mechanismfor regulatingcellular DNA replication. Ac_ S t r a n dl i g a t i o n

replication. In the caseof SV40 DNA, replication is initiated by binding of two large T-antigen hexamiric helicasesto the

into two daughtercells.We discussthe various regulatory mech_ anismsthat determinethe rate of cell division in Chapter 20.

DNA Replication Each strand in a parental duplex DNA acts as a template r synthesisof a daughter strand and remains base-paired the new strand, forming a daughter duplex (using a 144

C H A P T E R4

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B A S T CM O L E C U L A RG E N E T T M C ECHANTSMS

semiconservativemechanism).New strands are formed the 5'--+3'direction. r Replication begins at a sequencecalled an origin. Each eukaryotic chromosomal DNA molecule contains multiple replication origins. r DNA polymerases,unlike RNA polymerases,cannot unwind the strandsof duplex DNA and cannot initiate synthesis of new strandscomplementaryto the templatestrands. r At a replication fork, one daughter strand (the leading strand) is elongated continuously. The other daughter strand (the lagging strand) is formed as a seriesof discontinuous Okazaki fragments from primers synthesizedevery few hundred nucleotides(Figure 4-30). r The ribonucleotidesat the 5' end of each Okazaki fragment are removed and replacedby elongation of the 3' end of the next Okazaki fragment. Finally adjacent Okazaki fragments are joined by DNA ligase. r Helicasesuse energyfrom ATP hydrolysis to separatethe parental (template)DNA strands. Primase synthesizesa short RNA primer, which remains base-pairedto the template DNA. This initially is extendedat the 3' end by DNA polymerase a (Pol c), resulting in a short (S')RNA(3' )DNA daughterstrand. r Most of the DNA in eukaryotic cells is synthesizedby Pol 6, which takes over from Pol ct and continues elongation of the daughterstrandin the 5'+3' direction.Pol 6 remains stably associatedwith the template by binding to Rfc protein, which in turn binds to PCNA, a trimeric protein that encirclesthe daughter duplex DNA (seeFigure 4-31). r DNA replication generally occurs by a bidirectional mechanism in which two replication forks form at an origin and move in opposite directions, with both template strandsbeing copied at eachfork (seeFigure4-33)' r Synthesisof eukaryotic DNA in vivo is regulatedby controlling the activity of the MCM helicasesthat initiate DNA replication at multiple origins spacedalong chromosomal DNA.

![

DNARepairand Recombination

Damage to DNA is unavoidable and arises in many ways. DNA damage can be caused by spontaneouscleavageof chemicalbonds in DNA, by environmental agentssuch as ultraviolet and ionizing radiation, and by reaction with genotoxic chemicalsthat are by-products of normal cellular metabolism or occur in the environment' A mutation in the normal DNA sequencecan occur during replication when a DNA polymerase inserts the wrong nucleotide as it reads a damagedtemplate. Mutations also occur at a low frequency as the result of copying errors introduced by DNA polymeraseswhen they replicate an undamagedtemplate. If such mutations were left uncorrected, cells might accumulate so many mutations that they could no longer function properly, In addition, the DNA in germ cells might incur too many

mutations for viable offspring to be formed' Thus the prevention of DNA sequenceerrors in all types of cellsis important for survival, and several cellular mechanismsfor repairing damagedDNA and correcting sequenceerrors have evolved' One mechanismfor repairing double-strandedDNA breaks' by a processcalled recombination, is also used by eukaryotic cells to generatenew combinations of maternal and paternal geneson each chromosome through the exchangeof segments of the chromosomesduring the production of germ cells (e'g', sperm and eggs). Significantln defects in DNA repair mechanisms and cancerare closelyrelated. !7hen repair mechanismsare compromised, mutations accumulate in the cell's DNA. If these mutations affect genes that are normally involved in the careful regulation of cell division, cells can begin to divide uncontrollably, leading to tumor formation, and cancer' Chapter 25 outlines in detail how cancer arisesfrom defects in DNA repair.We will encountera few examplesin this section, as well, as we first consider the ways in which DNA integrity can be compromised' and then discussthe repair mechanismsthat cells have evolved to ensurethe fidelity of this very important molecule'

IntroduceCopyingErrors DNA Polymerases and Also CorrectThem The first line of defensein preventingmutations is DNA polymerase itself. Occasionally, when replicative DNA polymerasesprogressalong the template DNA, an incorrect nuto the growing 3' end of the daughterstrand cleotideit for instance,in(seeFigure"da.a 4-31,).E. coll DNA polymerases., troduce about 1 incorrect nucleotideper 104 polymerizednucleotides.Yet the measuredmutation rate in bacterial cells is much lower: about 1 mistake in 10e nucleotidesincorporated into a growing strand. This remarkable accuracy is largely due to proofreading by E. coli DNA polymerases' Pro-ofreadingdependson a 3'-+5' exonucleaseactiuity of some DNA polymerases.Sfhen an incorrect baseis incorporated during DNA synthesis,base-pairingbetweenthe 3' nucleotideof the nascentstrand and the templatestrand doesnot occur.As a result,the polymerasepauses'then transfersthe 3' end of the growing chain to its exonucleasesite, where the incorrectmispairedbaseis removed (Figure4-34)' Then the 3' end is transferredback to the polymerasesite' where this region is copied correctly. Like the E. coli DNA polymerases' I*o enkaryotic DNA polymerases,6 and e, used for replication of most chromosomal DNA in animal cells' also have proofreading activity. It seemslikely that proofreading is indispensablefor all cellsto avoid excessivemutatlons'

C h e m i c aal n d R a d i a t i o nD a m a g et o D N A Can Leadto Mutations DNA is continually subiectedto a baffage of damaging chemical reactions; estimatesof the number of DNA damin a singlehuman cell range from 10a to 105 per "g..,r..t,, day! Even if DNA were not exposedto damaging chemicals, ..it"in aspectsof DNA structure are inherently unstable' D N A R E P A I RA N D R E C O M B I N A T I O N O

145

Frngers

Thumb

t^

Thumb

Fingers urowtng strand

Pol

Template strand

Exo

FIGURE 4-34 Proofreading by DNApolymerase. All DNA polymerases havea similar three-dimensional structure, which resembles a half-opened righthandThe,,fingers,, bindthesinglestranded segment of thetemplatestrand,andthe polymerase catalytic (Pol)liesin thejunctionbetween activity thefingers andpalm.As long asthecorrect nucleotides areaddedto the3, endof thegrowing strand,it remains in the polymerase site Incorporation of an incorrect

baseat the3' endcauses melting of thenewlyformedendof the duplexAsa result, pauses, thepolymerase andthe3,endof the growingstrandistransferred to the 3,-+5,exonuclease site(Exo) about3 nm away,wherethe mtspaired baseandprobably otherbases areremoved. Subsequently, the 3' endflipsbackintothe polymerase siteandelongation resumes. fromC M Joyce andT.T.Steltz, [Adapted 1995, 1 Bacteriol 177:6321,and S Bellandl Baker, 1998, Ceil92:2951

For example, the bond connecting a purine baseto deoxyribose is prone ro hydrolysis at low rate under physiological conditions, leaving a sugar without an attached base.Thus coding information is lost, and this can lead to a mutarion during DNA replication. Normal cellular reacrions,including the movement of electronsalong the electron-transportchain in mitochondria and lipid oxidation in peroxisomei, produce severalchemicalsthat react with and damage DNA, including hydroxyl radicals and superoxide (O2 ). These too can causemutations, including those that lead to cancers. Many spontaneous mutations are point mutations, which involve a changein a single basepair in the DNA se-

quence. One of the most frequent point mutations comes ftom deamination of a cytosine (C) base,which converts it into a uracil (U) base. In addition, the common modified baseS-methylcytosineforms thymine when it is deaminated. If thesealterationsare not correctedbefore the DNA is replicated,the cell will usethe strandcontainingU or T as template to form a U.A or T.A basepair, thus creating a permanent changeto the DNA sequence(Figure 4-35). Radiation from the environment can also have dramatic consequences for DNA. High-energy ionizing radiation such as x-rays and gamma rays causedouble-strandedbreaks in DNA. Uy radiation found in sunlight causesdistortions in

NH.

U

l' N'c-c-cr. ttl ozc'-r-cH I

Deamination

(.tl HNz"\c-cH"

trl

o-rctN.c

2-Deoxyribose

2-Deoxyribose

5-Methylcytosine

Thymine

5',

?21

Deamination

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5',

J

Replication

z

E .* Base-excision repatr 3',

3',

Witd-type DNA FIGURE 4-35 Deamination leadsto point mutations.A spontaneous pointmutation canformbydeamination of 5-methylcytosine (C)to formthymine(T).lf the resulting T.Gbasepair isnot restored to the normalC.Gbasepairby baseexcision_repair 146

.

Mutant DNA

5',

Wild-type DNA

(step[), it willleadto a permanent mechanisms changeIn sequence (r.e., a mutation) following (stepZ). Afteroneround DNAreplication of replication, onedaughter DNAmolecule willhavethe mutantT.A basepairandtheotherwillhavethewild-type C.Gbasepair.

c H A p r E4R | B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

the DNA double helix that interfere with proper replication and transcription.

High-FidelitD y N A E x c i s i o n - R e p aSi ry s t e m s a n d R e p a i rD a m a g e Recognize In addition to proofreading,cellshaveother repair systemsfor preventingmutations due to copying errors, spontaneousmutation, and exposureto chemicalsand radiation. SeveralDNA excision-repair systemsthat normally operate with a high degree of accvracy have been well studied. These systemswere first elucidated through a combination of genetic and biochemicalstudiesin E. coli. Homologs of the key bacterialproteins exist in eukaryotes from yeast to humans, indicating that theseerror-freemechanismsaroseearly in evolution to protect DNA integrity. Each of these systems functions in a similar manner-a segmentof the damagedDNA strand is excised, and the gap is filled by DNA polymeraseand ligaseusing the complementary DNA strand as template. I7e will now turn to a closer look at some of the mechanisms of DNA repair, ranging from repair of single basemutations to repair of DNA broken acrossboth strands. Some of theseaccomplish their repairs with great accuracy;others are lessprecise.

BaseExcisionRepairsT.G Mismatchesand DamagedBases In humans, the most common type of point mutation is a C to T, which is causedby deamination of S-methyl C to T (see Figure 4-35). The conceptual problem with base excision repair in this case is determining which is the normal and which is the mutant DNA strand, and repairing the latter so that it is properly base-pairedwith the normal strand. Sincea G.T mismatch is almost invariably causedby chemical conversion of C to U or S-methyl C to T, the repair system evolvedto removethe T and replaceit with a C (Figure4-36). The G'T mismatch is recognized by a DNA glycosylase that flips the thymine base out of the helix and then hydrolyzes the bond that connects it to the sugar-phosphate DNA backbone. Following this initial incision, an apurinic (AP) endonucleasecuts the DNA strand near the abasicsite. The deoxyribose phosphate lacking the base is then removed and replaced with a C by a specializedrepair DNA polymerasethat reads the G in the template strand. As mentioned earlier, this repair must take place prior to DNA replication becausethe incorrect base in this pair, T, occurs naturally in normal DNA. Consequently,it would be able to engagein normal \Tatson-Crick basepairing during replication, generatinga stablepoint mutation that is now unable to be recognizedby repair mechanisms(seeFigure 4-35, s t e pZ ) . Human cells contain a battery of glycosylases'each of which is specific for a different set of chemically modified DNA bases.For example,one removesS-oxyguanine,an oxidized form of guanine, allowing its replacementby an undamagedG, and others remove basesmodified by alkylating agents.The resultingnucleotidelacking a baseis then replaced

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4-36 Baseexcisionrepair of a T'G mismatch.A DNA A FIGURE formedby usually for G'Tmismatches, glycosylase specific (see flipsthethymine 4-35), Figure residues of 5-mC deamination baseout of the helixandthencutsit awayfromthesugar-phosphate (blackdot).An justthe deoxyribose (stepn), leaving DNAbackbone site[apurinic baseless icfor the resultant specif endonuclease (stepE), and thencutsthe DNAbackbone | (APE1)l endonuclease apurinic endonuclease, by an phosphate is removed thedeoxyribose with DNApolymerase associated B, a specialized lyase(APlyase), usedin repair(stepB). Thegapisthenfilledin by DNApolymerase (stepZl),restoring theoriginal by DNAligase DNAPolB andsealed 42:2946] Chemie Angewandte pair 2003, O Schdrer, G.Cbase [After

by the repair mechanismjust discussed.A similar mechanism functions in the repair of lesions resulting from depwrination, the loss of a guanine or adenine base from DNA resulting from hydrolysis of the glycosylic bond between deoxyribose and the base.Depurination occursspontaneouslyand is fairly common in mammals. The resulting abasicsites,if left unrepaired, generatemutations during DNA replication because they cannot specifythe appropriatepaired base.

MismatchExcisionRepairsOther Mismatches a n d S m a l lI n s e r t i o n sa n d D e l e t i o n s Another process,also conservedfrom bacteria to man' principally eliminates base-pair mismatches and insertions or deletionsof one or a few nucleotidesthat are accidentallyintroduced by DNA polymerasesduring replication. As with base excision repair of a T in a T'G mismatch, the conceptual problem with mismatch excision repair is determining whic^his the normal and which is the mutant DNA strand, and repairing the latter. How this happensin human cellsis not known with certainty. It is thought that the proteins that bind to the mismatchedsegmentof DNA distinguishthe template AND RECOMBINATION . DNA REPAIR

147

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Figure 4-39 illustrates how the nucleotide excisionrepair system repairs damaged DNA. Some 30 proteins are involved in this repair process,the first of which were identified through a study of the defectsin DNA repair in cultured cellsfrom individuals with xeroderma pigmentosum,a hereditarydiseaseassociatedwith a predisposition to cancer.Individuals with this diseasefrequently develop the skin cancers called melanomas and squdmous cell carcinomasif their skin is exposedto the UV rays in sunlight. Cells of affected patients lack a functional nucleotide excision-repair system. Mutations in any of at least seven different genes, called XP-A through Xp-G,

FIGURE 4-37 Mismatchexcisionrepairin humancells.The mismatch excision-repair pathway corrects errorsintroduced during replication A complex of the MSH2andMSH6proteins (bacterial MutShomologs 2 and6) bindsto a mispaired segment of DNAin sucha wayasto distinguish between thetemplate anonewty synthesized (steptr) Thistriggers daughter strands bindingof MLH.j andPMS2(bothhomologs of bacterial MutL)Theresulting DNAproteincomplex thenbindsan endonuclease thatcutsthe newly synthesized daughter strandNexta DNAhelicase unwinds the helix, andan exonuclease removes several nucleotides fromthecutendof the daughter strand,including the mismatched base(stepf,l) Finally, aswith baseexcision repair, thegapisthenfilledin by a DNA (Pol6, in thiscase) polymerase andsealed (stepS) by DNAligase

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and daughter strands; then the mispaired segmentof the daughter strand-the one with the replication error-is excisedand repaired to becomean exact;omplement of the templatestrand (Figure4-37).In contrastto baseexcisionrepair, mismatch excision repair occurs after DNA replication.

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Predispositionto a colon cancerknown as bereditary nonpolyposiscolorectalcancerresultsfrom an inherited loss-of-function mutation in one copy of either the MLHL or the MSH2 gene. The MSH2 and MLH1 proteins are essentialfor DNA mismatch repair (seeFigure 4-37). Cells with ar least one functional copy of each of these

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mon in noninherited forms of colon cancer.I 148

CHAPTER 4

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FIGURE4-38 Formation of thymine-thymine dimers. The most commontype of DNA damagecausedby UV irradiation, thymine-thymine drmerscan be repairedby an excision-repair mechanism

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vast majority of women with inherited susceptibility to breast cancer have a mutation in one allele of either the rttttttl BRCA-1 or the BRCA-2 genesrhat encodeproteins participating in this repair process.Loss or inactivation of the second allele inhibits the homologous recombination repair pathway and thus tends to induce cancerin mammary or ovarian epithelialcells,although at presentit is not clear why these estrogen-responsive tissuesare favored sites of carcinogenesis. Yeastscan repair double-strandbreaks induced by 1-irradiation. Isolation and analysisof radiationsensitive(RAD) mutants that are deficient in this reoair system facilitated study of the process. Virtually all the proteins a yeast Rad proteins have homologs in the human genome, Jo,n". and the human and yeastproteins function in an essentially identical fashion. A variety of DNA lesions not repaired by mechanisms discussedearlier can be repaired by mechanismsin which the damagedsequenceis replaced by a segmentcopied from the same or a highly homologous DNA sequenceon the homologous chromosome of diploid organisms,or the sister chromosome following DNA replication in all organisms.These mechanismsinvolve an exchange of strands between separate DNA moleculesand henceare collectivelyreferred to as FIGURE 4-40 Nonhomologous end-joining.Whensister D N A r ecombination mechanisms. chromatids arenot available to helprepairdouble-strand breaks, In addition to providing a mechanism for DNA repair, nucleotide sequences arebuttedtogetherthatwerenot apposed in similar recombination mechanismsgenerategeneticdiversity the unbroken DNA TheseDNAendsareusually fromthesame among the individuals of a speciesby causing the exchange chromosome locus,andwhenlinkedtogether, several basepairsare of large regions of chromosomesbetween the maternal and lost Occasionally, endsfromdifferent chromosomes areaccidentally paternal pair of homologous chromosomesduring the spejoinedtogetherA complex of two proteins, KuandDNA-dependent cial type of cellular division that generatesgerm cells (sperm protern kinase, bindsto theendsof a double-strand break(steptr) and eggs),meiosis (Figure 5-3). In fact the exchange of reAfterformation of a synapse, the endsarefurtherprocessed by gions of homologous chromosomes,called crossing over, is nucleases, resulting in removal (stepE), andthetwo of a few bases required for proper segregationof chromosomesduring the double-stranded molecules areligatedtogether(stepB) Asa result, first the double-strand meiotic cell division. Meiosis and the consequencesof breakis repaired, but several basepairsat thesite of the breakareremoved[Adapted generating new combinations of maternal and paternal fromG Chu,1997, J Biot Chem 272:24097; M Lieber et al, 1997,Curr. geneson one chromosome by recombination are discussed OpinGenet. Devel. T:99;andD van G a n t e t a l , 2 O O 1 ,N a t u r eR e v .G e n e t 2 : 1 9 6 I further in Chapter 5. The mechanismsleading to proper segregation of chromosomes during meiosis are discussedin Chapter 20. Here we will focus on the molecular mechanisms of DNA recombination, highlighting the exchangeof DNA strands betweentwo recombining DNA molecules.

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chromosome to another. Such translocations may generare chimeric genesthat can have drastic effects on normal cell function, such as uncontrollable cell growth, which is the hallmark of cancer.The devastatingeffectsof double-strand breaks make this the "most unkindest cut of all," to borrow a phrase from Shakespeare's Julius Caesar.

H o m o l o g o u sR e c o m b i n a t i oC n a n R e p a i rD N A Damageand GenerateGeneticDiversity

150

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cHAprER 4

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Repair of a Collapsed Replication Fork An exampleof recombinationalDNA repair is the repair of a "collapsed,'replication fork. If a break in the phosphodiesterbackboneof one DNA strand is not repaired before a replication fork passes, the replicatedportions of the daughterchromosomesbecome separatedwhen the replication helicasereachesthe ,,nick,, in the parental DNA strand becausethere are no covalent bonds between the two fragments of the parental strand on either side of the nick. This processis called replication fork collapse (Figure4-41, stepn). If it is not repaired,it is generallylethal to at leastone daughtercell following cell division becauseof the loss of genetic information between the nick and the end of the chromosome.The recombination processthat repairs the resultingdouble-strandedbreak and regenerat.s r.pli."" tion fork involves multiple enzymesand other proteins, only some of which are mentionedhere.

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(Figure 4-41.,steptr ). The lagging nascentstrand (pink) created on the unnicked, homologous' parent strand is ligated to the unreplicated portion of the parent chromosome, as shown in Figure 4-41, step Z). A critical protein required for the next step is RecA in bacteria, or the homologous Rad51 in S. cereuisiae and other eukaryotes. Multiple RecA/RadS1 molecules bind to the single-stranded DNA (now considered the invading strand) and catalyze its hybridization to a perfectly or nearly perfectly complementary sequencein another, homologous, double-strandedDNA molecule, either the ligated molecule created after fork collapse (as shown in the figure) or the other homologous chromosome in diploid organisms.The other strand (dark blue) of this target double-stranded DNA (the strand not basepairing with the invading strand) is displaced as a singlestranded loop of DNA along the region of hybridization between its complement and the invading strand (refer to Figure 4-4L, step B). The RecA/RadS1-catalyzedinvasion of a duplex DNA by a single-strandedcomplement of one of the strands is key to the recombination process. Since no basepairs are lost or gained in this process,calledstrand inuasion,it doesnot requirean input of energy. Next, the hybrid region between target DNA (pink) and the invading strand (dark red) is extended in the direction away from the break by proteins that use energy from ATP hydrolysis. This process is called branch migration (Ftgure 4-41,, step 4) becausethe point at which the target DNA strand (pink) crossesfrom one complementary strand (dark blue) to its complement in the broken DNA molecule (dark red), is called a branch in the DNA structure. In this diagram, the diagonal lines represent only one phosphodiesterbond. Molecular modeling and other studiesshow that the first baseon either side of the branch is base-pairedto a complementary nucleotide. As this branch miSrates to the Ieft, the number of base pairs remains constant; one new base pair formed with the (red) invading strand is matched by the loss of one base pair with the parental (dark blue)

strand. When the region of hybridization extends beyond the 5' 3', end of the broken strand (light blue), this broken parental DNA strand becomes increasingly single-stranded, as its repairof a collapsed A FIGURE 4-41 Recombinational complement, the invading (dark red) strand' base-pairsinfork. Parental strands arelightanddarkblueTheleading replication steadwith the target (pink) DNA strand. This single-stranded pink. red, lagging daughter strand is dark and the daughter strand (light blue) parcntal strand then base-pairswith the complephosphodiester mentary region of the other parental strand (dark blue) that represent a single linesin stepB andbeyond Diagonal colorSmallblack bondfromtheDNAstrandof theconesponding has likewise become single-strandedas the branch migrates of the phosphodiester cleavage arrowsfollowingstep@ represent to the left (Figure 4-41., step 4). The resulting structure is See in the Holliday structure. of DNAstrands bondsat the crossover called a Hotliday strwcture, after the geneticist who first prornalVm bb/ruva. htmI f or an animationof sdscedu/jou http://www. posed it as an intermediatein Seneticrecombination. Again, RuvAandRuvB. See catalyzed by E coliproteins branchmigration the diagonal lines in the diagram following step 4 represent genetics.wisc.edu/Holliday/holliday3D htmlfor an http://engels single phosphodiesterbonds (not a stretch of DNA), and all Seethetextfor a of theHolliday structure anditsresolution animation basesin the Holliday structure are base-pairedto compleLehninger fromD L Nelson andM M Cox,2005, discussion. [Adapted mentary basesin the parental strands. Cleavageof the phos4thed, W H Freeman andCompany.l of Biochemistry Principles phodiesterbonds that crossover from one parental strand to ' the other and ligation of the 5' and 3 ends base-pairedto the The first step in the repair of the double-strand break is same parental strands (stepsE and 6) result in the generaexonucleolytic digestionof the strand (light blue) that has its (dark tion of a structure similar to a replication fork. Rebinding of red) 5' end at the break, leaving a portion of the other replication fork proteins results in extension of the leading (the single-stranded one with the 3' end at the break) strand D N A R E P A I RA N D R E C O M B I N A T I O N

151

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A FIGURE 4-42 Double-strand DNAbreakrepairby homologousrecombination. Forsimplicity, eachDNAdoublehelix is represented by two parallel lineswith the polarities of the strands indicated by arrowheads at their3, endsTheuppermolecule hasa double-strand breakNotethat in the diagram of the upperDNA strand past the point of the original strand break and reinitiation of lagging-strandsynthesis(step Z), thus regenerating a replication fork. The overall processallows the undamagedupper strand in the lower molecule following step E (pink/light blue) to serveas template for extensionof the leading (dark red) strand in step Z. Double-Stranded DNA Break Repair by Homologous Recombination A similar mechanism called bomolopous 152

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molecule the strandwith its3' endat the rightison thetop,whilein thediagram of the lowerDNAmolecule thisstrandisdrawnon the bottom Seethetextfordiscussion. L Orr-WeaverandJ W [AfterT. Szostak, 1985,MicrobiolRev. 49:33.1 recombination can repafua double-strand break in a chromosome and can also exchangelarge segmentsof two double-strandedDNA molecules (Figure 4-42). Homologous recombination is also dependenton strand invasion catalyzed principally by RecA in bacteriaand Rad51 in eukaryotes(steps Il and tr). The 3' end of the invading DNA strand is then extended by a DNA polymerase,displacingthe parental strand as an enlargingsingle-stranded loop of DNA (dark blue, step E). Vhen DNA synthesis extends sufficiently far, the displaced

B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

parentalstrand (dark blue)that is complementaryto the 3' single-strandedregion generatedat the other broken end of DNA (the pink single-stranded region on the left following step n), base-pair(step B). This 3' end the complementarysequences (pink) is then extendedby a DNA polymerase,using the displaced single-strandedloop of parental DNA (dark blue) as template(step4). Next, the two 3' ends generatedby DNA synthesisare ligated(stepE)to the 5'ends generatedin step I by 5'-exonucleasedigestion of the broken ends. This generatestwo Holliday structuresin the paired molecules(stepE).Branch migration of theseHolliday structurescan occur in either direction (not diagrammed).Finally, cleavageof the strands at the positions shown by the arrows, and ligation of the alternative 5' and 3' ends at each cleavedHolliday structure generatestwo recombinant chromosomesthat each contain the DNA of one parental DNA molecule (pink and red strands) on one side of the break point and the DNA of the other parental DNA molecule (light and dark blue) on the other sideof the break point (step6). Eachchromosomecontains a third region, located in the immediate vicinity of the initial break point, that forms a heterodwplex;here one strand from one parent is base-pairedto the complementary strand of the other parent (pink or red base-pairedto dark or light blue). Base-pair mismatches between the two parental strandsare usually repairedby repair mechanismsdiscussed above to generatea complementary basepair. In the process, sequencedifferencesbetween the two parents are lost, a processreferredto as geneconuersion. Figure 4-43 diagrams how cleavageof one or the other pair of strandsat the four-way strand junction in the Holliday structure generatesparental or recombinant molecules.This process,calledresolution of the Holliday structure,separates DNA molecules initially joined by RecA/RadS1-catalyzed strand invasion. Each Holliday structure in the intermediate following step El of Figure 4-42 can be cleavedand religated in the two possibleways shown by the two setsof small black arrows. Consequently,there are four possibleproducts of the

recombination process.Two of theseregeneratethe parental chromosomes(with the exceptionof the heteroduplexregion at the break point that is repaired into the sequenceof one parent or the other (geneconversion),and t'wo generaterecombinant chromosomesas shown inFigure 4-42. Meiotic Recombination Meiosis is the specializedform of cell division in eukaryotesthat generateshaploid germ cells (e.g.,sperm and eggs)from a diploid cell (Figure 20-38). At least one recombination occurs between the paternal and maternal homologous chromosomesbefore the first meiotic cell division. Recombination is initiated by an enzyme that makes a double-strandedbreak in the DNA of one chromosome at any one of a very large number of sites.The process diagrammed in Figure 4-42 is then followed. The entire process from cleavageof the DNA of one chromosome through resolution of the Holliday structuresis repeateduntil at least one recombination, also called a crossouer'occurs between one pair of each of the homologous chromosomes. As mentioned earlier,the resulting link betweenhomologous chromosomesis required for their proper segregationduring the first meiotic cell division (seeChapter 20). As a consequence,every germ cell contains multiple recombinant chromosomes made of large segmentsof either the maternal or the oaternal chromosome.

DNA Repair and Recombination Changesin the DNA sequenceresult from copying errs and the effects of various physical and chemical agents. r Many copying errors that occur during DNA replication are corrected by the proofreading function of DNA polymerasesthat can recognize incorrect (mispaired) basesat the 3' end of the growing strand and then remove them by activity (seeFigure4-34). an inherent3'-+5' exonuclease

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153

r Eukaryotic cells have three excision-repair systemsfor correcting mispaired basesand for removing W-induced thymine-thymine dimers or large chemical adducts from DNA. Base excision repair, mismatch repair, and nucleotide excision repair operate with high accuracy and generally do not introduce errors. r Repair of double-strand breaks by the nonhomologous end-joining pathway can link segmentsof DNA from different chromosomes, possibly forming an oncogenic translocation. The repair mechanismalso producesa small deletion, even when segmentsfrom the same chromosome are joined. r Inherited defectsin the nucleotideexcision-repairpathway, as in individuals with xeroderma pigmentosum, predisposethem to skin cancer.Inherited colon cancer frequently is associatedwith mutant forms of proteins essentialfor the mismatch repair pathway. Defects in repair by homologous recombination are associatedwith inheritanceof one murant alleleof the BRCA-1 or BRCA2 gene and result in predispositionto breast and uterine cancer. r Error-free repair of double-strand breaks in DNA is accomplished by homologous recombination using the undamaged sister chromatid as a template. This processcan lead to recombination of parental chromosomesand is exploited by eukaryotes to generate genetic diversity by recombination of paternal and maternal chromosomes in developinggerm cells.

of the Cellutar Wl Viruses:Parasites GeneticSystem Virusesare obligate,intracellularparasites.They cannot reproduce by themselvesand must commandeera host cell's machineryto synthesizeviral proteins and in some casesto replicate the viral genome. RNA viruses, which usually replicate in the host-cell cytoplasm, have an RNA genome, and DNA viruses,which commonly replicate in the hostcell nucleus, have a DNA genome (seeFigure 4-1). Viral genomesmay be single-or double-stranded,dependingon the specific type of virus. The entire infectious virus particle, called a virion, consistsof the nucleicacid and an outer shell of protein that both protects the viral nucleic acid and functions in the processof host-cell infection. The simplest viruses contain only enough RNA or DNA to encode four proteins; the most complex can encode=200 proteins. In addition to their obvious importanceas causesof disease,viruses are extremely useful as researchtools in the study of basic biological processes,such as thosediscussedin this chaoter.

Most Viral Host RangesAre Narrow The surfaceof a virion contains many copies of one type of protein that binds specificallyto multiple copies of a receptor protein on a host cell. This interaction determines the

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host range-the group of cell types that a virus can infectand beginsthe infection process.Most viruses have a rather limited host range. A virus that infects only bacteria is called a bacteriophage, or simply a phage. Viruses that infect animal or plant cells are referred to generally as animal uiruses or plant uiruses.A few viruses can grow in both plants or animals and the insects that feed on rhem. The highly mobile insects serve as vectors for transferring such viruses between susceptibleplant or animal hosts. Wide host ranges are also characteristicof some strictly animal viruses,such as vesicularstomatitis virus, which grows in insect vectors and in many different types of mammals. Most animal viruses,however, do not cross phyla, and some (e.g.,poliovirus) infect only closely related speciessuch as primates. The host-cell range of some animal viruses is further restricted to a limited number of cell types because only these cells have appropriare surface receptors to which the virions can attach.

V i r a l C a p s i d sA r e R e g u l a rA r r a y so f O n e o r a Few Typesof Protein The nucleic acid of a virion is enclosedwithin a protein coat, or capsid, composed of multiple copies of one prorein or a few different proteins, each of which is encoded by a single viral gene.Becauseof this structure, a virus is able to encode all the information for making a relatively large capsid in a small number of genes.This efficient use of geneticinformation is important, since only a limited amount of DNA or RNA, and therefore a limited number of genes,can fit into a virion capsid. A capsid plus the enclosednucleic acid is called a nucleocapsid. Nature has found two basic ways of arranging the multiple capsid protein subunits and the viral genome into a nucleocapsid.In some viruses,multiple copies of a single coat protein form a helical structure that enclosesand protects the viral RNA or DNA, which runs in a helical groove within the protein tube. Viruses with such a helical nucleocapsid, such as tobacco mosaic virus, have a rodlike shape (Figure 4-44a). The other major structural type is based on the icosahedron, a solid, approximately spherical object built of 20 identical faces,each of which is an equilateral triangle (Figure 4-44b). During infection, some icosahedral viruses interact with host cell-surfacereceptors via clefts in between the capsid subunits; others interact via long fiberlike proteins extending from the nucleocapsid. In many DNA bacteriophages,the viral DNA is located within an icosahedral "head" that is attached to a rodlike "tail." During infection, viral proteins at the tip of the tail bind to host-cellreceptors,and then the viral DNA passesdown the tail into the cytoplasm of the host cell (Figure 4-44c). In someviruses,the symmetricallyarrangednucleocapsid is coveredby an external membrane,or envelope,which consistsmainly of a phospholipid bilayer but also conrains one or two types of virus-encodedglycoproteins (Figure 4-44d).

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The phospholipidsin the viral envelopeare similar to thosein the plasmamembraneof an infectedhost cell.The virai envelope is, in fact, derivedby budding from that membrane,but contains mainly viral glycoproteins,as we discussshortly.

V i r u s e sC a n B e C l o n e da n d C o u n t e d i n P l a q u eA s s a y s The number of infectiousviral particlesin a samplecan be quantifiedby a plaque assay.This assayis performedby culturing a dilute sample of viral particleson a plate covered with host cells and then counting the number of local Iesions,called plaques,that develop (Figure 4-45). A plaque developson the plate wherevera singlevirion initially infects

a singlecell. The virus replicatesin this initial host cell and then lyses(ruptures)the cell, releasingmany progenyvirions that infect the neighboring cells on the plate. After a few such cyclesof infection, enough cells are lysed to produce a visibleclear area,the plaque,in the layer of remaininguninfectedcells. Sinceall the progeny virions in a plaque are derived from a single parental virus, they constitute a virus clone. This type of plaque assayis in standardusefor bacterialand animal viruses.Plant virusescan be assayedsimilarly by counting local lesions on plant leavesinoculated with viruses. Analysis of viral mutants' which are commonly isolated by plaque assays,has contributedextensivelyto current understandingof molecularcellularprocesses.

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3. Replication-Yiral mRNAs are produced with the aid of the host-cell transcription machinery (DNA viruses)or by viral enzymes(RNA viruses).For both types of viruses, viral mRNAs are translated by the host-cell translation machinery. Production of multiple copies of the viral genome is carried out either by viral proteins alone or with the help of host-cell proteins. 4. Assembly-Yiral proteins and replicated genomesassociate to form progeny virions. 5. Release-Infected cell either ruptures suddenly (lysis), releasingall the newly formed virions at once, or disintegratesgraduallS with slow releaseof virions. Both cases lead to the death of the infected cell.

Eachplaque representscell lysis initiatedby one viral particle(agar restrictsmovement so that virus can infectonly contiguouscells)

Figure 4-46 rllustratesthe lytic cycle forT4 bacteriophage,a nonenveloped DNA virus that infects E. coli. Viral capsid proteins generally are made in large amounts becausemany

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A EXPERIMENTAL FTGURE 4-45 The plaqueassaydetermines the numberof infectiousparticlesin a viral suspension. (a)Each lesion, or plaque, whichdevelops wherea singlevirioninitially infected a singlecell,constitutes a pureviralclone.(b)plaques on a lawnof Pseudomonasfluorescens bacteriamadeby bacteriophage polytechnique of Dr Pierre Rossi, $S1 [Part(b)Courtesy Ecole F6d€rale de (LBE-EPFL) Lausanne l Replication of viralDNA Expression of virallateproteins

LyticViral Growth CyclesLeadto the Death of Host Cells Although details vary among different types of viruses,those that exhibit a lytic cycle of growth proceed through the following general stages: l, Adsorption-Virion interacts with a host cell by binding of multiple copies of capsid protein to specificreceptors on the cell surface. 2. Penetration-Yiral genome crossesthe plasma membrane. For some viruses,viral proteins packagedinside the capsid also enter the host cell.

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A FIGURE 4-46 tytic replicationcycleof a nonenveloped, bacterialvirus.E.colibacteriophage T4 hasa double-stranded DNA genomeandlacksa membrane envelope Afterviralcoatproteins at thetip of thetailin T4 interact with specific receptor proteins on the exterior of the hostcell,theviralgenomeisinjected intothe host (stepIl). Host-cell enzymes thentranscribe viral"early"genesinto mRNAs andsubsequently translate theseintoviral"early"proteins (stepZ) Theearlyproteins replicate theviralDNAandinduce expression of viral"late"proteins (stepS) The by host-cell enzymes virallateproteins include capsid andassembly proteins andenzymes thatdegrade the host-cell DNA,supplying nucleotides for synthesis of moreviralDNA Progeny virions areassembled in thecell(stepZl) (step5) whenviralproteins andreleased lysethecell.Newly liberated viruses initiateanothercycleof infection in otherhost ceils

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FIGURE4-47 Lytic replication cycle of an enveloped, animal e dN A v i r u s . R a b i evsi r u si s a n e n v e l o p evdi r u sw i t h a s i n g l e - s t r a n d R genome The structuralcomponentsof this virusare depictedat the top After a virionadsorbsto multiplecopiesof a specifichost membraneprotein(steptr), the cellengulfsit in an endosome ( s t e pE ) A c e l l u l apr r o t e i ni n t h e e n d o s o m em e m b r a n ep u m p s H* ionsfrom the cytosolinto the endosomeinterior.The resulting s c o n f o r m a t i o n cahl a n g ei n t h e d e c r e a sien e n d o s o m apl H i n d u c e a leadingto fusionof the viralenvelopewith the viralglycoprotein, r e m b r a n ea n d r e l e a soef t h e n u c l e o c a p s i d e n d o s o m al il p i db i l a y em uses into the cytosol(stepsB and 4) ViralRNApolymerase in the cytosolto replicatethe viralRNA ribonucleoside triphosphates genome(stepE) and to synthesize viralmRNAs(step6) One of the glycoprotein, which is viralmRNAsencodesthe viraltransmembrane ( E R a) s i t i s r n s e r t e idn t o t h e m e m b r a n eo f t h e e n d o p l a s m ri ce t i c u l u m

(stepZ). Carbohydrate isadded ribosomes on ER-bound synthesized t h e i n s i d e E Rl u m e na n di sm o d i f i eads t o t h el a r g ef o l d e dd o m a i n passthroughthe glycoprotein andtheassociated the membrane fuse (stepE) Vesicles with matureglycoprotern Golgiapparatus on the viralglycoprotein depositing membrane, with the hostplasma the cell domainoutside withthe largereceptor-binding cellsurface on host-cell aretranslated (step9) Meanwhile, otherviralmRNAs andviralRNA protein, matrixprotein, intonucleocapsid ribosomes with replicated areassembled polymerase GtepIE). Theseproteins nucleocapsids GtepI[), RNA(brightred)intoprogeny viralgenomic domainof viraltransmembrane withthecytosolic whichthenassociate (stepIE) Theplasma membrane in the plasma glycoproteins t h en u c l e o c a p sf oi dr ,m i n ga " b u d "t h a t m e m b r a ni sef o l d e da r o u n d (stePIE) isreleased eventually

copiesof them are requiredfor the assemblyof eachprogeny virion. In eachinfectedcell, about 100-200 T4 progenyvirions are producedand releasedby lysis. The lytic cycle is somewhatmore complicatedfor DNA virusesthat infect eukaryoticcells.In most suchviruses,the DNA genome is transported (with some associated proteins)into the cell nucleus.Once inside the nucleus,the viral DNA is transcribedinto RNA by the host'stranscription machinery.Processingof the viral RNA primary transcript by host-cell enzymesyields viral mRNA, which is transportedto the cytoplasmand translatedinto viral pro-

teins by host-cellribosomes,tRNA, and translationfactors. The viral proteins are then transported back into the nucleus,where some of them either replicate the viral DNA directly or direct cellular proteinsto replicatethe viral DNA' as in the case of SV40 discussedearlier.Assembly of the capsid proteins with the newly replicated viral DNA occurs in the nucleus,yieldingthousandsto hundredsof thousands of progenyvirions. Most plant and animal viruseswith an RNA genome do not require nuclear functions for lytic replication. In some of these viruses, a virus-encoded enzyme that enters the host

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d u r i n g p e n e t r a t i o n t r a n s c r i b e st h e g e n o m i c R N A i n t o mRNAs in the cell cytoplasm. The mRNA is directly translated into viral proteins by the host-cell translation machinery. One or more of theseproteins then produces additional copies of the viral RNA genome. Finally, progeny genomes are assembledwith newly synthesizedcapsid proteins into p r o g e n yv i r i o n si n t h e c y t o p l a s m . After the synthesisof hundreds to hundreds of thousandsof new virions has beencompleted,dependingon the type of virus and host cell, most infecredbacterialcellsand some infectedplant and animal cellsare lysed,releasingall the virions at once. In many plant and animal viral infections, however, no discretelytic event occurs; rather, the dead host cell releasesthe virions as it gradually disintegrates. As noted previously,envelopedanimal virusesare surrounded by an outer phospholipid layer derived from the plasma membrane of host cells and containing abundant viral glycoproteins. The processesof adsorption and release of envelopedviruses differ substantially from these p r o c e s s e sf o r n o n e n v e l o p e dv i r u s e s . T o i l l u s t r a t e l y t i c replication of enveloped viruses, we consider the rabies v i r u s , w h o s e n u c l e o c a p s i dc o n s i s t so f a s i n g l e - s t r a n d e d RNA genome surrounded by multiple copies of nucleocapsid protein. Like most other lytic RNA viruses,rabies virions are replicated in the cytoplasm and do not require host-cell nuclear enzymes.As shown in Figure 4-47, a rabies virion is adsorbedto a host cell by binding to a specific cell-surfacereceptor moleculeand then entersthe cell by endocytosis.Progeny virions are releasedfrom a host cell by budding from the host-cellplasma membrane.Budding virions are clearly visible in electron micrographs of infected cells, as illustrated in Figure 4-48. Many tens of thousands of progeny virions bud from an infected host cell before it dies.

V i r a l D N A l s I n t e g r a t e di n t o t h e H o s t - C e l l G e n o m ei n S o m eN o n l y t i cV i r a l G r o w t h C y c l e s Some bacterial viruses, called temperate phages,can establish a nonlytic associationwith their host cellsthat doesnot kill the cell. For example,when bacteriophageinfectsE. coli, the viral DNA may be integrated into the host-cell chromosome rather than being replicated. The integrated viral DNA, called a prophage, is replicated as part of the cell's DNA from one host-cell generation to the next. This phenomenon is referred to as lysogeny. Under certain conditions, the prophage DNA is activated,leading to its excision from the host-cell chromosome and entrance into the lytic cycle, with subsequentproduction and releaseof progeny vlrtons. The genomesof a number of animal viruses also can rntegrateinto the host-cell genome. One of the most important are the retroviruses,which are envelopedviruses with a genomeconsistingof two identical srrandsof RNA. These viruses are known as retrouiruses because their RNA genome acts as a template for formation of a DNA

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A EXPERIMENTAL FIGURE 4-48 Progenyvirionsare released by budding.Progeny virions of enveloped viruses arereleased by buddingfrominfected cellsIn thistransmission electron micrograph of a cellinfected with measles virus,virionbudsareclearly visible protruding fromthe cellsurfaceMeasles virusisan enveloped RNA viruswith a helical nucleocapsid, likerabies virus,andreplicates as illustrated in Figure4-47 [From A Levine. 1991 Scientific , Viruses, p 221 American tibrary,

molecule-the opposite flow of geneticinformation compared with the more common transcription of DNA into RNA. In the retroviral life cycle (Figure 4-49), a viral enzyme called reversetranscriptaseinitially copies the viral RNA genome into single-strandedDNA complementary to the virion RNA; the same enzyme then catalyzessynthesisof a complementaryDNA strand. (This complex reaction is detailed in Chapter 6 when we consider closely r e l a t e d i n t r a c e l l u l a r p a r a s i t e sc a l l e d r e t r o t r a n s p o s o n s . ) The resulting double-strandedDNA is integrated into the chromosomal DNA of the infected cell. Finally, the integrated DNA, called a provirus, is transcribed by the cell's own machinery into RNA, which either is translated into viral proteins or is packagedwithin virion coat proteins to form progeny virions that are releasedby budding from the host-cell membrane. Becausemost retrovirusesdo not kill their host cells, infected cells can replicate,producing daughter cells with integrated proviral DNA. These daughter cells continue to rranscribe the proviral DNA and bud progeny virions. Some retrovirusescontain cancer-causing genes (oncogenes),and cellsinfectedby suchretrovirusesare oncogenically transformedinto tumor cells.Studiesof oncogenicretroviruses (mostly virusesof birds and mice) have revealeda great deal about the processes that leadto transformationof a normal cell into a cancercell (Chapter25).

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Among the known human retrovirusesare human Tc e l l l y m p h o t r o p h i c v i r u s ( H T L V ) , w h i c h c a u s e sa form of leukemia, and human immunodeficiency virus ( H I V ) , w h i c h c a u s e sa c q u i r e d i m m u n e d e f i c i e n c y s y n drome (AIDS). Both of these viruses can infect only specific cell types, primarily certain cells of the immune system and, in the caseof HIV, some central nervous system neurons and glial cells. Only these cells have cell-surface receptors that interact with viral envelope proteins, accounting for the host-cell specificity of these viruses.Unlike most other retroviruses,HIV eventually kills its host cells. The eventual death of large numbers of immunesystem cells results in the defectiveimmune response characteristicof AIDS. Some DNA viruses also can integrate into a host-cell chromosome. One example is the human papillomaviruses (HPVs), which most commonly cause warts and other benign skin lesions. The genomes of certain HPV serotypes, however,occasionallyintegrate into the chromosomal DNA of infected cervical epithelial cells, initiating developmentof cervical cancer. Routine Pap smears can detect cells in the early stagesof the transformation processinitiated by HPV integration, permitting effectivetreatment. I

sitesin the hostintooneof manypossible andintegrated nucleus chromosome onlyonehost-cell DNA.Forsimplicity, cellchromosomal istranscribed viralDNA(provirus) isdepictedStepZl: Theintegrated (darkred)and generating mRNAs RNApolymerase, bythe host-cell (brightred).Thehost-cell machinery genomicRNAmolecules andnucleocapsid intoglycoproteins theviralmRNAs translates by andarereleased virions thenassemble proteins. StepEt: Progeny 4-47. in Figure buddingasillustrated

Viruses: Parasitesof the Cellular Genetic System r Viruses are small parasitesthat can replicate only in host cells. Viral genomes may be either DNA (DNA viruses)or RNA (RNA viruses)and either single-or doublestranded. r The capsid, which surrounds the viral genome, is composed of multiple copiesof one or a small number of virusencodedproteins. Somevirusesalso have an outer envelope' which is similar to the plasma membrane but contains viral transmembraneproteins. r Most animal and plant DNA viruses require host-cell nuclear enzymesto carry out transcription of the viral genome into mRNA and production of progeny genomes' In contrast, most RNA viruses encode enzymesthat can transcribe the RNA genomeinto viral mRNA and produce new copies of the RNA genome. r Host-cell ribosomes, tRNAs, and translation factors are used in the synthesisof all viral proteins in infected cells.

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r Lytic viral infection entails adsorption, penetration, synthesisof viral proteins and progeny genomes(replication), assemblyof progeny virions, and releaseof hundreds to thousandsof virions, leadingto death of the host cell (seeFigure 4-46).Releaseofenveloped vrrusesoccurs by budding through the host-cell plasma membrane (see Figure 4-471. r Nonlytic infection occurs when the viral genome is integrated into the host-cell DNA and generally does not lead to cell death. r Retroviruses are enveloped animal viruses containing a single-stranded RNA genome. After a host cell is penetrated, reversetranscriptase,a viral enzymecarried in the virion, converts the viral RNA genome into doublestranded DNA, which integratesinto chromosomal DNA (seeFigure4-49). r Unlike infection by other retroviruses,HIV infection eventually kills host cells, causing the defects in the immune responsecharacteristicof AIDS. r Tumor viruses, which contain oncogenes,may have an RNA genome (e.g.,human T:cell lymphotrophic virus) or a DNA genome (e.g., human papillomaviruses).In the case of theseviruses,integration of the viral genomeinto a hostcell chromosome can causetransformation of the cell into a tumor cell.

In this chapter we first reviewed the basic structure of DNA and RNA and then describedfundamental aspectsof the transcription of DNA by RNA polym.rur.t. RNA polymerases are discussed in greater detail in Chapter 7, along with additional factors required for transcriotion i n i t i a t i o n i n e u k a r y o t i cc e l l sa n d i n t e r a c t i o n sw i t h . e g u l a tory transcription factors that control transcription initiation in both bacterial and eukaryotic cells. Next, we discussedthe geneticcode and the participation of IRNA and the protein-synthesizingmachine, the ribosome, in decoding the information in mRNA to allow accurateassembly of protein chains. Mechanisms that regulate protein synthesis are consideredfurther in Chapter 8. Then, we consideredthe molecular detailsunderlying the accuratereplication of DNA required for cell division. Chapter 20 coversthe mechanismsthat regulatewhen a cell replicates its DNA and that coordinate DNA replication with the complex process of mitosis that distributes the daughter DNA molecules equally to each daughter cell. The next section addressedmechanisms for repairing damage to DNA, including recombination mechanismsthat also lead to the generationof geneticdiversity among individuals of a species.This genetic recombination contributes to the diversity of traits subjectedro natural selectionduring the evolution of contemporary species.In Chapter 20, we discuss the mechanisms that segregatechromosomes into haploid germ cells, a processthat requires recombination 160

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between maternal and paternal chromosomes.Finally, we discussed viruses, parasites of the cellular molecular genetic system and important model systemsand useful tools for studying multiple aspects of molecular cell biology. The basicmolecular geneticprocessesdiscussedin this chapter form the foundation of contemporary molecular cell biology. Our current understandingof theseprocesses is grounded in a wealth of experimentalresults and is not likely to change.However, the depth of our understanding will continue to increaseas additional details of the structures and interactionsof the macromolecularmachinesinvolved are uncovered. The determination in recent years of the three-dimensionalstructures of RNA polymerases, ribosomal subunits, and DNA replication proteins has allowed researchersto design ever more penerraung experimental approachesfor revealinghow thesemacromoleculesoperate at the molecular level. The detailed level of understanding currently being developed may allow the design of new and more effective drugs for treating illnessesof humans, crops, and livestock.For example,the recent high-resolutionstructuresof ribosomesare providing insights into the mechanism by which antibiotics inhibit bacterial protein synthesiswithout affecting the function of mammalian ribosomes. This new knowledge may allow the design of even more effective antibiotics. Similarln detailed understanding of the mechanisms regulating transcription of specifichuman genesmay lead to therapeutic strategies that can reduce or prevent inappropriate immune responsesthat lead to multiple sclerosisand arthritis, the inappropriate cell division that is the hallmark of cancer, and other pathological processes. Much of current biological researchis focusedon discovering how molecular interactions endow cells with decision-making capacity and their special properties. For this reason severalof the following chapters describecurrent knowledge about how such interactions regulate transcription and protein synthesisin multicellular organisms and how such regulation endows cells with the capacity to become specializedand grow into complicated organs. Other chapters deal with how protein-protein interactions underlie the construction of specialized organellesin cells, and how they determine cell shapeand movement. The rapid advancesin molecular cell biology in recent years hold promise that in the not too distant future we will understand how the regulation of specialized cell function, shape,and mobility coupled wirh regulared c e l l r e p l i c a t i o n a n d c e l l d e a t h ( a p o p t o s i s )l e a d t o t h e growth of complex organisms like flowering plants and human beings.

KeyTerms anticodon127 codons127 complementary L14

B A S tM c o L E c u L AGRE N E TM t cE c H A N t s M s

crossingover 150 deamination 146 depurination 147

DNA end-joining 149

polyribosomes138

DNA polymerases141

primary transcript 121

double helix 114 envelope(viral) 154

primer 141

excision-repair systems147

reading frame 1.28

exons 123 geneconversion153

recombination112

genetic code 127

retroviruses152

Holliday structure154

reversetranscriptase152

homologousrecombination repair L50

ribosomal RNA (rRNA) 112

introns 123

ribosomes127

lagging strand 141

RNA polymerase120

leading strand 141

thymine-thymine dimers 148

messengerRNA (mRNA) 112

promoter 1-21-

replicationfork 141

mutation 138

transcription1 12 transferRNA (IRNA) 112

Okazaki fragments 141 phosphodiesterbond 1 14

translation 112 'Watson-Crick basepairs 114

Review the Concepts l. \fhat are'Watson-Crickbasepairs? lWhy are they important? 2, TAIA box-binding protein binds to the minor groove of DNA, resulting in the bending of the DNA helix (seeFigure 4-5). Vhat property of DNA allows the TAIA box-binding protein to recognizethe DNA helix? 3. Preparing plasmid (double-stranded,circular) DNA for sequencinginvolves annealing a complementary,short, single-strandedoligonucleotide DNA primer to one strand of the plasmid template. This is routinely accomplishedby heating the plasmid DNA and primer to 90 "C and then 'C. \ilhy does slowly bringing the temperature down to 25 this protocol work? 4. \fhat difference between RNA and DNA helps to explain the greater stability of DNA? What implications does this have for the function of DNA? 5. What are the major differencesin the synthesisand structure of prokaryotic and eukaryotic mRNAs? 6, 'While investigatingthe function of a specificgrowth factor receptor gene from humans, researchersfound that two types of proteins are synthesizedfrom this gene. A larger protein containing a membrane-spanningdomain functions to recognizegrowth factors at the cell surface,stimulating a specific downstream signaling pathway. In contrast, a related, smaller protein is secretedfrom the cell and functions to bind availablegrowth factor circulating in the blood, thus inhibiting the downstream signaling pathway. Speculateon how the cell synthesizesthesedisparateproteins. 7. The transcription of many bacterial genesrelieson functional groups called operons,such as the tryptophan operon

'$7hat advantagesare (Figure 4-1.3a).'What is an operon? there to having genesarrangedin an operon' compared with the arrangementin eukaryotes? 8. Contrast how selectionof the translational start site occurs on bacterial,eukaryotic, and poliovirus mRNAs. 9. \fhat is the evidence that the 23S rRNA in the large rRNA subunit has a peptidyltransferaseactivity? 10. How would a mutation in the poly(A)-binding protein I gene affect translation? How would an electron micrograph of polyribosomesfrom such a mutant differ from the normal pattern? L1. What characteristic of DNA results in the requirement that some DNA synthesis is discontinuous? How are Okazaki fragments and DNA ligase utilized by the cell? L2. Eukaryotes have repair systems that prevent mutations due to copying errors and exposure to mutagens. 'What are the three excision-repair systemsfound in eukaryotes, and which one is responsible for correcting thymine-thymine dimers that form as a result of UV light damageto DNA? 13. DNA-repair systemsare responsiblefor maintaining genomic fidelity in normal cellsdespitethe high frequencywith which mutational eventsoccur.'Sfhattype of DNA mutation is generatedby (a) UV irradiation and (b) ionizing radiation? Describe the system responsiblefor repairing each of these types of mutations in mammalian cells. Postulatewhy a loss of function in one or more DNA-repair systems typifies many cancers. 14. lVhat is the name given to the processthat can repair DNA damage and generate genetic diversity? Briefly describe the similarities and differences of the two processes. 15. The genome of a retrovirus can integrate into the host-cellgenome.What geneis unique to retroviruses,and why is the protein encoded by this gene absolutely necessary for maintaining the retroviral life cycle?A number of retroviruses can infect certain human cells. List two of them, briefly describe the medical implications resulting from these infections, and describewhy only certain cells are infected.

Analyze the Data Protein synthesisin eukaryotes normally begins at the first AUG codon in the mRNA. Sometimes,however' the ribosomes do not begin protein synthesisat this first AUG but scan past it (leaky scanning),and protein synthesisbegins instead at an internal AUG. In order to understandwhat features of an mRNA affect efficiency of initiation at the first AUG, studies have been undertaken in which the synwas examined. thesisof chloramphenicolacetyltransferase protein referred a give rise to Translation of its messagecan protein, CAT smaller a slightly give rise to to as preCM or (see M. Kozak. 2005. Gene 367:1'3). The two proteins differ in that CAT lacks several amino acids found at the A N A L Y Z ET H E D A T A

T

161

N-terminus of preCAT. CAT is not derived by cleavageof preCAT but, instead, by initiation of translation of the mRNA at an internal AUG:

precAT Start

CAT Start

Stop

I

ll

vt m 7 c p p p " , ' ' , ", A U G 12

,:r [J[[*;,[[[[n

AUG

a. Resultsfrom a number of studieshave given rise to the hypothesisthat the sequence(-3)ACCAUGG(+4), in which the start codon AUG is shown in boldface,provides an optimal context for initiation of protein synthesisand ensures that ribosomesdo not scanpast this first AUG to begin initiation insteadat a downstreamAUG. In the numbering scheme used here, the A of the AUG initiation is designated(*1); bases5' of this are given negarivenumbers [so that the first b a s eo f t h i s s e q u e n cies ( - 3 ) ] , a n d b a s e s3 ' t o t h e ( + 1 ) A a r e given positive numbers [so that the last baseof this sequence is (+4)]. To test the hypothesisthat the start sire sequence (-3)ACCAUGG(+4) preventsleaky scanning,the chloramphenicol acetyltransferase mRNA sequencewas modified and the resulting effectson translation assessed. In the following figure, the sequence(red) surrounding the first AUG codon (black) of the mRNA that gives rise to the synthesisof preCAT is shown above lane 3. Modification of this messageis shown above the other gel lanes (altered nucleotides are in blue), and the completedproteins generatedfrom eachmodified messageappear as bands on the SDS-polyacrylamide gel below. The intensity of each band is an indication of the amount of that protein synthesized.Analyzethe alterations to the wild-type sequence,and describe how they affect translation. Are the positions of some nucleotidesmore important than others? Do the data shown in this figure provide support for the hypothesisthat the context in which the first AUG is presentaffectsefficiencyof translation from this site? Is ACCAUGG an optimal contexr for initiation from the first AUG?

CCCCA -3UUAAC UUCCC UUCCA (t

AUG1 I A A A A A U U U U ^-^^"-1U

PrtrLAr

G G IC +4UGGAA

preCAT+ * CAT+*

***{} s

G

G

b. What are some additional alterations to this message, other than those shown in the figure, that would further elucidate the importance of the ACCAUGG .

c H A p r E R4

|

c. A mutation causing a severe blood diseasehas been found in a single family (seeT. Matthes et a1.,2004, Blood 104:2181). The mutation, shown in red in the fig, ure below, has been mapped to the 5' untranslatedregion of the gene encoding hepcidin and has been found to alter the gene'smRNA. The shadedregions indicate the coding sequenceof the normal and mutant genes.No hepcidin is produced from the altered mRNA, and lack of hepcidin resultsin the disease.Can you provide a reasonableexplanation for the lack of synthesisof hepcidin in the family memberswho have inherited this mutation? $fhat can you deduce about the importance of the context in which the start site for initiation of protein synthesisoccurs in this c a s e?

Starthepcidin .....cCAGUGGGACAGCCAGACAGACGGcncCnricccncUG..... Norma

I

Y .....GCAAUGGGACAGCCAGACAGACGGCACGAUGGCACU........ Mutant

References Structure of Nucleic Acids Arnott, S. 2006. Historical article: DNA polymorphism and the earfy history of the double helix. TrendsBiochem. Sci.3l:349-354. Berger,J. M., and J. C. Wang. 1996. Recentdevelopmentsin DNA topoisomeraseII structureand mechanism.Curr. Opin. Struc. Biol. 6:84-90. Dickerson,R. E. 1983. The DNA helix and how it is read. Sci. Am. 249:94-1.'1.1.. Dickerson,R. E., and H. L. Ng. 2001. DNA srructurefrom A to B. Proc. Nat'l Acad. Sci.USA.98:6986-69888. Doudna, J. A., and T. R. Cech.2002.The chemicalrepertoireof natural ribozymes.N ature 418:222-228. Kornberg, A., and T. A. Baker.2005. DNA Replication.University Science,chap. 1. A good summary of the principlesof DNA structure. Lilley, D. M. 2005. Structure,folding and mechanismsof ribozymes.Curr. Opin. Struc.Biol. t5:313-323. Vicens,Q., and T. R. Cech.2005. Atomic level architectureof group I introns revealed.TrendsBiochem. Sci.3l:41-51. 'Wang, J. C. 1980. SuperhelicalDNA. TrendsBiochem. Sci. 5:279-221. Wigley,D.B. 1995. Srructureand mechanismof DNA topoisomerases.Ann. Reu.Biophys. Biomol. Struc.24:L85-208.

+

Lane12345

'162

sequence as an optimal context for synthesis of preCAT rather than CAT? How would you further examine w h e t h e rA a t t h e ( - 3 ) p o s i t i o n a n d G a t t h e ( + 4 ) p o s i t i o n are the most important nucleotidesto provide context for the AUG start?

Transcription of Protein-Coding Genes and Formation of Functional mRNA Brenner,S., F. Jacob,and M. Meselson.1,96L.An unstableintermediatecarrying information from genesto ribosomesfor protein synthesis.Natur e 190:576- 5 8'1,. Murakami, K. S., and S. A. Darst. 2003. BacterialRNA polvmerases:the whole story.Curr. Opin. Strwc.Biol. t3:31-39.

B A S t cM o L E c u L A R G E N E I cM E c H A N t s M s

Okamoto K., Y. Sugino,and M. Nomura. 1962. Synthesisand turnover of phagemessengerRNA in E. coli infectedwith bacteriophageT4 in the presenceof chloromycetin.J. Mol. Biol. 5:527-534. Steitz,T. A. 2006. Visualizingpolynucleotidepolymerase machinesat work. EMBO J.25:3458-3468. The Decoding of mRNA by tRNAs Alexander,R. !(., and P. Schimmel.2001. Domain-domain comProg. Nucl. Acid Res. munication in aminoacyl-tRNA synthetases. Mol. Biol. 69:31,7-349. Hatfield, D. L., and V. N. Gladyshev.2002.How seleniumhas altered our understanding of the genetic code.Mol. Cell Biol. 22:3565-3576. Hoagland, M. B., et al. 1958. A solubleribonucleicacid intermediatein protein synthesis./. Biol. Chem.23l:241-257. Ibba, M., and D. Soll. 2004. Aminoacyl-tRNAs: settingthe limits of the geneticcode.GenesDeu. 18:731-738. Khorana, G. H., et al. 1966. Polynucleotidesynthesisand the geneticcode. Cold Spring Harbor Symp. Quant. Biol.3l:3949. Nakanishi, K., and O. Nureki. 2005. Recentprogressof structural biology of IRNA processingand modification. MoL Cells l91.57-166. Nirenberg, M., et al. 1966.The RNA code in protein synthesis. Cold Spring Harbor Symp. Quant. Biol. 3t:ll-24. Rich. A.. and S.-H. Kim. 1978. The three-dimensionalstructure of transfer RNA. Scl.Am.240(1,1:52-62(offprint 1.377). Stepwise Synthesis of Proteins on Ribosomes Abbott, C. M., and C. G. Proud. 2004. Translationfactors:in sicknessand in health. TrendsBiocbem.Sci.2925-131. Auerbach,T., A. Bashan,and A. Yonath. 2004. Ribosomal antibiotics:structural basisfor resistance,synergismand selectivity. TrendsB iotech nol. 22:570- 576. Frank, J., et al. 2005. The role of IRNA as a molecularspring in decoding,accommodation,and peptidyl transfer.FEBSLett. 579:959-962. Ganoza,M. C., M. C. Kiel, and H. Aoki. 2002. Evolutionary conservationof reactionsin translation.Microbiol. Mol. Biol. Reu. 66:460485. Gualerzi,C. O., et al. 2001. Initiation factorsin the early events of mRNA translation in bacteria.Cold Spring Harbor Symp. Quant. Biol. 66:363-376. Hellen, C. U., and P. Sarnow.2001. Internal ribosomeentry sitesin eukaryotic mRNA molecules.Genet.Deuel. 15:1593-161'2. Kahveiian,A., G. Roy, and N. Sonenbery.200l. The mRNA closed-loopmodel: the function of PABPand PABP-interactingproteins in mRNA translation.Cold Spring Harbor Symp. Quant. Biol. 66:293-300. Kapp, L. D., and J. R. Lorsch. 2004. The molecularmechanics of eukaryotictranslation.Ann. Reu.Biochem. 73:657-704. Mitra, K., and J. Frank. 2006. Ribosomedynamics:insights from atomic structuremodeling into cryo-electronmicroscopy maps.Ann. Reu.Biophys.Biomol. Struc.35:299-3t7. Noller, H. F. 2005. RNA structure:readingthe ribosome. Science309:1508-15 14. Noller, H. F., et al. 2002. Translocationof IRNA during protein synthesis.FEBSLett. 514i1.1.-16. Polacek,N., and A. S. Mankin. 2005. The ribosomal peptidyl transferasecenter:structure,function, evolution, inhibition' Cdr. Reu.Biocbem.Mol. Biol. 4O:285-31.1. Preiss,T., and M. W. Hentze.2003. Startingthe protein synthesis machine:eukaryotic translation initiation. BioEssays 25:1201-1.211'. Richter,J. D., and N. Sonenberg.2005. Regulationof capdependenttranslation by eIF4E inhibitory proteins.Nature 433:477480.

Scheper,G. C., C. G. Proud, and M. S' van der Knaap. 2005' Defectivetranslation initiation causesvanishingof cerebralwhite matter.TrendsMol. Med. t2:1'59-1'66. Sonenberg,N. 2006. TranslationalControl in Biology and Medicine (CSH monograph).Cold SpringHarbor Press. Sonenberg,N., and T. E. Dever.2003. Eukaryotic translation initiation factors and regulators.Curr. Opin. Struc.Biol' 13:56-63' Steitz,T. A. 2005. On the structural basisof peptide-bondformation and antibiotic resistancefrom atomic structuresof the large ribosomal subunit. FEBSLett. 579:955-958' Taylor, S. S., N. M. Haste' and G. Ghosh. 2005. PKR and eIF2ct:integration of kinasedimerization,activation' and substrate docking. Cell 122:823-82 5. DNA Replication Brautigam, C. A, and T. A. Steitz. 1998' Structural and functional insights provided by crystal structures of DNA polymerases and their snbtti"t. complexes.Curr. Opin. Struc. Biol. 8:54-63. Bullock, P.A. 1997. The initiation of simian virus 40 DNA replication in vitro. Crit. Reu.Biochem.Mol. Biol' 32:503'568. DePamphilis,M. L., ed. 2006 DNA Replicationand Human Disease.Cold SpringHarbor Laboratory Press. Kornberg. A., and T. A. Baker.2005. DNA Replication' University Science. Langston,L. D., and M. O'Donnell. 2005. DNA replication: keep moving and don't mind the gap.Mol. Cells23:155-160' Mendez,J., and B. Stillman.2003. Perpetuatingthe double helix: molecularmachinesat eukaryotic DNA replication origins' 25:11'58-I1'67. BioEssays O'Donnell, M.2006. Replisomearchitectureand dynamicsin Eschericbia coli. l. B iol. Cbem. 281:106 5 3-106 56. Sclafani,R. A., R. J. Fletcher,and X. S' Chen' 2004' Two heads are better than one: regulation of DNA replication by hexameric helicases.GenesDeu. 18:2039-2045. DNA Repair and Recombination Andressoo,J. O., and J. H. Hoeijmakers'2005. Transcriptioncoupled repair and premature aging.Mutat. Res.577:179-1'94' Barnes,D. 8., and T. Lindahl. 2004. Repair and geneticconsequencesof endogenousDNA basedamagein mammalian cells'Ann' Reu.Genet.38:445476. Bell, C. E. 2005. Structureand mechanismoI Escherichiacoli RecA ATPase.Mol. Microbiol. 58:358-366. Friedberg,E. C., et al' 2006. DNA repair: from molecularmechanism to human disease.DNA Repair 5:986-996. Haber,J. E. 2000. Partnersand pathwaysrepairing a doublestrand break. Trends Genet. 16:259-264. system'Na/' Jiricny,1.2006. The multifacetedmismatch-repair Reu.Mol. Cell Biol.7:335-346. Khuu. P.A.. et al. 2005. The stacked-XDNA Holliday iunction and protein recognition.l. Mol. Recog.192234-242. Lilley, D. M., and R. M. Clegg' t993-The structureof the four--ay jonction in DNA. Ann. Reu-Biophys. Biomol' Struc' 22:299-328. Mirchandani, K. D., and A. D. D'Andrea- 2006. The Fanconi anemia/BRCAp"lh*"yt a coordinator of cross-link repail Exp' Cell Res.372:2647-2653' Mitchell, J. R., J. H. Hoeijmakers,and L. J. Niedernhofer'2003' Divide and.*qu.t, nucleotideexcisionrepair battlescancerand aging.Curr. Opin. Cell Biol.l5:232-240. Orr-Weaver,T. L., and J. I7. Szostak.1985. Fungal recombination. Microbiol. Reu. 49:33-58. Shin, D. S., et al. 2004. Structureand function of the double strand break repair machinery.DNA Repair 32863-873' '!food, R. D., M. Mitchell, and T. Lindahl. Human DNA repair Res.577:275-283. Mutat. senes. R E F E R E N C E 5o

16 3

Yoshida,K., and Y. Miki. 2004. Role of BRCA1 and BRCA2 as re-gulatorsof DNA repair, transcription, and cell cycle in responseto DNA damage.CancerSci. 95,:865-87t.

Viruses:Parasites of the CellularGeneticSystem Flint,S.J.,et al. 2000.Principles of Virology: Molecular Biology, Pathogenesis,and Control. ASM press. Hull, R. 2002. Matheuts' Plant Virology. Academic press.

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Klug, A. 1999.The tobacco mosaic virus particle: structure and assembly.Phil. Trans, R. Soc.Lond. B Biol. SZi.354:531-535. Knipe, D. M., and P. M. Howley eds. 2001. Fields Virology. Lippincott Villiams & Ifilkins. Kornberg, A., and T. A. Baker. 1992. DNA Replication,2d, ed,. W. H. Freeman and Company. Good summary of bacteriophagemolecular biology.

CHAPTER

GENETIC MOLECULAR TECHNIQUES (RNA|) mostgenesin the canbe usedto silence RNAinterference worm on the right (markedby C. elegansgenome Thetransgenic expresses dsRNAto the muscle a GFPreporterin headneurons) geneunc-\5, resultingin the potentdegradationof the unc-/5 of the worm ln contrast, mRNAand leadingto completeparalysis body the typicalsinusoidal the wild-typeworm on the leftexhibits of JohnKim] movement[Courtesy

I n previous chapters, we were introduced to the variety of I tasks that proteins perform in biological systems.Indeed' I the very field of molecular cell biology seeksto understand the molecular mechanismsof individual proteins and how groups of proteins work together to perform their biological functions. In studying a newly discoveredprotein' cell biologistsusually begin by asking three questionsabout it: what is its function, where is it located, and what is its structure?To answer thesequestions,investigatorsemploy three tools: the genethat encodesthe protein, a mutant cell line or organism that lacks the function of the protein, and a sourceof the purified protein for biochemicalstudies.In this chapter we consider various aspectsof two basic experimentalstrategiesfor obtaining all three tools (Figure5-1). The first strategy,often referred to as classicalgenetics, beginswith isolation of a mutant that appearsto be defective in some process of interest. Genetic methods then are used to identify and isolate the affected gene. The isolated genecan be manipulatedto produce large quantitiesof the protein for biochemical experiments and to design probes for studies of where and when the encoded protein is expressedin an organism.The secondstrategyfollows essentially the same stepsas the classicalapproach but in reverse order, beginning with isolation of an interesting protein or its identification based on analysis of an organism's genomic sequence.Once the corresponding gene has been isolated, the gene can be altered and then reinsertedinto an organism. In both strategies,by examining the phenoof mutations that inactivate a particutypic consequences lar gene, geneticistsare able to connect knowledge about the sequence,structure, and biochemical activity of the

encoded protein to its function in the context of a living cell or multicellular organism. An important component in both strategiesfor studying a protein and its biological function is isolation of the corresponding gene. Thus we discussvarious techniques by which res.archits can isolate, sequence'and manipulate specific re-

we discusstechniquesthat abolish normal protein function in order to analyzethe role of the protein in the cell.

OUTLIN 5.1

GeneticAnalysisof Mutationsto ldentify and StudYGenes

166

5.2

DNA Cloningand Characterization

176

5,3

UsingClonedDNA Fragmentsto StudyGene ExPression

5.4

ldentifying and LocatingHuman DiseaseGenes

5.5

Inactivatingthe Functionof Specific Genesin EukarYotes

165

> FIGURE 5-1 Overviewof two strategies for relatingthe function,location,and structureof gene products,A mutant organism isthestarting pointfor the classical geneticstrategy(greenarrows)Thereverse (orange strategy arrows) usually beginswith identification of a protein-codrng sequence by analysis of genomesequence databases In bothstrategies, the actualgeneisisolated eitherfroma DNAlibrary or by specific amplification of the genesequence from genomicDNA Oncea clonedgeneis isolated, it canbe usedto produce theencoded protein in bacterial or eukaryotic expression systems Alternatively, a clonedgenecanbe rnactivated by oneof various techniques andusedto generate mutantcellsor orqanisms

Mutant organism/cell Comparisonof mutant and wild-type function G e n e t i ca n a l y s i s Screeningof DNA library

Clonedgene DNA sequencing

to ldentifyand StudyGenes As describedin Chapter 4, the information encoded in the DNA sequenceof genesspecifiesthe sequence-and therefore the structure and function-of every protein moleculein a cell. The power of geneticsas a tool foi studying cells and organismslies in the ability of researchersto selectivelyalter every copy of just one type of protein in a cell by making a change in the gene for that protein. Genetic analysesof mutants defectivein a particular processcan reveal (a) new genes required for the process to occur, (b) the order in which gene products act in the process,and (c) whether the proteins encoded by different genes interact with one another. Before seeinghow geneticstudiesof this rype can provide insights into the mechanism of complicated cellular or developmental process,we first explain some basic genetic terms used throughout our discussion. The different forms, or variants, of a geneare referred to as alleles.Geneticistscommonly refer to the numerous naturally occurring genetic variants that exist in populations, particularly human populations, as alleles.The term mutation usually is reserved for instances in which an allele is known to have been newly formed, such as after treatment of an experimental organism with a muragen, an agent that causesa heritable changein the DNA sequence. Stricdyspeaking,the particular serof alielesfor all the genes carried by an individual is its genotype.However, this term also is usedin a more restrictedsenseto denoteiust the allelesof the

type usually denoresan allele that is present at a much higher frequency than any of the other possible alternatives. 166

.

cHAprER 5

|

Databasesearchto identify protein-codingsequence PCRisolationof corresponding gene

Expression in cultured cells

Genetic Analysis of Mutations

ff,t

Gene inactivation

M o L E c u L AG R E N E T tr cE c H N t e u E s

Protein Localization Biochemical studies Determ i nati o n of structu re

Geneticistsdraw an important distinction between the genotype and the phenotype of an organism. The phenotype refers to all the physical attributes or traits of an individual that are the consequenceof a given genotype. In practice, however, the term phenotype often is used to denote the physical consequencesthat result from just the alleles that are under experimental study. Readily observable phenotypic characteristicsare critical in the geneticanalysisof mutations.

R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e s GenerallyHave OppositeEffectson G e n eF u n c t i o n A fundamental genetic difference between experimental organismsis whether their cells ca:i::ya single set of chromosomes or two copies of each chromosome. The former are referred to as haploid; the latter, as diploid. Complex multicellular organisms (e.g., fruit flies, mice, humans) are diploid, whereasmany simple unicellular organismsare haploid. Someorganisms,notably the yeastSaccharomycescereuisiae, can exist in either haploid or diploid states. Many cancer cells and rhe normal cells of some organisms, both plants and animals) caffy more than two copies of each chromosome.However, our discussionof genetictechniques and analysis relates to diploid organisms, including dipioid yeasts. Although many different allelesof a gene might occur in different organisms in a population, any individual diploid organism will carry two copiesof eachgeneand thus at most can have two different alleles.An individual with two different allelesis heterozygousfor a gene, whereas an individual that carriestwo identical allelesis homozygousfor a gene.A recessivemutant allele is defined as one in which both alleles must be mutant in order for the mutant phenotype to be observed;that is, the individual must be homozygous for the mutant allele to show the mutant phenotype.In contrast, the phenotypic consequences of a dominant mutant allelecan be

;;-;-'*i

=

GENOTYPE I l-----t DIPLOID PHENOTYPE

Wild type

Dominant =

i M uta nt

mutant alleles 5-2 Effectsof dominantand recessive FIGURE on phenotypein diploidorganisms.A singlecopyof a dominant bothcopies whereas a mutantphenotype, to produce alleleissufficient observedin a heterozygousindividual carrying one mutant and one wild-type allele(Figure5-2). 'Whether a mutant allele is recessiveor dominant provides valuable information about the function of the affectedgene and the nature of the causativemutation' Recessivealleles usually result from a mutation that inactivates the affected gene,leadingto a partial or completelossof fwnctioz. Suchrecessivemutations may remove part of the gene or the entire genefrom the chromosome,disrupt expressionof the gene,or alter the structure of the encoded protein, thereby altering its function. Conversely,dominant alleles are often the consequenceof a mutation that causessome kind of gain of function. Such dominant mutations may increase the activity of the encodedprotein, confer a new function on it, or lead to its inappropriate spatial or temporal pattern of expression. Dominant mutations in certain genes,however,are associated with a lossof function. For instance,somegenesatehaploinswfficient, meaning that both allelesare required for normal function. Removing or inactivating a singleallele in such a gene leadsto a mutant phenotype.In other rare instancesa dominant mutation in one allelemay lead to a structuralchangein the protein that interferes with the function of the wild-rype protein encoded by the other allele. This type of mutation, referred to as a dominant-negatiue,producesa phenotype similar to that obtainedfrom a loss-of-functionmutation. Someallelescan exhibit both recessiveand dominant properties.In such cases,statementsabout whether is dominant or recessivemust specify the phenoallele an type. For example, the allele of the hemoglobin gene in humans designatedHb'has more than one phenotypicconsequence.Individuals who are homozygousfor this allele (Hb'/Hb') have the debilitating diseasesickle-cellanemia, but heterozygousindividuals (Hb'/Hb") do not have the disease.Therefore, Hb' is recessiuefor the trait of sicklecell disease.On the other hand, heterozygous(Hb'/Hb') individuals are more resistantto malaria than homozygous (Hb"/Hb") individuals, revealing that Hb' is dominant for the trait of malaria resistance.I

to causea mutantphenotype allelemustbe present of a recessive causea lossof function;dominant usually mutations Recessive causea gainof functionor an alteredfunction usually mutations

mutationsthan dominant mutations.

Segregationof Mutations in Breeding ExperimentsRevealsTheir Dominance or RecessivitY Geneticistsexploit the normal life cycle of an organism to test for the dominance or recessivityof alleles' To see how this is done, we need first to review the type of cell division that gives rise to gametes (sperm and egg cells in higher plants and animals). l7hereas the body (somatic)cells of -ort *.tl,i.ellular organisms divide by mitosis, the germ

their genesmay exist in different allelic forms' Figure 5-3 depicts the major eventsin mitotic and meiotic cell division' InLitosis DNA replication is always followed by cell division, yielding two diploid daughter cells' In meiosis oze round'of DNe replication is followed by two separatecell divisions, yieldingfour haploid (12) cells that contain only one chromosome of each homologous pair' The apportion-

A commonly used agent for inducing mutations (mutagenesis) in experimental organisms is ethylmethane sulfonate (EMS). Although this mutagen can alter DNA sequencesin severalways, one of its most common effectsis to chemically modify guanine basesin DNA, ultimately leading to the conversionof a G'C basepair into an A T basepair. Such an alteration in the sequenceof a gene,which involves only a singlebasepair, is known as a point mutation. A silent A N D s T U D YG E N E S TO IDENTIFY OF MUTATIONS ANALYSIS GENETIC

167

Focus Animation: Mitosisftttt Focus

Meiosis

MEIOTIC CELL DIVISION

Paternal homolog Maternal homolog

S o m a t i cc e l l( 2 n )

Premeioticcell l2nl

I DNA reptication

vI

v

Replicated cnromosomes (4nl

DNA reptication

Replicated cnromosomes

I

I Homologous chromosomes + align; synapsisand crossing over

v

Mitotic apparatus

Mitotic appara

;

MetaphaseI

o o o

I

I

v

v

/\

/ Ceil division \

++

D a u g h t e cr e l l s( 2 n )

o o o o

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FIGURE 5-3 Comparison of mitosisand meiosis.Both somatrc cellsandpremeiotic germcellshavetwo copies of each (2n),onematernal chromosome andonepaternal. In mitosis, the replicated chromosomes, eachcomposed of two sister chromatids, alignat the cellcenterin sucha waythatboth daughter cellsreceive a maternal andpaternal fromolog of each morphological typeof chromosome. Durinqthefirstmerctrc division, however, eachreplicated chromosome pairswith its nomotogous partnerat the cellcenter; thispairingoff isreferred Io assynapsis, andcrossing overbetweenhomoloqous

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chromosomes is evidentat this stage One replicatedchromosomeof eachmorphological type then goesinto eachdaughtercell The resultingcellsundergoa seconddivisionwithout interveningDNA replication, with the srsterchromatidsof eachmorphological type beingapportionedto the daughtercells.In the secondmeiotic divisionthe alignmentof chromatidsand their equalsegregation into daughtercellsis the sameas in mttoticdivision.The alignmentof pairsof homologouschromosomein metaphaseI is randomwith respectto other chromosomepairs,resultingln a mix of paternally and maternallyderivedchromosomes in eachdaughtercell.

(a) Segregationof dominant mutation Mutant

Wild-tYPe

Gametes

F i r s tf i l i a l g e n e r a t i o nF, . : all offspringhave mutant phenotype

Gametes S e c o n df i l i a l g e n e r a t i o nF, r : 3/aof offspringhave mutant phenotype Normal

(b) Segregationof recessivemutation Mutanl

(Figure 5-4). If the F1 progeny exhibit the mutant trait' then ih."r.r.rr"n, allele is dominant; if the F1 progeny exhibit the wild-type trait, then the mutant is recessive'Further crossing b"t*.Ln F1 individualswill also revealdifferent patternsof inheritanceaccording to whether the mutation is dominant or recessive.When F1 individuals that are heterozygousfor a dominant allele are crossedamong themselves,three-fourths of the resulting F2 progeny will exhibit the mutant trait' In contrast, when F1 individuals that are heterozygous for a recessiveallele are crossedamong themselves,only one-fourth of the resultingF2 progeny will exhibit the mutant tratt' As noted earlier,the yeast S. cereuisiae,an lmportant experimental organism' can exist in either a haploid or a iiotoid state.1n these unicellular eukaryotes' crossesbetween haploid cells can determinewhether a mutant allele is dominani or recessive.Haploid yeast cells' which carry one copy of each chromosome' can be of two different mating types k.town as a and ct. Haploid cells of opposite mating type can mate to produce a/ct diploids, which carry two of each chromosome. If a new mutation with an ob.*i., servablephenotypeis isolatedin a haploid strain, the mutant strain can be mated to a wild-type strain of the opposite mating type to produce a/o diploids that are heterozygousfor thl mutant ull.t.. ff these diploids exhibit the mutant trait, then the mutant allele is dominant, but if the diploids appear as wild-type, then the mutant allele is recessive'I7hen a/ct diploids aie placed under starvation conditions, the cells undergo -eiosfu, giving rise to a tetrad of four haploid spores' t*Jof type a and two of type ct. Sporulation of a heterozygo.rsdipiiid cell yields two sporescalryils the mutant allele ina t*o carrying the wild-type allele (Figure 5-5)' Under Wild type (type a)

Gametes

Mutant (type o)

F i r s tf i l i a l g e n e r a t i o nF, r . no offspringhave mutant phenotype Diploid cells: w i l l n o t e x h i b i tm u t a n t phenotypeif mutation is recessive

Gametes S e c o n df i l i a l g e n e r a t i o nF, r : 1/+of offspringhave mutant phenotype

Haploid spores in tetrad: 2 will be mutant 2 will be wild tYPe

5-4 Segregationpatternsof dominantand A FIGURE recessivemutationsin crossesbetweentrue-breedingstrains o f d i p l o i do r g a n i s m sA. l l t h e o f f s p r i nign t h e f i r s t( F r ) the F1 lf the mutantalleleisdominant, generation areheterozygous. ( a ) p a r t l f t he i n p h e n o t y p a e s , m u t a n t t h e e x h i b i t o f f s p r i nw g ill g i l le x h i b itth e ee , F 1o f f s p r i nw m u t a na t l l e l ei s r e c e s s i vt h a ,si n p a r t( b ) C r o s s i nogf t h e F 1 w i l d - t y pp eh e n o t y p e different alsoproduces amongthemselves heterozygotes m u t a na t lleles r e c e s s i v e a n d f d o m i n a n t s e g r e g a t i or ant i o so r intheF,qeneration

of allelesin yeast'Haploid 5-5 Segregation FIGURE cellsof oppositematingtype(i e , oneof matingtype Saccharomyces an a/ctdiploidlf ctandoneof matingtypea) canmateto produce theothercarries and allele wild-type a dominant carries onehaploid gene, resulting the same of the allele mutant a recessive trait Undercertaln the dominant diploidwillexpress heterozygous haploidspores' four of tetrad a diploidcellwillforma conditions, trait and recessive the express will tetrad the Twoof the sporesin tratt dominant the express two will

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appropriate conditions, yeastsporeswill germinate,produc_ rng vegetativehaploid srrains of both mating rypes.

(a)

p Incubate at 23 .C for 5 n

-+

C o n d i t i o n aM l u t a t i o n sC a n B e U s e dt o S t u d y E s s e n t i aGl e n e si n y e a s t

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00001000 [000t[00

VVVV

The proceduresusedto identify and isolate mutants, referred to as genettc screens,depend on whether the experimental organism is haploid or diploid and, if the latter, whether the mutation is recessiveor dominant. Genes that encode proteins essentialfor life are among the most interestingand important ones ro study. Since phenotypic expressionof mutationsin essentialgenesleadsto death of the individual, ingeniousgeneticscreensare neededto isolateand maintain organismswith a lethal mutation.

rI V V V

Colonies

Incubate at23'C

ff5fi:JJ:ru

Temperature-sensitive for growth; growth at 23., no growth at 36.

(b) Wild type

cdc28 mutants

t

t

tagenizedyeastcellsthat could grow normally at23 "C but that could not form a colony when placedat 36;C (Figure5_6a). Once temperature-sensitive murants were isolated,fu.th., analysisrevealedthat some indeed were defectivein cell divi-

l

o cdcZmutants

Cg

6 ,G

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170

o

c H A p r E5R |

MoLEcuLA GRE N E TrtEcc H N t e u E S

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FIGURE 5-6 Haploidyeastscarrying < EXPERIMENTAL lethalmutationsare maintainedat temperature-sensitive permissive temperatureand analyzedat nonpermissive cellfor temperature-sensttive screen temperature.(a)Genetic grow form (cdc) and yeast that in Yeasts mutants divisioncycle but not at 36 "C temperature) at 23 'C (permissive colonres thatblocks (nonpermissive maycarrya lethalmutation temperature) for blocksat (b)Assay colonies of temperature-sensitive celldivision. of wildin thecellcycle.Shownherearemicrographs stages specific after mutants different temperature-sensitive yeast two and type cells, for 6 h Wild-type temperature at the nonpermissive incubation sizes of buds, to grow,canbe seenwith alldifferent whichcontinue cellsin the of the cellcycleIn contrast, stages different reflecting in thecell stage at a specific a block exhibit lowertwo micrographs of a cycleThecdc28mutantsarrestat a pointbeforeemergence cellsThecdcZmutants, appearasunbudded newbudandtherefore justbeforeseparation of the mothercellandbud whicharrest (a)see (emerging cell),appearascellswith largebudslPart daughter L H H a r t w e l l ,1 9 6 -,l J B a c t e r i o9l 3 j 6 6 2 ; p a r t ( b ) f r o m L M H e r e f o r d a n d L H Hartwell.1914,J Mol Biol 84.4451

ComplementationTestsDetermineWhether Mutations Are in the Different Recessive S a m eG e n e In the genetic approach to studying a particular cellular process, researchersoften isolate multiple recessivemutaiion, that produce the same phenotype. A common test for

ganism heterozygousfor both mutations (i'e', carrying one a one b allele)will exhibit the mutant phenotype beIll.le "nd causeneither allele provides a functional copy of the gene' In contrast, if mutation a and b arein separategenes,then heterozygotescarrying a single copy of each mutant allele will not;;hibit the mutant phenotype becausea wild-type allele

theseyeastmutants did not simply fail to grow, as they might if they carried a mutation affecting generalcellular metabolism. Rather,at the nonpermissivetemperature,the mutants of interest grew normally for part of the cell cycle but then arrestedat a particular stageof the cell cycle,so that many cells at this stagewere seen(Figure 5-6b). Most cdc mutations in that is, when haploid cdc strainsare mated yeastare recessive; to wild-type haploids,the resulting heterozygousdiploids are nor defectivein cell division. neither temperature-sensitive

R e c e s s i vLee t h a lM u t a t i o n si n D i p l o i d sC a n B e l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e d in Heterozygotes In diploid organisms,phenotypesresulting from recessive mutations can be observedonly in individuals homozygous for the mutant alleles.Sincemutagenesisin a diploid organism typically changesonly one allele of a gene' yielding heterozygousmutants, geneticscreensmust include inbreeding stepsto generateprogeny that are homozygous for the mutanl alleles. The geneticist H. Muller developed a general and efficient procedure for carrying out such inbreeding experiments in the fruit fly Drosophila. Recessivelethal mutaiions in Drosophila and other diploid organisms can be maintained in heterozygousindividuals and their phenotypic consequencesanalyzedin homozygotes. The Muller approach was used to great effect by C' Niisslein-Volhard and E. \Tieschaus, who systematically lethal mutations affectingembryogenesis screenedfor recessive in Drosophila. Dead homozygousembryos carrying recessive lethal mutations identifiedby this screenwere examinedunder the microscope for specific morphological defects in the embryos.Current understandingof the molecularmechanisms underlyingdevelopmentof multicellularorganismsis based,in large part, on the detailedpicture of embryonic development rWe revealedby characterizationof theseDrosoprila mutants. will discusssome of the fundamental discoveriesbased on thesegeneticstudies\n Chapter 22.

lar characterization of the CDC genes and their encoded proteins, as describedin detail in Chapter 20, has provided a ?r"-.*ork for understanding how cell division is regulated in organismsranging from yeast to humans'

Double Mutants Are Useful in Assessing t h e O r d e ri n W h i c h P r o t e i n sF u n c t i o n Based on careful analysis of mutant phenotypes associated with a particular cellular process' researchersoften can deduce the order in which a set of genesand their protein products function. Two general types of processesare amenable to such analysis: (a) biosynthetic pathways in which a precursor material is convertedvia one or more intermediatesto a final product and (b) signaling pathways that regulate other processesand involve the flow of information rather than chemical intermediates.

A N D S T U D YG E N E S TO IDENTIFY OF MUTATIONS ANALYSIS GENETIC

171

> EXPERIMENTA FLI G U R E5 - 7 Complementation analysisdetermines whether recessivemutations are in the same or different genes. Complementation testsin yeastare performedby matinghaploida and o cellscarryingdifferentrecessrve mutations to producediploidcells In the analysis of cdc mutations,pairsof differenthaploid temperature-sensitive cdc strainswere systematically matedand the resultingdiploids testedfor growth at the permissive and nonpermrsstve temperaturesIn this hypothetical example,the cdcX and cdcy mutants c o m p l e m e net a c ho t h e ra n d t h u s h a v e mutationstn differentgenes,whereasthe cdcX and cdcZmutantshavemutationsin the s a m eg e n e

M a t e h a p l o i d so f oppositemating types and carryingdifferent recessivetemperaturesensitivecdc mutations

Mutant (type a)

Mutant (type cr)

(cdcX)

(cdcY\

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\_-/ \/

Mutant (typea)

Mutant (typeo)

\cdcx)

(cdcz)

\_-./

\_-,i

\/

\r'

cdcXlcdcY (type a/o)

\r'

cdcXlcdcZ (type a/o) P l a t ea n d i n c u b a t e at permissive temperature

T e s tr e s u l t i n gd i p l o i d s for a temperaturesensitivecdc phenotype

2C

aa

Growth

No growth

6A

PHENOTYPE: Wild type INTERPRETATION:

36'C

Growth indicatesthat mutations cdcX and cdcY are in differentgenes

Y9

M utant Absenceof growth indicatesthat mutations c d c Xa n d c d c Z a r e i n t h e s a m eg e n e -Ir-V----l...t-^_ia"'-l___LF

Respectivewild-typealleles p r o v i d en o r m a lf u n c t i o n

next. In E. coLi,the genesencodingtheseenzymeslie adja_ cent to one another in the genome, constituting the trp operon (seeFigure 4-73a). The order of action of the differ_ ent genesfor theseenzymes,hencethe order of the biochemi_ cal reactions in the pathway, initially was deducedfrom the types of intermediatecompoundsthat accumulatedin each

In Chapter 14 we discussthe classicuse of the double_ mutant.strategyto help elucidatethe secretorypathway. In this pathway proteins to be secreredfrom the cell rnou. i.otheir site of synthesison the rough endoplasmicreticulum (ER) to the Golgi complex. then to ,..r.toiy vesicles, and fi_ n a l l y t o r h ec e l l s u r f a c e . Ordering of Signaling pathways As we learn in later chapters,expressionof many eukaryoticgenesis regulated by signaling pathways that are initiateJ by extracellular 172

.

c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

B o t h a l l e l e sn o n f u n c t i o n a l

hormones, growth factors, or other signals. Such signaling pathways may include numerouscomponents,and doublemutant analysisofren can provide insight into the functions and interactionsof thesecompon.nrr.th. only prerequisite for obtaining useful information from this type of analysisis that the two mutations must have opposite effects on the output of the sameregulatedpathway. Most commonl5 one mutatio_nrepressesexpressionof a particular reporrer gene even when the signal is present, while another mutation resultsin reporter geneexpressionevenwhen the signalis absent(i.e.,constirutiveexpression).As illustratedin Figure5_gb, two simple regulatory mechanismsare consistentwith such

Note that this technique differs from complementation analysisjust describedin that when testingtwo recessive mu_ tations, the double mutant created is homozygolzsfor both mutations.Furthermore,dominant mutantscan be subiected t o d o u b l e - m u t a natn a l v s i s .

(a)Analysisof a biosyntheticpathway 1. A m u t a t i o ni n A a c c u m u l a t e isn t e r m e d i a t e e. A m u t a t i o ni n B a c c u m u l a t e isn t e r m e d i a t 2 OF PHENOTYPE D O U B L EM U T A N T

A d o u b l em u t a t i o ni n A a n d B a c c u m u l a t e s 1. intermediate INTERPRETATION: The reaction catalYzedby A precedes the reaction catalYzedbY B. I

: '

Eo'Et'E (b)Analysisof a signalingpathway

mutant allelewould have a mutant phenotype(Figure5-9a)' The observation of genetic suppressionin yeast strains carrying a mutant actin allele (act1-1) and a secondmutation (sic5) in another gene provided early evidence for a

A mutation in A gives repressedreporterexpresslon. A mutation in B gives constitutivereporterexpression. PHENOTYPE OF : D O U B L EM U T A N T : A d o u b l em u t a t i o ni n A a n d B g i v e s repressedreporterexPression. I , INTERPRETATION: A positively regulates repofter expression and is negativelYregulated bY B' .

(a)Suppression GenotyPe AB Phenotype Wildtype

aB Mutant

ab Ab Mutant SuPPressed

rNrERPRErAloP H E N O T Y PO EF D O U B L EM U T A N T

INr ERPRErArIoN:

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A d o u b l em u t a t i o ni n A a n d B g i v e s constitutivereporterexpression.

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B

(b) Synthetic lethality 1 Genotype

AB Phenotype Wild type

5-8 Analysisof doublemutantsoften canorderthe FIGURE in stepsin biosyntheticor signalingpathways'Whenmutations process but have genesaffectthe samecellular two different of the doublemutant phenotypes, the phenotype different distinctly genes mustfunction(a) which the two in the order canoftenreveal pathway, a thataffectthe samebiosynthetic Inthe caseof mutations t em e d t a t e l y t i l la c c u m u l attheei n t e r m e d i ai m d o u b l em u t a nw in the by the proteinthatactsearlier preceding the stepcatalyzed of a signaling analysis organism(b)Double-mutant wild-type on effects haveopposite if two mutations pathwayispossible phenotype gene In thiscase,the observed of a reporter expression aboutthe orderin which information of the doublemutantprovides regulators or negative actandwhethertheyarepositive the proteins

G e n e t i cS u p p r e s s i o an n d S y n t h e t i cL e t h a l i t y t roteins C a n R e v e a Il n t e r a c t i n go r R e d u n d a n P Two other types of genetic analysis can provide additional clues about how proteinsthat function in the samecellular processmay interactwith one anotherin the living cell. Both of thesemethods,which are applicablein many experimental organisms,involve the use of double mutants in which the phenotypic effects of one mutation are changed by the presenceof a secondmutation. Suppressor Mutations The first type of analysisis basedon geneticsuppression.To understandthis phenomenon,suppose

aB Partial defect

ab

Ab

Severe defect

Partial defect

rNrERPRErArto-

,fii\

( | 11I ) U,/

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i

(cl Synthetic lethality 2 Genotype

AB

Phenotype Wildtype

aB Wild type

ab

Ab Wild tYPe

or 5-9 Mutationsthat resultin geneticsuppression A FIGURE proteins' redundant or interacting reveal syntheticlethality proteins with two defective (a)Observation thatdoublemutants phenotype butthatsinglemutantsgive (A andB)havea wild-type thatthefunctionof eachprotein indicates a mutantphenotype thatdouble other.(b)Observation the with on interaction depends mutants single than defect phenotypic severe more a have mutants (e g , subunits of a heterodimer) thattwo proteins alsoisevidence (c)Observation thata double to functionnormally. mustinteract mUtantisnonviab|ebutthatthecorrespondingsing|emutants f unctionin thattwo proteins indicates phenotype thewild-type product an essential to produce pathways redundant

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173

direct interaction in vivo between the proteins encoded by the two genes.Later biochemical studies showed that these two proteins-Act1 and Sac6-do indeed inreract in the construction of functional actin structureswithin the cell. Synthetic Lethal Mutations Another phenomenon,called synthetic lethality, produces a phenotypic effect opposrte to that of suppression.In this case,the deleteriouseffectof one mutation is greatly exacerbated(rather than suppressed)by a secondmutation in a relatedgene.One situation in which such synthetic lethal mutations can occur is illustrated in Figure 5-9b. In this example,a heterodimericprotein is partiall5 but not completely,inactivatedby mutations in eiiher one of the nonidentical subunits.However, in double mutants carrying specificmurationsin the genesencodingboth subunits,little interaction betweensubunits occurs,iesulting in severephenotypic effects. Synthetic lethal mutations

product cannor be synthesizedand the double mutanrs will be nonviable.

(a)

G e n e sC a nB e l d e n t i f i e db y T h e i r M a p p o s i t i o n on the Chromosome The preceding discussionof genetic analysisillustrates how a geneticistcan gain insight into gene function by observing the phenotypic effectsproduced by joining together different combinations of mutant allelesin the samecell or organism. For example, combinations of different alleles of the same gene in a diploid can be used to determine whether a mutation is dominant or recessiveor whether two different recessive mutations are in the same gene.Furthermore, combinations of mutations in different genes can be used to determine the order of gene function in a pathway or to identify functional relationships between genessuch as suppressionand synthetic enhancement.Generally speaking,all these methods can be viewed as analytical tests baseJ on gene functioz. \7e will now consider a fundamentally different type of genetic analysis based on gene positioz. Studies designedto determine the position of a gene on a chromosome, often referred to as genetic mapping studies, can be usedto identify the geneaffected by a particular mutation or to determine whether two mutations are in the samegene. In many organisms generic mapping studies rely on exchangesof genetic information that occur during meiosis. As discussedin Chapter 4 and as shown in Figure 5-10a,

(b)Consider two linkedgenesA and Bwith recessive allelesa and b.

-'-lf m2

Replicated chromosomes (4nl

Crossof two mutantsto construct a doublyheterozygous strain:

and over

AlAblb

x alaBlB

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A i

A n a p h a s eI

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a

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v AbaBABab abuA"brb \___________-Y_

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Recombinanttypes

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Parental gamete

tn

Recombinantgametes

tn

parental gamete

A FIGURE5-10 Recombinationduring meiosis can be used to map the position of genes. (a)Shown is an individualthat carries two mutations,designatedm/ (yellow)andm2 (green),that are on the maternaland paternalversionsof the samechromosomelf crosslngoveroccursat an intervalbetweenml andm2 beforethe first meloticdivision,then two recombinantgametesare produced; one carriesboth m / and m2, whereasthe other carriesneither mutation The longerthe distancebetweentwo mutationson a 174

.

cHAprER s

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M o l E c u L A RG E N E T tTcE C H N t o u E S

GeneticdistancebetweenA and B can be determinedfrom frequencyof parentaland recombinantgametes: gametes Geneticdistancein cM = 100, Itotbi.nant total gametes chromatid,the more likelythey are to be separatedby recombination and the greaterthe proportionof recombinantgametesproduced (b) In a typicalmappingexperiment,a strainthat is heterozygous for two differentgenesis constructedThe frequencyof parentalor recombinantgametesproducedby thls straincan be determjned from the phenotypesof the progenyin a testcross to a homozygous recessive strain The geneticmap distancein centimorgans (cM) is givenas the percentof the gametesthat are recombinant

genetic recombination takes place before the first meiotic cell division in germ cells,when the replicatedchromosomes of each homologous pair align with each other. At this time, homologous DNA sequenceson maternally and paternally derived chromatids can exchangewith each other, a process 'We now know that the resulting known as crossing over. crossovers between homologous chromosomes provide structural links that are important for the proper segregation of pairs of homologous chromatids to opposite poles during the first meiotic cell division (for discussionseeChapter 20). Considertwo different mutations,one inheritedfrom each parent, that arc located close to one another on the same chromosome.Two different typesof gametescan be produced according to whether a crossoveroccurs betweenthe mutations during meiosis. If no crossoveroccurs between them, gametesknown as parental types,which contain either one or the other mutation, will be produced. In contrast' if a crossoveroccurs betweenthe two mutations' gametesknown as recombinant types will be produced. In this example recombinant chromosomeswould contain either both mutations, or neither of them. The sites of recombination occur more or lessat random along the length of chromosomes;thus the closertogethertwo genesare, the lesslikely that recombination will occur between them during meiosis. In other words, the lessfrequently recombination occursbetweentwo genes on the same chromosome, the more tightly they are Iinked and tbe closer together they are. Two genesthat are sufficiently close together such that there are significantly fewer recombinant gametes produced than parental gametes are consideredto be geneticallylinked. The technique of recombinational mapping was devised in 1911 by A. Sturtevantwhile he was an undergraduate working in the laboratory of T. H. Morgan at Columbia University. Originally used in studies on Drosophila, this technique is still used today to assessthe distance between two genetic loci on the same chromosome in many experlmental organisms. A typical experiment designedto determine the map distancebetweentwo geneticpositions would involve two steps. In the first step' a strain is constructed that carriesa different mutation at eachposition, or locus. In to dethe secondstep, the progeny of this strain are assessed termine the relative frequency of inheritance of parental or recombinant types.A typical way to determinethe frequency of recombination between two genesis to cross one diploid parent heterozygousat each of the genetic loci to another parent homozygousfor each gene.For such a cross,the proportion of recombinant progeny is readily determined becauserecombinant phenotypeswill differ from the parental phenotypes.By convention, one genetic map unit is defined as the distance between two positions along a chromosome that results in one recombinant individual in 100 total progeny. The distance corresponding to this 1 percent recombination frequency is called a centimorgan (cM) in honor of Sturtevant'smentor,Morgan (seeFigure 5-10b). A complete discussion of the methods of genetic mapping experiments is beyond the scope of this introductory discussion;however,two featuresof measuringdistancesby recombination mapping need particular emphasis.First, the

frequency of genetic exchange between two loci is strictly proportional to the physical distancein basepairs separating ih.- o.tly for loci that are relatively close together (sa5 less than about 10 cM). For loci that are farther apart than this' a distance measured by the frequency of genetic exchange tends to underestimatethe physical distance becauseof the possibility of two or more crossoversoccurring within an inie.ual. In the limiting casein which the number of recombinant types will equal the number of parental types, the two loci under considerationcould be far apart on the same chromosome or they could be on different chromosomes, and in such casesthe loci are consideredto be unlinked' A secondimportant concept neededfor interpretation of

recombination frequency (i.e.' a genetic distance of 1 cM) representsa physical distanceof about 2.8 kilobasesin yeast distance of about 400 kilobases in compared *lth " Drosophila and about 780 kilobasesin humans' One of the chief uses of genetic mapping studies is to

human diseasescan be identified using such methods' A second general use of mapping experiments is to determine *hethe. two different mutations are in the samegene' If two mutations are in the same gene, they will exhibit tight linkage in mapping experiments,but if they are in different g..r.{ they will usually be unlinked or exhibit weak linkage'

Genetic Analysis of Mutations to ldentify and Study Genes r Diploid organisms carry two copies (alleles)of each gene,whereashaploid organismscarry only one copy' r Recessivemutations lead to a loss of function, which is masked if a normal allele of the gene is present' For the mutant phenotype to occur' both alleles must carry the mutatl0n. r Dominant mutations lead to a mutant phenotype in the presenceof a normal allele of the gene.The phenotypesassociatedwith dominant mutations often representa gain of function but in the caseof some genesresult from a loss of function. r In meiosis,a diploid cell undergoesone DNA replication and two cell divisions, yielding four haploid cells in which maternal and paternal alleles are randomly assorted (see Figure5-3).

GENES G E N E T I CA N A L Y S I SO F M U T A T I O N ST O I D E N T I F YA N D S T U D Y

175

r Dominant and recessivemutations exhibit characteristic segregatlonpatternsin geneticcrosses(seeFigure5_4). r I n h a p l o i d y e a s t .t e m p e r a t u r e - s e n s i t i vmeu t a t i o n s a r e particularly useful for identifying and studying genesessential to survival. r The number of functionally related genesinvolved in a process can be defined by complemenration analysis (see Figure-5-7). r The order in which genesfunction in a signaling pathway can be deducedfrom the phenotype of double mutants defectivein two stepsin the affectedprocess. r Functionally significant interactions between Drorelns can be deduced from the phenotypic effects o? allelespecificsuppressormutations or syntheticlethal mutations. r Genetic mapping experimentsmake use of crossingover between homologous chromosomes during meiosis to measurethe genetic distance between two different muta_ tions on the samechromosome.

DNACloningand Characterization

f[

is simply any DNA molecule composed of sequencesde_ rived from different sources. The key to cloning a DNA fragment of interest is to link it to a vector DNA molecule that can replicate within a host cell. After a singlerecombinant DNA molecule,composedof a vector plus an insertedDNA fragmenr, is introdu.id irrto " host cell, the inserted DNA is replicated along with the vec_ tor, generating a large number of identical DNA molecules. The basic schemecan be summarizedas follows:

vectorsexist, our discussionwill mainly focus on plasmid vectors in E. coli host cells,which are commonly used. Various techniquescan then be employed to identify the sequenceo{ interest from this collection of DNA fragments,known as a DNA library. Once a specificDNA fragment is isolated,it is typically characterizedby determining the exact sequenceof nucleotidesin the molecule. We end with a discussionof the polymerasechain reaction (PCR).This powerful and versatile techniquecan be usedin many ways to generatelarge quantities of a specificsequenceand otherwisemanipulate ONR m the laboratory.The various usesof cloned DNA frasmentsare discussed in subsequent sections.

RestrictionEnzymesand DNA Ligases A l l o w I n s e r t i o no f D N A F r a g m e n t si n t o CloningVectors A major objective of DNA cloning is to obtain discrete, small regions of an organism'sDNA that constitute specific genes.In addition, only relatively small DNA moleculescan be cloned in any of the availablevectors.For thesereasons, the very long DNA molecules that compose an organism's genome must be cleavedinto fragments that can be inserted into the vector DNA. Two types of enzymes-restriction enzymes and DNA ligases-facilitate production of such recombinantDNA molecules. Cutting DNA Molecules into Small Fragments Restric_ tion enzymesare endonucleasesproduced by bacteria that typically recognizespecific 4- to 8-bp sequences,calledrestrictin sites, and then cleaveboth DNA strandsat this site. Restriction sites commonly are short palindromic sequences;that is, the restriction-sitesequenceis the sameon each DNA strand when read in the 5'-+3' direction (Figure5-11). For each restriction enzyme) bacteria also produce a modification enzyme, which protects a bacterium's own DNA from cleavageby modifying it at or near eachpotenrial cleavagesite. The modification enzymeadds a methyl group to one or two bases,usually within the restriction site. When

Vector + DNA fragment EcoRl

J

+

RecombinantDNA

J

J'

r(-

|

J

Cleavage EcoRl I

Isolation, sequencing,and manipulation of purified DNA fragment Although investigatorshave devisednumerous experimental variations, this flow diagram indicatesthe essentialsteDsin DNA cloning. In this section,we first describemethods for isolating a specificsequenceof DNA from a seaof other DNA sequences. This processoften involvescutting the genomernto fragmentsand then placing each fragment ii a vector so that the entire collection can be propagatedas recombinantmole_ cules in separatehost cells. While many different types of '176 .

c H A p r E5R I M o L E c u L AGRE N E Tr tEcc H N t e u E s

|

ti +

Replication of recombinant DNA within host cells

Stickyends

s',.),

-------r

G . TTAA

c ----r

3'

\l

5

FIGURE 5-11 Cleavage of DNAby the restrictionenzyme EcoRf.Thisrestriction enzymefromE colimakesstaqqered cutsat the specific 6-bppalindromic sequence shown,yielding fragments withsingle-stranded, "sticky,, complementary ends.Manyother restriction enzymes alsoproduce fragments with stickyends

a methyl group is presentthere, the restriction endonuclease is prevented from cutting the DNA. Together with the restriction endonuclease,the methylating enzyme forms a restriction-modification system that protects the host DNA while it destroysincomingforeign DNA (e.g.,bacteriophage DNA or DNA taken up during transformation) by cleaving it at all the restriction sitesin the DNA. Many restriction enzymesmake staggeredcuts in the two DNA strands at their recognition site, generatingfragments that have a single-stranded"tail" at both ends,sticky ends (seeFigure 5-11). The tails on the fragmentsgeneratedat a given restriction site are complementaryto those on all other fragments generated by the same restriction enzyme. At room temperature, these single-strandedregions can transiently base-pairwith thoseon other DNA fragmentsgenerated with the same restriction enzyme. A few restriction enzymes,such as Alul and SmaI, cleaveboth DNA strandsat the same point within the restriction site, generating fragmentswith "blunt" (flush)endsin which all the nucleotides at the fragment ends are base-pairedto nucleotides in the complementarystrand.

The DNA isolated from an individual organism has a specificsequence,which purely by chancewill contain a specific set of restriction sites' Thus a given restriction enzyme will cut the DNA from a particular source into a reproducible set of fragmentscalled restriction fragments.The frequency with which a restriction enzymecuts DNA, and thus the average size of the resulting restriction fragments, depends largely on the length of the recognition site. For example, a restriction enzyme that recognizesa 4-bp site will cleaveDNA an averageof once every4", or 256, basepairs, whereas an enzyme that recognizesan 8-bp sequencewill cleaveDNA an averageof onceevery4E basepairs (=65 kb)' Restriction enzymes have been purified from several hundred different speciesof bacteria, allowing DNA molecules to be cut at a large number of different sequencescorresponding to the recognitionsitesof theseenzymes(seeTable5-1)' Inserting DNA Fragments into Vectors DNA fragments with either sticky endsor blunt endscan be insertedinto vector DNA with the aid of DNA ligases.During normal DNA replication, DNA ligase catalyzestheend-to-endioining (ligation) of

ENZYME

MICRO()RGANISM SOURCE

SITERECOGNITION

BamHl

B a cillu s amy lo liq u efaciens

-G-G-A-T-C.C. -C-C-T-A-G-G.

PBODUCED ENOS

J Sticky

1 J Sau3A

aureus Staphylococcus

-G-A-T-C.C-T-A-G-

Sticky

t EcoRl

Escherichia coli

Hindlll

Haemophilus influenzae

J -G-A-A-T-T-C-C.T-T-A-A-G t J -A,A-G.C-T-T.

Sticky

Sticky

-T-T-C-G-A-4.

T Smal

Serratia mdrcescens

J -C-C-C-G-G-G-G-G-G-C-C-C-

Blunt

1

Notl

No cardia otiti di s-cauiarum

-G-C_G-G.C-C-G-C-C-G-C-C-G-G-C-G-

Sticky

t Many of these recognition sequencesare included in a common polylinker sequence(seeFigure 5-13). D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

177

short fragments of DNA called Okazaki fragments. For purposesof DNA cloning, purified DNA ligaseis usedto covalently join the ends of a restriction fragment and vector DNA that have complementaryends (Figure5-12). The vector DNA and restriction fragment are covalently ligated rogetherthrough the standard3'-+5' phosphodiesterbonds of DNA. In addition to Iigating complementary sticky ends, the DNA ligasefrom bacteriophage T4 can ligate any two blunt DNA ends. However, blunt-end ligation is inherently inefficient and requires a higher concentration of both DNA and DNA ligasethan doesligation of sticky ends.

E. coliPlasmidVectorsAre Suitablefor Cloning lsolatedDNA Fragments Plasmidsare circular, double-strandedDNA (dsDNA) molecules that are separatefrom a cell's chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g.,yeast),exist in a

Genomic DNA fragments (a)

3' P-AATT oH__r-----::-.---r 5,

VectorDNA (a')

(b)

s', oH 3'r---------r-TTAA-p

+

P-C GHO-:5'

3' (c)

P-A G CT HOj-:-:r

3' 5,

I

I Complementary I endsbase-pair I

v

OHP

lltr 3'r------]-TTAA

/\ PHO 2 ATP T4 DNA ligase 2AMP+2PPi (a')

5'I-AATT

(a)

3',---------F++ii

3'

s,

FIGURE 5-12 Ligationof restrictionfragmentswith complementary stickyends.In thisexample, vectorDNAcutwith EcoRl ismixedwith a sample containing restriction fragments produced by cleaving genomicDNAwith several different restriction enzymes Theshortbasesequences composing the stickyendsof eachfragment typeareshownThestickyendon the cutvectorDNA (a')base-pairs onlywith thecomplementary stickyendson theEcoR/ fragment(a)in the genomic sample. Theadjacent 3, hydroxyl and5, phosphate groups(red)on the base-paired fragments thenare joined(ligated) covalently byT4 DNAligase. 178

.

c H A p r E R5

|

MoLEcuLAR G E N E T trcE c H N t o u E s

Polylinker

Plasmid cloning vector

FIGURE 5-13 Basiccomponentsof a plasmidcloningvector that can replicatewithin an E. coli cell. plasmid vectorscontaina genesuchasamp',whichencodes selectable the enzyme andconfers resistance to ampicillin. Exogenous P-lactamase DNAcan be inserted intothe bracketed regionwithoutdisturbing theabilityof the plasmidto replicate or express theamp,gene plasmid vectors alsocontaina replication origin(ORl) sequence whereDNA replication isinitiated by host-cell enzymes. Inclusion of a synthetic polylinker containing the recognition sequences for several different restriction enzymes increases the versatility of a plasmidvectorThe vectorisdesigned sothateachsitein the polvlinker is unioueon t h ep l a s m i d .

parasitic or symbiotic relationship with their host cell. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, copies of the plasmid DNA segregateto each daughtercell, assuring continued propagation of the plasmid through successive generationsof the host cell. The plasmidsmost commonly usedin recombinant DNA technology are those that replicate in E. col/. Investigators have engineeredtheseplasmids to optimize their use as vectors in DNA cloning. For instance, removal of unneeded portions from naturally occurring E. coli plasmids yields plasmid vectors, (=1.2-3 kb in circumferential length, that contain three regions essentialfor DNA cloning: a replication origin; a marker that permits selection,usually a drugresistancegene;and a region in which exogenousDNA fragments can be inserted (Figure 5-13). Host-cell enzymes replicate a plasmid beginning at the replication origin (ORI), a specific DNA sequenceof 50-100 basepairs. Once DNA replication is initiated at the ORI, it continues around the circular plasmid regardlessof its nucleotide sequence.Thus any DNA sequenceinsertedinto such a plasmid is replicated along with the rest of the plasmid DNA. Figure 5-14 outlines the general procedure for cloning a DNA fragment using E. coli plasmid vectors. \lhen E. coli cells are mixed with recombinant vector DNA under certain conditions, a small fraction of the cells will take up the plasmid DNA, a process known as transformation. Typically, 1, cell in about 10,000 incorporates a single plasmld DNA molecule and thus becomes transformed. After plasmid vectors are incubated with E. coli, those cells that take up the plasmid can be easily selectedfrom the much larger number of cells. For instance,if the plasmid carries a gene that confers resistance to the antibiotic ampicillin,

> EXPERIMENTAL FIGURE 5-14 DNAcloningin a plasmid vector permitsamplificationof a DNAfragment.A fragmentof an intoa plasmid vectorcontaining DNAto be clonedisfirstinserted gene(amp'),suchasthatshownin Figure 5-13 ampicillin-resistance molecule by incorporation of a plasmid Onlythefew cellstransformed cells, the mediumIntransformed willsurvive on ampicillin-containing plasmid in intodaughter cells,resulting DNAreplicates andsegregates formation of an ampicillin-resistant colonv

transformed cells can be selected by growing them in an ampicillin-containing medium. DNA fragments from a few base pairs up to =10 kb commonly are insertedinto plasmid vectors. When a recombinant plasmid with an inserted DNA fragment transforms an E. coli cell, all the antibiotic-resistantprogeny cells that arise from the initial transformed cell will contain plasmids with the sameinsertedDNA. The insertedDNA is replicated along with the rest of the plasmid DNA and segregatesto daughter cells as the colony grows. In this way, the initial fragment of DNA is replicated in the colony of cells into a large number of identical copies. Since all the cells in a colony arise from a single transformed parental cell, they constitute a clone of cells, and the initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA or a DNA clone. The versatility of an E. coli plasmid vector is increased by the addition of a polylinker, a synthetically generated sequencecontaining one copy of severaldifferent restriction sitesthat are not presentelsewherein the plasmid sequence (seeFigure 5-13). \fhen such a vector is treated with a restriction enzyme that recognizesa restriction site in the polylinker, the vector is cut only once within the polylinker. Subsequentlyany DNA fragment of appropriate length produced with the same restriction enzymecan be insertedinto the cut plasmid with DNA ligase.Plasmids containing a polylinker permit a researcher to use the same plasmid vector when cloning DNA fragmentsgenerated with different restriction enzymes,which simplifies experimentalprocedures. For some purposes,such as the isolation and manipulation of large segmentsof the human genome, it is desirable to clone DNA segments as large as several megabases [L megabase(Mb) : 1 million nucleotides).For this purpose specializedplasmid vectors known as BACs (bacterialartificial chromosomes)have been developed.One type of BAC uses a replication origin derived from an endogenousplasmid of E. coli known as the F factor. The F factor and cloning vectors derived from it can be stably maintained at a single copy per E. coli cell even when they contain inserted sequencesof up to about 2 Mb. Production of BAC libraries requires special methods for the isolation, ligation, and transformation of large segmentsof DNA becausesegments of DNA larger than about 20 kb are highly vulnerable to mechanical breakage by even standard manipulations such as prpetung.

DNA fragment to be cloned Enzymaticallyinsert DNA into plasmid vector

Recombinant plasmid

Mix E. coliwith olasmids in presenceol CaCl2;heat-pulse C u l t u r eo n n u t r i e n ta g a r p l a t e sc o n t a i n i n ga m p i c i l l i n

E. coli cnromosome

Transformedcell SUTVIVES

Cellsthat do not t a k e u p p l a s m i dd i e o n a m p i c i l l i np l a t e s

I P l a s m i replication d I

+

certmurtiotication I

g Colony of cells,each containingcopies of the same recombinantPlasmid

GDNALibrariesRepresentthe Sequences of Protein-CodingGenes A collection of DNA molecules each cloned into a vector molecule is known as a DNA library. !7hen genomic DNA from a particular organism is the source of the starting DNA, the set of clones that collectively represent all the DNA sequencesin the genome is known as a genomic D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N .

179

I lJ

+F*ry65' TranscriUe RNAintocDNA

< FIGURE 5-15 A cDNAlibrary containsrepresentative copiesof cellularmRNAsequences. A mixtureof mRNAsis pointfor preparing thestarting plasmid recombinant clones eachcontaining a cDNA.Transforming E coliwith the plasmids recombinant generates a setof cDNAclones representing allthecellular mRNAs. Seethetextfor a step-bysteodiscussion.

g I n " . o u " R N Aw i t ha t k a t i E J A d dp o t y ( d c ) t a i l Single-stranded 3' G GGGT-___--I cDNA

T T T T 5' t,,,.,.

EI lil$:fl6"#lli",

Y 5'I 3'GGGGI_---__--lTTTT5' -

complementary | Synttresize

sJ strand Double-stranded cDNA r (Jbbb a

-

i ^ F

3', - - r

I fTT5

oRl sites 5', 3'GGGG

E

I

C T T A A G E G G G G I _ _ _ _ _ _ - _ _T_ IT T T E C

lf Gf]GGGGI-.-_lT Stickyend

.

.,*"" withEcoRl T T TECTTAA

I Individual clones

180

T TAAGE

c H A p r E Rs

I

rJl:ru:HT:;,0?,,

MoLEcuLAR G E N E I Cr E c H N t e u E s

nJ.,,with EcoRl

library. Such genomic libraries are ideal for representingthe geneticcontent of relatively simple organismssuch as bacteria or yeast, but presentcertain experimental difficulties for higher eukaryotes.First, the genesfrom such organismsusually contain extensiveintron sequencesand therefore can be too large to be inserted intact into plasmid vectors. As a result, the sequencesof individual genesare broken apart and carried in more than one clone. Moreover, the presenceof introns and long intergenic regions in genomic DNA often makes it difficult to identify the important parts of a gene that actually encode protein sequences.For example, only about 1.5 percentof the hunran genomeactually represents protein-coding genesequences. Thus for many studies,cellular mRNAs, which lack the noncoding regionspresentin genomic DNA, are a more useful starting material for generating a DNA library. In this approach, DNA copies of mRNAs, called complementaryDNAs (cDNAs), are synthesized and cloned into plasmid vectors. A large collection of the resulting cDNA clones, representingall the mRNAs expressedin a cell type, is called a cDNA library.

cDNAsPreparedby ReverseTranscription o f C e l l u l a rm R N A sC a n B e C l o n e dt o G e n e r a t ec D N AL i b r a r i e s The first stepin preparing a cDNA library is to isolatethe total mRNA from the cell type or tissueof interest.Becauseof their poly(A) tails, mRNAs are easily separated from the much more prevalent rRNAs and tRNAs presentin a cell extract by use of a column to which short strings of thymidylate (oligo-dTs) are linked to the matrix. The generalprocedure for preparing a cDNA library from a mixture of cellular mRNAs is outlined in Figure 5-15. The enzymereversetranscriptase,which is found in retroviruses,is usedto synthesize a strand of DNA complementary to each mRNA molecule, starting from an oligo-dT primer (steps1 and2). The resulting cDNA-mRNA hybrid moleculesare converted in several stepsto double-strandedcDNA moleculescorrespondingto all the mRNA molecules in the original preparation (steps 3-5). Each double-stranded cDNA contains an oligodC.oligo-dG double-strandedregion at one end and an oligo-dT.oligo-dA double-strandedregion at the other end. Methylation of the cDNA protects it from subsequent restrictionenzymecleavage(step6). To prepare double-stranded cDNAs for cloning, short double-strandedDNA moleculescontaining the recognition site for a particular restriction enzymeare ligatedto both ends of the cDNAs using DNA ligasefrom bacteriophageT4 (Figure 5-15, step 7). As noted earlier,this ligasecan join "bluntended" double-strandedDNA moleculeslacking sticky ends. The resulting moleculesare then treated with the restriction enzymespecificfor the attachedlinker, generatingcDNA moleculeswith sticky endsat eachend (step8a). In a separateprocedure,plasmid DNA first is treatedwith the samerestriction enzymeto produce the appropriate sticky ends (step8b). The vector and the collection of cDNAs, all containing complementary sticky ends, then are mixed and joined covalently by DNA ligase(Figure 5-15, step 9). The resulting

DNA moleculesare transformed into E. coli cellsto generate individual clones;each clone carrying a cDNA derived from a singlemRNA. Becausedifferent genesare transcribed at very different rates,cDNA clonescorrespondingto abundantly transcribed genes will be representedmany times in a cDNA library, whereas cDNAs corresponding to infrequently transcribed geneswill be extremely rare or not presentat all. This property is advantageousif an investigatoris interestedin a gene that is transcribedat a high rate in a particular cell type' In this case,a cDNA library preparedfrom mRNAs expressedin that cell type will be enriched in the cDNA of interest, facilitating isolation of clonescarrying that cDNA from the library. Howeve! to have a reasonablechance of including clones corresponding to slowly transcribedgenes'mammalian cDNA libiaries must contain 1.06-107individual recombinantclones.

D N A L i b r a r i e sC a n B e S c r e e n e db y H y b r i d i z a t i o n e robe to an OligonucleotidP Both genomic and cDNA libraries of various organisms contain hundredsof thousandsto upwards of a million individual clones in the case of higher eukaryotes. Two general approaches are available for screening libraries to identify clonescarrying a geneor other DNA region of interest: (1) detectionwith oligonucleotideprobes that bind to the clone of interest and (2) detection basedon expression of the encoded protein. Here we describe the first method; an example of the secondmethod is presentedin the next section. The basis for screeningwith oligonucleotideprobes is hybridization, the ability of complementary singlestrandedDNA or RNA moleculesto associate(hybridize) specificallywith each other via basepairing. As discussed in Chapter 4, double-stranded(duplex) DNA can be denatured (melted) into single strands by heating in a dilute salt solution. If the temperature then is lowered and the ion concentration raised, complementary single strands will reassociate(hybridize) into duplexes.In a mixture of nucleic acids, only complementary single strands (or strands containing complementary regions) will reassociate; moreover,the extent of their reassociationis virtually unaffectedby the presenceof noncomplementarystrands. As we will seelater in this chapter,the ability to identify a particular DNA or RNA sequencewithin a highly complex mixture of moleculesthrough nucleic acid hybridization is the basis for many techniquesemployed to study gene expresslon. The stepsinvolved in screeningan E. coli plasmid cDNA library are depicted in Figure 5-16. First' the DNA to be screenedmust be attachedto a solid support. A replica of the petri dish containing a large number of individual E. coli clones is reproduced on the surfaceof a nitrocellulose membrane. The DNA on the membrane is denatured, and the membrane is then incubated in a solution containing a radioactively labeled probe specificfor the recombinant DNA containing the fragment of interest. Under hybridization conditions (near neutral pH, 40-65 "C' 0.3-0'6 M NaCl), D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N .

181

*'**

5eu

I n d i v i d u acl o l o n i e s

B o u n ds i n g l e - s t r a n d eDdN A

Master plate of E. coli colonies

Filter

P l a c en i t r o c e l l u l o sfei l t e ro n D l a t e t o p i c ku p c e l l sf r o m e a c hc o l o n y Hvbridized c o m p l e m e n t a rD y NAs

N i t r o c e l l u l o sfei l t e r

Wash away labeledDNA that does not hybridizeto DNA bound to filter

r"uorr autoradiography f

I

Performaut o r a d i o g r a p h y

o)

S i g n a la p p e a r so v e r p l a s m i dD N A t h a t i s complementary to probe

A EXPERIMENTAL FIGURE 5-16 cDNAlibrariescan be screened with a radiolabeledprobeto identifya cloneof interest.The a p p e a r a noc fea s p o to n t h ea u t o r a d i o g r ai nmd i c a t et hs ep r e s e n c e o f a r e c o m b i n acnl to n ec o n t a i n i nDgN Ac o m p l e m e n t a t or yt h e probeTheposition of the spoton the autoradioqram isthe mirror

i m a g eo f t h ep o s i t i oonf t h a tp a r t i c u l a c lro n eo n t h eo r i g i n aple t r i dish(although for easeof comparison, it isnot shownreversed h e r e )A. l i g n i ntgh ea u t o r a d i o g r awm i i s hw i l l i t ht h eo r i g i n aple t r d locatethe corresponding clonefromwhichE colicellscanbe recovered

this labeledprobe hybridizesro any complemenrarynucleic acid strandsbound to the membrane.Any excessprobe that doesnot hybridize is washedaway, and the labeledhybrids are detectedby autoradiography of the filter. This technique can be usedto screenboth genomicand cDNA libraries,but is most commonly usedto isolatespecificcDNAs. ClearlS identification of specific clones by the membrane-hybridizationtechnique depends on the availa b i l i t y o f c o m p l e m e n r a r yr a d i o l a b e l e d p r o b e s . F o r a n oligonucleotideto be useful as a probe, ir must be long enough for its sequenceto occur uniquely in the clone of interest and not in any other clones. For most purposes, this condition is satisfied by oligonucleotidescontaining a b o u t 2 0 n u c l e o t i d e s .T h i s i s b e c a u s ea s p e c i f i c2 0 - n u c l e o t i d e s e q u e n c eo c c u r s o n c e i n e v e r y 4 2 0 ( = 1 0 1 2 )n u cleotides.Since all genomesare much smaller (=3 x t0e nucleotidesfor humans),a specific20-nucleotidesequence in a genomeusually occurs only once. With automated ins t r u m e n t s n o w a v a i l a b l e , r e s e a r c h e r sc a n p r o g r a m t h e chemicalsynthesisof oligonucleotidesof specificsequence up to about 100 nucleotideslong. Longer probes can be preparedby the polymerasechain reaction (pCR), a widely

usedtechniquefor amplifying specificDNA sequences rhat is describedlater. How might an investigator design an oligonucleotide probe to identify a clone encoding a particular protein? It helps if all or a portion of the amino acid sequenceof the protein is known. Thanks to the availability of the complete genomic sequencesfor humans and some important model organisms such as the mouse, Drosophila, and the roundworm Caenorbabditis elegans,a researchercan use an appropriate computer program to searchthe genomic sequence database for the coding sequencethat corresponds to the amino acid sequenceof the protein under study. If a match is found, then a single, unique DNA probe based on this known genomic sequencewill hybridize perfectly with the clone encoding the protein of interest.

182

.

cHAprER s

I

M o l E c u L A RG E N E T rI cE c H N t e u E S

YeastGenomicLibrariesCan Be Constructed with Shuttle Vectorsand Screenedby F u n c t i o n aC l omplementation In somecasesa DNA library can be screenedfor the ability to express a functional protein that complements a recessive

Polylinker

(a)

Shuttlevector

CEN (b)

l^

dNos mil$

mmrs

Yeastgenomic DNA

Transform E. coli Screenfor amoicillinresistance 'as\ +

*

***

I lsolateandpoolrecombinant from 105transformed I plasmids { F. colicolonies complementation Assayyeastgenomiclibraryby functional

5-17 A yeastgenomiclibrarycan be < EXPERIMENTAL FIGURE plasmid vector that can replicatein shuttle in a constructed shuttlevector of a typicalplasmid yeastand E.coli.(a)Components genesThepresence of a yeastoriginof for cloningSaccharomyces (CEN) (ARS) allowsstable anda yeastcentromere DNAreplication isa yeast yeast included Also in andsegregation replication markersuchasURA3,whichallowsa ura3 mutantto selectable sequences thevectorcontains growon mediumlackinguracilFinally, in E.coli(ORlandamp')anda polylinker andselection for replication (b)Typical protocol for of yeastDNAfragments. for easyinsertion yeast of total digestion genomic Partial yeast library a constructing with an fragments to generate genomic DNAwith5au3Aisadjusted to acceptthe sizeof about10 kb Thevectorisprepared average the whichproduces with BamHl, genomic by digestion fragments E. coli that clone of transformed samestickyendsas5au3A.Each a singletype contains resistance for ampicillin growsafterselection of yeastDNAfragment in a genomic DNA fragment inserted into a plasmid vector. To construct a plasmid genomic library that is to be screenedby functional complementationin yeast cells,the plasmid vector must be capable of replication in both E. coli cells and yeast cells. This type of vector' capable of propagation in two different hosts, is called a shuttle vector. The structure of a typical yeast shuttle vector is shown in Figure 5-L7a. This vector contains the basic elements that permit cloning of DNA fragmentsin E- coli.In addition, the shuttle vector contains an autonomouslyreplicating sequence(ARS), which functions as an origin for DNA replication in yeast; a yeast centromere (called CEN), which allows faithful segregationof the plasmid during yeastcell division; and a yeastgeneencodingan enzymefor uracil synthesis (URA3), which serves as a selectable marker in an approprlate yeastmutant. To increasethe probability that all regions of the yeast genome are successfullycloned and represented in the plasmid library, the genomic DNA usually is only partially digest.d to yield overlapping restriction fragments of =tb t U. These fragments are then ligated into the shuttle vector in which the polylinker has been cleavedwith a restriction enzymethat produces sticky ends complementary to those on the yeast DNA fragments (Figure 5-17b). Becausethe 10-kb restriction fragmentsof yeastDNA are incorporated into the shuttle u.ito., randomly, at least 10s E. coli colonies,each containing a particular recombinant shuttle vector, are necessaryto assurethat each region of yeast DNA has a high probability of being representedin

the library at least once. mutation. Sucha screeningstrategywould be an efficientway Figure 5-18 outlines how such a yeast genomic library to isolate a cloned genethat correspondsto an interestingrecan be screenedto isolate the wild-type gene corresponding cessivemutation identified in an experimental organism. To one of the temperature-sensitivecdc mutations mentioned to illustrate this method, referredto as functional complementain this chapter. The starting yeast strain is a double earlier E. coli tion, we describehow yeast genescloned in special that requires uracil for growth due to a ura3 mttamutant identify yeast cells to plasmidscan be introduced into mutant is temperature-sensitivedue to a cdc28 mutation and tion strain. in the mutant the wild-type genethat is defective by its phenotype (see Figure 5-6). Recombinant identified purpose of screening for the Libraries constructed from the yeast genomic library are mixed isolated plasmids from constructed are among yeast gene sequencesusually genomic DNA rather than cDNA. BecauseSaccharomyces with yeast cells under conditions that promote transformation of the cells with foreign DNA. Sincetransformed yeast genesdo not contain multiple introns, they are sufficiently cells carry a plasmid-borne copy of the wild-type URA3 compact that the entire sequenceof a genecan be included D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

183

Libraryof yeast genomic DNA carrying URA3selectivemarker

Temperature-sensitive cdc-mutant yeast; ura3 (requiresuracil)

Transformyeast by treatmentwith LiOAC,PEG.and heat shocr Plateand incubateat permissivetemperature o n m e d i u m l a c k i n gu r a c i l

Only colonies carrYtng a URA3 marker are able to grow

23'C

R e p l i c a - p l a taen d incubateat nonpermissive te m perature

Only colonies carrying a wild-type CDC gene are able to grow

36'C

EXPERIMENTAL FIGURE 5-18 Screening of a yeastgenomic libraryby functionalcomplementation can identifyclones carryingthe normalform of a mutantyeastgene.Inthis example, a wild-type CDCgeneis isolated by complementation of a cdcyeastmutant TheSaccharomyces strainusedfor screentng rne yeastlibrary carries ura3 anda temperature-sensitive cdcmutation Thismutantstrainisgrownandmaintained at a permisstve (23"C) Pooled temperature plasmids prepared recombinant as

shownin Figure 5-17arerncubated with the mutantyeastcellsunder conditions thatpromotetransformation Therelatively few transformed yeastcells, plasmid whichcontainrecombinant DNA. cangrowin theabsence of uracilat 23 'C Whentransformed yeast colonies arereplica-plated andplacedat 36'C (a nonpermissrve temperature), onlyclones carrying a libraryplasmid thatcontains the wild-type copyof the CDCgenewillsurviveL|OAC= lithiumacetate; PEG= polyethylene glycol

gene,they can be selectedby their ability to grow in the absenceof uracil. Typically, about 20 petri dishes, each containing about 500 yeast transformants, are sufficient to represent the entire yeast genome. This collection of yeast transformants can be maintained at 23"C, a temperature permissivefor growth of the cdc28 mutant. The entire colIection on 20 plates is then transferredto replica plates, which are placed at 36 "C, a nonpermissivetemperature for cdc mutants. Yeastcoloniesthat carry recombinant plasmids expressinga wild-type copy of the CDC28 genewill be able to grow at 36'C. Once temperature-resistant yeastcolonies have been identified, plasmid DNA can be extracted from the cultured yeast cells and analyzed by subcloning and DNA sequencing,topics we take up next.

the well into which the original DNA mixture was placed at the start of the electrophoreticrun. Smaller moleculesmove through the gel matrix more readily than larger molecules, so that molecules of different length migrate as distinct bands. Smaller DNA moleculesfrom about 10 to 2000 nucleotides can be separatedelectrophoretically on polyacrylamide gels, and larger molecules from about 200 nucleotidesto more than 20 kb on agarose gels. A common method for visualizingseparatedDNA bands on a gel is to incubatethe gel in a solution containing the fluorescentdye ethidium bromide. This planar moleculebinds to DNA by intercalating berweenthe base pairs. Binding concentratesethidium in the DNA and also increasesits intrinsic fluorescence.As a result, when the gel is illuminated with ultraviolet light, the regions of the gel containing DNA fluoresce much more brightly than the regionsof the gel without DNA. Once a cloned DNA fragment, especiallya long one, has beenseparatedfrom vector DNA, it often is treated with various restriction enzymesto yield smallerfragments.After separation by gel electrophoresis,all or some of these smaller fragments can be ligated individually into a plasmid vector and cloned in E. coli by the usual procedure. This process, known as subcloning,is an important step in rearranging parts of genesinto useful new configurations. For instance, an investigator who wants to change the conditions under which a geneis expressedmight usesubcloningto replacethe normal promoter associatedwith a cloned genewith a DNA segmentcontaining a different promoter. Subcloningalso can be used to obtain cloned DNA fragments that are of an appropriate length for determining the nucleotidesequence.

Gel Electrophoresis Allows Separationof Vector DNA from ClonedFragments In order to manipulate or sequencea cloned DNA fragment, it sometimesmust first be separatedfrom the vector DNA. This can be accomplished by cutting the recombinant DNA clone with the samerestriction enzymeusedto produce the recombinant vectors originally. The cloned DNA and vector DNA then are subjected to gel electrophoresis, a powerful method for separatingDNA moleculesof different size(Figure5-19). Near neutral pH, DNA molecules carry a large negative charge and therefore move toward the positive electrode during gel electrophoresis.Becausethe gel marrix restricrs random diffusion of the molecules, molecules of the same length migrate together as a band whose width equalsthat of '184 .

c H A p r E5 R | M o L E c u L AGRE N E TrtEc c H N t e u E s

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dKs

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separates 5-19 Gel electrophoresis FIGURE < EXPERIMENTAL by DNA moleculesof different lengths.(a)A gel is prepared p o u r i n ga l i q u i dc o n t a i n i negi t h e rm e l t e da g a r o soer t w o g l a s sp l a t e sa f e w u n p o l y m e r i zaecdr y l a m i dbee t w e e n m i l l i m e t ear sp a r t A s t h e a g a r o sseo l i d i f i eosr t h e a c r y l a m i d e e v a l sf)o r m s p o l y m e r i z ienst op o l y a c r y l a m i ad eg ,e lm a t r i x( o r a n g o p o l y m e r s T h e d i m e n s i o nosf o f c h a i n s c o n s i s t i nogf l o n g ,t a n g l e d s ,r p o r e sd, e p e n do n t h e t h e i n t e r c o n n e c t icnhga n n e l o f t h e a g a r o soer a c r y l a m i dues e dt o f o r mt h e g e l concentration T h es e p a r a t ebda n d sc a nb e v i s u a l i z ebdy a u t o r a d i o g r a p(hi fyt h e n yt e f r a g m e n tasr er a d i o l a b e l eodr)b y a d d i t i o no f a f l u o r e s c e d ( e g , e t h i d i u mb r o m i d et )h a t b i n d st o D N A ( b )A p h o t o g r a pohf a ( E t B r )E t B rb i n d st o D N Aa n d g e ls t a i n e w d i t h e t h i d i u mb r o m i d e f l u o r e s c eusn d e rU Vl i g h t ,T h eb a n d si n t h ef a r l e f ta n df a r r i g h t f r a g m e n tosf k n o w ns i z e l a n e sa r ek n o w na s D N Al a d d e r s - D N A h e l e n g t ho f t h e D N A f o r d e t e r m i n i nt g t h a ts e r v ea sa r e f e r e n c e f r a g m e n tisn t h e e x p e r i m e n tsaal m p l eI P a r(tb )S c i e n cPeh o t o L i b r alr y

o- o - PI : o

-O-P:O

I o

I

o

- O - P I: O

- O - P I: O

I o Subjectto autoradiography or incubatewith fluorescentdye

I

-O-P:O

l o

ase

- O - PI : O I O

I

CH,

QH,

HH Signals corresponding to DNA bands

Deoxyribonucleoside triphosPhate (dNTP)

Dideoxyribonucleoside triphosPhate (ddNTP)

of deoxyribonucleoside 5-20 Structures FIGURE triphosphate (dNTP) dideoxyribonucleoside and triphosphate intoa growingDNA residue (ddNTP). of a ddNTP Incorporation at thatpoint elongation strandterminates D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

185

Technique Animation:DideoxySequencing of DNA{tttt (b)

P r i m e r5 ' T e m p l a t e3 ' -

5'

(a) 5'TAGCTGACTC3' 3' A T C G A C T G A G T C A A G A A C T A T T G G G C T T A A

I DNApolymerase

+ ddGTP

I | + dATB dGTB dCTB dTTp | + ddGTPin low concentration I

v 5'TAGCTGACTCAG3' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

-A

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5 ' T A G C T G A CT C A GT T C + T T G 3 ' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

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5' TAGCTGACTCAGTTJTTCNTNACCCG3' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

-T

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etc.

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etc.

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Denatureand separatedaughterstrandsby electrophoresis I N N N N A A T G A A TA G A T A T A T A G G G G A A T T G A G T 20 30 40

AAATAG 110

TTGG GTAA 120

ATGGT 130

GGTA

ATAG

GGGGAT

TGTTT 140

a EXPERIMENTAL FTGURE 5-21 ClonedDNAscan be sequencedby the Sangermethod, usingfluorescent-tagged dideoxyribonucleoside (ddNTps). triphosphates (a)A single (template) strandof the DNAto be sequenced (blueletters) is hybridized to a synthetic primer(blackletters) deoxyribonucleotide Theprimeriselongated in a reaction mixture containing thefour normaldeoxyribonucleoside triphosphates plusa relatively small amountof oneof thefourdideoxyribonucleoside triphosphates. ln thisexample, (yellow) ddGTP ispresentBecause of the relatively low concentration of ddGTP, incorporation of a ddGTBandthus chaintermination, occurs at a givenposition in the sequence only about1 percent of thetime Eventually the reaction mixture will containa mixture of prematurely (truncated) terminated dauqhter

186

CHAPTER 5

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MOLECULAG RENETIC TECHNIQUES

T

TAGAGT

GA

TG

AGG

GTGAAATTGTTATGTAAA 150 160

AIG 80

AAG TTGAGTATT 90-

170

A

A

AA '180

I

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fragments endingat everyoccurrence of ddGTp(b)Toobtainthe complete sequence of a templateDNA,four separate reactions are performed, eachwith a different dideoxyribonucleoside triphosphate (ddNTP) TheddNTP thatterminates eachtruncated fragment canbe identified by useof ddNTPs taggedwith four differentfluorescent dyes(indicated (c)In an automated bycolored highlights). sequencing machine, thefourreaction mixtures aresubjected to gel electrophoresis, andtheorderof appearance of eachof thefour drfferent fluorescent dyesat the endof the gelis recordedShown hereisa sample printoutfroman automated sequencer fromwhich thesequence of theoriginal template DNAcanbe deduced fromthe sequence of thesynthesized strandN = nucleotide thatcannotbe (c)fromGriffiths assigned[Part et al, Figure 14-27 I

lsolation of a genomic library spanning the genome of interest A l i g n i n gl i b r a r yc l o n e s by hybridizationor restriction-site mapping

Ordered set of clones spanningthe genome

Sequencing of orderedclones

Genomicsequence > FIGURE 5-22 Two Strategies for Assembling WholeGenome Sequences. Onemethoddepends on isolating andassembling a set thatspanthe genomeThiscanbe doneby of clonedDNAsegments or by alignment of matching clonedsegments by hybridization restriction sitemapsTheDNAsequence of the ordered clones can genomic intoa complete The thenbe assembled sequence

of Sequencing r a n d o ml i b r a r yc l o n e s

Sequenceof unordered fragments, for about lO-fold coverageof eachgenomic segment

Aligning sequenced clonesby comPuter Assembled genomlcsequence DNA easeof automated on the relative methoddepends alternative the library stepof ordering the laborrous andbypasses sequencing sothateachsegment clones enoughrandomlibrary Bysequencing to it ispossible to 10 times genome from 3 is represented of the of the alignment by computer sequence thegenomrc reconstruct fragments verylargenumberof sequence

guished by their corresponding fluorescent label (Figure 521b). For example,all truncated fragmentsthat end with a G would fluoresce one color (e.g., yellow), and those ending with an A would fluoresceanother color (e.g',red), regardless The complete characterizationof any cloned DNA fragment requires determination of its nucleotide sequence.F. Sanger of their lengths. The mixtures of truncated daughter fragments from each of the four reactionsare subjectedto elecand his colleaguesdeveloped the method now most comtrophoresison specialpolyacrylamidegels that can separate monly used to determine the exact nucleotide sequenceof single-strandedDNA moleculesdiffering in length by only 1 DNA fragmentsup to =500 nucleotideslong. The basic idea nucleotide.A fluorescencedetector that can distinguish the behindthis method is to synthesizefromthe DNA fragmentto four fluorescenttags is located at the end of the gel. The sebe sequenceda set of daughterstrandsthat are labeledat one quence of the original DNA template strand can be deterend and differ in length by one nucleotide.Separationof the mined from the order in which different labeledfragmentsmitruncated daughterstrandsby gel electrophoresiscan then esgrate past the fluorescencedetector (Figure 5-21'c). tablish the nucleotidesequenceof the original DNA fragment. In order to sequencea long continuousregion of genomic Synthesisof truncated daughterstandsis accomplishedby triphosphates(ddNTPs). DNA or even the entire genomeof an organism, researchers use of 2',3'-dideoxyribonucleoside These molecules,in contrast to normal deoxyribonucleotides usually employ one of the strategiesoutlined in Figure 5-22. (dNTPs), lack a 3' hydroxyl group (Figure 5-20). Although The first method requiresthe isolation of a collection of cloned overlap. Once the sequence DNA fragmentswhose sequences ddNTPs can be incorporated into a growing DNA chain by oligonucleotidesbased is determined, fragments of these of one DNA polymerase,once incorporated they cannot form a for use as synthesized can be chemically sequence on that phosphodiesterbond with the next incoming nucleotide In fragments. overlapping the adjacent in sequencing primers triphosphate.Thus incorporation of a ddNTP terminates is determined DNA of long stretch of a sequence way, the this resulting at in a daughterstrand truncated chain synthesis, incrementally by sequencingof the overlapping cloned DNA specific positions correspondingto the basecomplementary fragments that compose it. A second method, which is called added ddNTP on the template strand. to the the time-consumbypasses whole genomeshotgunseqwencing, Sequencingusing the Sangerdideoxy chain-termination ing step of isolating an ordered collection of DNA segments method is usually carried out using an automated DNA sethat span the genome.This method involves simply sequencing quencing machine. The reaction begins by denaturing a random clones from a genomic library. A total number of double-strandedDNA fragment to generatetemplate strands for in vitro DNA synthesis.A syntheticoligodeoxynucleotide clonesare chosenfor sequencingso that on averageeach segabout 10 times.This degreeof ment of the genomeis sequenced is usedas the primer for the polymerizationreactionthat concoverageensuresthat eachsegmentof the genomeis sequenced tains a low concentrationof eachof the four ddNTPs in addimore than once.The entiregenomicsequenceis then assembled tion to higher concentrationsof the normal dNTPs. The using using a computeralgorithm that alignsall the sequences, ddNTPs are randomly incorporated at the positions of the their regionsof overlap.\7hole genomeshotgunsequencingis correspondingdNTP, causingtermination of polymerization of the fastestand most cost-effectivemethod for sequencinglong at thosepositionsin the sequence(Figure5-21,a).Inclusion stretchesof DNA, and most genomes'including the human fluorescenttagsof different colors on eachof the four ddNTPs genome,have beensequencedby this method. allows each set of truncated daughterfragmentsto be distin-

Rapidly C l o n e dD N A M o l e c u l e sA r e S e q u e n c e d b y t h e D i d e o x yC h a i n - T e r m i n a t i oMne t h o d

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

187

TechniqueAnimation: PolymeraseChain Reaction{tttt

c Y c l e1

, Denaturationof DNA I A n n e a l i n go f p r i m e r s

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c Y c l e2

, Denaturationof DNA I A n n e a l i n go f p r i m e r s

Elongationof primers

{

G Y c l e3

< EXPERIMENTAL FIGURE 5-23 The polymerase chainreaction(PCR) is widely used to amplifyDNAregionsof known sequences. Toamplifya specific regionof DNA,an investigator willchemically primers synthesize two different oligonucleotrde complementary to (designated sequences of approximately 18 bases flankingtheregionof interest aslightblue anddarkbluebars)Thecomplete reaction iscomposed of a complex mixture of doublegenomic stranded DNA(usually DNAcontaining thetargetsequence of interest), a stoichiometric excess of bothprimers, thefourdeoxynucleoside triphosphates, anda heatstableDNApolymerase knownasTaqpolymerase. DuringeachPCRcycle, the reaction mixture isfirstheatedto separate thestrands andthencooledto allowtheprimers to bindto complementary sequences flanking the regionto beamplified. Iaq polymerase thenextends eachprimerfromits3' end,generating newlysynthesized strands thatextendin the3' direction to the 5' endof thetemplate strand.Duringthethirdcycle, two double-stranded DNAmolecules aregenerated equalin lengthto thesequence of theregionto beamplified Ineachsuccessive cyclethetargetsegment, whichwillannealto theprimers, isduplicated, andwilleventually vastlyoutnumber allotherDNAsegments in thereaction mixture Successive PCRcycles canbeautomated bycycling the reaction for timedintervals at high temperature for DNAmeltingandat a definedlowertemperature for theannealing and portions elongation of thecycleA reaction thatcycles 20 timeswillamplifythespecific target sequence 1-million-fold

, Denaturationof DNA I n n n e a t i n so f p r i m e r s

E l o n g a t i o no f p r i m e r s

I

L

Cycles4, 5, 6, etc.

T h e P o l y m e r a sC e h a i nR e a c t i o nA m p l i f i e sa SpecificDNA Sequencefrom a ComplexMixture If the nucleotide sequencesat the ends of a parricular DNA region are known, the intervening fragment can be amplified directly by the polymerase chain reaction (PCR). Here we describethe basic PCR technique and three situations in which it is used. 188

.

cHAprER s

I

M o L E c u L AG R E N E I cr E c H N t e u E s

The PCR dependson the ability to alternately denature (melt) double-strandedDNA moleculesand hybridizecomplementary singlestrandsin a controlled fashion. As outlined in Figure5-23, a typical PCR procedurebeginsby heat-denaturation of a DNA sampleinto singlestrands.Next, two synthetic oligonucleotidescomplementaryto the 3' ends of the target DNA segmentof interestare addedin greatexcessto the denatured DNA, and the temperarure is lowered to 50-60 'C. Thesespecificoligonucleotides,which are at a very high concentration,will hybridizewith their complementarysequences in the DNA sample, whereas the long strands of the sample DNA remain apart becauseof their low concentration.The hybridized oligonucleotidesthen serveas primers for DNA chain synthesisin the presenceof deoxynucleotides(dNTPs) and a temperature-resistantDNA polymerase such as that from Thermusaquaticus(a bacteriumthat livesin hot springs).This enzymq called Taq polymerase, can remain active even after being heatedto 95 oC and can extend the primers at temperaturesup to72"C. When synthesisis complete,the whole mixture is then heatedto 95 "C to denaturethe newly formed DNA duplexes.After the temperatureis lowered again, another cycle of synthesistakes place becauseexcessprimer is still present. Repeated cycles of denaturation (heating) followed by hybridization and synthesis(cooling) quickly amplify the sequence of interest. At each cycle, the number of copies of the sequencebetweenthe primer sitesis doubled; therefore, the desired sequenceincreasesexponentially-about a million-fold after 20 cycles-whereas all other sequencesin the original DNA sampleremain unamplified. Direct lsolation of a Specific Segment of Genomic DNA For organismsin which all or most of the genome has been sequenced,PCR amplification starting with the total genomic DNA often is the easiestway to obtain a specificDNA region of interest for cloning. In this application, the two oligonucleotideprimers are designedto hybridize to sequences flanking the genomic region of interest and to include sequences

Regionto be amplified 5'

3'

P r i m e r1

D N As y n t h e s i s

s'

5'

P r i m e r2

5-24 A specifictarget region in total FIGURE < EXPERIMENTAL genomicDNAcan be amplifiedby PCRfor usein cloning.Each to oneendof thetargetsequence primerfor PCRiscomplementary that enzyme for a restriction sequence the recognition andincludes primer doesnot havea sitewithinthetargetregion.In thisexample, primer2 contains a Hindlll whereas a BamHl sequence, 1 contains (Notethatfor clarity, icationof only in anyround,amplif sequence isshown,the onein brackets oneof thetwo strands ) After aretreatedwith appropriate thetargetsegments amplification, generating with stickyendsThese fragments restriction enzymes, plasmid vectors andcloned into complementary canbe incorporated (seeFigure 5-13). in E colibythe usualprocedure

c

Prime1 r

C o n t i n u ef o r = 2 0 PCRcycles Cut with restriction enzymes

Stickyend

-Stickyend Ligatewith plasmidvector with stickyends

that are recognizedby specificrestriction enzymes(Figure 524). After amplification of the desired target sequencefor about 20 PCR cycles,cleavagewith the appropriate restriction enzymesproduces sticky ends that allow efficient ligation of the fragment into a plasmid vector cleaved by the same restriction enzymesin the polylinker. The resulting recombinant plasmids,all carrying the identical genomic DNA segment,can then be cloned in E. coli cells.\fith certain refinements of the PCR, even DNA segmentsgreater than 10 kb in length can be amplified and cloned in this way. Note that this method does not involve cloning of large numbers of restriction fragments derived from genomic DNA and their subsequentscreeningto identify the specificfragment of interest. In effect, the PCR method inverts this traditional approach and thus avoids its most tedious aspects.The PCR method is useful for isolating genesequencesto be manipulated in a variety of useful ways describedlater. In addition the PCR method can be used to isolate gene sequencesfrom mutant organismsto determinehow they differ from the wild type. A variation on the PCR method allows PCR amplification of a specificcDNA sequencefrom cellular mRNAs. This

method, known as reuersetranscriptase-PcR /R?PCR/, begins with the same procedure describedpreviously for isolation of cDNA from a collection of cellular mRNAs' Typically' an oligo-dT primer, which will hybridize to the 3' poly(A) tail of the mRNA, is used as the primer for the first strand of cDNA synthesisby reversetranscriptase.A specific cDNA can then be isolatedfrom this complex mixture of cDNAs by PCR amplification using two oligonucleotide primers designedto match sequencesat the 5' and 3' ends of the corresponding mRNA. As described previously, these primers could be designedto include restriction sitesto facilitate the insertion of amplified cDNA into a suitableplasmid vector. Preparation of Probes Earlier we discussedhow oligonucleotide probes for hybridization assayscan be chemically synthesized.Preparation of such probes by PCR amplification requires chemical synthesisof only two relatively short primers corresponding to the two ends of the target sequence. The starting sample for PCR amplification of the target sequencecan be a preparation of genomic DNA, or a preparation of cDNA synthesizedfrom the total cellular 32PmRNA. To generatea radiolabeled product from PCR, labeled dNTPs are included during the last several amplification cycles.Becauseprobes prepared by PCR are relatively 3'P atoms incorporated into long and have many radioactive them, theseprobes usually give a stronger and more specific signal than chemically synthesizedprobes. Tagging of Genes by Insertion Mutations Another useful application of the PCR is to amplify a "tagged" gene from the genomic DNA of a mutant strain. This approach is a simpler method for identifying genesassociatedwith a particular mutant phenotype than screening of a library by functionalcomplementation(seeFigure5-18). The key to this use of the PCR is the ability to produce mutations by insertion of a known DNA sequenceinto the genome of an experimental organism. Such insertion mutations can be generated by use of mobile DNA elements, which can move (or transpose)from one chromosomal site to another. As discussedin more detail in Chapter 5, these DNA sequencesoccur naturally in the genomesof most organisms and may give rise to loss-of-function mutations if they transposeinto a protein-coding region. For example, researchershave modified a Drosophila mobile DNA element, known as the P element, to optimize D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

r89

> EXPERIMENTAL FIGURE 5-25 The genomic sequenceat the insertionsite of a transposon is revealedby PCRamplificationand Restriction sites:t sequencing. Toobtainthe DNAsequence of the insertion siteof a P-element transooson it is necessary to PCR-amplify thejunctionbetween knowntransposon sequences andunknown flankingchromosomal sequences Onemethodto achieve thisisto cleave genomic DNAwith a restriction enzyme thatcleaves oncewithinthe transposon sequence Ligation of the resulting restriction fragments willgenerate circular DNA molecules. Byusingappropriately designed DNA primers thatmatchtransposon sequences it is possible junction to PCR-amplify the desired fragmentFinally, (see a DNAsequencing reaction Figure 5-21)is performed usrngthe PCR-amplified fragmentasa template andan oligonucleotide primerthat matches sequences neartheendof the transposon, to obtainthesequence of thejunction between thetransposon andchromosome

I ransDoson

I cr, *it, I

restriction enzyme

Ligate to circularize

PCRprimers

Sequencing pflmer

I PCRamplification with Orimers to transposon I

-+

its use in the experimental generation of insertron mutations. Once it has been demonstratedthat insertion of a P element causesa mutation with an interestingphenotype, the genomic sequencesadjacentto the insertion site can be amplified by a variation of the standardPCR protocol that usessyntheticprimers complementaryto the known P-element sequencebut that allows unknown neighboring sequencesto be amplified. One such method, depicted in Figure 5-25, beginsby cleavingDrosophila genomic DNA containing a P-elementinsertion with a restriction enzyme that cleavesonce within the P-elementDNA. The collection of cleaved DNA fragmenrs treated with DNA ligase yields circular molecules,some of which will contain P-element DNA. The chromosomal region flanking the P element can then be amplified by PCR using primers that match P-elementsequencesand are elongatedin opposite directions. The sequenceof the resulting amplified fragment can then be determined using a third DNA primer. The crucial sequencefor identifying the site of P-element insertion is the junction between the end of the P-element and genomic sequences.Overall, this approach avoids the cloning of large numbers of DNA fragments and their screeningto detect a cloned DNA correspondingto a mutated geneof interest. Similar methods have been applied to other organrsms for which insertion mutations can be generatedusing either mobile DNA elements or viruses with sequencedgenomes that can insertrandomly into the genome. 190

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DNA Cloning and Characterization r In DNA cloning, recombinant DNA molecules are formed in vitro by inserting DNA fragments into vecror DNA molecules.The recombinant DNA molecules are then introduced into host cells, where they replicate, producing large numbers of recombinant DNA molecules. r Restriction enzymes (endonucleases)typically cut DNA at specific4- to 8-bp palindromic sequences,producing defined fragments that often have self-complementarysinglestrandedtails (stickyends). I Two restriction fragments with complementary ends can be joined with DNA ligase to form a recombinant DNA molecule(seeFigure 5-12). t E. coli cloning vectors are small circular DNA molecules (plasmids)that include three functional regions:an origin of replication, a drug-resistancegene, and a site where a DNA fragmentcan be inserted.Transformedcellscarrying a vector grow into colonieson the selectionmedium (seeFigure5-13). r A cDNA library is a set of cDNA clones prepared from the mRNAs isolated from a particular type of tissue. A genomic library is a set of clones carrying restriction fragments produced by cleavageof the entire genome. r In cDNA cloning, expressed mRNAs are reversetranscribedinto complementaryDNAs, or cDNAs. By a series of reactions,single-strandedcDNAs are converted into

double-strandedDNAs, which can then be ligated into a plasmidvector (seeFigure5-15). r A particular cloned DNA fragment within a library can be detected by hybridization to a radiolabeledoligonucleotidewhose sequenceis complementaryto a portion of the fragment(seeFigure5-16). r Shuttle vectors that replicate in both yeast and E. coli can be used to construct a yeastgenomic library. Specificgenes can be isolated by their ability to complementthe correspondingmutant genesin yeastcells(seeFigure5-17). r Long cloned DNA fragments often are cleavedwith restriction enzymes,producing smallerfragmentsthat are then separatedby gel electrophoresisand subclonedin plasmid vectorsprior to sequencingor experimentalmanipulation. r DNA fragmentsup to about 500 nucleotideslong are sequenced in automated instruments based on the Sanger (dideoxychain-termination)method (seeFigure5-21). r lil/hole genome sequencescan be assembledfrom the sequencesof a large number of overlappingclones from a genomiclibrary (seeFigure 5-22). r The polymerasechain reaction(PCR)permitsexponential amplification of a specificsegmentof DNA from just a single initial template DNA molecule if the sequenceflanking the DNA regionto be amplifiedis known (seeFigure5-23). r PCR is a highly versatilemethod that can be programmed to amplify a specificgenomicDNA sequence,a cDNA, or a sequenceat the junction betweena transposableelement and flanking chromosomalsequences.

EE UsingClonedDNAFragments to StudyGeneExpression In the last sectionwe describedthe basictechniquesfor using recombinantDNA technologyto isolatespecificDNA clones, and ways in which the clones can be further characterized.

Now we considerhow an isolatedDNA clone can be usedto study geneexpression.We discussseveralwidely usedgeneral techniquesthat rely on nucleic acid hybridization to elucidate when and where genesare expressed,as well as methods for generatinglarge quantitiesof protein and otherwisemaniputo determinetheir expressionpatlating amino acid sequences terns,structure,and function. More specificapplicationsof all thesebasictechniquesare examinedin the following sections.

s e r m i tD e t e c t i o no f H y b r i d i z a t i o nT e c h n i q u eP F r a g m e n t s a n d mRNAs S p e c i f i cD N A Two very sensitivemethodsfor detectinga particular DNA or RNA sequencewithin a complex mixture combine separation and hybridizationwith a complementary by gel electrophoresis probe. A third method involveshybridizing DNA radiolabeled labeledprobesdirectly onto a preparedtissuesample.\Wewill encounter referencesto all three of these techniques,which have numerousapplications,in other chapters. Southern Blotting The first hybridizationtechniqueto detect DNA fragments of a specific sequenceis known as Southern blotting after its originator E. M. Southern.This techniqueis capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavageof the entire human genomewith a restrictionenzyme.When sucha complex mixture is subjectedto gel electrophoresis,so many different fragmentsof nearly the same length are presentit is not possibleto resolveany particular DNA fragmentsas a discrete band on the gel. Neverthelessit is possibleto identify a particular fragmentmigrating as a band on the gel by its ability to hybridize to a specificDNA probe. To accomplish this, the restriction fragments present in the gel are denatured with alkali and transferredonto a nitrocellulosefilter or nylon membraneby blotting (Figure 5-26). This procedurepreservesthe distribution of the fragmentsin the gel, creatinga replica of the gel on the filter. (The blot is usedbecauseprobesdo not readily diffuseinto the original gel.)The filter then is incubatedunder hybridization conditions with a specificradiolabeledDNA probe, which usually is

DNA I I Cleavewith restriction enzymes I Gel V

Autoradiogram

Ni t r o c e l l luo s e Ni t r o c e l l luo s e Gel ----.f----

Hybridizewith l a b e l e dD N A o r R N Ap r o b e

---r---T---T

A l k a l i n es o l u t i o n C a p i l l a r ya c t i o nt r a n s f e r s DNAfrom gelto nitrocellulose

FIGURE A EXPERIMENTAL 5-25 Southernblot techniquecan detecta specificDNAfragmentin a complexmixtureof threedifferent restrictionfragments.Thediagram depicts to fragments in the gel,butthe procedure canbe applied restriction

that of DNAfragmentsOnlyfragments of millions a mixture probewillgivea signalon an autoradiogram to a labeled hybridize mRNAs specifrc blottingdetects calledtVorthern technique A similar J Mol Blol98:508 1975, l withina mixturelseeE M Southern,

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generatedfrom a cloned restriction fragment. The DNA restriction fragmentthat is complementaryto the probe hybridizes,and its location on the filter can be revealedby autoradiography. Northern Blotting One of the most basicways to characterize a cloned geneis to determinewhen and where rn an organism the geneis expressed.Expressionof a particular genecan be followed by assayingfor the corresponding mRNA by Northern blotting, named, in a play on words, after the related method of Southern blotting. An RNA sample, often the total cellular RNA, is denatured by treatment with an agent such as formaldehyde that disrupts the hydrogen bonds between base pairs, ensuring that all the RNA moleculeshave an unfolded, linear conformation. The individual RNAs are separated according to size by gel electrophoresisand transferred to a nitrocellulose filter to which the extendeddenatured RNAs adhere.As in Southernbloning, the filter then is exposedto a labeled DNA probe that is complementaryto the geneof interest; finally, the labeled filter is subjected to autoradiography. Becausethe amount of a specificRNA in a samplecan be estimated from a Norrhern blot, the procedureis widely used ro compare the amounts of a particular mRNA in cells under differentconditions(Figure5-27). In Situ Hybridization Northern blotting requiresextracring the mRNA from a cell or mixture of cells,which meansthat the cellsare removedfrom their normal location within an orsan-

UN

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ism or tissue.As a result, the location of a cell and its relation to its neighbors is lost. To retain such positional information in precisestudiesof geneexpression,a whole or sectionedtissueor evena whole permeabilizedembryo may be subjectedto in situ hybridization to detect the mRNA encoded by a particular gene.This technique allows genetranscription to be monitored in both time and space(Figure5-28).

D N A M i c r o a r r a y sC a nB e U s e dt o E v a l u a t et h e E x p r e s s i o on f M a n y G e n e sa t O n e T i m e Monitoring the expressionof thousandsof genessimultaneously is possiblewith DNA microarray analysis,another technique based on the concept of nucleic acid hybridization. A DNA microarray consists of an organized array of thousands of individual, closely packed gene-specific sequencesattached to the surfaceof a glassmicroscopeslide. By coupling microarray analysis with the results from genome sequencing projects, researchers can analyze the global patterns of gene expression of an organism during specificphysiological responsesor developmentalprocesses. Preparation of DNA Microarrays In one methodfor preparing microarrays,an =1-kb portion of the coding region of each gene analyzedis individually amplified by the PCR. A robotic deviceis used to apply each amplified DNA sample to the surface of a glass microscope slide, which then is chemically processedto permanently attach the DNA sequencesto the glasssurfaceand to denature them. A typical array might contain =6000 spotsof DNA in a2 x 2- cm grid. In an alternative merhod, multiple DNA oligonucleotides, usually at least 20 nucleotides in length, are synthesizedfrom an initial nucleotide that is covalently bound to the surface of a glass slide. The synthesisof an oligonucleotide of specific sequencecan be programmed in a small region on the surfaceof the slide. Severaloligonucleotidesequencesfrom a singlegeneare thus synthesizedin neighboring regions of the slide to analyzeexpressionof that gene. Vith this method, oligonucleotidesrepresentingthousands of genescan be produced on a singleglassslide. Becausethe methods for constructing these arrays of synthetic oligonucleotideswere adapted from methods for manufacturing microscopic integrated circuits used in computers, these types of oligonucleotidemicroarrays are often called DNA chips.

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A EXPERIMENTAL FIcURE5-27 Northernblot analysisreveals increased expression of p-globinmRNAin differentiated erythroleukemia cells.ThetotalmRNAin extracts of erythroleukemia cellsthatweregrowingbut uninduced andin cells induced to stopgrowingandallowedto differentiate for 4g hoursor 96 hourswasanalyzed by Northern blottingfor B-globin mRNAThe density of a bandisproportional to theamountof mRNApresent TheB-globin mRNAisbarelydetectable in uninduced cells(UNlane) but increases morethan1000-fold by 96 hoursafterdifferentiation is induced[Courtesy of L Kole] 192

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Using Microarrays to Compare Gene Expression under Different Conditions The initial stepin a microarrayexpression study is to preparefluorescentlylabeledcDNAs corresponding to the mRNAs expressedby the cellsunder study.When the cDNA preparation is applied to a microarray,sporsrepresenting genesthat are expressedwill hybridize under appropriate conditions to their complementarycDNAs in the labeledprobe mix, and can subsequentlybe detectedin a scanninglasermicroscope. Figure 5-29 depicts how this method can be applied to examine the changes in gene expression observed after starved human fibroblasts are transferred to a rich, serumcontaining, growth medium. In this type of experiment, the separatecDNA preparations from starvedand serum-grown

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A EXPERIMENTAL FIGURE 5-28 In situ hybridizationcandetect activityof specificgenesin whole and sectionedembryos.The s p e c i m ei n s p e r m e a b i l i zbeydt r e a t m e nwt i t h d e t e r g e natn da protease to expose the mRNAto the probeA DNAor RNAprobe, s p e c i f ifco r t h e m R N Ao f i n t e r e sits, m a d ew i t h n u c l e o t i daen a l o g s c o n t a i n i ncgh e m i c aglr o u p tsh a tc a nb e r e c o g n i z ebdy a n t i b o d i e s A f t e rt h e p e r m e a b i l i zsepde c i m ehna sb e e ni n c u b a t ewdi t ht h e p r o b eu n d e cr o n d i t i o nt h s a tp r o m o t e hybrid z a t i o nt,h ee x c e s s p r o b ei s r e m o v ew d i t ha s e r i eosf w a s h e sT h es p e c i m ei n st h e n i n c u b a t ei d n a s o l u t i ocno n t a i n i nagn a n t i b o dtyh a tb i n d st o t h e p r o b eT hs a n t i b o diysc o v a i e n tl loyi n e dt o a r e p o r t eern z y m (ee g , se h o r s e r asdh p e r o x i d a o r a l k a l i npeh o s p h a t a st hea) tp r o d u c eas productAfterexcess coloredreaction antibodyhasbeenremoved,

drecipitate o r t h e r e p o r t eern z y m iesa d d e dA c o l o r e p s u b s t r a tf e f o r m sw h e r et h e p r o b eh a sh y b r i d i z et od t h e m R N Ab e i n g d e t e c t e d( a )A w h o l em o u s e m b r y oa t a b o u t1 0 d a y so f probedfor Sonichedgehog mRNAThestainmarks development ( r e d a l o n gt h e m e s o d e r rmu n n i n g a r o d o f a r r o w ) , t h en o t o c h o r d f u t u r es p i n acl o r d ( b )A s e c t i oonf a m o u s e m b r y os i m i l atro t h a t i n o a r t( a ) T h ed o r s a l / v e n tar xailso f t h e n e u r atlu b e( N T c) a nb e (redarrow) notochord seen,with the Sonichedgehog-expressing ( b l u e v e n t r a (l c )A s t i l f l a r t h e r a r r o w ) e n d o d e r m b e l o wi t a n dt h e during embryoprobedfor an mRNAproduced wholeDrosophila is patternof bodysegments Therepeating tracheadevelopment of L visibleAnterior(head)is up,ventralisto the left Icourtesy andN/ P Scott Milenkovic l

fibroblastsare labeledwith differently colored fluorescent dyes.A DNA array comprising8600 mammaliangenesthen is incubatedwith a mixture containingequal amountsof the t w o c D N A p r e p a r a t i o n su n d e r h y b r i d i z a t i o nc o n d i t i o n s . After unhybridizedcDNA is washed away, the intensity of green and red fluorescenceat each DNA spot is measured microscopeand storedin computerfiles using a fluorescence under the name of eachgeneaccordingto its known position on the slide.The relativeintensitiesof red and greenfluorescencesignalsat eachspot are a ffreasureof the relativelevel of expressionof that genein resp()nseto serum.Genesthat are not transcribedunder thesegrowth conditions give no detectablesignal. Genesthat are transcribed at the same level under both conditions will hybridize equally to both r e d a n d g r e e n - l a b e l e dc D N A p r e p a r a t i o n s .M i c r o a r r a y analysisof geneexpressionin fibroblastsshowedthat transcriptionof about 500 of the 8600 genesexaminedchanged substantiallyafter addition of serum.

the many different changesin cell physiology that occur when cells are transferred from one medium to another. I n o t h e r w o r d s , g e n e st h a t a p p e a rt o b e c o - r e g u l a t e di n a s i n g l e m i c r o a r r a y e x p r e s s i o ne x p e r i m e n t m a y u n d e r g o changesin expressionfor very different reasonsand may a c t u a l l y h a v e v e r y d i f f e r e n t b i o l o g i c a l f u n c t i o n s .A s o l u t i o n t o t h i s p r o b l e m i s t o c o m b i n et h e i n f o r m a t i o n f r o m a set of expressionarray experimentsto find genesthat are similarly regulated under a variety of conditions or over a oeriod of time. This more informative use of multiple expressionarray experiments is illustrated by examining the relative expression of the 8600 genesat different times after serum addition, generatingmore than 104 individual piecesof data. A computer program, relatedto the one usedto determinethe can organizethese of differentprotein sequences' relatedness over the expression genes show similar that data and cluster cluster such Remarkably, serum addition. after time course particproteins genes whose encoded groups sets of analysis ipate in a common cellular process, such as cholesterol biosynthesisor the cell cycle (Figure5-30).

C l u s t e rA n a l y s i so f M u l t i p l e E x p r e s s i o n E x p e r i m e n t sl d e n t i f i e sC o - r e g u l a t e G d enes F i r m c o n c l u s i c l n sr a r e l y c a n b e d r a w n f r o m a s i n g l e m i c r o a r r a y e x p e r i m e n t a b o u t w h e t h e r g e n e st h a t e x h i b i t s i m i l a r c h a n g e si n e x p r e s s i o na r e c o - r e g u l a t e da n d h e n c e likely to be closely related functionally. For example, m a n y o f t h e o b s e r v e dd i f f e r e n c e si n g e n e e x p r e s s i o nj u s t e fs d e s c r i b e di n f i b r o b l a s t sc o u l d b e i n d i r e c t c o n s e < r u e n c o

analysiswill be a powerful F.i In the future, microarray di"enustic tool in medicine. For instance,particular fiil setsof .RXRt have beenfound to distinguishtumors with a poor prognosisfrom those with a good prognosis.Previously indistinguishablediseasevariations are now detectable' Analysisof tumor biopsiesfor thesedistinguishingmRNAs

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Technique Animation:Synthesizing an Oligonucleotide Array flllt TechniqueAnimation: Screeningfor Patternsof Gene Expression Fibroblasts w i t h o u ts e r u m

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< EXPERIMENTAL FIGURE 5-29 DNAmicroarrayanalysiscan revealdifferencesin gene expressionin fibroblastsunder differentexperimentalconditions.(a)In thisexample, cDNA prepared frommRNAisolated fromfibroblasts eitherstarved for serumor afterserumadditionislabeled with different fluorescent dyesA microarray composed of DNAspotsrepresenting 8600 genesisexposed mammalian to an equalmixture of thetwo cDNA preparations underhybridization conditions Theratioof the intensities of redandgreenfluorescence overeachspot,detected with a scanning confocal lasermicroscope, indicates the relative expression of eachgenein response to serum.(b)A micrograph of a smallsegment of an actualDNAmicroarray. Eachspotin this 16 x genehybridized 16arraycontains DNAfroma different to control andexperimental cDNAsamples labeled with redandgreen fluorescent dyes(A yellowspotindicates equalhybridization of greenandredfluorescence, indicating no changein gene (b)Alfred expression) Pasieka/Photo Researchers, Inc] [Part

Wash Measuregreen and red fluorescenceover eachspot

will help physiciansto selectthe most appropriate treatment. As more patternsof geneexpressioncharacteristicof various diseasedtissuesare recognized,the diagnostic use of DNA microarrays will be extendedto other conditions.I

E. coliExpressionSystemsCan ProduceLarge Quantitiesof Proteinsfrom ClonedGenes

A lf a spot is green,expressionof that gene decreasesin cells after serum addition

.E l f a s p o t i s r e d ,e x p r e s s i o no f t h a t g e n e i n c r e a s e isn c e l l s after,seru m,addition

Many protein hormones and other signalingor regulatory proteins are normally expressedat very low concentrations,precluding their isolation and purification in large quantities by standard biochemical techniques. Widespread therapeutic use of such proteins, as well as basicresearchon their structureand functions, dependson efficient proceduresfor producing them in large amounts at reasonablecost. RecombinantDNA techniquesthat turn E. coli cells into factories for synthesizinglow-abundance proteins now are used to commercially produce granulocyte colony-stimulating factor (G-CSF), insulin, growth hormone, and other human proteins with therapeuticuses. For example, G-CSF stimulatesthe production of granulocytes, the phagocytic white blood cells critical to defense against bacterial infections. Administration of G-CSF to cancer patients helps offset the reduction in granulocyte production caused by chemotherapeuticagents, thereby protecting patients againstseriousinfection while they are receiving chemotherapy.I The first step in producing large amounts of a lowabundanceprotein is to obtain a cDNA clone encoding the full-length protein by methods discussedpreviously.The second step is to engineerplasmid vectorsthat will expresslarge amounts of the encoded protein when it is inserted into E. coli cells.The key to designingsuch expressionvectors is inclusion of a promoter, a DNA sequencefrom which transcription of the cDNA can begin. Consider,for example, the

194

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A A EXPERIMENTAL FIGURE 5-30 Clusteranalysisof data from multiple microarrayexpressionexperimentscan identify cogeneswas of 8600mammalian regulatedgenes.Theexpression at timeintervals overa 24-hour detected by microarray analysis periodafterserum-starved wereprovided with serum.The fibroblasts that algorithm diagram shownhereisbasedon a computer cluster groupsgenesshowingsimilar with a compared changes in expression overtime.Eachcolumnof colored serum-starved controlsample a time boxesrepresents a singlegene,andeachrow represents point.A redboxindicates relative to the in expression an increase in expression; anda blackbox,no control;a greenbox,a decrease

relatively simple system for expressing G-CSF shown in Figure 5-31. In this case,G-CSF is expressedin E. coli transformed with plasmid vectors that contain the lac promoter adjacentto the cloned cDNA encoding G-CSF.Transcription from the lac promoter occurs at high rates only when lactose, or a lactose analog such as isopropylthiogalactoside (IPTG), is added to the culture medium. Even larger quantities of a desired protein can be produced in more complicated E. coli expressionsystems. To aid in purification of a eukaryotic protein produced in an E. coli expression system, researchersoften modify the cDNA encodingthe recombinant protein to facilitate its separation from endogenousE. coli proteins. A commonly used modification of this type is to add a short nucleotide sequenceto the end of the cDNA, so that the expressedprotein will have six histidine residuesat the C-terminus. Proteins modified in this way bind tightly to an affinity matrix > EXPERIMENTAL FIGURE 5-31 Someeukaryoticproteinscan be producedin E coli cellsfrom plasmidvectorscontainingthe vectorcontainsa fragment lac promoter.(a)Theplasmidexpression the /acpromoterandthe containing of the E colichromosome analogIPTG, of the lactose lacZgene.Inthe presence neighboring /acZ normallytranscribes the iacZgene,producing RNApolymerase protein, intotheencoded mRNA,whichistranslated B-galactosidase (b)The/acZgenecanbe cut out of the expression vectorwith andreplaced by a clonedcDNA,in thiscaseone restriction enzymes granulocyte Whenthe factor(G-CSF). colony-stimulating encoding plasmid intoE.collcells,additionof IPTG istransformed resulting produce G-CSF fromthe/acpromoter transcription andsubsequent protein intoG-CSF mRNA.whichistranslated

at thetop The"tree"diagram icantchangein expression. signif genescanbe patterns for individual showshowthe expression to grouptogetherthe geneswith fashion in a hierarchical organized overtime'Five of expression in theirpatterns similarity the greatest geneswereidentified in this regulated of coordinately clusters by the barsat the bottom.Eachcluster asindicated experiment, proteins functionin a geneswhoseencoded multiple contains (A),thecellcycle process: biosynthesis cholesterol particular cellular (C),signaling andangiogenesis (B),the immediate-early response (E).[Courtesy of Michael (D),andwoundhealing andtissueremodeling Laboratoryl National Berkeley Lawrence B Eisen,

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G E N EE X P R E S S I O N TO STUDY U S I N GC L O N E DD N A F R A G M E N T S

195

that contains chelated nickel atoms, whereas most E. coli proteins will not bind to such a matrix. The bound proteins can be releasedfrom the nickel atoms by decreasingthe pH of the surrounding medium. In most cases,this procedure yields a pure recombinant protein that is functional, since addition of short amino acid sequencesto either the Cterminusor the N-terminus of a protein usually doesnot interfere with the protein's biochemicalactivity.

{a) Transient transfection cDNA

I transtectcultured I c e l l sb y l i p i dt r e a t m e n t or electroporation J

PlasmidExpressionVectorsCan Be Designedfor U s ei n A n i m a lC e l l s \Vhile bacterial expressionsystemscan be used successfully to createlarge quantitiesof someproteins, bacteriacannot be used in all cases.Many experimenrsto examine the function of a protein in an appropriate cellular context requlre expression of a geneticallymodified protein in cultured animal cells. Genesare cloned into specializedeukaryotic expressionvec, tors and are introduced into cultured animal cells by a processcalled transfection.Two common methods for transfecting animal cells differ in whether the recombinant vecor DNA is or is not integratedinto the host-cellgenomic DNA. In both methods,culturedanimal cellsmust be treatedto facilitatetheir initial uptake of the recombinantplasmidvector. This can be done by exposingcells to a preparationof lipids that penetrarethe plasma membrane, increasingits permeability to DNA. Alternatively, subjecting cells to a brief electric shock of severalthousand volts, a technique known as electroporation, makes them transiently permeableto DNA. Usuallythe plasmidDNA is addedin sufficient concentrationto ensurethat a large proportion of the cultured cellswill receiveat leastone copy of the plasmidDNA. Researchers have also harnessedvirusesfor their use in the laboratory; virusescan be modified ro contain DNA of interest, which is then introduced into host cells by simply infecting them with the recombinant virus. Transient Transfection The simplestof the two expression methods, calledtransient transfectioz, employs a vector similar to the yeast shuttle vectors describedpreviously. For use in mammalian cells,plasmid vectorsare engineeredalso to carry an origin of replicationderivedfrom a virus that infecrsmammalian cells, a strong promoter recognized by mammalian RNA polymerase,and the cloned cDNA encodingthe protein to be expressedadjacentto the promoter (Figure5-32a). Once such a plasmid vector entersa mammalian cell, the viral origin of replication allows it to replicate efficientl5 generating numerousplasmidsfrom which the protein is expressed.However,duringcell divisionsuchplasmidsare nor faithfullysegregatedinto both daughtercellsand in time a substantialfraction of the cells in a culture will not contain a plasmid, hencethe name t ransi ent t ransfecti on. Stable Transfection (Transformation) If an introduced vector integratesinto the genomeof the host cell, the genomeis permanentlyalteredand the cell is said to be transformed.lntegration most likely is accomplishedby mammalian enzymes that normally function in DNA repair and recombination. 196

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Proteinis expressedfrom cDNA integrated i n t o h o s tc h r o m o s o m e A EXPERIMENTAL FIGURE5-32 Transientand stable transfection with specially designed plasmid vectors permit expression of cloned genes in cultured animal cells.Bothmethodsemployplasmid vectorsthat containthe usualelements-ORl,selectablemarker(e.g , amp'), and polylinker-that permitpropagationin E. coli and inserlionof a clonedcDNAwith an adjacentanimalpromoter.Forsimplicity, these elementsarenot depicted(a)In transient transfection, the plasmid vectorcontainsan originof replication for a virusthat can replicatern the culturedanimalcells Sincethe vectoris not incorporated into the genome of the culturedcells,production of the cDNA-encoded proteincontinues onlyfor a limitedtime (b) In stabletransfection, the vectorcarries a selectable markersuchas neo',whichconfersresistance to G-418,The relatively few transfectedanimalcellsthat integratethe exogenousDNA into theirgenomesareselected on mediumcontainingG-418 Because the vectoris integratedinto the genome,thesestablytransfected,or transformed, cellswill continueto producethe cDNA-encoded protein as longasthe cultureis maintainedSeethe textfor discussion

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AUTOSOMAL RECESSIVE Sickle-cellanemia

Abnormal hemoelobin causesdeformation of red blood cells, which can become lodged in capillaries; also confers resistanceto malaria'

11625of sub-SaharanAfrican origin

Cysric fibrosis

Defective chloride channel (CFTR) in epithelial cells leads to excessivemucus in lungs.

1'12500of European origin

Phenylketonuria (PKU)

Defective enzyme in phenylalanine metabolism (tyrosine hydroxylase) results in excessphenylalanine, leading to mental retardation, unless restricted by diet.

1/10,000 of European origin

Tay-Sachsdisease

Defective hexosaminidaseenzyme leads to accumulation of excesssphingolipids in the lysosomesof neurons, impairing neural development.

1/1000 easternEuropean Jews

Huntington's disease

Defective neural protein (huntingtin) may assembleinto aggregatescausing damage to neural ttssue.

1/10,000of Europeanorigin

Hypercholesterolemia

Defective LDL receptor leads to excessivecholesterol in blood and early heart attacks.

tlI22French Canadians

Duchenne muscular dystrophy (DMD)

Defective cytoskeletal protein dystrophin leads to impaired muscle function.

1/3500 males

Hemophilia A

Defective blood clotting factor VIII leads to uncontrolled bleedine.

1-2110,000 males

AUTOSOMAL DOMINANT

X-LINKED RECESSIVE

provide clues to the molecular and cellular causeof the disease.Historically, researchershave usedwhatever phenotypic clues might be relevant to make guessesabout the molecular basis of inherited diseases.An early example of successful guessworkwas the hypothesisthat sickle-cellanemia,known to be a diseaseof blood cells,might be causedby defectivehemoglobin. This idea led to identification of a specificamino acid substitution in hemoglobin that causespolymerization of the defectivehemoglobin molecules,causingthe sickle-like deformation of red blood cellsin individuals who have inherited two copiesof the Hb' allele for sickle-cellhemoglobin. Most often, however,the genesresponsiblefor inherited diseasesmust be found without any prior knowledge or reasonablehypothesesabout the nature of the affectedgeneor its encodedprotein. In this section,we will seehow human geneticistscan find the generesponsiblefor an inherited disease by following the segregationof the diseasein families' The segregationof the diseasecan be correlatedwith the segregation of many other genetic markers, eventually leading to identification of the chromosomalposition of the affected

gene.This information, along with knowledgeof the sequence of th. hn-".t genome,can ultimately allow the affectedgene mutations to be pinpointed. and the disease-causing

s howOneof Three M a n y I n h e r i t e dD i s e a s e S Major Patternsof Inheritance Human genetic diseasesthat result from mutation in one specific gene exhibit severalinheritance patterns depending on the nature and chromosomal location of the alleles that causethem. One characteristicpattern is that exhibited by a dominant allele in an autosome (that is, one of the 22 human chromosomes that is not a sex chromosome). Becausean autosomal dominant allele is expressed in the heterozygote,usually at least one of the parents of an affected individual will also have the disease'It is often the case that the diseasescaused by dominant alleles appear later in life after the reproductive age. If this were n o i t h e c a s e , n a t u r a l s e l e c t i o nw o u l d h a v e e l i m i n a t e d the allele during human evolution. An example of an EENES H U M A N D I S E A SG I D E N T I F Y I NAGN D L O C A T I N G

.

199

(al Autosomal dominant: Huntington's disease

d

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FIGURE 5-35 Threecommoninheritancepatternsfor human geneticdiseases. Wild-type (A)andsexchromosomes autosomal (X andY)areindicated by superscript plussigns(a)In an autosomal d o m i n a ndti s o r d es ru c ha sH u n t i n g t osnd i s e a soen, l yo n em u t a n t alleleisneeded to conferthe diseaself eitherparentis heterozygous for the mutantHDallele,hisor herchildren havea 50 percent c h a n coef i n h e r i t i nt h g em u t a nat l l e l e a n dg e t t i n g t h ed i s e a s(eb )l n an autosomal recessive disorder suchascystic fibrosis, two mutant alleles mustbe present to conferthe diseaseBothparents mustbe heterozygous carriers of the mutantCFIRgenefor theirchildren to be at riskof beingaffected or beingcarriers(c)An X-linked recessive disease suchasDuchenne muscular dystrophy iscauseo oy a recessive mutation on theX chromosome andexhibits thetypicalsex_ linkedsegregatron patternMalesbornto mothers heterozygous for a mutantDMDallelehavea 50 percent chance of inheriting the mutantalleleandbeingaffectedFemales bornto heterozvqous mothers havea 50 percent chance of beinqcarriers

a u t o s o m a l d o m i n a n t d i s e a s ei s H u n t i n g t o n ' s d i s e a s e ,a neural degenerativediseasethat generally strikes in midto late life. If either parenr carries a murant HD allele, e a c h o f h i s o r h e r c h i l d r e n ( r e g a r d l e s so f s e x ) h a s a 5 0 percent chance of inheriting rhe mutanr allele and being a f f e c t e d( F i g u r e5 - 3 5 a ) . A recessiveallelein an autosomeexhibits a quite different segregatronpattern. For an autosomal recessiueallele, both parentsmust be hererozygouscarriersof the allelein order for their children to be at risk of being affectedwith the disease. Each child of heterozygousparenrshas a 25 percentchanceof receiving both recessiveallelesand thus being affected,a 50 percentchanceof receivingone normal and one mutant allele and thus being a carrier, and a 25 percentchanceof receiving two normal alleles.A clear exampleof an autosomalrecessive diseaseis cystic fibrosis, which results from a defectivechloride-channelgeneknown as CFTR (Figure5-35b). Relatedindividuals (e.g.,first or secondcousins)have a relatively high 200

.

c H A p r EsR I

probability of being carriers for the same recessivealleles. Thus children born to related parents are much more likely than those born to unrelated parents to be homozygousfor, and thereforeaffectedby, an autosomalrecessivedisorder. The third common pattern of inheritanceis that of an XLinked recessiueallele. A recessiveallele on the X chromosome will most often be expressedin males,who receiveonly one X chromosome from their mother, but not in females, who receivean X chromosome from both their mother and their father. This leadsto a distinctive sex-linked segregation pattern where the diseaseis exhibited much more frequently in males than in females.For example, Duchenne muscular dystrophy(DMD), a muscledegenerative diseasethat specifically affects males, is causedby a recessiveallele on the X chromosome. DMD exhibits the typical sex-linked segregation pattern in which mothers who are heterozygous and therefore phenotypically normal can act as carriers, rransmitting the DMD allele, and therefore the disease,to 50 percent of their male progeny (Figure5-35c).

M o L E c u L AGRE N E Tr tEcc H N l o u E s

D N A P o l y m o r p h i s mAs r e U s e di n L i n k a g e M a p p i n gH u m a nM u t a t i o n s Once the mode of inheritancehas beendetermined,the next step in determining the position of a diseaseallele is to genetically map its position with respect to known genetic markers using the basic principle of genetic linkage as described in Section5.1. The presenceof many different already mapped genetic rraits, or markers, distributed along the length of a chromosome facilitatesthe mapping of a new mutation by assessingits possiblelinkage to these marker genesln appropriate crosses.The more markers that are available,the more preciselya mutarion can be mapped.The density of genetic markers neededfor a high-resolution human genetic map is about one marker every 5 centimorgans (cM) (as discussedpreviously,one geneticmap unir, or centimorgan, is defined as the distance between two positions along a chromosome that results in one recombinant individual in 100 progeny).Thus a high-resolutiongenericmap requires25 or so geneticmarkersof known position spread along the length of each human chromosome. In the experimentalorganismscommonly used in genetic studies,numerous markers with easily detectablephenotypes are readily availablefor geneticmapping of mutations.This is not the casefor mapping geneswhose mutant allelesare associated with inherited diseasesin humans. However" recombinant DNA technologyhas made availablea wealth of useful DNA-based molecular markers. Becausemost of the human genomedoesnot code for protein, alarge amount of sequence variation exists betweenindividuals. Indeed, it has been estimated that nucleotidedifferencesbetweenunrelatedindividuals can be detectedon an averageof every 103 nucleotides.If thesevariations in DNA sequence,referredto as DNA polymorphisms,can be followed from one generationto the next, they can serve as genetic markers for linkage studies. Currently, a panel of as many as 104 different known polymorphisms whose locations have been mapped in the human genomeis usedfor geneticlinkage studiesin humans.

Restriction fragment length polymorphisms (RFLPs) were the first type of molecular markers used in linkage studies.RFLPsarisebecausemutationscan createor destroy the sitesrecognizedby specificrestriction enzymesthat happen to lie in human DNA, leadingto variationsbetweenindividuals in the length of restriction fragments produced from identical regionsof the genome.Differencesin the sizes of restriction fragments betweenindividuals can be detected by Southern blotting with a probe specific for a region of DNA known to contain an RFLP (Figure5-36a).The segregation and meiotic recombination of such DNA polymorphisms can be followed like typical genetic markers. Figure 5-36b illustrates how RFLP analysis of a family can detect the segregaticlnof an RFLP that can be used to test for statistically significant linkage to the allele for an inherited diseaseor some other human trait of interest. The amassedgenomic sequenceinformation from different humans has led to identification of other useful DNA polymorphisms in recent years. Single-nwcleotidepolymorphisms (SNPs) constitute the most abundant type and are therefore useful for constructing geneticmaps of maximum resolution. Another useful type of DNA polymorphism consistsof a variable number of repetitionsof a one- two-, or Suchpolymorphisms,known as simple three-basesequence. sequencerepeats (SSRs)or microsatellites, presumably are formed by recombinationor a slippagemechanismof either the template or newly synthesizedstrands during DNA Hybridization banding pattern from individual with both allele 1 and allele2

lat

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replication. A useful property of SSRsis that different individuals will often have different numbers of repeats.The existenceof multiple versionsof an SSRmakes it more likely to produce an informative segregationpattern in a given pedigreeand therefore be of more generalusein mapping the positionsof diseasegenes.If an SNP or SSRaltersa restriction site, it can be detectedby RFLP analysis. More commonly' however, these polymorphisms do not alter restriction fragments and must be detectedby PCR amplification and DNA sequenclng.

Geneswith a LinkageStudiesCanMap Disease Resolutionof About 1 Centimorgan lVithout going into all the technical considerations,let's see how the allele conferring a particular dominant trait (e.9., familial hypercholesterolemia)might be mapped. The first step is to obtain DNA samplesfrom all the members of a family containing individuals that exhibit the disease.The DNA from each affected and unaffected individual then is analyzed to determine the identity of a large number of known DNA polymorphisms (either SSR or SNP markers can be used). The segregationpattern of each DNA polymorphism within the family is then compared with the segrigation of the diseaseunder study to find those polymorphisms that tend to segregatealong with the disease' Finaily, computer analysisof the segregationdata is used to (b) Grandparents

G randparents

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FIGURE5-36 Restrictionfragment length A EXPERIMENTAL can be followed like genetic markers. polymorphisms (RFLPs) (a) In the two homologouschromosomes shown,DNA is treatedwith enzymes(A and B),which cut DNA at two differentrestriction (a and b) The resultingf ragmentsare subjected differentsequences probe (seeFigure5-26)with a radioactive to Southernblot analysis that bindsto the indicatedDNA region(green)to detectthe betweenthe two homologous fragments Sinceno differences by the B enzyme, recoqnized occurin the sequences chromosomes by the probe,as indicatedby a only one fragmentis recognized band However,treatmentwith enzymeA singlehybridization producesradiographically distinctf ragmentsof two differentlengths

tr

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(a1-a2anda1-a3),and two bandsare seen,indicatingthat a mutationhascausedthe lossof one of the a sitesin one of the two of the DNA from a analysis chromosomes(b) Pedigreebasedon RFLP regionknown to be presenton chromosome5. The DNA samples enzymeIaql and analyzedby Southern were cut with the restriction of the genomeexistsin threeallelic region this family, blotting In this by Iaql sitesspaced10, 7 '7, or 6 5 kb apart forms characterized Eachindividualhastwo alleles;somecontainallele2 (7 7 kb) on both at this site Circles and othersare heterozygous chromosomes, gel lanesare in the The males indicate indicatefemales;squares and are generally pedigree above in the the subjects as order same , e l5l 1 : 3 1 ]9 l l tearl , 1 9 8 7C r D o n i s - K ee a l i g n e db e l o wt h e m l A f t e H . I D E N T I F Y IA NN GD L O C A T I NHGU M A ND I S E A SGEE N E S

201

calculatethe likelihood of linkage between each DNA polymorphism and the disease-causing allele. In practice, segregation data are collected from different families exhibiting the same diseaseand pooled. The more families exhibiting a parricular diseasethaican be examined, the greater the statistical significance of evidence for linkage that can be obtained and the grearerthe precisionwith which the distancecan be measuredbetween a linked DNA oolvmorphism and a diseaseallele.Most family studieshave a maximum of about 100 individualsin which linkage betweena diseasegene and a panel of DNA polymorphismscan be tested. This number of individuals setsthe practical upper limit on the resolutionof sucha mapping study to about 1 cenrimorgan,or a physicaldistanceof about 7.5 x 10s basepairs. A phenomenoncalledlinkagedisequilibriumisthe basisfor an alternative strategy,which in some casescan afford a higher degreeof resolution in mapping studies.This approach dependson the particular circumstancein which a genericdisease commonly found in a particular population resultsfrom a single mutation that occurredmany generationsin the past. The DNA polymorphismscarriedby this ancestralchromosomeare collectively known as the haplotype of that chromosome. As the diseasealleleis passedfrom one generationto the next, only the polymorphisms that are closestto the diseasegenewill not be separatedfrom it by recombination. After many generarlons the region that contains the diseasegenewill be evident because this will be the only region of the chromosome thar will carry the haplotype of the ancestralchromosome conservedthrough many generations(Figure5-37).By assessing the distributionof specificmarkersin all the affectedindividualsin a population, geneticistscan identify DNA markers tightly associatedwith

appearedon the ancestralchromosome-in somecasesthis can amount to finding markersthat are so closelylinked to the diseasegenethat even after hundredsof meiosesthey have never beenseparatedby recombinarion.

FurtherAnalysisls Neededto Locatea Disease G e n ei n C l o n e dD N A Although linkage mapping can usually locare a human diseasegene to a region containing about 105 base pairs, as many as 10 different genesmay be located in a region of this size. The ultimate objective of a mapping study is to locate the genewithin a cloned segmenrof DNA and then to determine the nucleotide sequenceof this fragment. The relative scalesof a chromosomalgenetic-ap anJphysicalmaps corresponding to ordered sers of plasmid clones and the nucleotidesequenceare shown in Figure5-38. One strategyfor further localizinga diseasegenewithin the genome is to identify mRNA encoded by DNA in the region of the geneunder study. Comparison of geneexpressionin tissues 2O2

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A FIGURE 5-37 Linkagedisequilibrium studiesof human populationscan be usedto map genesat high resolution.A newdisease mutation willarisein the contextof an ancestral chromosome amonga setof polymorphisms known asthehaplotype (indicated by pinkshading). Aftermanygenerations, chromosomes thatcarrythe disease mutation willalsocarrysegments of the ancestral haplotype thathavenot beenseparated fromthe disease mutationby recombination Thebluesegments of these chromosomes general represent haplotypes derived fromthegeneral population andnotfromtheancestral haplotype in whichthe mutatron originally aroseThisphenomenon is knownaslinkage disequilibrium Thepositionof the disease mutationcanbe located by scanning chromosomes containing thedisease mutation for highly polymorphisms conserved corresponding to theancestral haplotype from normal and affected individuals may suggesttissuesin which a particular diseasegenenormally is expressed.For instance,a mutation that phenotypically affects muscle, but no other tissue,might be in a genethat is expressedonly in muscle tissue. The expressionof mRNA in both normal and affected individuals generally is determined by Northern blotting or in situ hybridization of labeledDNA or RNA to tissuesections. Northern blots, in situ hybridization, or microarray experiments permit comparison of both the level of expression and the size of mRNAs in mutant and wild-type tissues.Although the sensitivity of in situ hybridization is lower than that of Northern blot analysis,it can be very helpful in identifying an mRNA that is expressedat low levels in a given tissue but at very high levels in a subclassof cells within that tissue.An mRNA that is altered or missing in various individuals affected with a diseasecomparedwith wild-type individualswould be an excellent candidate for encoding the protein whose disrupted function causesthat disease. In many cases,point mutations that give rise to diseasecausing allelesmay result in no detectablechange in the level of expression or electrophoretic mobility of mRNAs. Thus if comparison of the mRNAs expressedin normal and affected individuals reveals no detectabledifferencesin the candidate mRNAs, a search for point mutarions in the

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M E T H O DO F Linkageto restriction DETECTION:Chromosome b a n d i n gp a t t e r n f r a g m e n tl e n g t hp o l y morPhismsRFLPS, in Fluorescence s i t u h y b r i d i z a t i o n s i n g l en u c l e o t i d ep o l y (FISH) m o r P h i s m sS N P s ,a n d s l m p l es e q u e n c e repeatsSSRs

5-38 The relationshipbetweenthe geneticand A FIGURE a deptcts Thediagram physicalmapsof a humanchromosome' of detailThe levels at different analyzed humanchromosome when asa wholecanbeviewedin the lightmicroscope chromosome andthe at metaphase, statethatoccurs it isin a condensed by canbe determined ic sequences locationof specif approximate (FISH) At the nextlevelof detail, in sltuhybridization fluorescence

DNA regions encoding the mRNAs is undertaken. Now that highly efficientmethodsfor sequencingDNA are availfrequently determinethe sequenceof canable, researchers DNA isolatedfrom affectedindividualsto of regions didate identify point mutations. The overall strategyis to search for a coding sequencethat consistentlyshowspossiblydelereriousalterationsin DNA from individualsthat exhibit the disease.A limitation of this approachis that the region near the affectedgene may carry naturally occurring polymorphisms unrelated to the gene of interest. Such polymorphisms,not functionally related to the disease,can lead to misidentificationof the DNA fragmentcarrying the geneof interest.For this reason,the more mutant allelesavailable for analysis,the more likely that a gene will be correctly identified.

Sequence map

Hybridization Sanger (dideoxY) to plasmid sequenclng clones

2d to Genomes' FromGenes et al, 2003,Genetics: fromL Hartwell lAdapted Hill ed, McGraw I

s e s u l tf r o m M u l t i p l e M a n y I n h e r i t e dD i s e a s e R GeneticDefects Most of the inherited human diseasesthat are now understood at the molecular level are monogenetic traits; that is, a clearly discerniblediseasestate is produced by a defect in a single gene.Monogenic diseasescausedby mutation in one specific gene exhibit one of the characteristic inheritance patterns shown in Figure 5-35' The genes associatedwith most of the common monogenic diseases have already been mapped using DNA-based markers as describedpreviouslY. However, many other inherited diseasesshow more complicatedpatterns of inheritance,making the identification of the underlying genetic causemuch more difficult' EENES H U M A N D I S E A SG I D E N T I F Y I NAGN D L O C A T I N G

O

203

One type of added complexity that is frequently encountered is genetic heterogeneity.In such cases,mutations in any one of multiple different genes can cause the same disease.For example, retinitis pigmentosa,which is characterizedby degenerationof the retina usually leading to blindness,can be causedby mutations in any one of more than 60 different genes. In human linkage studies, data from multiple families usually must be combined to deter_ mine whether a statisricallysignificant linkage exists be_ t w e e n a d i s e a s eg e n e a n d k n o w n m o l e c u l a r m a r k e r s . Genetic heterogeneitysuch as rhar exhibited by retinitis pigmentosa can confound such an approach becauseany statisticaltrend in the mapping data from one family tends to be canceledout by the data obtained from another fam_ i l y w i t h a n u n r e l a t e dc a u s a t i v eg e n e . Human geneticisrsused two different approachesto _ identify the many genesassociatedwith retinitii pigmenrosa. The first approach relied on mapping studies in &ception_ ally large single families rhar contained a sufficient number of affectedindividuals to provide statisticallysignificant evidencefor linkage betweenknown DNA polymolphisms and a single causativegene. The genesidentified in such studies

retinitis pigmentosa.This approach of using additional in_ formation to focus screeningefforts on a s.rbiet of candidate genesled to identificationof additional rare causativemura_

ldentifying and Locating Human DiseaseGenes r Inherited diseasesand other traits in humans show t h r e e m a j o r p a t t e r n s o f i n h e r i t a n c e :a u t o s o m a l d o m i nant, autosomal recessive,and X-linked recessive(see F i g u r e5 - 3 5 ) . r Genesfor human diseasesand other trairs can be mapped by determining their cosegregation during meiosis with markers whose locations in the genome are known. The closer a geneis to a particular marker, the more likelv thev are to cosegregate, of human geneswith great precision requires of molecular markers distributed along the es. The most useful markers are differencesin the DNA sequence(polymorphisms) between individuals in noncoding regions of the genome. r DNA polymorphisms useful in mapping human genesinclude restriction fragment length polymorphisms (RFLps), single-nucleotidepolymorphisms (SNps), and simple sequencerepeats(SSRs). inkage mapping often can locate a human diseasegene a chromosomal region that includes as many as l0 genes.To identify the geneof interestwithin this candidate region_typicallyrequires expressionanalysis and compari_ son of DNA sequencesbetween wild-type and disiaseaffectedindividuals. r Some inherited diseasescan result from mutations in dif_ ferent genesin different individuals (geneticheterogeneity). The occurrence and severity of other diseasesdepend on the presenceof mutant allelesof multiple genesin the same individuals (polygenictraits). Mapping of the genesassociated with such diseasesis particularly difficult becausethe occurrenceof the diseasecannot readily be correlated to a singlechromosomallocus.

ff,l Inactivatingthe Functionof SpecificGenesin Eukaryotes The elucidation of DNA and protein sequencesrn recent y e a r s h a s l e d t o i d e n t i f i c a t i o no f m a n y g e n e s .u s i n g s e quencepatternsin genomicDNA and the sequencesimilar_ ity of the encodedproteins with proteins oi known function. As discussedin Chapter 6, the general functions of proteins identified by sequencesearchesmay be predicted by analogy with known proteins. However, the precisein vivo roles of such "new" proteins may be lrnclearin the ab_ senceof mutant forms of the correspondinggenes.In this

CHAPTER 5

J

MOLECULAG RENETIC TECHNIQUES

Three basic approachesunderlie these gene-inactivation techniques:(1) replacinga normal genewith other sequences, (2) introducing an allelewhose encodedprotein inhibits functioning of the expressednormal protein, and (3) promoting destructionof the mRNA expressedfrom a gene.The normal endogenousgeneis modified in techniquesbasedon the first approach but is not modified in the other approaches'

(a)

20-nt flanking sequence

.-f-hi"_l

s y n r h sr i s DNJA

-

NormalYeastGenesCan Be Replacedwith M u t a n t A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n Modifying the genomeof the yeastS. cereuisiaeis particularly easy for two reasons:yeast cells readily take up exogenous DNA under certain conditions, and the introduced DNA is efficiently exchangedfor the homologous chromosomal site in the recipient cell. This specific,targeted recombination of identical stretchesof DNA allows any genein yeastchromosomesto be replacedwith a mutant allele. (As we discussin Section5.1, recombination between homologous chromosomesalso occurs naturally during meiosis.) In one popular method for disrupting yeast genesin this fashion, the PCR is used to generatea disruption construct containing a selectablemarker that subsequentlyis transfectedinto yeastcells.As shown in Figure 5-39a,primersfor PCR amplification of the selectablemarker are designedto flankinclude about 20 nucleotidesidenticalwith sequences amplified resulting The replaced. ing the yeast gene to be constructcomprisesthe selectablemarker (e.g.,the kdnMX gene,which llke neo' confers resistanceto G-41 8 ) flanked by about 20 basepairs that match the ends of the target yeast gene. Transformeddiploid yeast cells in which one of the two copiesof the target endogenousgenehas beenreplaced by the disruption construct are identified by their resistance phenotype.Theseheterozygous to G-418 or other selectable diploid yeastcellsgenerallygrow normally regardlessof the function of the target gene,but half the haploid sporesderived from thesecellswill carry only the disruptedallele(Figure 5-39b). If a geneis essentialfor viability,then sporescarrying a disruptedallelewill not survive. Disruption of yeastgenesby this method is proving parthe role of proteinsidentifiedby ticularly usefulin assessing analysisof the entire genomic DNA sequence(seeChapter 6). A large consortiumof scientistshas replacedeach of the approximately 6000 genesidentified by this analysiswith the kanMX disruption construct and determinedwhich gene disruptionslead to nonviablehaploid spores.Theseanalyses have shown that about 4500 of the 6000 yeastgenesare not

gene may be viable becauseof operation of backup or compensatorypathways.To investigatethis possibility,yeast geneticistscurrently are searchingfor syntheticlethal mutations that might reveal nonessentialgeneswith redundant functions(seeFigure 5-9c).

*Fri-"|.

-

Z

Disruption construct

(b)

Four haploid spores

FLI G U R E5 - 3 9 H o m o l o g o u s r e c o m b i n a t i o n a EXPERIMENTA constructs can inactivate specific disruption with transfected ( a ) y e a s t . A s u i t a b l ec o n s t r u cfto r d i s r u p t i n ga g e n e s i n target . h et w o p r i m e r s t a r g e tg e n ec a n b e p r e p a r e db y t h e P C R T d e s i g n e df o r t h i s p u r p o s ee a c hc o n t a i na s e q u e n c eo f a b o u t 2 0 yeast n u c l e o t i d e(sn t )t h a t i s h o m o l o g o u st o o n e e n d o f t h e t a r g e t o f D NA a s e g m e n t a m p l i f y t o n e e d e d s e q u e n c e s a s g e n ea s w e l l carryinga selectablemarkergene such as kanMX, which confers to G-418. (b) When recipientdiploid Saccharomyces resistance c e l l sa r et r a n s f o r m e dw i t h t h e g e n e d i s r u p t i o nc o n s t r u c t , h o m o l o g o u sr e c o m b i n a t i obne t w e e nt h e e n d so f t h e c o n s t r u c t s i l l i n t e g r a t et h e a n d t h e c o r r e s p o n d i ncgh r o m o s o m asl e q u e n c ew

nonvrable

O F S P E C I F IGCE N E SI N E U K A R Y O T E S I N A C T I V A T I NTGH E F U N C T I O N

205

(a) Formation of ES cells carrying a knockout mutation neor

fuHSV

G e n eX r e p l a c e m e nct o n s t r u c t

Homologous ,/ r. recombination,? "il" ./

r'

ES-ceilDNA

;

G e n eX

O t h e rg e n e s

I I Gene-targeted linsertion

I II R a n d o m ltnserilon

v

v

M u t a t i o ni n g e n eX

N o m u t a t i o ni n g e n eX

Cellsare resistantto G - 4 1 8a n d g a n c i c l o v i r

C e l l sa r e r e s i s t a ntto G - 4 1 8b u t s e n s i t i v e to ganciclovir

(b) Positive and negative selection of recombinant ES cells O OO 6+

N o n r e c o m b i n a ncte l l s Recombinantswith gene-targetedinsertion

| - r " . , w i t hG - 4 1 8 ( p o s i t i vsee l e c t i o n ) J

o.',oo o-oo Ag

I fr"u, with ganciclovir ( n e g a t i v es e l e c t i o n ) |

*

o, -o rro

< EXPERIMENTAL FIGURE 5-40 lsolationof mouseEScellswith a gene-targeteddisruption is the first stage in productionof knockoutmice.(a)Whenexogenous DNAisintroduced rnto embryonic stem(ES) cells,randominsertion vianonhomologous recombrnation occursmuchmorefrequently thangene-targeted insertion viahomologous recombination Recombinant cellsin which onealleleof geneX (orange andwhite)isdisrupted canbe obtained by usinga recombinant vectorthatcarries geneX disrupted with neo'(green), whichconfers resistance to G-418,and,outside the regionof homology, tkHsv(yellow), thethymidine genefrom kinase herpes simplex vrrusTheviralthymidlne kinase, unlikethe endogenous mouseenzyme, canconvert the nucleotide analog ganciclovir intothe monophosphate form;thisisthenmodified to t h et r i p h o s p h af o t er m ,w h i c hi n h i b i tcse l l u l aDrN Ar e p l i c a t i o i nnE S cellsThusganciclovir iscytotoxic for recombinant EScellscarrying the tkHsv gene.Nonhomologous insertion includes the tkHsv gene, whereas homologous insertion doesnot;therefore, onlycellswith nonhomologous insertion aresensitive to ganciclovir (b) Recombinant cellsareselected by treatment with G-41g,since cellsthatfailto pickup DNAor integrate it intotheirgenomeare sensitive to thiscytotoxic compoundThesurvivrng recombinant cells aretreatedwith ganciclovir Onlycellswith a targeted disruption in geneX, andtherefore lacklng geneanditsaccompanying the fkHsv cytotoxicity, willsurvive[See S L Mansour etal, 1988,Nature336.348l

A useful promoter for this purpose is the yeast GAL1 promoter, which is active in cells grown on galactose but completely inactive in cells grown on glucose. In this approach, the coding sequenceof an essentialgene (X) ligated to the GALl promoter is inserted into a yeast shuttle u..to. (seeFigure 5-17a).The recombinant vector then is introduced into haploid yeastcellsin which geneX has beendisrupted. Haploid cells that are transformed will grow on galactosemedium, since the normal copy of gene X on the vector is expressedin the presenceof galactose.\fhen the cells are transferredto a glucose-containingmedium, geneX no longer is transcribed;as the cells divide, the amount of

c'l

o o-

E S c e l l sw i t h t a r g e t e dd i s r u p t i o ni n g e n eX

Transcriptionof GenesLigatedto a Regulated P r o m o t e rC a n B e C o n t r o l l e dE x p e r i m e n t a l l y Although disruption of an essenrialgene required for cell g r o w t h w i l l y i e l dn o n v i a h l es p o r e sr.h i i m e r h o dp r o v i d e sl i m l e infclrmation about what the encodedprotein actually does in cells.To learn more about how a specificgenecontributesto cell growth and viability investigarorsmust be able to selec_ tively inactivatethe genein a population of growing cells.One method for doing rhis empkrysa regularedpro-oi.. to selec_ tively shut off transcription of an essentialgene. 206

o

c H A p r E5R I

M o L E c u L AGRE N E Tr tEcc H N t o u E S

In an early application of this method, researchersex, plored the function of cytosolic Hsc70 genes ln yeasr. Haploid cells with a disruption in all four redundant Hsc70 geneswere nonviable, unlessthe cells carried a vector containing a copy of the Hsc70 gene that could be ex_ pressedfrom the GALI promoter on galactosemedium. On transfer to glucose,the vector-carryingcells eventually stopped growing becauseof insufficient Hsc70 acivity. C a r e f u l e x a m i n a r i o n o f t h e s e d y i n g c e l l s r e v e a l e dt h a t their secretory proteins could no longer enter the endoplasmic rericulum (ER). This study provided the first evi_ dencefor the unexpectedrole of Hsc70 protein in translocation of secretory proteins into the ER, a process e x a m i n e di n d e t a i l i n C h a p t e r 1 3 .

#

uia"o: Microinjectionof ESCellsinto a Blastocyst

for a FIGURE 5-41 EScellsheterozygous > EXPERIMENTAL disruptedgene are usedto producegene-targetedknockout for a knockout cellsheterozygous stem(ES) mice.Step1: Embryonic (X andhomozygous for a dominant mutationin a geneof interest alleleof a markergene(here,browncoatcolor,A) aretransplanted thatarehomozygous cavityof 4 5-dayembryos intothe blastocoel alleleof the marker(here,blackcoatcolor,a) Step2: for a recessive female intoa pseudopregnant thenareimplanted Theearlyembryos by indicated cellsarechimeras, ES-derived containing Thoseprogeny micethenare theirmixedblackandbrowncoats.Step3: Chimeric fromthismatinghaveE5to blackmice;brownprogeny backcrossed of DNAisolated 4-6: Analysis derived cellsin theirgermline Steps brownmice canidentify froma smallamountof tailtissue of thesemice alleleIntercrossing for the knockout heterozygous that allele, produces for the disrupted homozygous someindividuals 1989,Trends fromM R.Capecchi, mice.lAdapted is,knockout Genet5:70.1

E gffl'"? ::l[T;"0?]X'J"'""' Brown mouse (NA, X-lx+) B l a c km o u s e (ala, X+lX+l 4.5-dayblastocyst

I

EIl"[';:Xl'.ffi ['Ji:;i]:il:; * A F o s t e rm o t h e r

SpecificGenesCan Be PermanentlyInactivated i n t h e G e r m L i n eo f M i c e Many of the methods for disrupting genesin yeast can be applied to genesof higher eukaryotes.Thesealteredgenescan be introduced into the germ line via homologous recombination to produce animals with a gene knockout, or simply "knockout." Knockout mice in which a specificgeneis disrupted are a powerful experimental systemfor studying mammalian development, behavior, and physiology. They also are useful in studying the molecular basisof certain human geneticdiseases. Gene-targetedknockout mice are generatedby a two-stage procedure.In the first stage,a DNA construct containing a disrupted allele of a particular target gene is introduced into embryonic stem (ES)cells.Thesecells,which are derived from the blastocyst,can be grown in culture through many generations (seeFigure 21'-7).In a small fraction of transfectedcells, the introduced DNA undergoeshomologousrecombinationwith the target gene, although recombination at nonhomologous chromosomal sitesoccurs much more frequently' To selectfor cells in which homologous gene-targetedinsertion occurs' the recombinant DNA construct introduced into ES cells needsto include two selectablemarker genes (Figure 5-40). One of thesegenes(neo'),which confersG-418 resistance,is inserted within the target gene (X), thereby disrupting it. The other selectable gene, the thymidine kinase gene from herpes simplex virus (lAHsv),confers sensitivity to ganciclovir, a cytotoxic nucleotide analog; it is inserted into the construct outside the target-genesequence.Only ES cells that undergo homologous reIo-bitr"tiott, and therefore do not incorporate tkHsv, can survivein the presenceof both G-418 and ganciclovir.In these cellsone alleleof geneX will be disrupted. In the second stage in production of knockout mice' ES cellsheterozygousfor a knockout mutation in geneX are injected into a recipient wild-type mouse blastocyst,which subsequentlyis transferred into a surrogatepseudopregnant female mouse (Figure 5-41). The resultingprogeny will be chimeras, containing tissues derived from both the transplantedES cellsand the host cells.If the ES cellsalso are ho-

Black I S.t""t chimericmicefor to wild-typeblackmice I crosses

v

germcells: All germcells: Possible a/X+ A/X+tA/X-: a/X* =r I tt cell-deriveo ProgenY s I will be brown

v

a/a, X+/X* Progenyfrom ES cell-derived germcells !| | s.re"n brown ProgenYDNA -* to identify X'lX+ heterozygotes al EI

Mate X-lX* heterozYgotes

Screen Progeny DNA to identifY lil - I l, X tx- homozYgotes

Knockoutmouse

mozygous for a visible marker trait (e.g., coat color)' then chimeric progeny in which the ES cells survived and proliferated can te identified easily.Chimeric mice are then mated with mice homozygous for another allele of the marker trait to determine if the knockout mutation is incorporated into the germ line. Finally, mating of mice' each heterozygous for the knockout allele, will produce progeny homozygous for the knockout mutation.

C E N E SI N E U K A R Y O T E S T H E F U N C T I O NO F S P E C I F I G INACTIVATING

207

loxP mouse

Cre mouse

A l l c e l l sc a r r ye n d o g e n o u sg e n e Xwith /oxPsitesflankingexon 2

Heterozygousfor gene X knockout; all cellscarry cre gene

Cell{ype-specif ic promoter

Cellsnot expressingCre

CellsexpressingCre

-4lJ@

G e n ef u n c t i o ni s n o r m a r

IoxP-Cre mouse All cells carry one copy of loxpmodified gene X, one copy of g e n eX k n o c k o u ta, n d c r e g e n e s

EXPERIMENTAL FTGURE 5-42 The loxp-Crerecombination system can knock out genes in specificcell types. A /oxpsjte is insertedon eachsideof the essential exon2 of the targetgeneX (blue)by homologousrecombination, producinga /oxpmouse.Since the /oxPsitesare in introns,they do not disruptthe functionof X T h e C r e m o u s ec a r r i e o s n e g e n eX k n o c k o u at l l e l ea n d a n i n t r o d u c e d p'l linkedto a cell-type_specific cre gene (orange)from bacteriophage promoter(yellow)The cre gene is incorporatedinto the mouse genomeby nonhomologousrecombination and doesnot affectthe

Development of knockout mice that mimic certain human diseasescan be illustrated by cystic fibrosis. By methodsdiscussedin Section5.4, the recessrve murarion that causesthis diseaseeventuallywas shown to be located in a geneknown as CFTR, which encodesa chloride chan_

rnice are currently being used as a model systemfor stud ing this geneticdiseaseand developingeffectivetherapies

S o m a t i cC e l lR e c o m b i n a t i o C n a nI n a c t i v a t e G e n e si n S p e c i f i cT i s s u e s Investigatorsoften are interestedin examining the effects o f k n o c k o u t m u t a t i o n si n a p a r t i c u l a rt i s s u eo i t h e m o u s e , at a specificstagein development,or both. However, mice carrying a germ-line knockout may have defectsin numer_ ous tissuesor die before the developmentalstageof inter_ est. To addressthis problem, mouse geneticistshave de_ v i s e d a c l e v e r t e c h n i q u et o i n a c t i v a l er a r g e r g e n e s l n 208

.

c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

-

FE G e n ef u n c t i o n isdisrupted

functionof othergenesln the/oxp-Cre mtcethat resultfrom crossing, Creproteinisproduced onlyin thosecellsin whichthe promoter isactiveThusthesearethe onlycellsin which recombrnation between the/oxPsites catalyzed by Creoccurs, leading to deletion of exon2 Since the otheralleleisa constitutive geneX knockout, deletion between the/oxpsitesresults in complete l o s so f f u n c t i o on f g e n e X i na l lc e l l se x p r e s s iC ng r e B yu s i n g promoters, different researchers canstudytheeffects of knockinq out geneX in various typesof cells specifictypes of somatic cells or at particular times during development This techniqueemploys site-specificDNA recombination sites(calledloxP sires)and the enzymeCre that catalyzesrecombination between them. The loxP-Cre recombination system is derived from bacteriophageP1, but this sitespecificrecombination sysremalso functions when placed in mouse cells. An essentialfeature of this technique is that expression of Cre is controlled by a cell-type-specificpromoter. In loxP-Cre mice generatedby the procedure depicted in Figure 5-42,inactivation of the geneof interest (X) occurs only in cells in which the promoter controlling the cre gene is active. An early application of this techniqueprovided strong evidencethat a particular neurotransmitterreceptor is important for learning and memory. Previouspharmacological and physiologicalstudieshad indicated that normal learning requiresthe NMDA classof glutamatereceptorsin the hippocampus,a region of the brain. But mice in which the gene encoding an NMDA receptor subunit was knocked out died neonatally,precluding analysis of the receptor's role in learning. Following the protocol in Figure 5-42, researchersgeneratedmice in which the receptorsubunit gene was inactivatedin the hippocampusbut expressedin other tissues. These mice survived to adulthood and showed

llll+ Technique Mouse Animation:creatinga Transgenic z/ar\ \\"2

DNA injected into a Pronucleusof a Mouse

miceare produced FIGURE 5-43 Transgenic > EXPERIMENTAL by randomintegrationof a foreigngeneinto the mousegerm (themale intooneof thetwo pronuclei DNAinjected line.Foreign hasa good bythe parents) andfemalehaploidnucleicontributed of the intothe chromosomes integrated of beingrandomly chance intothe recipient is integrated a transgene diploidzygoteBecause it doesnot disrupt recombination, genomeby nonhomologous l l7 . 2 7 3 1 L B r i n s t e r e, t1a9l 8 1C e n d o g e n o u s g e n[ S e se e R , e2

Injectforeign DNA intooneofthe pronuclei

Pronuclei

Fertilizedmouse egg Prlor t o f u s i o no f m a l e a n d f e m a l ep r o n u c l e i

II Transferinjectedeggs I into foster mother

learning and memory defects,confirming a role for thesereceptors in the ability of mice to encode their experiences rnto memory.

I

+

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y l n h i b i t S o m eG e n e s In diploid organisms,as noted in Section5.1, the phenotypic effect of a recessiveallele is expressedonly in homozygous individuals,whereasdominant allelesare expressedin heterozygotes.Thus an individual must carry two copies of a recessiveallele but only one copy of a dominant allele to exhibit the corresponding phenotypes. Sfe have seen how strains of mice that are homozygous for a given recessive knockout mutation can be produced by crossingindividuals that are heterozygousfor the same knockout mutation (see Figure 5-41). For experimentswith cultured animal cells, however, it is usually difficult to disrupt both copies of a genein order to produce a mutant phenotype.Moreover, the difficulty in producing strainswith both copiesof a genemutated is often compounded by the presenceof related genes of similar function that must also be inactivated in order to reveal an observablephenotype. For certain genes,the difficulties in producing homozygous knockout mutants can be avoided by use of an allele carrying a dominant-negativemutation. Theseallelesare genetically dominant; that is, they produce a mutant phenotype even in cells carrying a wild-type copy of the gene. However, unlike other types of dominant alleles,dominantnegativeallelesproduce a phenotypeequivalentto that of a loss-of-function mutatton. Useful dominant-negativealleleshave been identified for a variety of genesand can be introduced into cultured cells by transfection or into the germ line of mice or other organisms. In both cases,the introduced geneis integratedinto the genome by nonhomologous recombination. Such randomly inserted genesare called transgenes;the cells or organisms carrying them are referred to as transgenic.Transgenescarrying a dominant-negative allele usually are engineeredso that the allele is controlled by a regulatedpromoter, allowing expressionof the mutant protein in different tissuesat different times. As noted above, the random integration of exogenousDNA via nonhomologousrecombinationoccursat

o About 10-30% of offspringwill contain foreign DNA in chromosomesof a l l t h e i r t i s s u e sa n d g e r m l i n e Breed mice expressing foreign DNA to proPagate DNA in germline

@

@

a much higher frequency than insertion via homologous recombination. Becauseof this phenomenon' the production of transgenicmice is an efficient and straightforward process

GTPasesfrom an inactive GDP-bound state to an active GTP-bound state dependson their interacting with a corresponding guanine nucleotide exchangefactor (GEF)' A ,rr,.riu.r,small GTPase that permanently binds to the GEF orotein will block conversion of endogenous wild-type imall GTPasesto the active GTP-bound state' thereby inhibiting them from performing their switching function ( F i g u r e5 - 4 4 ) .

C E N E SI N E U K A R Y O T E S T H E F U N C T I O NO F S P E C I F I G INACTIVATING

(a) Cellsexpressingonly wild-type allelesof a small GTPase

Inactive

j

fGo' \-/ Wildtype

(b) Cellsexpressingboth wild-type allelesand a dominant-negativeallele

D o m i n a n t - n e guve a mutant

FIGURE 5-44 Inactivationof the functionof a wild-type GTPase by the actionof a dominant-negative mutantallele. (a)Small(monomeric) (purple) GTPases areactivated by their interaction with a guaninenucleotide exchange factor(GEF), which catalyzes the exchange of GDpfor GTp(b)Introduction of a dominant-negative alleleof a smallGTpase geneintocultured cellsor transgenic animals leadsto expression of a mutantGTpase that binds to andinactivates the GEFAsa result, endogenous wild-type copies of thesamesmallGTPase aretrappedin the inactive GDp-bound stateA singledominant-negative allelethuscauses a loss-offunctionphenotype in heterozygotes similar to thatseenin homozygotes carrying two recessive loss-of-f unctionalleles

R N AI n t e r f e r e n c e C a u s e sG e n eI n a c t i v a t i o nb y Destroyingthe CorrespondingmRNA A recently discoveredphenomenon known as RNA interference (RNAi) is perhaps the mosr straightforward method to inhibir the function of specificgenes.This approach is technically simpler than the methods described above for disrupting genes.First observed in the roundworm C. elegans,RNAi refers to rhe ability of doublestranded RNA to block expression of its corresponding single-strandedmRNA but not that of mRNAs with a difl ferent sequence. As describedin Chapter 8, the phenomenon of RNAi rests on the general ability of eukaryotic cells to cleave double-strandedRNA inro short (23-nt) double-stranded segmentsknown as small inhibitory RNA (siRNA). The RNA endonuclease that catalyzesthis reaction, known as

tween one strand of the siRNA and its complementaryse_ quenceon the target nRNA; subsequently,specificnucle_ asesin the RISC complex then cleave the mRNA/siRNA hybrid. This model accounts for the specificity of RNAi, since it dependson basepairing, and for its potency in si_ lencing gene function, since the complem entary mRNA is 21O

.

cHAprER 5

|

M o L E c u L AG R E N E T tr cE c H N t e u E s

permanently destroyed by nucleolytic degradation. The normal function of both Dicer and RISC is to allow for gene regulation by small endogenous RNA molecules known as micro RNAs (miRNAs). Researchersexploit the micro RNA pathway for intentional silencing of a gene of interest by using either of two generalmethods for generatingsiRNAs of defined sequence. In the first method a double-strandedRNA corresponding to the target genesequenceis produced by in vitro transcription of both senseand antisensecopiesofthis sequence(Figure5-45a). This dsRNA is injected into the gonad of an adult worm. where it is converted to siRNA by Dicer in the developing embryos. In conjunction with the RISC complex, the siRNA molecules causethe corresponding mRNA molecules to be destroyedrapidly. The resulting worms display a phenotype similar to the one that would result from disruption of the corresponding gene itself. In some cases,entry of just a few moleculesof a particular dsRNA into a cell is sufficient to inactivate many copies of the corresponding mRNA. Figure 5-45b illustrates the ability of an injected dsRNA to interfere with production of the corresponding endogenousmRNA in C. elegansembryos.In this experiment, the mRNA levels in embryos were determined by incubating the embryos with a fluorescently labeled probe specific for the mRNA of interest. This technique,in situ hybridization, is useful in assayingexpressionof a particular mRNA in cells and tissuesections. The second method is to produce a specific doublestranded RNA in vivo. An efficient way ro do this is to expressa synthetic gene thar is designedto contain tandem segmentsof both senseand anti-sensesequencescorresDonding to the target gene (Figure 5-45c). \X/henthis gene is transcribed, a double-strandedRNA "hairpin" srructure forms, known as small hairpin RNA, or shRNA. The shRNA will then be cleaved by Dicer to form siRNA molecules. The lentiviral expressionvectors are particularly useful for introducing synrhericgenes for the expressionof shRNA constructsinto animal cells. Both RNAi methods lend themselves to systematic studiesto inactivateeach of the known genesin an organism and to observewhat goeswrong. For example, in initial studies with C. elegans,RNA interferencewith 16,700 genes(about 86 percent of the genome)yielded 1722 visibly abnormal phenotypes.The geneswhose functional inactivation causesparticular abnormal phenotypescan be grouped into sets;each member of a set presumably controls the same signals or events.The regulatory relations between the genes in the set-for example, the genes that control muscledevelopment-can then be worked out. Other organismsin which RNAi-mediated geneinactivation has been successfulinclude Drosophila, many kinds of plants, zebra fish, the frog Xenopus, and mice, and are now the subjects of large-scaleRNAi screens.For example, lentiviral vectors have been designedto inactivate by RNAi more than 10,000 different genes expressedin cultured mammalian cells. The function of the inactivated genescan be inferred from defects in growth or morphology of cell clones transfectedwith lentiviral vectors.

fi| ,oo."rt: RNAInterference (a) In vitro production of double-stranded RNA Antisensetranscript

--\(-

Injected

Noninjected

(c) In vivo production of double-stranded RNA

(RNA|)can 5-45 RNAinterference FIGURE < EXPERIMENTAL functionallyinactivategenesin C.elegansand other for RNA(dsRNA) of double-stranded organisms.(a)Invitroproduction gene, the gene of sequence The coding target RNA|of a specific DNA,is of genomic fromeithera cDNAcloneor a segment derived to a strong vectoradjacent in a plasmid placedin two orientations in vitrousingRNA of bothconstructs promoterTranscription in yields manyRNAcoptes triphosphates polymerase andribonucleoside (identical or sequence) mRNA with the orientation thesense these conditions, orientationUndersuitable antisense complementary to formdsRNAWhenthe willhybridize RNAmolecules complementary by DicerintosiRNAs(b) intocells,it iscleaved isinjected dsRNA by RNA|(seethe worm embryos in of mex3RNAexpression lnhibition was (Left) RNA in embryos mex3 of Expression mechanism) for the text for thismRNA,that with a probespecific by in situhybridization assayed product(Rtgtht) a colored(purple) thatproduces is,linkedto an enzyme mex3 double-stranded with froma worminjected Theembryoderived indicated by mRNA, as mex3 no endogenous produces little or mRNA embryois=50 Fm in length of color.Eachfour-cell-stage theabsence viaan engineered RNAoccurs (c)Invivoproduction of double-stranded geneconstruct isa intocellsThesynthetic directly plasmid introduced of the sequences and antisense sense of both tandemarrangement RNA smallhairpin double-stranded targetgeneWhenit istranscribed, (b) by Dicerto formsiRNAlPart iscleaved TheshRNA forms(shRNA) Nature 391:806 etal. 1998, l fromA Fire

Sensetranscript

r The /oxP-Cre recombination system permits production of mice in which a geneis knocked out in a specifictissue.

siRNA 2+

Inactivating the Function of SpecificGenes in Eukaryotes r Once a gene has beencloned, important clues about its normal function in vivo can be deducedfrom the observed phenotypic effectsof mutating the gene. r Genescan be disrupted in yeast by inserting a selectable marker geneinto one allele of a wild-rype genevia homologous recombination, producing a heterozygousmutant. \7hen such a heterozygoteis sporulated, disruption of an essential genewill producetwo nonviablehaploid spores(Figure5-39). r A yeastgenecan be inactivatedin a controlledmannerby using the GALI promoter to shut off transcription of a genewhen cellsare transferredto glucosemedium.

r In the production of transgeniccells or organisms,exogenous DNA is integrated into the host genome by nonhomologous recombination (seeFigure 5-43). Introduction of a dominant-negativeallele in this way can functionally inactivate a genewithout altering its sequence. r In many organisms,including the roundworm C. elegans, double-stranded RNA triggers destruction of the all the mRNA moleculeswith the samesequence(seeFigure 5-45). This phenomenon, known as RNAI (RNA interference), provides a specificand potent means of functionally inactivating geneswithout altering their structure.

As the examplesin this chapter and throughout the book illustrate, genetic analysis is the foundation of our understanding of many fundamental processesin cell biology. By examining the phenotypic consequencesof mutations that inactivate a particular gene' geneticistsare able to connect knowledge about the sequence'structure' and biochemical

r In mice, modified genescan be incorporated into the germ line at their original genomic location by homologous recombination,producingknockouts (seeFigures5-40 and 5-41).Mouse knockoutscan providemodelsfor human genetic diseases such as cysticfibrosis. P E R S P E C T I VF EO S RT H E F U T U R E

211

Although scientistscontinue to use this classicalgenetic approachto dissectfundamentalcellular processes and biochemical pathways, the availability of iomplete genomic sequenceinformation for most of the common experimental organisms has fundamentally changed the way genetic experiments are conducted. Using various computational methods, scientistshave identified the protein-coding gene sequences in most experimentalorganismsincludingE. coli, yeast, C. elegans,Drosophila, Arabidopsis, mouse, and humans.The genesequences, in turn, revealthe primary amino acid sequenceof the encodedprotein products, providing us with a nearly complete list of the proteins found in each of the major experimentalorganisms. The approach taken by most researchershas thus shifted from discoveringnew genesand proteins to discoveringthe functions of genesand proteins whose sequencesare already known. Once an interestinggenehas beenidentified,genomic sequenceinformation greatly speedssubsequentgeneticmanipulations of the gene,including its designedinactivation, to learn more about its function. Already sets of vectors for RNAi inactivation of most defined senesin the nematode C. elegansnow allow efficientgeneric,ir..n, to be performed in this multicellular organism.Thesemethodsare now being applied to largecollectionsof genesin cultured mammalian cells and in the near future either RNAi or knockout methodswill have beenusedto inactivateeverygenein the mouse. In the past, a scientistmight spend many years studying only a single gene, but nowadays scientistscommonly study whole setsof genesar once. For example,with DNA microarrays the level of expressionof all genesin an organismcan be measuredalmost as easily as the expressionof a singlegene. One of the great challengesfacing geneticistsin the twentyfirst century will be to exploit the vast amount of available data on the function and regulation of individual genesto understandhow groups of genesare organizedto form complex biochemicalpathways and regulatory nerworks.

KeyTerms alleles 166

linkage175

ctone I /')

tlntation 765 Northern blotting 192 phenotype155

|

4

- ^

complementary DNAs (cDNAs) 181 complementation183 DNA cloning 126 DNA library 179 DNA microarray 192 dominant L66 geneknockoul'207 genomics129 genotype166 heterozygous166 homozygous165 hybridization 181 in situ hybridizatton 192 212

.

c H A p r EsR

plasmids178 polymerasechain r e a c t i o n( P C R )1 8 8 p r o b e s1 8 1 recessive165 recombinantDNA 126 recombination175 restriction enzymes176 RNA interference (RNAi)210 segregation167 Southern blotting 191 MOLECULAG RENETIC TECHNIQUES

temperature-sensrtrve mutations 170

transgenes209 vector 1-76

transfection 196 transformationL78

Review the Concepts 1, Genetic mutations can provide insights into the mechanisms of complex cellular or developmentalprocesses.How might your analysisof a geneticmutation be different depending on whether a particular mutation is recessiveor dominant? 2. Give an example of how and why temperature-sensitive mutations might be usedto study the function of essentialgenes. 3. Describehow complementation analysiscan be used to reveal whether two mutations are in the sameor in different genes.Explain why complementation analysiswill not work with dominant mutations. 4, Compare the different usesof suppressorand synthetic lethal mutations in geneticanalysis. 5. Restriction enzymesand DNA ligaseplay essentialroles in DNA cloning. How is it that a bacterium that produces a restriction enzyme does not cut its own DNA? Describe some general features of restriction enzymesites.ril/hat are the three types of DNA ends that can be generatedafter cutting DNA with restriction enzymes?\{hat reaction is catalyzedby DNA ligase? 6. Bacterial plasmids often serve as cloning vectors. Describethe essentialfeaturesof a plasmid vector. Sfhat are the advantagesand applicationsof plasmidsas cloning vectors? 7. A DNA library is a collectionof clones,eachcontaining a different fragment of DNA, inserted into a cloning vector. What is the differencebetweena cDNA and a genomic DNA library? How can you use hybridization or expressionto screen a library for a specific gene? How many different oligonucleotideprimers would need to be synthesizedas probes to screena library for the gene encoding the peptide Met-Pro-Glu-Phe-Tyr? 8. In 1993, Kerry Mullis won rhe Nobel prize in chemistry for his invention of the PCR process.Describethe three steps in each cycle of a PCR reaction. $fhy was the discoveryof a thermostableDNA polymerase(e.g.,Taq polymerase)so important for the developmentof PCR? 9. Southernand Northern blotting are powerful tools in molecular biology basedon hybridization of nucleic acids. How are these techniques the same? How do they differ? Give some specific applications for each blotting technique. 10. A number of foreign proteins have been expressedin bacterial and mammalian cells. Describe the essential featuresof a recombinantplasmid that are required for expressionof a foreign gene.How can you modify the foreign protein to facilitate its purification? What is the advantage of expressinga protein in mammalian cellsversusbacteria? 11. What is a DNA microarray? How are DNA microarrays usedfor studying geneexpression?How do experimentswith microarrays differ from Northern blotting experiments?

12. In determining the identity of the protein that corresponds to a newly discoveredgene, it often helps to know the pattern of tissueexpressionfor that gene.For example, researchers have found that a genecalledSERPINA6 is expressedin the liver, kidney, and pancreasbut not in other tissues.!7hat techniques might researchersuse to find out which tissuesexpressa particular gene?

b. A wild-type yeastcDNA library, preparedin a plasmid that contains the wild-type URA3* gene' is used to transformX cells,which are then culturedas indicated.Each black spot below representsa singleclone growing on a petri '$7hat plate. are the molecular differencesbetweenthe clones growing on the two plates?How can theseresults be used to identify the geneencodingX?

13. DNA polymorphisms can be used as DNA markers. Describethe differencesbetweenRFLR SNR and SSRpolymorphisms.How can thesemarkersbe usedfor DNA mapping studies? 14. How can linkage disequilibrium mapping sometimcs provide a much higher resolutionof genelocation than classicallinkagemapping? 15. Genetic linkage studies can usually only roughly locate the chromosomalposition of a "disease"gene.How can expression analysis and DNA sequenceanalysis help locate a diseasegenewithin the region identified by linkage mapping? 16. Gene targetingby siRNA techniquesexploits a normal micro RNA pathway that is found in all metazoansbut not in simpler eukaryotessuch as yeast.\7hat are the roles of Dicer and RISC in this pathway? 77. The ability to selectivelymodify the genome in the mousehas revolutionizedmousegenetics.Outline the procedure for generatinga knockout mouse at a specificgenetic locus.How can the loxP-Cresystembe usedto conditionally knock out a gene?\Vhat is an important medicalapplication of knockout mice? 18. Two methods for functionally inactivating a gene withmuout alteringthe genesequenceare by dominant-negative tations and RNA interference(RNAi). Describehow each method can inhibit expressionof a gene.

A culture of yeast that requiresuracil for growth (ura3-) was mutagenized,and two mutant colonies,X and Y, have been isolated.Mating type a cells of mutant X are mated with mating type o cellsof mutant Y to form diploid cells. The parental (ura3 ), X, Y, and diploid cells are streaked onto agar platescontaininguracil and incubatedat 23 "C or 32'C. Cell growth was monitored by the formation of colonieson the culture platesas shown in the figure below. Denotesgrowth of cells

ura3 '/'

ura

w

Parental X

Growthat 23'C

Parental X PCR

d. A construct of the wild-type geneX is engineeredto encode a fusion protein in which the green fluorescentprotein (GFP) is presentat the N-terminus (GFP-X) or the Cterminus (X-GFP) of protein X. Both constructs'presenton a URA3* plasmid, are used to transform X cells grown in the absenceof uracil. The transformants are then monitored for growth at 32oC, shown below at the left. At the right are typical fluorescentimagesof X-GFP and GFP-X cells grown at 23 "C in which green denotesthe presenceof green fluorescentprotein. What is a reasonableexplanation for growth o f G F P - X b u t n o t X - G F P c e l l sa t 3 2 ' C ?

\X \

\

/,

G r o w t ha t 3 2 ' C o n m e d i al a c k i n gu r a c i l

c. DNA is extracted from the parental cells, from X cells, and from Y cells and digested with a restriction enzyme.The digests are analyzedby Southern blot analysis, shown at the left below, using a probe obtained from the gene encoding X. In addition, PCR primers are used to amplify the gene encoding X in both the parental and the X cells.The primersare complementaryto regionsof DNA just external to the geneencoding X. The PCR results are shown in the gel at the right. What can be deducedabout the mutation in the X genefrom thesedata?

Southern

Analyzethe Data

Diploid

G r o w t ha t 2 3 ' C o n m e d i al a c k i n gu r a c i l

Diploid

G F P - Xc e l l

/, G r o w t ha t 3 2 ' C X-GFP

What can be deducedabout mutants X and Y from

a. the data provided?

Growth at 32 "C

G F Pl o c a l i z a t i oinn c e l l s grown at 23 'C THE DATA ANALYZE

O

213

e. Haploid offspring of the diploid cells from part (a) above are generated.XY double mutants constitute 114 of theseoffspring.Haploid X cells,Y cells,and XY cellsin liquid culture are synchronizedat a stagejust prior to budding and then are shifted from 23'C to 32 oC. Examination of the cells 24 hours later revealsthat X cellsare arresredwith small buds, Y cellsare arrestedwith large buds, and XY cellsare arrested with small buds.\Whatis the relationshipbetweenX and Y?

References Genetic Analysis of Mutations to ldentify and Study Genes Adams,A. E. M., D. Botstein,and D. B. Drubin. 1989.A yeast actin-bindingprotein is encodedby sac6,a genefound by suppression of an actin muration. Science243:231. Griffiths, A. G. F., et al. 2000. An Introduction to Genetic Analysis,Tth ed. W. H. Freemanand Company. Guarente,L.1993. Synthericenhancement in geneinteraction:a genetictoof comesof age.TrendsCenet.g:362-3o6. Hartwell, L. H. 1967.Macromolecularsynthesis of temperature-sensitive mutanrs of yeast.J. Bacteriol. 93:1662. Hartwell, L.H. 1974. Geneticcontrol of the cell divisioncycle in yeast.Science183:46. Niisslein-Volhard, C., and E.'Wieschaus. 1980.Mutations affectins segmentnumber and polariryin Drosophila.Nature 287:795-801. Simon,M. A., er al. 1991. Rasl and a putativeguaninenucleotide exchangefactor perform crucialstepsin signalingby the sevenless protein tyrosinekinase.Cell 67:701-776. Tong,A. H., et al. 2001. Systematicgeneticanalysiswith ordered arraysof yeastdeletionmurants.Science294:2364)368. DNA Cloning and Characterization Ausubel,F. M., et al.2002. Current Protocolsin Molecular Biology.Vlley. Gubler,U., and B. J. Hoffman. 1983.A simpleand very efficient method for generatingcDNA libraries. Gene 25:263-289. Han, J. H., C. Stratowa,and W. I. Rutter.1987.Isolationof fulllengthputativerat lysophospholipase cDNA usingimprovedmethods for mRNA isolationand cDNA cloning.Biochem.26:.161.7-1632. Itakura,K., J.J. Rossi,and R. B. Wallace.1984.Synthesis and use of syntheticoligonucleotides.Ann. Reu.Biochem. 53:323-356. Maniatis, T., et al. 1978.The isolation of structural senesfrom libraries of eucaryoticDNA. Cel/ 15:687-701. Nasmyth,K. A., and S. I. Reed.1980.Isolationof senesby complementationin yeast:molecularcloning of a cell-Jyclegene. Proc. Nat'l Acad. Sci.USA 77:21.19-2123. Nathans,D., and H. O. Smith. 1975.Restrictionendonucleases in the analysisand restructuringof DNA molecules.Ann. Reu.Biochem. 44:273193. Roberts,R. J., and D. Macelis.1997.REBAsE-restricrion enzymesand methylases.Nacl. Acids Res.25:248-262. Information on accessinga continuouslyupdateddatabaseon restrictionand modification enzymesat http://www.neb.com/rebase.

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M O L E C U L AG R E N E T TTCE C H N T Q U E S

Using Cloned DNA Fragments to Study Gene Expression Andrews, A.T. 1986. Electrophoresis,2d ed. Oxford University Press. Erlich, H., ed. 1.992.PCR Technology:Principlesand Applicationsfor DNA Amplification. W. H. Freemanand Company. Pellicer,A., M. Wigler, R. Axel, and S. Silverstein.1978. The transfer and stableintegration of rhe HSV thymidine kinasegene into mousecells.Cel/ 4t:1,33-1{1,. Saiki,R. K., et al. 1988. Primer-directed enzymaticamplification of DNA with a thermostableDNA polymerase.Science239:487491. Sanger,F. 1981. Determinationof nucleotidesequences in DNA. Science214:720 5-'1210. Souza,L. M., et al. 1986.Recombinanthuman granulocyte-colony stimulating factor: effectson normal and leukemic myeloid cells. Science232:61-65. 'Wahl, G. M., J. L. Meinkoth, and A. R. Kimmel. 1987. Northern and Southern6lots. Metb. Enzymol. 152:572-581. Wallace,R. B., et al. 1,987.The useof syntheticoligonucleotides as hybridizationprobes.II: Hybridization of oiigonucleotides of mixed sequence to rabbit B-globinDNA. Nzcl. Acids Res.9:879-887. ldentifying and Locating Human Disease Genes Botstein,D., et al. 1980. Constructionof a geneticlinkage map in man using restriction fragmentlength polymorphisms.Az. /. Genet.32:31.4-331.. Donis-Keller,H., et al. 1,987.A geneticlinkage map of the human genome.Cell 5l:319-337. Hartwell, et al. 2000. Genetics:From Genesto Genomes. McGraw-Hill. Hastbacka,T., et al. 1994.The diastrophicdysplasiagene encodesa novel sulfatetransporter:positional cloning by finestructurelinkage disequilibriummapping. Cell 781.073. Orita, M., et al. 1989. Rapid and sensitivedetecrionof point mutations and DNA polymorphismsusing the polymerasechain reaction. Genomics5:874. Tabor,H. K., N.J. Risch,and R. M. Myers.2002. Opinion: candidate-gene approachesfor studyingcomplex generictrairs: practicalconsiderations.Nat. Reu.Genet.3:391,-397. Inactivating the Function of Specific Genes in Eukaryotes Capecchi,M. R. 1989. Altering the genomeby homologousrecombination. Science 244 :'1,288-1,292. Deshaies,R. J., et al. 1988. A subfamily of stressproteins facili, tatestranslocationof secretoryand mitochondrial precursor polypeptides.Nature 332:800-805. Fire, A., et al. 1,998.Porentand specificgeneticinterferenceby double-strandedRNA in Caenorhabditiselegans.Nature 391:806-8'1.1. Gu, H., et al. 1994. Delerion of a DNA polymerasebeta gene segmentin T cellsusing cell type-specificgenetargeting.Science 265:L03-106. Zamore, P. D., T. Tuschl,P.A. Sharp,and D. P. Bartel.2000. RNAi: double-strandedRNA directsthe ATP-dependentcleavageof mRNA at 21 to 23 nucleotideinrervals.Cell 101:25-33. Zimmer, A. 1992. Manipulating the genomeby homorogous recombinationin embryonic stem cells.Ann. Reu.Neurosci. 15:115

CHAPTER

GENES, AND GENOMICS, CHROMOSOMES ThesebrightlycoloredRxFISH-painted chromosomes areboth anomalies and in beautifuland usefulin revealing chromosome of Clinical comparingkaryotypes of differentspecies[@Department Researchers, Incl Cytogenetics, Addenbrookes Hospital/Photo

I n previous chapterswe learnedhow the structure and comI position of proteins allow them to perform a wide variety I of cellular functions. We also examined another vital component of cells, the nucleic acids, and the processby which information encoded in the sequenceof DNA is translated into protein. In this chapter,our focus again is on DNA and proteins as we consider the characteristicsof eukaryotic nuclear and organellar genomes:the features of genesand the other DNA sequencethat comprise the genome, and how this DNA is structured and organized by proteins within the cell. By the beginning of the twenty-first century, molecular biologists had completed sequencingthe entire genomesof hundredsof viruses,scoresof bacteria,and one unicellular eukaryote, the budding yeast S. cereuisiae.In addition, the vast majority of the genome sequenceis also known for the fission yeast S. pombe, and severalmulticellular eukaryotes including the roundworm C. elegans, the fruit fly D. melanogastel,mice, and humans. Detailed analysis of these sequencingdata has revealedinsights into genome organtzation and genefunction. It has allowed researchersto identify previously unknown genesand to estimatethe total number of protein-coding genesencoded in each genome. Comparisons betweengenesequencesoften provide insight into possible functions of newly identified genes. Comparisons of genomesequenceand organizationbetweenspeciesalso help us understand the evolution of organisms. Surprisingly,DNA sequencingrevealedthat a large portion of the genomesof higher eukaryotes does not encode mRNAs or any other RNAs required by the organism. Remarkably, such noncoding DNA constitutes :98.5 percent

of human chromosomal DNA! The noncoding DNA in multicellular organisms contains many regions that are similar but not identical. Variations within some stretchesof this repetitious DNA between individuals are so great that every person can be distinguishedby a DNA "fingerprint" based on these sequencevariations. Moreover, some repetitious DNA sequencesare not found in the same positions in the genomesof different individuals of the same species.At one "junk time, all noncoding DNA was collectively termed 'We now DNA" and was considered to serve no purpose. understandthe evolutionary basisof all this extra DNA, and the variation in location of certain sequencesbetween

OUTLINE 6.1

EukaryoticGene Structure

217

6.2

C h r o m o s o m aOl r g a n i z a t i o no f G e n e s a n d N o n c o d i n gD N A

223

5.3

(Mobile) DNA Elements Transposable

226

6.4

O r g a n e l l eD N A s

236

5.5

G e n o m i c sG: e n o m e - w i d eA n a l y s i so f G e n e Structureand ExPression

6.6

StructuralOrganizationof Eukaryotic Chromosomes

6.7

M o r p h o l o g ya n d F u n c t i o n aEl l e m e n t s of EukaryoticChromosomes

215

individuals. Cellular genomesharbor symbiotic transposable (mobile) DNA elements,sequencesthat can copy themselves and move throughout the genome.Although transposable DNA elementsseemro have little funcrion in the life cycleof an individual organism, over evolutionary time they have shapedour genomesand contributedto the rapid evolution of multicellularorganisms. In higher eukaryores,DNA regions encoding proteins or functional RNAs-that is, genes-lie amidst this expanseof apparently nonfunctional DNA. In addition ro the nonfunctional DNA between genes,noncoding introns are common within genesof multicellularplants and animals.Sequencing of the same protein-coding gene in a variety of eukaryotic specieshas shown that evolutionary pressureselectsfor maintenanceof relativelysimilar sequences in the coding reglons,or exons.In contrast,wide sequence variation,evenincludingtotal loss,occursamon€lintrons,suggesting that most intron sequenceshave little functional significance.However, as we shall see,although most of the DNA sequenceof introns is not functional,the existenceof introns has favored the evolution of multidomain proteins that are common ln highereukaryotes.It also allowed the rapid evolutionof proteinswith new combinationsof functionaldomains. Mitochondria and chloroplastsalso contain DNA that encodesproteins essentialto the function of thesevital organelles.!7e shall see that mitochondrial and chloroplast DNAs are evolutionaryremnantsof the origins of theseorganelles.Comparisonof DNA sequences betweendifferent classes of bacteria and mitochondrial and chloroolast genomeshas revealedthat theseorganellesevolvedfrom in-

tracellular bacteria that developed symbiotic relationships with ancient eukaryotic cells. The sheerlength of cellular DNA is a significant problem with which cells must contend. The DNA in a single human cell, which measuresabout 2 meters in total length, must be contained within cells with diametersof less than 10 pm, a compactionratio of greaterthan 105to 1.In relativeterms,if a cell were 1 centimeter in diameter, the length of DNA packed into its nucleuswould be about 2 kilometers! Specialized eukaryotic proteins associatedwith nuclear DNA exquisitely fold and organizethe DNA so thar it fits into nuclei. And yet at the same time, any given portion of this highly compacted DNA can be accessedreadily for transcription, DNA replication, and repair of DNA damage without the long DNA moleculesbecoming tangled or broken. Furthermore, the integrity of DNA must be maintained during the processof cell division when it is partitioned into daughter cells. In eukaryotes,the complex of DNA and the proteins that organize it, called chromatin, can be visualized as individual chromosomesduring mitosis (seechapter opening figure). As we will seein this and the following chapter,the organization of DNA into chromatin allows a mechanism for regulation of geneexpressionthat is not availablein bacteria. In the first five sections of this chapter, we provide an overview of the landscapeof eukaryotic genes and genomes. First we discussthe structure of eukaryotic genesand the complexities that arise in higher organismsfrom the processingof mRNA precursorsinto alternatively spliced mRNAs. Next we discussthe main classes of eukaryoticDNA includingthe special properties of transposableDNA elementsand how they shaped

> FIGURE6-1 Overview of the structure of genes and chromosomes.DNA of higher eukaryotes consistsof uniqueand repeated s e q u e n c eO s n l y: 1 5 p e r c e not f h u m a nD N A encodesproteinsand functionalRNAsand the regulatorysequences that controltheir expression; the remainderis merelyintrons within genesand intergenicDNA between g e n e s M u c ho f t h e i n t e r g e n iD c N A ,- 4 5 percentin humans,is derivedf rom transposable (mobile)DNA elements,geneticsymbiontsthat havecontributedto the evolutionof contemporary genomes Eachchromosome consrsts of a single,long moleculeof DNA up t o : 2 8 0 M b i n h u m a n so, r g a n i z e d into increasing levelsof condensation by the histone a n d n o n h i s t o n pe r o t e i n sw i t h w h i c h i t i s intricately complexedMuch smallerDNA m o l e c u l easr e l o c a l i z eidn m i t o c h o n d r iaan d chloroplasts

" B e a d so n a s t r i n g "

Nucleosome

Major Types of DNA Sequence S i n g l e - c o pg yenes S i m p l e - s e q u e n cDeN A G e n ef a m i l i e s T r a n s p o s a b lD e N Ae l e m e n t s T a n d e m l yr e p e a t e dg e n e s S p a c e rD N A Introns

216

C H A P T E6R |

GENES G,E N O M t CASN, D C H R O M O S O M E S

contemporarygenomes.We then considerorganelleDNA and how it differs from nuclear DNA. This background preparesus to discussgenomics,computer-basedmethods for analyzingand interpreting vast amounts of sequencedata. The final two sections of the chapter addresshow DNA is physically organizedin eukaryoticcells.'Sfeconsiderthe packagingof DNA and histone proteins into compact complexes(nucleosomes)that are the fundamental building blocks of chromatin, the large-scalestructure of chromosomes,and the functional elementsrequired for chromosomeduplication and segregation.Figure 6-1 provides an overview of theseinterrelatedsubjects.The understandingof genes,genomics,and chromosomesgained in this chapter will prepareus to explore current knowledge about how the synthesis and concentration of each protein and functional RNA in a cell is regulatedin the following two chapters.

lil

Eukaryotic GeneStructure

In molecular terms, a genecommonly is defined as tbe entire nucleic acid seqwencethat is necessaryfor the synthesisof a fwnctionalgeneproduct (polypeptideor RNA). According to this definition, a geneincludesmore than the nucleotidesencoding an amino acid sequenceor a functional RNA, referred to as the coding region. A genealso includesall the DNA sequencesrequired for synthesisof a particular RNA transcript, no matter where those sequences are locatedin relation to the coding region.For example,in eukaryoticgenes,transcriptioncontrol regions known as enhancerscan lie 50 kb or more from the coding region.As we learnedin Chapter4, other critical noncoding regions in eukaryotic genesinclude the promoter, as well as sequencesthat specify 3' cleavage and polyadenylation,known as poly(A) sites, and splicing of primary RNA transcripts,known as splicesiles(seeFigure4-15). Mutations in thesesequences, which control transcriptioninitiation and RNA processing,affect the normal expressionand function of RNAs, producing distinct phenotypesin mutant organisms.We examine these various control elementsof genesin greaterdetail in Chapters7 and 8. Although most genes are transcribed into mRNAs, which encode proteins, some DNA sequencesare transcribedinto RNAs that do not encodeproteins (e.g.,tRNAs and rRNAs described in Chapter 4 and micro RNAs that regulatemRNA stability and translationdiscussedin Chapter 8). Becausethe DNA that encodestRNAs, rRNAs and micro RNAs can cause specific phenotypeswhen mutated, theseDNA regions generallyare referred to as tRNA, rRNA and micro RNA genes, even though the final products of thesegenesare RNA moleculesand not proteins. In this section,we will examine the structure of genesin bacteria and eukaryotesand discusshow their respective gene structuresinfluence geneexpressionand evolution.

M o s t E u k a r y o t i cG e n e sC o n t a i nI n t r o n s a n d P r o d u c em R N A sE n c o d i n gS i n g l eP r o t e i n s As discussedin Chapter4, many bacterialmRNAs (e.g.,the mRNA encodedby the trp operon) include the coding region

for several proteins that function together in a biological process.SuchmRNAs are said to be polycistronic. (A cistron is a genetic unit encoding a single polypeptide.) In contrast' most eukaryotic mRNAs are monocistronic; that is, each mRNA molecule encodes a single protein. This difference betweenpolycistronic and monocistronic mRNAs correlates with a fundamental differencein their translatton. Within a bacterial polycistronic mRNA a ribosome-binding site is locatednear the start site for eachof the protein-coding regions,or cistrons,in the mRNA. Translation initiation can beginat any of thesemultiple internal sites,producing multiple proteins (seeFigure 4-13a).In most eukaryoticmRNAs' however,the 5'-cap structuredirectsribosome binding, and translation beginsat the closestAUG start codon (seeFigure4-13b). As a result, translation beginsonly at this site. In many cases, the primary transcriptsof eukaryoticprotein-codinggenesare processedinto a singletype of mRNA, which is translatedto give a singletype of polypeptide(seeFigure4-15). Unlike bacterialand yeastgenes,which generallylack introns, most genesin multicellular animals and plants contain introns, which are removedduring RNA processingin the nucleus beforethe fully processedmRNA is exported to the cytosol for translation. In many cases,the introns in a geneare considerablylonger than the exons. Although many introns are :90 bp long, the median intron length in human genesis 3.3 kb. Some,however,are much longer: the longestknown human intron is 17,1.06bp, and lies within titan, a gene encoding a structural protein in muscle cells. In comparison, most human exonscontain only 50-200 basepairs. The typiprotein is :50,000 cal human geneencodingan average-size bp long, but more than 95 percentof that sequenceis present in introns and flanking noncoding 5' and 3' regions. Many large proteins in higher organisms that have repeated domains are encoded by genesconsisting of repeats of similar exons separatedby introns of variable length. An example of this is fibronectin, a component of the extracellular matrix. The fibronectin gene contains multiple copies of five types of exons (seeFigure 4-16). Suchgenesevolved by tandem duplication of the DNA encoding the repeated exon, probably by unequal crossingover during meiosisas shown in Figure6-2a.

n nits S i m p l ea n d C o m p l e xT r a n s c r i p t i o U A r e F o u n di n E u k a r y o t i cG e n o m e s The cluster of genesthat form a bacterial operon comprises a single transcription unit that is transcribed from a specific promoter in the DNA sequenceto a termination site. producing a singleprimary transcript. In other words, genesand transcription units often are distinguishablein prokaryotes since a single transcription unit contains severalgeneswhen they are part of an operon. In contrast' most eukaryotic genes are expressedfrom separatetranscription units' and each mRNA is translated into a single protein. Eukaryotic transcription units, however, are classified into two types, dependingon the fate of the primary transcript. The primary transcript produced from a simple transcription unit is processedto yield a singletype of mRNA' encoding E U K A R Y O T IG C E N Es T R U C T U R E .

217

(a) Exon duplication L1

E x o n1

Parental chromosomes

===

E x o n2

Exon 3

L1

Exon 2

Exon 1

Exon 3

===

I

I R e c o m b i n a t i o(nu n e q u acl r o s s i n go v e r )

+ L1 Recombinant cnromosomes

Exon 1

Exon 2

Exon 3

Exon 3

Exon 1

Exon 2

(b) Gene duplication B - g l o b i ng e n e

Parentat I chromosomes I I * Recombinant cnromosomes

R e c o m b i n a t i o(nu n e q u acl r o s s i n g B - s l o b i ng e n e

t

A FIGURE 5-2 Exonand geneduplication.(a)Exonduplication results fromunequal crossing overduringmeiosrs Eachparental chromosome contains oneancestral genecontaining threeexons ( n u m b e r e1d- 3 )a n dt w o i n t r o n sH o m o l o g o n uo s n c o d i nLg1 r e p e a t esde q u e n c lei es5 ' a n d3 ' o f t h eg e n ea, n da l s oi n t h ei n t r o n between exons2 and3 As discussed in Section 6 3, L1sequences havebeenrepeatedly transposed to newsitesin thegenomeoverrne course of humanevolution, sothatallchromosomes arepeppered withthem Theparental chromosomes areshowndisplaced relative to eachother,sothatthe L1sequences arealignedHomologous recombination betweenL1sequences asshownwouldgenerate one recombinant chromosome in whichthegenehasfourexons(two

copies of exon3) andonechromosome in whichthe geneis missing exon3 (b)Unequal crossing overbetweenL'1sequences alsocan generate duplications of entiregenesInthisexample, eachparental chromosome gene,andoneof the contains oneancestral B-globin recombinant chromosomes contains two duplicated genes B-globin S u b s e q u ei n d t e p e n d em nu t t a t i o ni ns t h ed u p l i c a t egde n e cs o u l d l e a dt o s l i g hct h a n g ei sn s e q u e n ct hea tm i g h tr e s u litn s l i g h t l y properties different functional proteinsUnequal of theencoded crossing overalsocanresultfromrarerecombinations between unrelated sequences Notethatthe scalein part(b)is muchlarger (b)seeD H A Fitch thanin part(a) [Part eral, 1991,ProcNat'|. AcadSci. USA88:7396 I

a singleprotein.Mutations in exons,introns,and transcriptioncontrol regions all may influence expressionof the protein encodedby a simpletranscriptionunit (Figure6-3a). In the caseof complex rranscription units, which are quite common in multicellular organisms,the primary RNA transcript can be processedin more than one way, leading to formation of mRNAs containing different exons. Each alternate mRNA, however, is monocistronic, being translated into a single polypeptide, with translation usually initiating ar the first AUG in the mRNA. Multiple mRNAs can arise from a primary transcriptin threeways, as shown in Figure6-3b. Examplesof all three types of alternariveRNA processing occur in the genesthat regulate sexual differentiation in Drosophila (see Figure 8-16). Commonly, one mRNA is produced from a complex transcription unit in some cell types, and a different mRNA is made in other cell types. For example, alternative splicing of the primary fibronectin tran-

script in fibroblasts and hepatocytesdetermineswhether or not the secretedprotein includes domains that adhereto cell surfaces(seeFigure 4-16). The phenomenonof alternative splicing greatly expands the number of proteins encoded in the genomesof higher organisms. It is estimated that -60 percent of human genesare contained within complex transcription units that give rise to alternatively spliced mRNAs encoding proteins with distinct functions, as for the fibroblast and hepatocyteforms of fibronectin. The relationshipbetweena mutation and a gene is not always straightforward when it comes to complex transcription units. A mutation in the control region or in an exon shared by alternatively spliced mRNAs will affect all the alternative proteins encoded by a given complex transcription unit. On the other hand, mutations in an exon present in only one of the alternative mRNAs will affect only the protein encodedby that mRNA. As explained in

218

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cHAprER 6

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G E N E sG, E N o M t c sA,N Dc H R o M o s o M E s

Transcription Units ffi ,od."rt: Eukaryotic > FIGURE 6-3 Simpleand complexeukaryotictrans€ription Simple transcription unit units.(a)A simple transcription unitincludes a regionthatencodes , 50kb , oneprotein, extending fromthe 5' capsiteto the 3' poly(A) site, l-l CaPsite andassociated controlregionsIntrons liebetween exons(light bluerectangles) andareremoved duringprocessing of the primary (dashed transcripts redlines); thustheydo notoccurin thefunctional Gene m o n o c i s t r o nmi cR N AM u t a t i o ni sn a t r a n s c r i p t i o n - c o nr et rgoilo n (a b) mayreduce or prevent transcription, thusreducing or eliminatrng Control regions protein. synthesis of theencoded A mutation withinan exon(c)may proteinwith diminished 5' resultin an abnormal activityA mutation nRNA withinan intron(d) thatintroduces a newsplicesiteresults in an protein(b) abnormally spliced mRNAencoding a nonfunctional primary transcription unitsproduce thatcanbe (b) Complex transcription units Complex transcripts processed in alternative ways.(Iop)lf a primary transcript contains Cap site alternative splicesites,it canbe processed intomRNAs with thesame 5' and 3' exonsbut differentinternalexons(Middle)lf a primary transcript hastwo poly(A) sites,it canbe processed intomRNAs with Gene (f or g) are promoters alternative 3' exons(Botton)lf alternative Exon1 produced in a celltypein which activein different celltypes,mRNA1, f isactivated, hasa different firstexon(1A)thanmRNA2 has,which mRNAT S'f----a (andwhereexon1B isproduced in a celltypein whichg isactivated or (aandb) andthosedesignated isused)Mutations in controlregions nRNA2 5' c withinexonssharedbythealternative mRNAs affectthe proteins processed encoded by bothalternatively mRNAsIn contrast, (designated mutations d ande)withinexonsuniqueto oneof the Cap site processed from alternatively mRNAs affectonlythe proteintranslated promoters thatmRNAForgenesthataretranscribed fromdifferent in differentcelltypes(bottom),mutations in differentcontrolregions (f andg) affectexpression onlyin thecelltypein whichthatcontrol Gene Exon1 regionisactive mRNAT

S'f

Poly(A)site

Exon2

f_l-

Exon3

Exon4

-___---l3, T_

Poly(A)

E x o n3

J

or Chapter 5, genetic complementationtests commonly are nRNA2 5'[---f .T used to determine if two mutations are in the same or different genes(seeFigure5-7). However,in the complex transcription unit shown in Figure 6-3b (middle), mutations d Cap site Cap site poty(A) and e would complement each other in a genetic complementation test, even though they occur in the same gene. This is becausea chromosomewith mutation d can express Gene E x o n3 Exon2 ExonlB E x o n1 A a normal protein encoded by mRNA2 and a chromosome with mutation e can express a normal protein encoded by nRNAI 5' mRNA1. Both mRNAs produced from this gene would be presentin a diploid cell carrying both mutations,generating or both protein products and hence a wild-type phenotype. mRNA2 However, a chromosome with mutation c in an exon common to both mRNAs would not complement either mutation d or e. In other words, mutation c would be in the same complementation groups as mutations d and e, even though P r o t e i n - C o d i nG g e n e sM a y B e S o l i t a r yo r B e l o n g d and e themselveswould not be in the same complementaF a m i ly to a Gene tion group! Given these complications with the genetic defThe nucleotidesequenceswithin chromosomal DNA can be inition of a gene, the genomic definition outlined at the of classified on the basis of structure and function' as shown in beginning of this section is commonly used. In the case 'We will examinethe propertiesof eachclass'beginThble 6-1. genes, gene DNA tranprotein-coding a is the sequence genes,which comprise two groups. protein-coding with ning pre-mRNA precursor, equivalent to a transcribed into a roughly 25-50 percent of the organisms, In multicellular plus required unit, any other regulatory elements scription protein-codinggenesare representedonly once in the haploid for synthesisof the primary transcript.The various proteins encoded by the alternatively spliced mRNAs expressed genome and thus are termed solitary genes.A well-studied example of a solitary protein-coding gene is the chicken from one gene are called isoforms. G E N ES T R U C T U R E . EUKARYOTIC

219

CLASS

ITNGTH

(0/o) C0PY NUMBEB lNHUMAN GEN0ME FRACTI0N 0FHUMAN GEN()ME

Protein-coding genes

0.5-2200kb

-25,000

:55" (1.8)1

Tandemlyrepeatedgenes U2 snRNA rRNAs

5 . 1k b + 43 kb+

:20 :300

<0.001 0.4

Variable

:$

300,000 440,000

3 8

850,000 1,600,000 1- : 1 0 0

2T 13 -0.4

n.a.

-25

RepetitiousDNA Simple-sequence DNA 1-500 bp Interspersed repeats(mobileDNA elements) DNA transposons 2-3 kb LTR retrotransposons 5-11kb Non-LIR retrotransposons LINEs 5-8 kb SINEs 100-400 bp Processed pseudogenes Variable Unclassified spacerDNA$

Variable

"' Complete transcription units including introns. t Transcription units not including introns. Protein-coding regions (exons) total 1.1% ofthe genome. + Length of tandemly repeated sequence. s Sequencesbetween transcription units that are not repeated in the genome; n.a. : not applicable. SoURcE:International Human Genome SequencingConsortium, 200L, Nature 409860 and 2004, Nature 431:931.

lysozymegene. The 15-kb DNA sequenceencoding chicken lysozyme constitutes a simple transcription unit containing four exons and three introns. The flanking regions,extending for about 20 kb upstreamand downstreamfrom the transcription unit, do not encodeany detectablemRNAs. Lysozyme,an enzymethat cleavesthe polysaccharidesin bacterial cell walls, is an abundant component of chicken egg-whiteprotein and also is found in human tears.Its activity helps to keep the surface of the eye and the chicken egg sterile. Duplicated genesconstitute the secondgroup of proteincoding genes.These are geneswith close but nonidentical sequencesthat often are located within 5-50 kb of one another. A set of duplicated genesthat encodeproteins with similar but nonidentical amino acid sequencesis called a gene family; the encoded,closely related, homologous proteins constitute a protein family. A few protein families, such as protein kinases, vertebrate immunoglobulins, and olfactory receptors include hundreds of members. Most protein families, however, include from iust a few to 30 or so members; common examples are cytoskeletal proteins, the myosin heavy chain, and the o- and B-globinsin vertebrates. The genesencodingthe B-like globins are a good example of a gene family. As shown in Figure 6-4a, the B-like globin gene family contains five functional genes designated B, E, A.y, G], and e; the encoded polypeptides are similarly designated.Two identical B-like globin polypeptides combine with two identical o-globin polypeptides (encoded by another gene family) and four small heme groups to form a hemoglobin molecule (seeFigure 3-13). AII the hemoglobins formed from the different B-like glo220

.

bins carry oxygen in the blood, but they exhibit somewhat different properties that are suited to specific roles in human physiology. For example, hemoglobins containing either the A" or G, polypeptides are expressedonly during fetal life. Becausethese fetal hemoglobins have a higher affinity for oxygen than adult hemoglobins, they can effectively extract oxygen from the maternal circulation in the placenta. The lower oxygen affinrty of adult hemoglobins, which are expressed after birth, permits better release of oxygen to the tissues,especiallymuscles,which have a high d e m a n df o r o x y g e nd u r i n g e x e r c i s e . The different B-globin genesarose by duplication of an ancestralgene, most likely as the result of an "unequal crossover" during meiotic recombination in a developing germ cell (egg or sperm) (Figure 6-2b). Over evolutionary time the rwo copies of the gene that resulted accumulated random mutations; beneficialmutations that conferred some refinementin the basicoxygen-carryingfunction of hemoglobin were retained by natural selection,resulting in sequence drift. Repeatedgene duplications and subsequentsequence drift are thought to have generatedthe contemporary globinlike genesobservedin humans and other mammals today. Two regionsin the human B-like globin genecluster contain nonfunctional sequences,called pseudogenes,similar to those of the functional B-like globin genes(seeFigure 6-4a). Sequenceanalysis shows that these pseudogeneshave the sameapparent exon-intron structure as the functional B-like globin genes,suggestingthat they also arose by duplication of the same ancestral gene. However, there was little selective pressureto maintain the function of thesegenes.Consequently

c H A p r E6R I G E N EG s ,E N o M t cAsN , Dc H R o M o s o M E s

(a) Human B-globingene cluster (chromosome111 I t"on

l--l

Pseudogene I nlu site

lbl S. cerevisiae(chromosome lll) l--l

o p " n r e a d i n gf r a m e

t R N Ag e n e

80 kb

A FIGURE 6-4 Structureof p-globingeneclusterand comparisonof genedensityin higherand lower eukaryotes. (a)Inthediagram genecluster of the B-globin on humanchromosome genesExons 11,thegreenboxesrepresent exonsof B-globin-related spliced together to formonemRNAareconnected bycaret-like spikes (white); genecluster ThehumanB-globin contains two pseudogenes genesbutare globin-type arerelated theseregions to thefunctional nottranscribed. Eachredarrowindicates thelocation of anAlu

thatis sequence repeated an :300-bpnoncoding sequence, (b)In the diagram of yeastDNA in the humangenome, abundant frames openreading lll,the greenboxesindicate fromchromosome protein-coding arefunctional sequences Mostof thesepotential of Notethe muchhigherproportion geneswithoutintrons. in the humanDNAthanin theyeast sequences noncoding-to-coding ProgNuclAcid 1984, andS M Weissman, DNA lPart(a),seeF 5 Collins N O l i v e r e t a l P a r t ( b ) , s e e S G B i o l . 3 1 : 3 1 5 , 1 9 9 2 , ature357'-78]l R e sM o l

sequencedrift during evolution generatedsequencesthat either terminate translation or block mRNA processing, rendering such regions nonfunctional. Becausesuch pseudogenes are not deleterious, they remain in the genome and mark the location of a geneduplication that occurred in one of our ancestors. Duplications of segmentsof a chromosome (called segmentdl duplication) occurredfairly often during the evolution of multicellular plants and animals. As a result, a large fraction of the genesin these organismstoday have been duplicated,allowing the processof sequencedrift to generategene families and pseudogenes. The extent of sequencedivergence betweenduplicatedcopiesof the genomeand characterization in relatedorganismsalof the homologousgenomesequences low an estimateof the time in evolutionaryhistory when the duplication occurred.For example,the human fetal 1-globin genes(G" and A") evolvedfollowing the duplicationof a 5.5-kb region in the B-globin locus that included the single1-globin genein the common ancestorof caterrhineprimates(old world monkeys, apes, and humans) and platyrrhine primates (new world monkeys)about 50 million yearsago. Although members of gene families that arose relatively recently during evolution are often found near each other on the samechromosome,as for genesof the human B-globin locus, members of gene families may also be found on different chromosomesin the same organism.This is the case for the human ct-globin genes,which were separatedfrom the B-globin genesby an ancient chromosomal translocation. Both the a- and B-globin genesevolvedfrom a single ancestralglobin genethat was duplicated(seeFigure 6-2b) to generatethe predecessorsof the contemporary cr- and B-globin genesin mammals.Both the primordial ct- and Bglobin genesthen underwent further duplicationsto generate the different genesof the cr- and B-globin gene clusters found in mammalstoday.

Severaldifferent genefamiliesencodethe various proteins that make up the cytoskeleton.Theseproteins are presentin varying amounts in almost all cells. In vertebrates,the major cytoskeletalproteins are the actins, tubulins, and intermediate filament proteins like the keratins discussedin Chapters 'We examine the origin of one such family, the 17, 18 and 19. tubulin family, in Section6.5. Although the physiologicalrationale for the cytoskeletalprotein families is not as obvious as it is for the globins, the different members of a family probably have similar but subtly different functions suited to the particular type of cell in which they are expressed.

HeavilyUsedGene ProductsAre Encoded b y M u l t i p l eC o p i e so f G e n e s In vertebratesand invertebrates'the genesencoding ribosomal RNAs and some other nonprotein-coding RNAs such as those involved in RNA splicing occur as tandemly repeated arldys. These are distinguishedfrom the duplicated genesof gene families in that the multiple tandemly repeated genes encode identical or nearly identical proteins or functional RNAs. Most often copiesof a sequenceappear one after the other, in a head-to-tail fashion, over a long stretch of DNA. Within a tandem array of rRNA genes,each copy is nearly exactly like all the others. Although the transcribed portions of rRNA genesare the same in a given individual, the nontranscribed spacer regions between the transcribed regions can vary. These tandemly repeatedRNA genesare neededto meet the great cellular demand for their transcripts. To understand why, consider that a fixed maximal number of RNA copies can be produced from a single gene during one cell generation when the gene is fully loaded with RNA polymerase molecules.If more RNA is required than can be transcribed from one gene, multiple copies of the gene are G E N ES T R U C T U R E EUKARYOTIC

221

necessary.For example, during early embryonic development in humans, many embryonic cells have a doubling time of :24 hours and contain 5-10 million ribosomes.To produce enough rRNA to form this many ribosomes,an embryonic human cell needs at least 100 copies of the large and small subunit rRNA genes,and most of thesemust be close to maximally active for rhe cell to divide every 24 hours. That is, multiple RNA polymerasesmust be transcribing each rRNA gene at the same time (seeFigure 8-33). Indeed, all eukaryotes,includingyeasts,contain 100 or more copres of the genesencoding 55 rRNA and the large and small subunit rRNAs. Multiple copies of IRNA genesand the genesencoding the histone proteins also occur.As we'll seelater in this chapter, histones bind and organize nuclear DNA. Just as the cell requiresmultiple rRNA and IRNA genesto support efficient translation, multiple copies of the histone genesare required to produce sufficient histone protein to bind the large amount of nuclear DNA. While IRNA and histone genesoften occur in clusters,they generally do not occur in tandem arrays in the human genome.

N o n p r o t e i n - C o d i nG g e n e sE n c o d e F u n c t i o n aR l NAs In addition to rRNA and IRNA genes,there are hundreds of additional genesthat are transcribedinto nonprotein-coding RNAs, some with various known functions, and many whose functions are not yet known. For example, small nuclearRNAs (snRNAs) function in RNA splicing,and small nucleolar RNAs (snoRNAs) function in rRNA processing and basemodification in the nucleolus. The RNase P RNA functionsin IRNA processing,and a largefamily (-1000) of short micro RNAs (miRNAs) regulate the stability and translation of specific mRNAs. The functions of these nonprotein-coding RNAs are discussedin Chapter 8. An RNA found in telomerase(Chapter 4) functions in maintaining the sequenceat the ends of chromosomes, and the 7SL RNA functions in the import of secretedproteins and most membrane proteins into the endoplasmicreticulum (Chapter 13). These and other nonprotein-coding RNAs encoded in the human genome and their functions, when known, are listed inTable 6-2.

(]FGENTS NUMBER INI|UMAN GTNI)MI FUNCTI()N

RNA rRNAs

-300

Protein synthesis

tRNAs

:500

Protein synthesis

:80

snRNAs U7 snRNA

1 :85

snoRNAs

:1 000

miRNAs

mRNA splicing Histone mRNA 3' processing Pre-rRNA processingand rRNA modification Regulation of gene expressron

Xist

1

X-chromosomeinactivation

7SK

I

Transcription control

RNAse P

1

IRNA 5' processing

7SL RNA

Protein secretion (component of signal recognition particle, SRP)

RNAseMRP

1

rRNA processing

Telomerase RNA

1

Template for addition of telomeres

Vault RNAs

3

Components of Vault ribonucleoproteins (RNPs), function unknown

:30

hY1, hY3, hY4, hY5 Hl9

I

Components of Ro ribonucleoproteins (RNPs), function unknown Unknown

souRcE: International Human Genome SequencingConsortium, 2001, Nature 409:860, and P. D. Zamore and B. Haley, 2005. Science 309':7519.

222

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c H A p T E R6

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G E N E 5G , E N O M t C SA,N D C H R O M O S O M E S

Eukaryotic Gene Structure r In molecular terms, a gene is the entire DNA sequence required for synthesisof a functional protein or RNA molecule.In addition to the coding regions(exons),a geneincludescontrol regions and sometimesintrons. r A simple eukaryotic transcription unit produces a single monocistronic mRNA. which is translated into a single proteln. r A complex eukaryotic transcription unit is transcribed into a primary transcript that can be processedinto two or more different monocistronic mRNAs depending on the choice of splice sites or polyadenylation sites. A complex transcription unit with alternate promoters also generates two or more different mRNAs (seeFigure 6-3b). r Many complex transcription units (e.g., the fibronectin gene) expressone mRNA in one cell type and an alternate mRNA in a different cell type. r About half the protein-coding genes in vertebrate genomic DNA are solitary genes,each occurring only once in the haploid genome. The remainder are duplicated genes, which arose by duplication of an ancestralgeneand subsequent independentmutations (seeFigure 6-2b). The proteins encoded by a gene family have homologous but nonidentical amino acid sequencesand exhibit similar but slightly di fferentproperties. r In invertebratesand vertebrates,rRNAs are encoded by multiple copies of genes located in tandem arrays in genomic DNA. Multiple copies of IRNA and histone genes also occur, often in clusters, but not generally in tandem arrays. r Many genesalso encode functional RNAs that are not translated into protein but nonethelessperform significant functions, such as rRNA, IRNA and snRNAs. Among these are micro RNAs, possibly up to 1000 in humans, whose biological significancein regulating geneexpression has only recentlybeenappreciated.

ChromosomalOrganization @ of Genesand NoncodingDNA Having reviewed the relationship between transcrlptlon units and genes,we now consider the organization of genes on chromosomesand the relationshipof noncodingDNA sequencesto coding sequences.

G e n o m e so f M a n y O r g a n i s m sC o n t a i nM u c h N o n f u n c t i o n aD l NA Comparisons of the total chromosomal DNA per cell in various speciesfirst suggestedthat much of the DNA in certain organismsdoes not encodeRNA or have any apparent regulatory function. For example, yeasts,fruit flies, chickens,

and humans have successivelymore DNA in their haploid c h r o m o s o m es e t s ( 1 2 ; 1 8 0 ; 1 3 0 0 ; a n d 3 3 0 0 M b , r e s p e c tively), in keepingwith what we perceiveto be the increasing complexity of these organisms.Yet the vertebrateswith the greatestamount of DNA per cell are amphibians, which are surely less complex than humans in their structure and behavior. Even more surprising, the unicellular protozoan speciesAmoeba dubia has 200 times more DNA per cell than humans. Many plant speciesalso have considerably more DNA per cell than humans have. For example, tulips have 10 times as much DNA per cell as humans. The DNA content per cell also varies considerably between closely related species.All insectsor all amphibians would appear to be similarly complex, but the amount of haploid DNA in specieswithin each of thesephylogenetic classesvaries by a factor of 100. Detailed sequencingand identification of exons in chromosomal DNA have provided direct evidence that the genomesof higher eukaryotescontain large amounts of noncoding DNA. For instance, only a small portion of the Bglobin gene cluster of humans, about 80 kb long' encodes protein (seeFigure 6-4a).In contrast,a typical 8O-kbstretch of DNA from the yeast S. cereuisiae,a single-celledeukaryote, contains many closely spacedprotein-coding sequences without introns and relatively much less noncoding DNA (seeFigure 6-4b). Moreover, compared with other regions of vertebrate DNA, the B-globin gene cluster is unusually rich in protein-coding sequences,and the introns in globin genes are considerablyshorter than those in many human genes. The density of genesvaries greatly in different regions of human chromosomal DNA, from "gene-rich" regions, such as the B-globin cluster,to large gene-poor "gene deserts."Of the 96 percent of human genomic DNA that has been sequenced,only :1.5 percentcorrespondsto protein-coding sequences(exons). We learned in the previous section that the intron sequencesof genesare often significantly longer Approximately one-third of human than the exon sequences. genomic DNA is thought to be transcribed into pre-mRNA precursors or nonprotein-coding RNAs in one cell or another, but some 95 percent of this sequenceis intronic, and thus removed by RNA splicing. This amounts to a large fraction of the total genome. The remaining two-thirds of human DNA is noncoding DNA between genesas well as regions of repeated DNA sequencesthat make up the centromeresand telomeres of the human chromosomes' Consequently,-98.5 percent of human DNA is noncoding' Different selectivepressuresduring evolution may account, in part, for the remarkable difference in the amount of least at nonfunctionalDNA in unicellularand multicellularorganisms. For example, microorganisms must compete for limited amounts of nutrients in their environment, and metabolic economythus is a critical characteristic.Sincesynthesisof nonfunctional (i.e.,noncoding)DNA requirestime, nutrients and energy,presumablythere was selectivepressureto lose nonfunctional DNA during the evolution of microorganisms. On the other hand, natural selectionin vertebratesdependslargely on their behavior. The energy invested in DNA synthesis is trivial compared with the metabolic energy required for the

DNA NF G E N E SA N D N O N C O D I N G C H R O M O S O M AOL R G A N I Z A T I OO

223

movementof muscles;thus therewas little selectivepressureto eliminatenonfunctionalDNA in vertebrates.

(a) Normal replication

M o s t S i m p l e - S e q u e n cDeN A sA r e C o n c e n t r a t e d i n S p e c i f i cC h r o m o s o m aLl o c a t i o n s (b) Backwardslippage

Besidesduplicated protein-codinggenesand tandemly repeated genes,eukaryotic cells contain multiple copies of other DNA sequences in the genome,generallyreferredto as repetitiousDNA (seeTable 6-1). Of the two main types of repetitiousDNA, the lessprevalentis simple-sequence DNA, or satelliteDNA, which constiruresabout 6 percent of the human genomeand is composedof perfector nearly perfect repeats of relatively short sequences.The more common type of repetitious DNA, collectively called interspersed repeats,is composedof much longer sequences. These sequences,consisting of several types of transposableele, ments,are discussedin Section6.3. The length of each repeat in simple-sequence DNA can range from 1 to 500 basepairs. Simple-sequence DNAs in which the repearscontain 1-13 basepairs are often called microsatellites. Most microsateiliteDNA has a repeatlength ol 1-4 base pairs and usually occurs in tandem repeatsof 150 repeatsor fewer. Microsatellitesare rhought to have originatedby "backward slippage"of a daughrerstrand on its templatestrand during DNA replicarionso that the same short sequenceis copiedtwice (Figure6-5).

a'

u, I

Second replication

I (c)Secondreplication Dau 53' 3',

5'

N o r m a ld a u g h t e rD N A

Microsatellitesoccasionallyoccur within transcription units. Some individuals are born with a larser number of repeatsin specificgenesthan observedin the generalpopulation, presumablybecauseof daughter-strandslippageduring DNA replication in a germ cell from which they developed. Such expanded microsatelliteshave been found to cause at least 14 different types of neuromusculardiseases,depending on the genein which they occur.In some casesexpandedmicrosatellites behavelike a recessive mutation becausethey interferewith just the function or expressionof the encoded gene.But in the more common types of diseases associated with expanded microsatelliterepeats,such as myotonic dystropby and spinocerebelldrataxia, the expanded repeatsbehave like dominant mutarions becausethey interfere with all RNA processingin the musclecellsand neuronswhere the affectedgenesare expressed.For example,in patientswith myotonic dystrophy,transcripts of the DMPK gene contain between 100-4000 repeats of the sequenceCUG in the 3, untranslatedregion, compared to 50-100 repeatsin normal individuals. The extendedstrerchof CUG reDeatsin affected individuals forms long RNA hairpins (seeFigure 4-9) that interferewith normal RNA processingand export of transcripts from the nucleusto rhe cyrosol. The double-stranded(ds) regions of these long RNA hairpins bind nuclear dsRNAbinding proteins,interferingwith their normal function in regulating the alternative splicing of other specificpre-mRNAs essentialfor normal muscleand nerve cell function. I Most satelliteDNA is composedof repeatsof 14-500 base pairs in randem arrays of 20-700 kb. In situ hybridizarion

224

CHAPTER 6

I

u' 3',

r, s,

FIGURE 6-5 Generationof microsatellite repeatsby backwardslippageof the nascentdaughterstrandduring DNA (a),the nascent replication.lf duringreplication daughter strand "slips"backward relative to thetemplate strandby onerepeat, one newcopyof the repeatisaddedto the daughter strandwhenDNA (b) Thisextracopyof the repeatformsa singlereplication continues stranded loopin the daughter strandof thedaughter duplexDNA moleculelf thissingle-stranded loopisnot removed by DNArepair proteins (c),theextracopy beforethe nextroundof DNAreplication of the repeatisaddedto oneof the double-stranded daughter DNA m o l e c u l et h s ,eo t h e rd a u g h t emr o l e c u w l ei l lb e n o r m a l

studieswith metaphasechromosomeshave localizedthese simple-sequenceDNAs to specific chromosomal regions. Much of this DNA lies near centromeres,the discretechromosomal regions that attach to spindle microtubules during mitosis and meiosis(Figure6-6). Experimentsin the fission yeast Schizosaccharomyces pombe indicate that these sequencesare required to form a specializedchromatin structure called centromeric heterochromatin, necessary for the proper segregationof chromosomes to daughter cellsduring mitosis. Simple-sequence DNA is also found in l o n g t a n d e m r e p e a t s a t t h e e n d s o f c h r o m o s o m e s ,t h e telomeres,where they function to maintain chromosome ends and prevent their joining ro the ends of other DNA molecules,as discussedfurther in the last section of this chaoter.

G E N E SG , E N O M I C SA, N D C H R O M O S O M E S

DNA fingerprinting, which is superior to conventional fingerprinting for identifying individuals (Figure 6-7). The use of PCR methods allows analysis of minute amounts of DNA, and individuals can be distinguished more precisely and reliably than by conventional fingerprinting.

U n c l a s s i f i eS d p a c e rD N A O c c u p i e sa S i g n i f i c a n t Portion of the Genome As Table5.1 shows,:25 percentof humanDNA liesbetween transcription units and is not repeatedanywhere else in the genome. Much of this DNA probably arose from ancient transposable elements that accumulated so many mutations over evolutionary time they can no longer be recognized as having arisen from this source (Section 6.3).

(a) Paternity determination

(b) Criminal identification Ih

MCF1F2

6

-o

FIGURE A EXPERIMENTAL 6-6 Simple-sequence DNAis localizedat the centromerein mousechromosomes.Purified simple-sequence DNAfrommousecellswascopiedin vitrousingE I andfluorescently labeled dNTPs to generate a collDNApolymerase fluorescently labeled DNAprobefor mousesimple-sequence DNA fromcultured Chromosomes mousecellswerefixedanddenatured slide,andthenthechromosomal DNAwas on a microscope probe(blue-green) Thesltdewas hybridized in situto the labeled with DAPI,a DNA-binding dye,to visualize thefullalsostained (darkblue)Fluorescence microscopy lengthof thechromosomes probehybridizes primarily to one showsthatthe simple-sequence (i e . chromosomes in endof thetelocentric mousechromosomes whichthe centromeres arelocated nearoneend) [Courtesy of Sabine

--!t<-

tr'^A +

6P

e

-= --E = --= 3--

, a n a d al M a l , P h D , M a n i t o b aI n s t i t u t eo f C e l lB i o l o g yC

--

D N A F i n g e r p r i n t i n gD e p e n d so n D i f f e r e n c e s DNAs in Length of Simple-Sequence \(ithin a species,the nucleotidesequencesof the repeat units composing simple-sequenceDNA tandem arrays are highly conservedamong individuals. In contrast, the nwmber of repeats, and thus the length of simple-sequence tandem arrays containing the same repeat unit, is quite variable among individuals. These differencesin length are thought to result from unequal crossing over within regions of simplesequenceDNA during meiosis.As a consequenceof this unequal crossing over, the lengths of some tandem arrays are unique in each individual. In humans and other mammals, some of the simplesequenceDNA exists in relatively short 1- to 5-kb regions made up of 20-50 repeat units, each containing :14-1.00 basepairs. Theseregionsare called minisatellites.Even slight differencesin the total lengths of various minisatellitesfrom different individuals can be detectedby the polymerasechain reaction (PCR), using a mixture of severalprimers that hybridize to unique sequencesflanking multiple minisatellites. TheseDNA polymorphisms (i.e., di{ferencesin sequencebetween individuals of the same species)form the basis of

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5-7 DNAfingerprintingis used FIGURE A EXPERIMENTAL to identify individualsin paternity casesand criminal is a singlePCRreaction In DNAfingerprinting, investigations. setsof usingseveral DNAfroman individual performed on template repeat the minisatellite primers flanking to uniquesequences generates a DNA of the PCRproducts Gelelectrophoresis sequences of thatis,a setof minisatellites for the individual: fingerprint in the gel (a)Inthis andhencemobility repeatlengths different products usingthe mother's the paternity, lane M shows of analysis DNA;and C, usingthechild's in the PCRreaction; DNAastemplate Thechildhas fathers. F1andF2usingDNAfromtwo potential fromeitherthe motheror F1, inherited repeatlengths minisatellite PCRproducts indicate Arrows father thatF1isthe demonstrating DNA.(b)In theseDNA fromF1.but not F2,foundin the child's froma rapevictimandthreemen isolated of a specimen fingerprints in repeatlengths of the crime,it isclearthat minisatellite susoected DNAwas 1 Thevictim's matchthoseof suspect the specimen DNAwasnot thatthe specimen to ensure in the analysis included andA P T.Strachan with DNAf romthevictimlFrom contamtnated 2, 1999, JohnWiley& Sons l Genetics Molecular Human Read,

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Transcription-control regions on the order of 50-200 base pairs in length that help to regulate transcription from distant promoters also occur in theselong stretchesof unclassified spacerDNA. In some cases,sequencesof this seemingly nonfunctional DNA are nonethelessconservedduring evolution, indicating that they may perform a significant function that is not yet understood.For example, they may contribute to the structuresof chromosomesdiscussedin Section6.7.

Chromosomal Organization of Genes a n d N o n c o d i n gD N A r In the genomesof prokaryotes and most lower eukaryotes, which contain few nonfunctional sequences,coding regions are densely arrayedalong the genomic DNA. r In contrast, vertebrateand higher plant genomescontain many sequencesthat do not code for RNAs or have any regulatory function. Much of this nonfunctional DNA is composedof repeatedsequences. In humans, only about 1.5 percentof total DNA (the exons)actuallyencodesproteins or functional RNAs. r Variation in the amount of nonfunctional DNA in the genomes of various speciesis largely responsible for the lack of a consistent relationship between the amount of DNA in the haploid chromosomesof an animal or plant and its phylogeneticcomplexity. r Eukaryotic genomic DNA consistsof three major classes of sequences:genes encoding proteins and functional RNAs, repetitiousDNA, and spacerDNA (seeTable 6-1). r Simple-sequence DNA, short sequencesrepeatedin long tandem arrays, is preferentially located in centromeres, telomeres,and specificlocations within the arms of particular chromosomes. r The length of a particular simple-sequence tandem array is quite variable betweenindividuals in a species,probably becauseof unequal crossing over during meiosis. Differencesin the lengthsof some simple-sequence tandem arrays form the basisfor DNA fingerprinting (seeFigure 6-7).

(Mobile) Transposable !p DNAElements Interspersedrepeats,the secondtype of repetitious DNA in eukaryotic genomes,is composed of a very large number of copies of relatively few sequencefamilies (seeTable 6-1). AIso known as moderately repeatedDNA, or intermediaterepeat DNA, these sequencesare interspersedthroughout mammalian genomes and make up :25-50 percent of mammalian DNA (-45 percentof human DNA). Becauseinterspersedrepeatshave the unique ability to "move" in the genome,they are collectivelyreferred to as transposableDNA elementsor mobile DNA elements(we use these terms interchangeably).Although transposable DNA elementsoriginally were discoveredin eukaryotes.

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they also are found in prokaryotes.The processby which these sequencesare copied and insertedinto a new site in the genomeis called transposition.TransposableDNA elements are essentiallymolecular symbionts that in most casesappear to have no specific function in the biology of their host organisms, but exist only to maintain themselves.For this reason, Francis Crick referred to them as "selfish DNA." 'When transposition occurs in germ cells, the transposed sequencesat their new sitesare passedon to succeedinggenerations. In this wag mobile elementshave multiplied and slowly accumulated in eukaryotic genomesover evolutionary time. Sincemobile elementsare eliminated from eukaryotic genomesvery slowly, they now constitute a significant portion of the genomesof many eukaryotes. Not only are mobile elementsthe source for much of the DNA in our genomes,but they also provided a secondmechanism, in addition to meiotic recombination, for bringing about chromosomal DNA rearrangementsduring evolution (seeFigure 6-2). This is becauseduring transposition of a particular mobile element, adjacent DNA sometimesalso is mobilized. Transpositions occur rarely: in humans, about one new germ-line transposition for every eight individuals. Since98.5 percent of our DNA is noncoding, most transpositions have no deleteriouseffects.But over time they played an essentialpart in the evolution of geneshaving multiple exons and of geneswhose expressionis restrictedto specific cell types or developmentalperiods. In other words, although transposableelementsprobably evolved as cellular symbionts,they have played a central role in the evolution of complex, multicellularorganisms. Transposition also may occur within a somatic cell; in this casethe transposedsequenceis transmitted only to the daughter cells derived from that cell. In rare cases,such somatic-cell transposition may lead to a somatic-cellmutation with detrimental phenotypic effects,for example, the inactivation of a tumor-suppressorgene (Chapter 25). In this section, we first describethe structure and transposition mechanisms of the major types of transposable DNA elements and then consider their likelv role in evolution.

M o v e m e n to f M o b i l e E l e m e n t sI n v o l v e sa D N A o r a n R N AI n t e r m e d i a t e Barbara McClintock discovered the first mobile elemenrs while doing classical generic experimenrs in maize (corn) during the 1940s. She characterizedgenetic entities that could move into and back out of genes, changing the phenotype of corn kernels. Her theories were very controversial until similar mobile elementswere discoveredin bacteria, where they were characterized as specific DNA sequences, and the molecular basisof their transposition was deciphered. As researchon mobile elementsprogressed,they were found to fall into two categories:(L) those that transposedirectly as DNA and (2) those that transpose via an RNA intermediate transcribed from the mobile element by an RNA polymerase and then convertedback into double-strandedDNA bv a reverse

c E N E S ,G E N O M t C SA, N D C H R O M O S O M E S

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6-8 Two maior classesof mobile < FIGURE DNAtransposons DNA elements.(a)Eukaryotic whichis (orange) moveviaa DNAintermediate, fromthe donorsite (b)Retrotransposons excised (green) intoan RNAmolecule, arefirsttranscribed intodoublewhichthenis reverse-transcribed thedouble-stranded DNA.In bothcases, stranded intothetargetis integrated DNAintermediate movementThusDNA siteDNAto complete mechanism, movebya cut-and-paste transposons moveby a copy-andwhereasretrotransposons oastemechantsm.

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m o b i l ee l e m e n t s transcriptase(Figure 5-8). Mobile elementsthat transpose directly as DNA are generallyreferredto as DNA transposons,or simply transposons.Eukaryotic DNA transposonsexcisethemselvesfrom one place in the genome,leaving that site and moving to another.Mobile elementsthat transposeto new sitesin the genomevia an RNA intermediateare called retrotransposons. Retrotransposonsmake an RNA copy of themselvesand introduce this new copy into another site in the genome, while also remaining at their original location. The movement of retrotransposons is analogous to the infectious processof retroviruses.Indeed,retrovirusescan be thought of as retrotransposonsthat evolved genesencoding viral coats, thus allowing them to transpose between cells. Retrotransposons can be further classifiedon the basis of their specific mechanism of transposition. To summarize, DNA transposonscan be thought ofas transposingby a "cut-and-paste" mechanism, while retrotransposonsmove by a "copy-andpaste" mechanismin which the copy is an RNA intermediate.

Are Presentin Prokaryotes DNA Transposons and Eukaryotes Most mobile elementsin bacteriatransposedirectly as DNA. In contrast, most mobile elementsin eukaryotes are retrotransposons, but eukaryotic DNA transposons also occur. Indeed, the original mobile elementsdiscoveredby Barbara McClintock are DNA transposons. Bacterial Insertion Sequences The first molecular understanding of mobile elementscame from the study of

certain E. coli mutations causedby the spontaneousinsertion of a DNA sequence,-1-2 kb long, into the middle of a gene. These inserted stretches of DNA ate called insertiin seqwences,or 15 elements. So far, more than 20 different IS elementshave been found in E. coli and other bacteria. Transposition of an IS element is a very rare event' occurring in only one in 105-107 cells per generation' depending on the IS element. Often, transpositions inactivate essentialgenes,killing the host cell and the IS elementsit carries. Therefore,higher rates of transposition would probably result in too great a mutation rate for the host organism to survive, However, since IS elementstranspose more or less randomly, some transposedsequencesenter nonessentialregions of the genome (e.g.' regions between genes),allowing the cell to survive. At a very low rate of transposition, most host cells survive and therefore propagate the symbiotic IS element. IS elementsalso can insert into plasmids or lysogenic viruses, and thus be transferred to other cells' In this way, IS elementscan transposeinto the chromosomesof vir-

on the other strand, such as:

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M O B I L E )D N A E L E M E N T S T R A N S P O S A B L( E

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lS element {=1-2 kb)

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responsiblefor the second classdissociation (Ds) elements becausethey also tended to be associatedwith chromosome breaks. Many years after McClintock's pioneering discoveries, cloning and sequencingrevealedthat Ac elementsare equivalent to bacterial IS elements.Like IS elements,they contain inverted terminal repeat sequencesthat flank the coding region for a transposase,which recognizesthe terminal repeats and catalyzestranspositionto a new site in DNA. Ds elements

Target-site direct repeal (5-11bp)

A FIGURE 6-9 Generalstructureof bacteriallS elements.The relatively largecentralregionof an lSelement, whichencodes oneor two enzymes required for transposition, isflankedby an inverted repeatat eachend.Thesequences of the inverted repeats arenearly identical, but theyareoriented in opposite directions Theinvertedrepeatsequence ischaracteristic of a particular lSelementThe5, and 3' shortdlrect(asopposed to inverted) repeats arenottransposed with the insertion element; rather, theyareinsertion-site sequences that become duplicated, withonecopyat eachend,duringinsertion of a mobileelement. Thelengthof thedirectrepeats isconstant for a given lSelement, buttheirsequence depends on thesiteof insertion and therefore varies witheachtransposition of the lSelementArrows indicate sequence orientation Theregions in thisdiagram arenotto scale; thecodingregionmakesup mostof thelenqthof an lSelement

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Betweenthe inverted repeatsis a region that encodestransposase,an enzyme required for transposition of the IS element to a new site. The transposaseis expressedvery rarely accounting for the very low frequency of transposition. An important hallmark of IS elementsis the presenceof a short direct-repeatsequence,containing 5-1 1 basepairs, immediately adjacent to both ends of the inserted element. The length of the direct repeat is characteristicof each type of IS element, but its sequencedependson the target site where a particular copy of the IS element inserted. When the sequence of a mutated gene containing an IS element is compared with the wild-type genesequence,only one copy of the short direct-repeatsequenceis found in the wild-type gene. Duplication of this target-sitesequencero createthe second direct repeat adjacent to an IS elemenroccurs during the insertlonprocess. As depicted in Figure 6-10, transposition of an IS ele-

5'

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donor DNA. Finally, a host-cell DNA polymerasefills in the single-strandedgaps, generatingthe short direct repearsthat flank IS elements,and DNA ligasejoins the free ends. Eukaryotic DNA Transposons McClintock's original discovery of mobile elementscame from observation of sDonta-

unlessthey occur in the presenceof the first class of mutations. McClintock called the agent responsiblefor the first class of mutations the actiuator (Ac) element and those

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A FIGURE 5-10 Modelfor transpositionof bacterialinsertion sequences. Step[: Transposase, whichisencoded by the lS (lS/0 in thisexample), element cleaves bothstrands of the donor DNAnextto the inverted (darkred),excising repeats the lS/0 elementAt a largely randomtargetsite,transposase makes staggered cutsin thetargetDNA.In thecaseof lSI0,thetwo cuts are9 bp apart.Step[: Ligation of the 3, endsof theexcised lS element to the staggered sitesin thetargetDNAalsoiscatalyzed by transposase Stepf,l: The9-bpgapsof single-stranded DNAleftin the resulting intermediate arefilledin by a cellular DNApolymerase; finallycellular DNAligase formsthe 3'-+5,phosphodiester bonds between the 3' endsof theextended targetDNAstrands andthe 5, endsof the lS70strandsThisprocess results in duplication of the target-site sequence on eachsideof the inserted lSelement. Note thatthe lengthof thetargetsiteandlS/0 arenotto scale[See HW Benjamrnand N Kleckner,1989, Cell59:373, and 1992, proc Nat'1.Acad Sci. USA 89:4648 I

G E N E SG, E N O M t C SA, N D C H R O M O S O M E S

are deletedforms of the Ac elementin which a portion of the sequenceencoding transposaseis missing. Becauseit does not encode a functional transposase,a Ds element cannot move by itself. However, in plants that carry the Ac element and thus expressa functional transposase,Ds elementscan be transposed becausethey retain the inverted terminal repeats recognizedby the transposase. Since McClintock's early work on mobile elements in corn, transposonshave been identified in other eukaryotes. For instance,approximately half of all the spontaneousmutations observed in Drosophila are due to the insertion of mobile elements.Although most of the mobile elementsin Drosophila function as retrotransposons,at leastone-the P element-functions as a DNA transposon, moving by a mechanism similar to that used by bacterial insertion sequences. Current methods for constructing transgenic Drosophila depend on engineered,high-level expressionof the P-elementtransposaseand use of the P-elementinverted terminal repeatsas targets for transposition, as discussedin Chapter 5 (seeFigure5-25). DNA transpositionby the cut-and-pastemechanismcan result in an increasein the copy number of a transposon if it occurs during the S phase of the cell cycle, when DNA synthesisoccurs. An increasein the copy number happens when the donor DNA is from one of the two daughter DNA molecules in a region of a chromosome that has replicated and the target DNA is in the region that has not yet repli'S7hen DNA replication is complete at the end of the cated. S phase, the target DNA in its new location is also replicated, resulting in a net increasein the total number of these transposonsin the cell (Figure6-11). Vhen such a transpo-

sition occurs during the S phase preceding meiosis' one of the four germ cells produced contains the extra copy of the transposon. Repetition of this process over evolutionary time has resulted in the accumulation of large numbers of DNA transposons in the genomesof some organisms. Human DNA contains about 300,000 copies of full-length and deletedDNA transposons,amounting to :3 percent of human DNA. As we will seeshortly, this mechanism can lead to the transposition of genomic DNA as well as the transposon itself.

BehaveLike LTRRetrotransposons l n t r a c e l l u l aR r etroviruses The genomesof all eukaryotes studied from yeast to humans contain retrotransposons' mobile DNA elements that transpose through an RNA intermediate utilizing a reversetranscriptase(seeFigure 6-8b). These mobile elements are divided into two major categories,those containing and those lacking long terminal repeats (LTRs). LTR retrotransposons'which we discusshere, are common in yeast (e.g.,Ty elements)and in Drosophila le.g., copia elements).Although less abundant in mammals than nonLTR retrotransposons, LTR retrotransposons nonetheless constitute -8 percent of human genomic DNA. In mammals, retrotransposonslacking LTRs are the most common type of mobile element; these are described in the next sectlon. The general structure of LTR retrotransposonsfound in eukaryotesis depicted in Figure 6-12'ln addition to short 5' and 3' direct repeatstypical of all transposons' these retro-

One copy of transposon before S phase

S phase:DNA replicationand DNA transposition

LTR retrotransposons encode all the proteins of the most common type of retroviruses, except for the envelope proteins. Lacking these envelope proteins' LIR retrotransDosons cannot bud from their host cell and infect other cells; however, they can transpose to new sites in the DNA

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6-12 Generalstructureof eukaryoticLTR FIGURE regionisflankedby Thecentralprotein-coding retrotransposons. direct (LTRs), whichareelement-specific repeats two longterminal retrotransposons integrated Likeothermobileelements, repeats. directrepeatsat eachend Notethat the haveshorttarget-site region arenot drawnto scaleTheprotein-coding regtons different andencodes 80 percentor moreof a retrotransposon constitutes proteins andotherretroviral integrase, transcriptase, reverse M O B I L E )D N A E L E M E N T S T R A N S P O S A B L( E

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229

of their host cell. Becauseof their clear relationship with retroviruses,this classof retrotransposonsare often called r etr ouirus-like elements. A key step in the retroviral life cycle is formation of retroviral genomic RNA from integratedretroviral DNA (see Figure 4-49). This processservesas a model for generationof the RNA intermediate during transposition of LTR retrotransposons.As depictedin Figure 6-13, the leftward retroviral LTR functions as a promorer that directs host-cell RNA polymeraseto initiate transcription at the 5, nucleotideof the R sequence.After the entire downstream retroviral DNA has been transcribed, the RNA sequencecorresponding to the rightward LTR direcrs host-cell RNA-processingenzymesto cleavethe primary transcript and add a poly(A) tail at the 3, end of the R sequence.The resultingrerroviral RNA genome, which lacks a completeLIR, exits the nucleusand is packaged into a virion that buds from the host cell. After a retrovirus infects a cell, reversetranscription of its RNA genome by the retrovirus-encodedreverie transcriptaseyields a double-strandedDNA containing complete LTRs (Figure 6-14). This DNA synthesistakes place in the cytosol. The double-strandedDNA with an LTR at each end is then transported into the nucleus in a complex with integrase, another enzyme encoded by retroviruses. Retroviral integrasesare closelyrelatedto the transposases encodedby DNA transposonsand use a similar mechanismto insert the double-strandedretroviral DNA into the host-cell genome. In this process,short direct repeats of the rarget-sltesequenceare generatedat either end of rhe insertedviral DNA sequence.Although the mechanism of reversetranscription

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is complex, it is a critical aspect of the retrovirus life cycle. The processgeneratesthe complete 5' LTR that functions as a promoter for initiation of transcription preciselyat the 5, nucleotide of the R sequence,while the complete 3, LTR functions as a poly(A) site leading to polyadenylation precisely at the 3' nucleotide of the R sequence.Consequentlg no nucleotidesare lost from a LTR retrotransposonas it undergoessuccessiverounds of insertion, transcription, reverse transcriprionand re-insertionat a new site. As noted above, LTR retrotransposons encode reverse transcriptase and integrase. By analogy with retroviruses, these mobile elementsmove by a "copy-and-paste" mechanism whereby reversetranscriptaseconverts an RNA copy of a donor elementinto DNA, which is inserted into a target site by integrase.The experiments depicted in Figure 6-15 provided strong evidencefor the role of an RNA intermediate in transposition of Ty elements. The most common LIR rerrotransposons in humans are calledERVs,for endogenousretroziruses.Most of the 443,000 ERV-relatedDNA sequencesin the human genome consist only of isolated LIRs. Theseare derived from fullJength proviral DNA by homologous recombination between the two LIRs, resultingin deletionofthe internal retroviral sequences. IsolatedLTRs suchas thesecannot be transposedto a new position in the genome, but recombination between homologous LTRs at different positions in the genome have likely contributed to the chromosomalDNA rearrangementsleadingto geneand exon duplications, the evolution of proteins with new combinationsof exons, and, as we will seein Chapter 7, the evolution of complex control of geneexpression.

U3-R

A FIGURE 6-13 Generationof retroviralgenomicRNAfrom integratedretroviral DNA.Theleft LTRdirectscellularRNA polymerase to initiate transcription at thefirstnucleotide of the leftR regionTheresulting primary transcript extends beyondthe rightLTR. TherightLTR, now present in the RNAprimary transcript, directs cellular enzymes to cleave the primary transcript at the last nucleotide of the rightR regionandto adda poly(A) tail,yielding a retroviral RNAgenomewiththe structure shownat thetop of F i g u r6e- 1 4 A . s i m i l am r e c h a n i si m st h o u g htto g e n e r a t eh eR N A intermedrate duringtransposition of retrotransposons Theshort drrect-repeat (black) sequences of target-site DNAaregenerated duringintegration of the retroviral qenome DNAintothe host-cell 230

> FIGURE 6-14 Model for reversetranscriptionof retroviral genomicRNAinto DNA.Inthismodel,a complicated series of nine eventsgenerates a double-stranded DNAcopyof the single-stranded (top)Thegenomic RNAgenomeof a retrovrrus RNAis packaged in thevirionwitha retrovirus-specific cellular IRNAhybridized to a complementary sequence nearits 5' endcalledIheprimer-binding site (PBS)Theretroviral RNAhasa shortdirect-repeat terminal sequence (R)at eachend.Theoverall reaction iscarried out by reverse transcriptase,whichcatalyzes polymerization of deoxyribonucleotides. RNaseH digests the RNAstrandin a DNA-RNA hybridTheentireprocess yieldsa double-stranded DNAmolecule thatislongerthanthetemplate RNA andhasa longterminalrepeat(LTR) at eachend Thedifferentregions arenotshownto scaleThePBS andRregions areactually muchshorter thantheU5andU3regions, andthecentral codingregionisverymuch longerthantheotherregions. E Gilboa etal, 1979, Ce// 18:93 [See ]

Non-LTRRetrotransposons Transpose b y a D i s t i n c tM e c h a n i s m The most abundant mobile elementsin mammals are retrotransposons that lack LTRs, sometimes called nonuiral retrotransposozs. These moderately repeated DNA sequences form two classesin mammalian genomes: LINEs (long interspersedelements)and SINEs (short interspersed elements).In humans, full-length LINEs are-6 kb long, and SINEsare:300 bp long (seeTable 6-1). Repeatedsequences with the characteristicsof LINEs have been observedin protozoans, insects,and plants, but for unknown reasonsthey

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EXPERIMENTAL FTGURE G-l5 The yeastTy element transposes throughan RNAintermediate. Whenyeastcellsare transformed with a Ty-containing plasmid, theTyelement can transpose to newsites,although normally thisoccursat a low rate Usingthe elements diagrammed at thetop,researchers engineered two differentrecombinant plasmid vectors containing recomornanr Tyelements adjacent to a galactose-sensitive promoterThese plasmids weretransformed intoyeastcells,whichweregrownin a g a l a c t o s e - c o n t a ranni dnagn o n g a l a c t om s e d i u mI ne x p e r i m e 1 n ,t growthof cellsin galactose-containing mediumresulted in many moretranspositions thanin nongalactose medium,indicating that transcription tntoan mRNAintermediate is required for Ty transposition In experiment 2, an intronfroman unrelated yeast genewasinserted intothe putative protein-coding regionof the recombinant galactose-responsive Tyelement. Theobserveo aosence of the intronin transposed Tyelements isstrongevidence that transposition involves an mRNAintermediate fromwhichthe intron wasremoved by RNAsplicing, asdepicted in the boxin the lower right In contrast, eukaryotic DNAtransposons, liketheAc element of maize,containintronswithinthetransposase gene,indicating that theydo nottranspose viaan RNAintermediate. J.Boeke etal.. [See 1985. Cell 4O:491 I

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are particularly abundant in the genomes of mammals. SINEs also are found primarily in mammalian DNA. Large numbers of LINEs and SINEs in higher eukaryoteshave accumulated over evolutionary time by repeatedcopying of sequencesat a few positions in the genome and insertion of the copiesinto new positions. LINEs Human DNA contains three major families of LINE sequencesthat are similar in their mechanismof transposition, but differ in their sequences: L1.,L2, and L3. Only membersof the L1 family transposein the contemporary human genome. Apparently there are no remaining functional copies of L2 or L3. LINE sequences arepresentat :900,000 sitesin the human genome,accounting for a staggering21 percent of total human DNA. The generalstructure of a completeLINE is diagrammed in Figure 6-15. LINEs usually are flanked by short direct repeats, the hallmark of mobile elements,and contain two long open readingframes (ORFs).ORF1, -1 kb long, encodesan RNA-binding protein. ORF2, -4 kb long, encodesa protein that has a long region of homology with the reversetranscriptases of retroviruses and LTR retrotransposons.but also exhibitsDNA endonuclease activiry. Evidence for the mobility of L1 elements first came from analysisof DNA cloned from humans with certain genetic diseasessuch as hemophilia and myotonic dystrophy. DNA from these patients was found to carry mutations resulting from insertion of an L1 elementinto a gene,whereasno such elementoccurredwirhin this genein either parent. About 1 in 600 mutarions rhat causesignificant diseasein humans are due to L1 transpositions or SINE transpositionsthat are catalyzedby L1-encodedproteins. Later experiments similar to those just described with yeastTy elements(seeFigure 6-15) confirmed that L1 elementstransposethrough an RNA intermediate.In these experiments,an intron was introducedinto a cloned mouse L1 element, and the recombinant L1 element was stably transformed into cultured hamster cells. After severalcell doublings, a PCR-amplified fragment corresponding to the L1 elementbut lacking the insertedintron was detectedin Long interspersedelement (LINE)(-6 161

FIGURE 6-16 Generalstructureof a UNE,a non-LTR retrotransposon. Mammalian DNAcarries two classes of non_LTR retrotransposons, (notshown)Thelengthof the LINES andSINES target-site directrepeats varies amongcopiesof a LINEat differentsites in thegenomeAlthough thefull-length L1sequence is=6 kb long, variable amountsof the leftendareabsentat over90 percentof the siteswherethismobileelement isfound Theshorter openreading -1 kb in length, frame(ORF1), encodes an RNA-binding proteinThe -4 kb in length,encodes longerORF2, a bifunctional protein with reverse transcriptase andDNAendonuclease activity. Notethat LINEs lackthe longterminalrepeats foundin LTRretrotransposons.

ORF2 protein C h r o m o s o m aD l NA AAATACT TTTATGA

5',

5',

L I N ER N A

E *,.n'"n f

Nicksite

Nicksite

5', 3',

a

P r i m i n g o f r e v e r s et r a n s c r i p t i o n b v c h r o m o s o m aD l NA

the cells. This finding strongly suggeststhat over time the recombinantL1 elementcontaining the insertedintron had transposedto new sitesin the hamster genomethrough an RNA intermediate that underwent RNA splicing to remove the intron. I

AAA

I S -

of LINEreverse 6-17 Proposedmechanism < FIGURE of ORFlandORF2 transcriptionand integration.Aftersynthesis (red), of ORFl copies multiple RNA LINE of a complex in thecytosol, into the moves tail Poly(A) to the bound ORF2 of copy andone is represented transcrlptase, a reverse nucleusOnlyORF2protein, LINEDNAisshownin blackStep[: ORF2makes Newlysynthesized DNAon eithersrdeof anyaccessible nicksin chromosomal staggered of transcription genome. Stepfl: Reverse in the A/f-richsequence T-rich sequence primed the single-stranded by is ORF2 RNA by LINE to the generated bythe nickin the bottomstrand,whichhybridizes RNA the LINE reverse-transcribes poly(A) tail StepsB-E: ORF2 LINE singleto the switching strand, thisnewDNA andthencontinues strandasa template regionof the upperchromosomal stranded the RNAandextendthe3' end thenhydrolyze enzymes Step6: Cellular RNAstrand theLINE replacing DNAtopstrand, of thechromosomal are DNA strands the of 3' ends the5' and withDNA StepZ: Finally, probably are steps two The last insertion the completing ligated, and that removeRNAprimers enzymes bythesamecellular catalyzed (seeFigure 4-30) duringDNAreplication fragments ligateOkazaki Cell72:5951 etal, 1993, {romD D Luan lAdapted

I R e v e r s et r a n s c r i p t i o n o t L I N ER N A b y O R F 2

I

5',

Since LINEs do not contain LTRs, their mechanism of transpositionthrough an RNA intermediatediffers from that of LTR retrotransposons.ORF1 and ORF2 proteins are translated from a LINE RNA. In vitro studies indicate that

5'

orotein then bind to the LINE RNA, and ORF2 protein tindr ,o the poly(A) tail. The LINE RNA is then transported back into the nucleus as a complex with ORF1 and ORF2 oroteins. and is reverse-transcribedinto LINE DNA in the .rr.r.letr,by ORF2. The mechanism involves staggeredcleavage of cellular DNA at the insertion site followed by priming o] ,.u.r.. transcription by the resulting cleaved cellular DNA as detailedin Figure 6-L7. The completeprocessresults in insertion of a copy of the original LINE retrotransooson into a new site in chromosomal DNA' A short direct ,.p.", is generatedat the insertion site becauseof the initial staggeredcleavageof the two chromosomal DNA strands' As noted abeady,the DNA form of an ITR retrotrans-

J

TGA

L I N ER N A TTTATGA.,..

orchromosomal E SR?';f I AAA TTTATGA

5' 3'

5',

by completed .,. I Insertion lI c e l l u l aer n z y m e s I 3. AAATACT!,\/^/>TNAAAA TTTATGA\AAAAATTTATGA

El

I

*

'rNE DNA

5', al

L I N ED N A Directrepeats

a reversetranscriptase,is primed by the 3'-end of cleaved chromosomalDNA' which basepairs with the poly(A) tail ) NA ELEMENTS T R A N S P O S A B(LMEO B I L E D

233

of the non-LTR retroviral RNA (seeFigure 6-17, step Z). Sinceits synthesisis primed by the cut end of a cleavedchromosome, and synthesisof the other strand of the non-LTR retrotransposonDNA is primed by the 3,-end of chromosomal DNA on the other side of the initial cut in chromosomal DNA (step E), rhe mechanismof synthesisresultsin integration of the non-LTR retrotransposon DNA. There is no need for an integraseto insert the non-LTR rerrorransposon DNA. The vast majority of LINEs in the human genome are truncated at their 5' end, suggestingthat reverserranscription terminated before completion, and the resulting fragments extending variable distancesfrom the poly(A) tail

intermediatein transposition.In addition to the fact that most L1 insertions are truncared, nearly all the full-length elementscontain stop codons and frameshift mutations in ORFl and ORF2; thesemutations probably have accumulated in most LINE sequences over eutl.rtionary time. As a result, only :0.01 percentof the LINE sequencesin the human genome are full-length with intact open reading frames for ORF1 and ORF2, representing-60-100 in total number. SINEs The secondmost abundanrclassof mobile elements in the human genome,SINEs constitute :13 percent of total human DNA. Varying in length from about 100 to 400 base pairs, theseretrotransposonsdo not encodeprotein, but most contain a 3' A/T:rich sequencesimilar to thar in LINEs. SINEs are transcribed by the same nuclear RNA polymerase that transcribes genes encoding tRNAs, 55 rRNAs, and other

ing and reverse transcription/integration by LINE encoded ORF1 and ORF2. SINEs occur at about 1.6 million sites in the human g e n o m e . O f t h e s e , - 1 . 1 m i l l i o n a r e A l u e l e m e n t s ,s o named becausemost of them contain a single recognition site for the restriction enzyme AluI. Alw elementsexhibit considerable sequencehomology with and probably evolved from 7SL RNA, a cytosolic RNA in a ribonucleoprotein complex called the signal recognition particle. This abundant cytosolic ribonucleoproteinparticle aids in targetlng cerrain polypeptides to the membranes of the endoplasmic reticulum (Chapter 13). Alu elemenrs are scattered throughout the human genome at sites where their insertion has not disrupted geneexpression:between genes,within introns, and in the 3, untranslatedregionsof some mRNAs. For instance,nine Alu elementsare located y1t\in the human B-globin gene clusrer (seeFigure 6-4a). Of the one new germ-line non-LTR retrotranspositionthat is estimated to occur in about every eight individuals, 234

.

-40 percent involve L1 and 50 percent involve SINEs, of which -90 percent areAlw elements.(Note that nearly all new insertionsin human DNA involve retrotransposons.) Similar to other mobile elements,most SINEs have accumulated mutations from the time of their insertion in the germ line of an ancient ancestor of modern humans. Like LINEs, many SINEs also are truncated at their 5, end.

Other Retrotransposed RNAsAre Found i n G e n o m i cD N A In addition to the mobile elementslisted in Table 6-1, DNA copies of a wide variety of mRNAs appear to have integrated into chromosomalDNA. Sincethesesequences lack introns and do not have flanking sequencessimilar to those of the functional genecopies,they clearly are nor simply duplicated genesthat have drifted into nonfunctionality and becomepseudogenes, as discussedearlier (seeFigure 6-4a). Instead, theseDNA segmentsappear to be retrotransposed copies of spliced and polyadenylatedmRNA. Compared with normal genes encoding mRNAs, these inserted segments generally contain multiple mutations, which are thought to have accumulated since their mRNAs were first reverse-transcribedand randomly integrated into the genome of a germ cell in an ancient ancestor.These nonfunctional genomic copies of mRNAs are referred to as processedpsewdogenes. Most processedpseudogenesare flanked by short direct repeats, supporting the hypothesis that they were generated by rare retrotransposition events involving cellular mRNAs. Other interspersedrepeatsrepresentingpartial or mutant copiesof genesencoding small nuclear RNAs (snRNAs) and tRNAs are found in mammalian genomes. Like processed pseudogenesderived from mRNAs, these nonfunctional copies of small RNA genes are flanked by short direct repeats and most likely result from rare retrotransposition events that have accumulated through the course of evolution. Enzymes expressedfrom a LINE are thought to have carried out all these retrotransposition events involving mRNAs, snRNAs. and tRNAs.

M o b i l e D N A E l e m e n t sH a v eS i g n i f i c a n t l y I n f l u e n c e dE v o l u t i o n Although mobile DNA elements appear to have no direct function other than to maintain their own existence,their presencehas had a profound impact on the evolution of modern-day organisms.As mentioned earlier,about half the spontaneousmutations in Drosophila result from insertion of a mobile DNA elemenrinto or near a transcription unit. In mammals, mobile elementscause a much smaller proportion of spontaneousmutations: -10 percent in mice, and only 0.1-0.2 percentin humans. Still, mobile elements have been found in mutant allelesassociatedwith several human genetic diseases.For example, insertions into the clotting factor IX genecausehemophilia, and insertionsinto the gene encoding the muscle protein dystrophin lead to

c H A p r E6R | G E N EG s ,E N o M t cAsN , Dc H R o M o s o M E s

myotonic dystrophy,commonly known as Duchennemuscular dystrophy. The genesencoding factor IX and dystrophin are both on the X chromosome. Becausethe male genome has only one copy of the X chromosome, transposition insertionsinto thesegenespredominantly affect males. In lineagesleading to higher eukaryotes'homologous recombination between mobile DNA elements dispersed throughout ancestralgenomesmay have generatedgene duplications and other DNA rearrangementsduring evolution (seeFigure 6-2b).For instance,cloning and sequencingof the B-globin gene cluster from various primate specieshas provided strong evidencethat the human G" and A" genesarose from an unequal homologous crossoverbetweentwo L1 sequencesflanking an ancestralglobin gene.Subsequentdivergenceof suchduplicatedgenescould leadto acquisitionof distinct, beneficialfunctions associatedwith each member of a gene family. Unequal crossing over between mobile elements located within introns of a particular gene could lead to the duplication of exons within that gene (seeFigure 6-2a). This processmost likely influencedthe evolution of genesthat contain multiple copiesof similar exons encodingsimilar protein domains,such as the fibronectin gene(seeFigure 4-16). Some evidence suggeststhat during the evolution of higher eukaryotes,recombination betweenmobile DNA elements (e.g., Alu elements)in introns of two sepdrdte genes also occurred, generatingnew genesmade from novel combinationsof preexistingexons (Figure6-18).This evolutionary process,termed exon shuffling, maY have occurred during evolution of the genes encoding tissue plasminogen activator, the Neu receptor, and epidermal growth factor' which all contain an EGF domain (seeFigure 3-11). In this case, exon shuffling presumably resulted in insertion of an EGF domain-encoding exon into an intron of the ancestral form of each of thesegenes. Both DNA transposonsand LINE retrotransposonshave been shown to occasionallycarry unrelated flanking sequenceswhen they transposeto new sitesby the mechanisms diagrammedin Figure 6-19. These mechanismslikely also contributed to exon shuffling during the evolution of contemporarygenes. In addition to causingchangesin coding sequencesin the genome,recombination between mobile elementsand transposition of DNA adiacent to DNA transposonsand retroi."nrporon, likely played a significantrole in the evolution of regulatory sequencesthat control gene expression'As noted earlier, eukaryotic genes have transcription-control regions > FIGURE6-18 Exon shuffling via recombination between homologous interspersedrepeats. Recombination repeatsin the intronsof betweeninterspersed separategenesproducestranscriptionunits with a new combinationof exons(greenand blue) ln the exampleshown here,a double crossoverbetween two setsof A/u elements, ( t h em o s ta b u n d a n S t I N E isn h u m a n s r) e s u l t s in an exchangeof exonsbetweenthe two

called enhancersthat can operate over distancesof tens of thousandsof basepairs. Transcription of many genesis controlled through the combined effectsof severalenhancerelements.Insertionof mobile elementsnear such transcriptioncontrol regionsprobably contributed to the evolution of new combinatr,onsof enhancersequences.These in turn control which specificgenesare expressedin particular cell types and the amount oi the encoded protein produced in modern organisms,as we discussin the next chapter' Theseconsiderationssuggestthat the early view of mobile DNA elements as completely selfish molecular parasites missesthe mark. Rather,they have contributed profoundly to the evolution of higher organismsby promoting (1) the generation of genefamilies via geneduplication, (2) the creation of new genesvia shuffling of preexistingexons' and (3) formation of -o.. complex regulatory regionsthat provide multifaceted control of gene expression.Today, researchersare attempting to harnesstranspositionmechanismsfor inserting therapeuticgenesinto patients as a form of genetherapy'

Transposable(Mobile) DNA Elements r Transposable DNA elements are moderately repeated sequencestnterspersedat multiple sites throughout the g.r,o-., of highir eukaryotes. They are present less frequently in prokarYotic genomes. r DNA transposonsmove to new sites directly as DNA; first transcribed into an RNA copy of retrotransporo.t. ".. then is reverse-transcribedinto DNA which the element, ( s e eF i g u r e6 - 8 ) ' r A common feature of all mobile elementsis the presence of short direct repeatsflanking the sequence' r Enzymesencodedby transposonsthemselvescatalyzeinsertion of thesesequencesat new sitesin genomic DNA' r Although DNA transposons'similar in structure to bacterial IS elements,occur in eukaryotes(e'g', the Drosophila P element), retrotransposonsgenerally are much more abundant, especiallyin vertebrates. r LTR retrotransposonsare flanked by long terminal repeats (LTRs), similar to those in retroviral DNA; like retroviruses,they encode reversetranscriptaseand integrase. They move in the genome by being transcribed into RNA, which then undergoesreversetranscription in

G e n e1 l l ] G e n e2 I I I

I Doublecrossover betweenA/uelements { +

9enes ) NA ELEMENTS T R A N S P O S A B (LM EO B I L E D

235

(a)

G e n e1 l l l

I

Trun.ooru."excisionfrom gene

tIsarfllo I S te

G e n e2 = : =

::: I I Trencnn,

y

s a s ei n s e r t i o ni n t o g e n e2

W e a kp o l y ( A ) srgnat

(b)

G e n e ' sp o l y ( A ) srgnat

G e n e1 l l ] 3'exon T r a n s c r i p t i oann d p o l y a d e n y l a t i o n at end of downstreamexon %AAAA l l t s c n t o r ts t e

G e n e2 : : :

::: I I ORF2reversetranscription and insertion I

:: =

:::

the cytosol, nuclear import of the resulting DNA with LTRs, and integration into a host-cell chromosome (see F i g u r e6 - 1 4 ) .

@

sequencesexhibit extensivehomology with small RNAs and are rranscribedby the sameRNA poly_ A/z elements,the most common SINEsin humans, 0-bp sequences found scatteredthroughout the hu_ man genome. r Some interspersedrepeats are derived from cellular RNAs that were reverse-transcribedand inserted rnto genomic DNA at some time in evolutionarv history. Processed pseudogenes derivedfrom mRNAs lack tntrons, a featurethat distinguishesthem from pseudogenes, which aroseby sequencedrift of duplicatedgenes. bile DNA elementsmost likely influencedevolution cantly by servingas recombinationsitesand by mo_ g adjacentDNA sequences.

CHAPTER 6

|

OrganelleDNAs

Although the vast majority of DNA in most eukaryotesis found in the nucleus,some DNA is presentwithin the mitochondria of animals, planrs, and fungi and within the chloroplastsof plants. Theseorganellesare the main cellular sitesfor ATP formation, during oxidative phosphorylation in mitochondria and photosynthesisin chloroplasts (Chapter 12). Many lines of evidenceindicate that mitochondria and chloroplastsevolvedfrom bacteriathat were endocytosedinto ancestral cells containing a eukaryotic nucleus,forming endosymbionts(Figure 6-20). Over evo_ lutionary time, most of the bacterialgeneswere lost from

( s e eF i g u r e6 - 1 7 ) .

236

< FIGURE 5-19 Exonshufflingvia transpositionof a DNAtransposonor LINE retrotransposon. (a)Transposition of an exon(blue)flankedby homologous DNA transposons intoan intronof a second gene As we sawin Figure 6-10,step[, transposase canrecognize andcleave the DNAat the ends of thetransposon inverted repeatsln gene1, if thetransposase cleaves at the leftendof the transposon on the leftandat the rightendof thetransposon on the right,it cantranspose a l lt h ei n t e r v e n i n DgN A i,n c l u d i nt g h ee x o n fromgene1,to a newsitein an intronof gene2 Thenetresultisan insertion of the exonfromgene1 intogene2 (b)lntegration of an exonintoanothergeneviatransposition of a LINE c o n t a i n i nagw e a kp o l y ( As)i g n a l .f s u c ha L I N E i s i n t h e3 ' - m o sitn t r o no f g e n e 1 , t r a n s c r i p t i o fnt h eL I N Ei n t oa n R N A i n t e r m e d i am t ea yc o n t i n ubee y o n di t so w n poly(A) siteandextendintothe 3, exon, t r a n s c r i b i tnhgec l e a v a gaen dp o l y a d e n y l a t i o n s i t eo f g e n e1 i t s e l fT h i sR N Ac a nt h e nb e reverse-transcribed andintegrated by the L I N EO R F 2 p r o t e i n( s e eF i g u r 6 e- ' 7 l ) i n t oa n i n t r o no n g e n e2 , i n t r o d u c i naqn e w3 , e x o n ( f r o mg e n e1 )i n t og e n e2

nucleus. However, mitochondria and chloroplasts in to_ day's eukaryotesretain DNAs encoding some prorernsessentialfor organellarfunction, as well as the ribosomal and transfer RNAs required for synthesis of these proteins. Thus eukaryotic cells have multiple geneticsysrems:a predominant nuclear systemand secondarysystemswith their own DNA, ribosomes, and tRNAs in mitochondria and chloroplasts.

G E N E SG , E N O M t C SA, N D C H R O M O S O M E S

Endocytosisof bacterium caoableof oxidative phosphorylation

,

Endocytosisof bacterium capableof photosYnthesis

Ancestral cell

ATPsynthase t,

Bacterial genome

BacteriaI genome

./

\/ E u k a r y o t i cp l a s m am e m b r a n e

Mitochondrial Mitochondrial genome matrix

Stroma

origin of 6-20 Modelfor endosymbiotic FIGURE by an of a bacterium Endocytosis mitochondriaand chloroplasts. with two an organelle cellwouldgenerate eukaryotic ancestral fromthe eukaryotic derived theoutermembrane membranes, plasma (gray) plasma andthe inneronefromthe bacterial membrane (red)Proteins bacterial localized to the ancestral membrane

suchthatthe portionof the retaintheirorientation, membrane spacenowfacesthe proteinoncefacingthe extracellular fromthe innerchloroplast of vesicles Budding space. intermembrane in of chloroplasts suchasoccursduringdevelopment membrane, of membranes thylakoid generate the plants, would contemporary indicated are DNAs organellar The chloroplasts

M i t o c h o n d r i aC o n t a i nM u l t i p l e m t D N AM o l e c u l e s

colonies. Genetic crossesbetween different (haploid) yeast strains showed that the petite mutation does not segregate with any known nuclear geneor chromosome' In later studies, most petite mutants were found to contain deletions of MtDNA.

Individual mitochondria arclarge enough to be seenunder the light microscope, and even the mitochondrial DNA (mtDNA) can be detectedby fluorescencemicroscopy.The mtDNA is located in the interior of the mitochondrion, the region known as the matrix (seeFigure 12-6). As judged by the number of yellow fluorescent "dots" of mtDNA' a Euglena gracilis cell contains at least 30 mtDNA molecules ( F i g u r e6 - 2 1 ) . Replication of mtDNA and division of the mitochondrial network can be followed in living cells using time-lapsemicroscopy.Such studiesshow that in most organismsmtDNA replicates throughout interphase. At mitosis each daughter cell receivesapproximately the same number of mitochondria, but since there is no mechanism for apportioning exactly equal numbers of mitochondria to the daughter cells, some cells contain more mtDNA than others. By isolating mitochondria from cells and analyztngthe DNA extracted from them, it can be seenthat each mitochondrion contains multiple mtDNA molecules. Thus the total amount of mtDNA in a cell dependson the number of mitochondria, the size of the mtDNA, and the number of mtDNA molecules per mitochondrion. Each of these parametersvaries greatly between different cell types.

m t D N A l s l n h e r i t e dC y t o p l a s m i c a l l y Studies of mutants in yeasts and other single-celledorganisms first indicated that mitochondria exhibit cytoplasmic inheritance and thus must contain their own genetic system (Figure 6-22). For instance,petite yeast mutants exhibit structurally abnormal mitochondria and are incapable of oxidative phosphorylation. As a result, petite cells grow more slowly than wild-type yeasts and form smaller

,

10tr-

t

6-21 Dualstainingrevealsthe FIGURE A EXPERIMENTAL muftipfemitochondrialDNAmoleculesin a growing Euglena of two dyes:ethidium graciliscell.Cellsweretreatedwith a mixture and red fluorescence, a emits and whichbindsto DNA bromide, emits and mitochondria into specifically is incorporated which D|OC6, and emitsa redfluorescence, Thusthe nucleus a greenfluorescence. of yellow-a combination DNAfluoresce areasrichin mitochondrial and Y Hayashi fluorescence [From redDNAandgreenmitochondrial J CellSci93:565 l K Ueda,1989, o R G A N E L L ED N A s

t

237

(a)

Haploidparentswith wild-type nucleargenes

Normal mitochondrion

< FIGURE 6-22 Cytoplasmic inheritanceof the pefite mutation in yeast.Petite-strain mitochondria aredefective in oxidative phosphorylation owingto a deletion in mtDNA(a)Haploid cellsfuse to produce a diploidcellthatundergoes meiosis, duringwhichrandom segregation of parental chromosomes andmitochondria containing mtDNAoccursNotethatalleles for genesin nuclear DNA(represented by largeandsmallnuclear chromosomes colored redandblue) segregate (seeFigure 2:2duringmeiosis 5-5) Incontrast, sinceyeast -50 mtDNAmolecules normally contain percell,allproducts of meiosis usually containbothnormalandpetitemtDNAs andarecapable of (b)Asthesehaploidcellsgrowanddividemitotically, respiration the (including cytoplasm themitochondria) israndomly distributed to the daughter cellsOccasionally, a cellisgenerated thatcontains only petitemtDNAandyieldsa petitecolony. defective Thusformation of suchpetitecellsisindependent of anynuclear geneticmarker.

"Petite" mitochondrion

Diploid zygore

In the mating by fusion of haploid yeastcells,both parents contribute equally to the cytoplasm of the resulting diploid; thus inheritance of mitochondria is biparental. In mammals and most other multicellular organisms,however,the sperm contributeslittle (if any) cytoplasmto the zygote,and virtually all the mitochondria in the embryo are derived from those in the egg, not the sperm. Studiesin mice have shown that 99.99 percentof mtDNA is maternally inherited, but a small part (0.01 percent)is inheritedfrom the male parent. In higher plants, mtDNA is inherited exclusivelyin a uniparental fashion through the femaleparent (egg),not the male (pollen).

M e i o s i s :r a n d o md i s t r i b u t i o n of mitochondriato d a u g h t e rc e l l s

The Size,Structure,and CodingCapacity of mtDNA Vary ConsiderablyBetweenOrganisms

(b)

\ \Mitosis

\

Respiratory-proficient

238

.

C H A P T E6R I

Petite

Respiratoryproficient

Surprisingly,the size of the mtDNA, the number and nature of the proteins it encodes,and eventhe mitochondrial genetic code itself vary gre^iy between different organisms. The mtDNAs of most multicellular animals are :16-kb circular moleculesthat encodeintron-lessgenescompactly arranged on both DNA strands. VertebratemtDNAs encode the two rRNAs found in mitochondrial ribosomes, the 22 tRNAs used to translatemitochondrial mRNAs, and 13 proteins involved in electron transport and AIP synthesis(Chapter 12). The smallestmitochondrial genomesknown arein plasmodium, single-celledobligate intracellular parasitesrhat cause malaria in humans. Plasmodium mtDNAs are only :6 kb, encoding five proteins and the mitochondrial rRNAs. The entire mitochondrial genomesfrom a number of different metazoan organisms (i.e., multicellular animals) have now been cloned and sequenced,and mtDNAs from all these sourcesencode essentialmitochondrial proteins (Figure 6-23). All proteins encodedby mtDNA are synthesized on mitochondrial ribosomes. Most mitochondriasynthesizedpolypeptidesidentified thus far are subunits of multimeric complexesused in electrontransport. ATp svnt h e s i s ,o r i n s e r r i o no f p r o r e i n si n t o t h e i n n e r m i t o c h o n d i i a l membrane or intermembranespace.However, most of the proteins localizedin mitochondria, such as those involved in the processeslisted at the top of Figure 6-23, are encoded by nuclear genes, synthesizedon cytosolic ribosomes, and imported into the organelle by processesdisc u s s e di n C h a p t e r1 3 .

G E N E SG , E N O M I C SA, N D C H R O M O S O M E S

L i p i dm e t a b o l i s m N u c l e o t i d em e t a b o l i s m A m i n o a c i dm e t a b o l i s m

C a r b o h y d r a tm e etabolism H e m es y n t h e s i s Fe-Ssynthesis

U b i q u i n o n es y n t h e s i s Co-factorsynthesis

Chaperones S i g n a l i n gp a t h w a y s

Proteases

DNA repair,replication,etc.

Heme ryase RNA polymerase

TIM

c Cvtochrome 6-23 Proteinsencodedin mitochondrialDNA A FIGURE Onlythe and their involvementin mitochondrialprocesses. a r e d e p i c t e dM o s tm i t o i n n e r m e m b r a n e m i t o c h o n d rm i aal t r i xa n d (blue); mitochondrial areencoded bythenucleus components chondrial components processes nucleus-encoded carriedout by exclusively a r el i s t e da t t h et o p M i t o c h o n d r ci aolm p o n e nst sh o w ni n p i n ka r e genomein but bythe nuclear by mtDNAin someeukaryotes encoded in encoded invariably few components The relatively othereukaryotes. in electron l-V areinvolved mtDNAareshownin orangeComplexes phosphorylation TlM,Sec,Tat,andOxal andoxidative transport

in proteinimportandexport,andinsertion areinvolved translocases e i sa o f p r o t e i nisn t ot h e i n n e rm e m b r a n(eC h a p t e1r3 ) R N a sP 8) lt should the 5'-endof tRNAs(Chapter that processes ribozyme havea multi-subunit be notedthat the majorityof eukaryotes encoded invariantly subunits three with here, I as depicted complex (e g , Saccharomyces' in a few organisms by mtDNA However, thiscomplexis replacedby andPlasmodium), Schizosaccharomyces, Formoredetailson enzyme. single-polypeptide a nucleus-encoded, C h a p t e r1s2 a n d1 3 s e e t r a n s p o r t , a n d m e t a b o l i s m mitochondrial 19:709 Genet Trends ] 2003, al et G Burger from , lAdapted

In contrast to metazoan mtDNAs, plant mtDNAs are many times larger,and most of the DNA doesnot encodeprotein. For instance,the mtDNA in the important model plant Arabidopsis thaliana is 366,924 base pairs, and the largest known mtDNA is =2 Mb, found in cucurbit plants (e.g., melon and cucumber).Most plant mtDNA consistsof long inmobile DNA elementsrestrictedto the mitrons, pseudogenes, and piecesof foreign (chloroplast, compartment, tochondrial nuclearand viral) DNA that were probably insertedinto plant mitochondrial genomesduring their evolution' Duplicated sequencesalso contributeto the greaterlength of plant mtDNAs. Differences in the number of genes encoded by the mtDNA from various organisms most likely reflect the movement of DNA between mitochondria and the nucleus during evolution. Direct evidencefor this movement comes from the observation that several proteins encoded by mtDNA in some speciesare encoded by nuclear DNA in other, closely related species.The most striking example of this phenomenon involves the cor 11 gene, which encodes subunit 2 of cytochrome c oxidase, which constitutes complex IV in the mitochondrial electron transport chain (see Figure 12-1.6).This gene is found in mtDNA in all multicellular plants studied except for certain related speciesof legumes,including the mung bean and soybeans,in which the cox 11geneis nuclear.The cox 1I geneis completely missing from mung beanmtDNA, but a defectivecox 1/ pseudo-

gene that has accumulatedmany mutations can still be recognized in soybeanmtDNA. Many RNA transcripts of plant mitochondrial genesare edited, mainly by the enzyme-catalyzedconversion of selected C residuesto U, and occasionallyU to C' (RNA editing is discussedin Chapter 8.) The nuclear cox lI gene of mrng bean corresponds more closely to the edited cox II RNA transcripts than to the mitochondrial cox 11 genes found in other legumes. These observations are strong evidencethat the cox ll genemoved from the mitochondrion to the nucleusduring mung bean evolution by a processthat involved an RNA intermediate. Presumablythis movement involved a reverse-transcriptionmechanism similar to that by which processedpseudogenesare generatedin the nuclear genome from nucleus-encodedmRNAs. In addition to the large differences in the sizes of mtDNAs in different eukaryotes' the structure of the mtDNA also varies greatly.As mentioned above, mtDNA in most animals is a circular molecule -16 kb' However' the mtDNA of many organismssuch as the protist Tetrahymena exists as linear head-to-tail concatomersof repeating sequence. In the most extreme examples, the mtDNA of the Amoebidium parasiticum is composed of several protist 'h,rndred distinct short linear molecules'And the mtDNA of Trypanosoma is comprised of multiple maxicircles concate,r"t.d (ittt.tlocked) to thousands of minicircles encoding o R G A N E L L ED N A s

o

239

guide RNAs involved in editing the sequenceof the mitochondrial mRNAs encodedin the maxicircles.

P r o d u c t so f M i t o c h o n d r i aG l enes Are Not Exported As far as is known, all RNA transcripts of mtDNA and their translationproducts remain in the mitochondrion in which they are produced, and all mtDNA-encoded proteins are synthesizedon mitochondrial ribosomes. Mitochondrial DNA encodesthe rRNAs that form mitochondrial ribosomes, although most of the ribosomal proteins are imported from the cytosol.In animalsand fungi, all the tRNAs used for protein synthesisin mitochondria also are encoded by mtDNAs. However,in plantsand many prorozoans,most mitochondrialtRNAs are encodedby the nuclearDNA and imported into the mitochondrion. Reflectingthe bacterialancestryof mitochondria, mitochondrial ribosomesresemblebacterial ribosomesand differ from eukaryotic cytosolic ribosomesin their RNA and protein compositions,their size,and their sensitivityto certain antibiotics (seeFigure 4-22). For insrance,chloramphenicol blocks protein synthesisby bacterialand mitochondrial ribosomesfrom most organisms,but cycloheximidedoesnot. This sensitivity of mitochondrial ribosomes ro the imoortant aminoglycosideclass of antibiotics that includ., .hlo."-phenicol is the main causeof the toxicity that theseantibiotics can cause. Conversely,cytosolic ribosomes are sensitiveto cycloheximideand resisrantto chloramohenicol.I

M i t o c h o n d r i aE v o l v e df r o m a S i n g l e E n d o s y m b i o t iE c v e n tI n v o l v i n ga -l Rickettsi a i ke Bacteriu m Analysisof the mtDNA sequences from various eukaryotes, including single-celledprotists that diverged from other eu, karyotes early in evolurion, provides rtrong support for the idea that the mitochondrion had a singleorigin. Mitochondria most likely arosefrom a bacterialsymbiontwhoseclosest contemporary relatives are in the Rickettsiacedegroup. Bacteria in this group are obligate intracellular parasitei. Thus, the ancestor of the mitochondrion probably also had an intracellular life style, putting it in a good location for evolving into an intracellularuylbion,. ihe mtDNA with the largest number of encoded genesso far found is in the protist speciesReclinomonasamericana. All other mtDNAs have a subset of the R. amertcana genes)strongly implying that they evolved from a common ancestor with R. i*rrlcqna, losing different groups of mitochondrial genesby deletion and/or transfer to the nucleusover time. In organismswhose mtDNA includesonly a limited num_ ber of genes, rhe same set of mitochondrial genes are retained, independent of the phyla that includes these organ_ rsms (seeFigure 6-23, orangeproteins). One hypothesisfor why thesegeneswere never successfullytransferredto the nuclear genome is that their encoded polypeptides are too hydrophobic to cross the outer mitochondrial membrane. and 240

.

thereforewould not be imported back into the mitochondria if they were synthesizedin the cytosol. Similarly,the large size of rRNAs may interferewith their transport from the nucleus through the cytosol into mitochondria. Alternatively, these genesmay not have been transferred to the nucleus during evolution becauseregulation of their expressionrn response to conditions within individual mitochondria may be advantageous.If thesegeneswere locatedin the nucleus,conditions within each mitochondria could not influencethe exoression of proteins found in that particular mitochondrion.

M i t o c h o n d r i aG l e n e t i cC o d e sD i f f e r f r o m t h e S t a n d a r dN u c l e a rC o d e The geneticcode used in animal and fungal mitochondria is different from the standard code used in l[ prokaryotic and eukaryotic nuclear genes;remarkably, the code even differs in mitochondria from different species(Table 6-3). '$7hyand how these differencesarose during evolution is mysterious. UGA, for example, is normally a stop codon, but is read as tryptophan by human and fungal mitochondrial translation systems;however,in plant mitochondria, UGA is still recognized as a stop codon. AGA and AGG, the standardnuclear codons for arginine, also code for arginine in fungal and plant mtDNA, but they are stop codons in mammalian mtDNA and serinecodons in Droso,bila mtDNA. As shown in Table 6-3, plant mitochondria appear ro E utilize the standard genetic code. However, comparisons of the amino acid sequencesof plant mitochondrial proteins with the nucleotidesequences of plant mtDNAs suggestedthat CGG could code for either arginine (the ,,stand a r d " a m i n o a c i d ) o r t r y p r o p h a n .T h i s a f p a r e n r n o n s p e c i ficity of the plant mitochondrial code is explained by editing of mitochondrial RNA rranscripts,which can converr cyrosine residuesto uracil residues.If a CGG sequenceis edited to UGG, the codon specifiestryptophan, the standard amino acid for UGG, whereas unedited CGG codons encode the standard arginine. Thus the translation systemin plant mitochondria doesutilize the standard qeneticcode. I

M u t a t i o n si n M i t o c h o n d r i aD l N A C a u s eS e v e r a l G e n e t i cD i s e a s e isn H u m a n s The severity of diseasecausedby a mutation in mtDNA depends on the nature of the mutation and on the proportion of mutant and wild-type mtDNAs presentin a particular cell type. Generally when mutations in mtDNA are found, cells contain mixtures of wild-type and mutant mtDNAs-a condition known as heteroplasmy.Each time a mammalian somatic or germ-line cell divides, the mutant and wild-type mtDNAs segregaterandomly into the daughter cells, as occurs in yeast cells (seeFigure 6-2Zb). Thus, the mtDNA genotype, which fluctuates from one generation and from one cell division to the next, can drift toward predominantlv w i l d - t y p eo r p r e d o m i n a n r l ym u r a n r m r D N A ; . S i n c ea l l e n zymes required for the replication and growth of mammalian mitochondria, such as the mitochondrial DNA and

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proteins. "For nuclear-encoded p' 239; ed', Springer-Verlag. S. Andersonet al., 1981,Nature 290:457;P.Borst,in InternationalCell Biology 1980-1981,H. G' Schweiger, souRCEs: Biol' Deu' Cell In Vitro 1'986, Levings, C' S. and K. Eckenrode V. 10:478483; Sci. Trends Biochem. Raj Bhandary, 1985, and U. L. C. Breitenberger S. Covelloand M. W. Gtay,1,989,Nature34l:662-666. 22:169-176;J.M. Gualberetal., 1989,Nature34'1.:660-662;andP.

RNA polymerases,are encodedin the nucleusand imported from the cytosol, a mutant mtDNA should not be at a "replication disadvantage"lmutants that involve large deletionsof mtDNA might even be at a selectiveadvantagein replication becausethey can replicate faster. Recent researchsuggeststhat the accumulation of mutations in mtDNA is an important component of aging in mammals. Mutations in mtDNA have been observedto accumulate with aging, perhaps due to a decreasein the proofreading ability of DNA polymerase.To study this hypothesis, researchersused gene "knock-in" techniquesto replace the nuclear gene encoding mitochondrial DNA polymerase with normal proofreading activity (seeFigure 4-34) with a mutant gene encoding a polymerasedefectivein proofreading. Mutations in mtDNA accumulatedmuch more rapidly in homozygous mutant mice than in wild-type mice, and the mutant mice aged at a highly acceleratedrate (Frgure6-24). With few exceptions,all human cells have mitochondria, yet mutations in mtDNA affect only some tisThose most commonly affectedare tissuesthat have a sues. high requirementfor ATP produced by oxidative phosphorylation and tissuesthat require most of or all the mtDNA in the cell to synthesizesufficient amounts of functional mitochondrial proteins. For instance, Leber's hereditary optic neuropathy (degeneration of the optic nerve) is causedby a missensemutation in the mtDNA geneencoding subunit 4 of the NADH-CoQ reductase(complex I)' a protein required for ATP production by mitochondria. Any of severallarge deletionsin mtDNA causesanother set of diseasesincluding chronic progressiueexternal ophthalmo' plegia, characterizedby eye defects, and Kearns-Sayresyndrome, characterized by eye defects, abnormal heartbeat and central nervous system degeneration.A third condition, causing "ragged" muscle fibers (with improperly assembled mitochondria) and associateduncontrolled jerky movements,is due to a single mutation in the TIICG loop

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6-24 Micewith a mitochondrial FIGURE a EXPERIMENTAL DNA polymerasedefectivefor proofreadingexhibit " i c ew e r ep r e p a r e d p r e m a t u r ea g i n g .A l i n eo f " k n o c k - i nm inthe mutation w i t h a b y m e t h o dds i s c u s s ei ndC h a p t e5r i nactivates p o l y m e r a t s h e a t D N A m i t o c h o n d r i a l g e n ee n c o d i n g e r' so o f r e a d i nf ugn c t i o n(.a ) W i l d - t y paen d t h e p o l y m e r a sp ) he t i c ea t 3 9 0d a y so l d( 1 3m o n t h s T h o m o z y g o umsu t a n m mutanm t o u s ed i s p l a yms a n yo f t h ef e a t u r eosf a n a g e dm o u s e versusage (>720 days,or 24 monthsof age) (b) Plotof survival muranls' homozygous and heterozygous and mice of wild-type T h eh o m o z y g o umsu t a n t cs l e a r lhy a v ea m u c hs h o r t elri f es p a n 309:481 et al, 2005,Sclence G C Kujoth thanwild-typemice [From Gregory and Wisconsin-Madison of of JeffMiller/Universrty Part(a)courtesy PhD l Kuioth DNAS ORGANELLE

241

of the mitochondrial lysine IRNA. As a result of this mutation, the translation of several mitochondrial proteins apparentlyis inhibited. I

ChloroplastsContain LargeDNAsOften E n c o d i n gM o r e T h a n a H u n d r e dp r o t e i n s Like mitochondria, chloroplasts are thought to have evolved from an ancestral endosymbiotic photosvnthetic bacterium(seeFigure6-20). However,the endosymbiotic event giving rise to chloroplasts occurred more recently (L2-1,.5 billion yearsago) than the eventleadingto the evolution of mitochondria (1.5-2.2 billion years ago). Consequentlg contemporary chloroplast DNAs show less structural diversity than do mtDNAs. Also similar to mitochondria, chloroplasts contain multiple copies of the organellar DNA and ribosomes, which synthesizesome chloroplast-encodedproteins using the standard genetic code. Like plant mtDNA, chloroplastDNA is inherited exclusively in a uniparental fashion through the female parent (egg). Other chloroplast proteins are encoded by nuclear genes,synthesizedon cytosolic ribosomes,and then incorporated into the organelle(Chapter 13). I In higher plants, chloroplast DNAs are 120-160 kb long, depending on the species.They initially were thought to be circular DNA molecules becausein genetically tractable organisms like the model plant protozoan Chlamydomonas reinhardtii, the geneticmap is circular. However, recentstudies have revealed that plant chloroplast DNAs are actually long head-to-tail linear concatomersplus recombination intermediates between these long linear molecules. In these studies,researchershave used techniquesthat minimize mechanical breakageof long DNA molecules during isolation and gel electrophoresis,permitting analysisof megabase-size DNA. The complere sequencesof several chloroplast DNAs from higher plants have been determined in the past several years. They contain 120-135 genes, 130 in the important model plant Arabidopsis thaliana. A. thaliana chloroplast DNA encodes 76 protein-coding genes and 54 geneswith RNA products such as rRNAs and tRNAs. Chloroplast DNAs encode the subunits of a bacterial-like RNA folymeraseand expressmany of their genesfrom polycistronic operons as in bacteria (seeFigure 4-l3a). Some chloroplast genescontain introns, but theseare similar to the soecialized introns found in some bacterial genesand in mirochondrial genesfrom fungi and protozoans, rather than the introns of nuclear genes. As in the evolution of mitochondrial genomes,many genesin the ancestralchloroplast endosym_ biont that were redundant with nuclear genes -g.n., have beenlost from chloroplast DNA. Also, many .rr..rtial for chloroplast function have been transferred to rhe nuclear genome of plants over evolutionary time. Recent estimates from sequenceanalysisof the A. thaliana and cyanobacterial genomesindicate that -4,500 geneshave been transferred from the original endosymbiont to the nuclear genome. 242

CHAPTER 5

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G E N E sG , E N O M t C SA, N D C H R O M O S O M E S

Methods similar to those used for the transformation of yeast cells (Chapter 5) have been developedfor stably introducing foreign DNA into the chloroplasts of higher plants. The large number of chloroplast DNA moleculesper cell permits the introduction of thousandsof copiesof an engineeredgeneinto eachcell, resulting in extraordinarily high levels of foreign protein production. Chloroplast transformation has recently led to the engineeringof plants that are resistant to bacterial and fungal infections, drought, and herbicides. The level of production of foreign proteins is comparable with that achievedwith engineeredbacteria, making it likely that chloroplast transformation will be used for the production of human pharmaceuticalsand possibly for the engineeringof food crops containing high levelsof all the amino acids essentialto humans. I

Organelle DNAs r Mitochondria and chloroplastsmost likely evolved from bacteria that formed a symbiotic relationship with ancestral cells containing a eukaryotic nucleus (seeFigure 6-20). r Most of the genes originally within mitochondria and chloroplastswere either lost becausetheir funcdons were redundant with nucleargenesor moved to the nucleargenome over evolutionary time, leaving different gene sets in the organellarDNAs of different organisms(seeFigure 6-23). r Animal mtDNAs are circular molecules,reflecting their probable bacterial origin. Plant mtDNAs and chloroplast DNAs generally are longer than mtDNAs from other eukaryotes, Iargely becausethey contain more noncoding regions and repetitive sequences. r All mtDNAs and chloroplast DNAs encoderRNAs and some of the proteins involved in mitochondrial or photosynthetic electron transport and ATP synthesis.Most animal mtDNAs and chloroplast DNAs also encode the tRNAs necessaryto translate the organellar mRNAs. r Becausemost mtDNA is inherited from egg cells rather than sperm, mutations in mtDNA exhibit a maternal cytoplasmic pattern of inheritance. Similarlg chloroplast DNA is exclusivelyinherited from the maternal parent. r Mitochondrial ribosomes resemble bacterial ribosomes in their structure, sensitivity to chloramphenicol, and resistanceto cycloheximide. r The genetic code of animal and fungal mtDNAs differs slightly from that of bacteria and the nuclear genome and varies between different animals and fungi (seeTable 6-3). In contrast, plant mtDNAs and chloroplast DNAs appear to conform to the standard geneticcode. r Severalhuman neuromusculardisordersresult from mutations in mtDNA. Patients generally have a mixture of wild-type and mutant mtDNA in their cells (heteroplasmy): the higher the fraction of mutant mtDNA, the more severe the mutant phenotype.

Analysis Genome-wide ff,| Genomics: of GeneStructureand Expression Using automated DNA sequencingtechniques,methods for cloning DNA fragments on the order of 100 kb in length, and computer algorithms to piece together the stored sequence data, researchershave determined vast amounts of DNA sequenceincluding nearly the entire genomic sequence of humans and many key experimental organisms. This enormous volume of data,which is growing at a rapid pace, has been stored and organized in two primary data banks: the GenBank at the National Institutes of Health, Bethesda, Maryland, and the EMBL SequenceData Base at the European Molecular Biology Laboratory in Heidelberg, Germany. These databasescontinuously exchange newly reported sequencesand make them available to scientists throughout the world on the Internet. By now, the genome sequenceshave been completeln or nearly completely,determined for hundreds of viruses and bacteria, scores of archaea,yeasts (eukaryotes),and model multicellular eukaryotes such as the roundworm C. elegans, the fruit fly Drosophila melanogaster,mice, and humans. The cost of sequencinga megabaseof DNA has fallen so low that projects are underway to sequencethe entire genome in cancer cells and compare it to the genomein normal cells from the samepatient in order to determine all the mutations that have accumulatedin that patient's tumor cells. This approach may reveal genes that are commonly mutated in all cancers,as well as genesthat are commonly mutated in tumor cells from different patients with the same type of cancer (e.g., breast versus colon cancer). Such detailed information also may eventually lead to highly individualized cancer treatments tailored to the specific mutations in the tumor cells of a particular patient. In this section, we examine some of the ways researchersare mining this treasuretrove of data to provide insights about gene function and evolutionary relationships, to identify new geneswhose encodedproteins have never been isolated, and to determine when and where genes are expressed.This use of computers to analyze sequencedata has led to the emergenceof a new field of biology: b ioinformatics.

The most widely usedcomputer program for this purpose is known as BLAST (basic/ocal alignment searchrool). The BLAST algorithm dividesthe "new" protein sequence(known as the query sequence) into shorter segments and then searchesthe databasefor significant matches to any of the The matching program assignsa high score stored sequences. to identically matched amino acids and a lower score to matchesbetweenamino acidsthat are related (e.g.,hydrophobic, polar, positively charged, negatively charged) but not identical.When a significantmatch is found for a segment'the BLAST algorithm will searchlocally to extend the region of similarity.After searchingis completed,the program ranks the matches betweenthe query protein and various known proteins accordingto their p-ualwes.This parameteris a measure of the probability of finding such a degreeof similarity between two protein sequencesby chance. The lower the pvalue, the greater the sequencesimilarity between two sequences.A p-value lessthan about 10-' usually is considered as significantevidencethat two proteins sharea common ancestor.Many alternativecomputer programs have beendeveloped in addition to BLAST that can detect relationshipsbetween proteins that are more distantly related to each other than can be detected by BLAST. The development of such methodsis currently an active areaof bioinformaticsresearch. To illustrate the power of this approach, we consider the human geneNF1 . Mutations in NF1 are associated with the inherited diseaseneurofibromatosis f in which multiple tumors develop in the peripheral nervous system' causinglarge protuberancesin the skin. After a cDNA clone of NFl was isolated and sequenced,the deducedsequenceof the NF1 protein was checked against all other protein sequencesin GenBank. A region of NF1 protein was discovered to have considerablehomology to a portion of the yeast protein calledIra (Figure6-25). Previousstudieshad shown that Ira is a GTPase-activatingprotein (GAP) that modulates the GTPaseactivity of the monomeric G protein called Ras (seeFigure 3-32). As we examine in detail in Chapter 16, GAP and Ras proteins normally function to control cell

StoredSequencesSuggestFunctionsof Newly ldentified Genesand Proteins As discussedin Chapter 3, proteins with similar functions often contain similar amino acid sequencesthat correspond to important functional domains in the three-dimensional structure of the proteins. By comparing the amino acid sequence of the protein encoded by a newly cloned gene with the sequencesof proteins of known function, an investigator can look for sequencesimilarities that provide clues to the function of the encoded protein. Becauseof the degeneracy in the geneticcode, related proteins invariably exhibit more sequencesimilarity than the genesencoding them. For this reason, protein sequencesrather than the corresponding are usuallvcompared. DNA seouences

characteristicof the disease.I Even when a protein shows no significant similarity to other proteins with the BLAST algorithm, it may nevertheless share a short sequencethat is functionally important' Such short segmentsrecurring in many different proteins, referred to as structural motifs, generally have similar functions. Several such motifs are described in Chapter 3 and illustratedin Figure3-9.To searchfor theseand other motifs in a new protein, researcherscompare the query protein sequencewith a databaseof known motif sequences.

243

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o n t h e t o p a n d b o t t o m l i n e so f e a c hr o w , r e s p e c t i v e liyn,t h e o n e l e t t e ra m i n o a c i dc o d e ( s e eF i q u r e2 - 1 4 ) A m i n o a c i d st h a t a r e i d e n t i c ailn t h e t w o p r o t e i n sa r e h i g h l i g h t e di n y e l l o w A m i n o a c i d s w i t h c h e m i c a l isyi m i l a rb u t n o n i d e n t i c asli d ec h a i n sa r e c o n n e c t e d

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lins present in different organisms today evolutionary relationshipscan be deduced,as illustratedin Figure 6-26b. Of the three types of sequencerelationships,orthologous sequencesare the most likely to share the same function.

BLAST searchesfor related protein sequencesmay reveal that proteins belong to a protein family. Earlier,we considered gene families in a single organism, using the B-globin genesin humans as an example (seeFigure 6-4a). But in a databasethat includes the genome sequencesof multiple organisms,protein families also can be recognizedas being shared among related organisms. Consider, for example, the tubulin proteins; rheseare the basic subunits of microtubules,which are important componentsof the cytoskeleton (Chapter 18). According to the simplified schemein Figure 6-26a, the earliest eukaryotic cells are thought to have contained a single tubulin gene that was duplicated early in evolution; subsequentdivergenceof the different copies of the original tubulin gene formed the ancestral versionsof the ct- and p-tubulin genes.As different species diverged from these early eukaryotic cells, each of these gene sequencesfurther diverged,giving rise ro the slightly different forms of ct-tubulin and B-tubulin now found in each species. All the different membersof the tubulin family of genes (or proteins)are sufficienrlysimilar in sequencero suggesta common ancestralsequence.Thus all these sequencesare consideredto be homologous.More specificallSsequences that presumably diverged as a result of gene duplication (e.g.,the ct- and B-tubulin sequences) are describedas paralogous. Sequences that arosebecauseof speciation(e.g.,the ct-tubulin genesin different species)are describedas irthologous.From the degreeof sequencerelatedness of the tubu244

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G e n e sC a nB e l d e n t i f i e dW i t h i n G e n o m i c DNASequences The complete genomic sequenceof an organism contains within it the information neededto deducethe sequenceof every protein made by the cells of that organis-. Fo. organisms such as bacteria and yeast, whose genomeshave few introns and short intergenic regions, mosr prorelncoding sequencescan be found simply by scanningthe genomic sequencefor open reading frames (ORFs) of significant length. An ORF usually is defined as a srretch of DNA containing at least 100 codons that begins with a start codon and ends with a stop codon. Becausethe probability that a random DNA sequencewill contain no stop codons for 100 codons in a row is very small, most ORFs encodeprotein. ORF analysiscorrectly identifiesmore than 90 percent of the genesin yeast and bacteria.Some of the very shortest genes,however,are missedby this method, and occasionally long open reading frames that are not actually genesarise by chance. Both types of mis-assignmentscan be corrected by more sophisticatedanalysis of the sequenceand by genetic tests for gene function. Of the Saccharomycesgenesidentified in this manner,about half were alreadyknown by some functional criterion such as mutant phenotype. The func, tions of some of the proteins encodedby the remainingputative (suspected)genes identified by ORF analysis have

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during FIGURE 6-26 Generationof diversetubulin sequences givingriseto mechanism the evolutionof eukaryotes.(a)Probable that foundin existing lt ispossible to deduce species thetubulingenes thecrbecause eventoccurred beforespeciation a geneduplication (eg , humans are andyeast) fromdifferent species tubulinsequences withina sequences andP-tubulin morealikethanarethect-tubulin (b)A phylogenetic therelationship between treerepresenting species points(nodes), indicated bysmall Thebranch thetubulinsequences genes at thetimethattwo represent common ancestral numbers,

theduplication node1 represents divergedForexample, sequences andnode2 families, andB-tubulin eventthatgaveriseto theo-tubulin Braces and species yeast multicellular from of the divergence represents which tubulin9enes, theorthologous respectively, arrowsindicate, genes, whichdiffer andthe paralogous drfferasa resultof speciatron, somewhat issimplified Thisdiagram of geneduplication asa result multiple contains actually represented eachof thespecies because fromlatergeneduplication genes thatarose andB-tubulin ct-tubulin events

been assignedbased on their sequencesimilarity to known proteinsin other organlsms. Identification of genesin organismswith a more complex genome structure requires more sophisticatedalgorithms than searching for open reading frames. Because most genesin higher eukaryotesare composedof multiple, relatively short exons separatedby often quite long noncoding introns, scanning for ORFs is a poor method for finding genes.The best gene-finding algorithms combine all the available data that might suggestthe presenceof a gene at a particular genomic site. Relevant data include alignment or hybridization of the query sequenceto a fulllength cDNA; alignment to a partial cDNA sequence,generally 200-400 bp in length, known as afl expressedse' quence tag (EST); fitting to models for exon, intron, and splice site sequences;and sequencesimilarity to other organisms.Using thesecomputer-basedbioinformatic methods, computational biologistshave identified approximately 25,000 genesin the human genome. However, for some 10,000 of these putative genesthere is not yet conc l u s i v e e v i d e n c et h a t t h e y a c t u a l l y e n c o d e p r o t e i n s o r RNAs. A particularly powerful method for identifying human genesis to compare the human genomic sequencewith that of the mouse. Humans and mice are sufficiently related to have most genesin common, although largely nonfunctional such as intergenicregionsand introns,will DNA sequences, are not untend to be very differentbecausethesesequences der strong selectivepressure.Thus correspondingsegments of the human and mousegenomethat exhibit high sequence similarity are likely to be functional coding regions,that is,

exons, transcription-control regions, or sequenceswith other functions that are not yet understood.

genes T h e N u m b e ro f P r o t e i n - C o d i nG s e n o m el s N o t D i r e c t l y in an Organism'G Complexity Biological lts Relatedto The combination of genomic sequencingand gene-finding computer algorithms has yielded the complete inventory of protein-coding genesfor a variety of organisms.Figure 6-27 iho*t the total number of protein-coding genesin several eukaryotic genomes that have been completely sequenced. The functions of about half the proteins encoded in these genomesare known or have been predicted on the basis of sequencecompansons.One of the surprising featuresof this comparison is that the number of protein-coding genes within different organisms does not seem proportional to our intuitive senseof their biological complexity. For example, the roundworm C. elegansapparently has more genes than the fruit fly Drosophila, which has a much more complex body plan and more complex behavior. And humans have fewer than one and one-half the number of genesas C' elegans.\fhen it first became apparent that humans have fewer than twice the number of protein-coding genesas the simple roundworm. it was difficult to understand how such a small increasein the number of proteins could generate such a staggeringdifferencein complexity. Clearly, simple quantitative differencesin the number of genes in the genomes of different organisms are inadequate for explaining differences in biological complexity' Howeveq severalphenomenacan generatemore complexity 245

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A FIGURE 6-27 Comparison of the numberand typesof proteinsencodedin the genomesof different eukaryotes.For eachorganism, the areaof theentirepiechartrepresents thetotal numberof protein-coding genes, allshownat roughly the same scaleIn mostcases, thefunctions of the proteins encoded by about halfthe genesarestillunknown(lightblue)Thefunctions of the remainder, whichareknownor havebeenpredicted by sequence similarity to genesof knownfunction, areclassified assnownIn the colorkey lAdapted fromInternational Human Genome Seouencino Consortium, 2001,Nature 409:860 l

kinds of proteins. Larger numbers of cells can interact in more complex combinations, as in comparing the cerebral cortex from mouse to man. Similar cells are presentin both the mouse and human cerebralcortex, but in humans more of them make more complex connections.Evolution of the increasing biological complexity of multicellular organisms likely required increasinglycomplex regulation of cell replication and geneexpression,leading to increasingcomplexity of embryological development. The specificfunctions of many genesand proteins identified by analysisof genomic sequencesstill have not been determined.As researchersunravel the functions of individual proteins in different organisms and further detail their interactions with other proteins, the resulting advanceswill becomeimmediately applicableto all homologous proteins in other organisms. When the function of every protein is known, no doubt, a more sophisticatedunCHAPTER 5

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P r o t e i nf o l d i n g a n d d e g r a d a t i o n Transport

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M u l t i f u n c t i o n apl r o t e i n s Cytoskeleton/structu re D e f e n s ea n d i m m u n i t y M i s c e l l a n e o ufsu n c t i o n Unknown

in the expressedproteins of higher eukaryotes than is predicted from their genomes.First, alternativesplicing of a

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derstandingof the molecular basis of complex biological systemswill emerge.

S i n g l eN u c l e o t i d eP o l y m o r p h i s masn d G e n e Copy-NumberVariationAre lmportant Determinantsof DifferencesBetween I n d i v i d u a l so f a S p e c i e s The DNA sequencebetweenindividual humans who are not closelyrelateddiffers at about L-2 percentof the 3 x 10ebase pairs in the human genome.Most of thesedifferences,called single nucleotide polymorpbisms (SNPs), are probably not functionally significant becausethey occur in long introns or betweengenes,or result in synonymouscodon changesin coding regions. Nonetheless,such SNPs are important markers for measuringthe frequencyof recombination betweengenes and can be used to link a specificgenewith a trait or phenotype as discussedin Chapter 5 (seeFigure 5-36). On the other hand, someSNPsmay be functionally significantbecausethey result in amino acid changes in protein-coding regions or base-pairchangesin control regionsthat affect the binding of transcription factors. Thesesingle nucleotidepolymorphisms clearly contribute to differencesbetweenindividuals.

c E N E S ,G E N O M | C SA, N D C H R O M O S O M E S

A secondhighly significant type of geneticvariation, differencesin gene-copynumber, was discoveredvery recently. Reper cent analysesof the number of copiesof DNA sequences cell in different individualsrevealedwidespreaddeletions,tandem duplications,and complex combinationsof deletionsand duplicationsthat vary benveenindividuals over a remarkably high:12 percentof the genome.The deletionsaverage:40 kb in length, and the tandem duplicationsavenge:120 kb, but some deletionsand duplicationsare much longer.Thesevarying deletionsand duplications probably arose from unequal crossingover befweenchromosomesduring meiotic recombination in a direct ancestor(seeFigure 6-2). This resultsin differencesin gene-copynumbers betweenindividuals. In some individuals. for instance.a deletion of DNA sequenceoccurson one chromosomebut the normal sequenceis presenton the homologouschromosome;as a result they have only a single copy of genesin the deletedregion. Likewise, some individuals contain a duplication of some geneson one chromosomethat is not presenton the homologous chromosome, resulting in three copies of genesin the duplicated region. Another possibility found in someindividuals is a duplication on both homologous chromosomes,generating four gene copies;additional duplications on one or both chromosomescan lead to genecopy numbersgreaterthan four. These copy-numberuariationsare inherited in a Mendelian manner, as for other alleles,and are occasionallygeneratedas a new variation not observedin the DNA of either parent. Copy-number variations are evenmore common between individuals than differencesin DNA sequence(SNPs).Since variations in genecopy number can affect the amount of protein expressedfrom a gene,copy-number variations may be among the most important determinantsof individual differencesbetweenhumans, including differencesin susceptibility to various diseases.Studiesare currently underway to determine the influence of gene copy number variations on individual traits including diseasesusceptibility.

Genomics:Genome-wide Analysis of Gene Structure and Expression r The function of a protein that has not been isolated (a query protein) often can be predicted on the basis of similarity of its amino acid sequenceto the sequencesof proteins of known function. r A computer algorithm known as BLAST rapidly searches databasesof known protein sequencesto find those with significantsimilarity to a query protein. r Proteinswith common functional motifs, which often can be quite short, may not be identified in a typical BLAST search.Such short sequencesmay be located by searchesof motif databases. r A protein family comprisesmultiple proteins all derived from the same ancestralprotein. The genesencoding these proteins, which constitute the corresponding gene family, arose by an initial gene duplication event and subsequent divergenceduring speciation (seeFigure 6-26).

r Relatedgenesand their encodedproteins that derive from a gene duplication event are paralogous' such as the o and B-globinsthat combine in hemoglobin (ct2B2);those that derive from mutations that accumulated during speciation are orthologous. Proteinsthat are orthologous usually have a similar function in different organisms,such as the mouse and human adult B-globins. r Open reading frames (ORFs) are regions of genomic DNA containing at least 100 codons located between a start codon and stop codon. r Computer search of the entire bacterial and yeast genomic sequencesfor open reading frames (ORFs) correctly identifies most protein-coding genes.Severaltypes of additional data must be used to identify probable (putative) genesin the genomic sequencesof humans and other higher eukaryotesbecauseof their more complex genestructure in which relatively short coding exons are separatedby relatively long, noncoding rntrons. r Analysis of the complete genome sequencesfor several different organisms indicates that biological complexity is not directly related to the number of protein-coding genes (seeFigure6'27).

StructuralOrganization f[ of EukaryoticChromosomes Now that we have examined the various types of DNA found in eukaryotic genomesand how they are orsequences ganizedwithin it, we turn to the question of how DNA molecules as a whole are organizedwithin eukaryotic cells. Because the total length of cellular DNA is up to a hundred thousand times a cell'sdiameter,the packing of DNA is crucial to cell architecture.It is also essentialto prevent the long DNA molecules from getting knotted or tangled with each other during cell division when they must be preciselysegregatedto daughter cells.The task of compacting and organizingchromosomal DNA is performed by abundant nuclear proteins called histones. As noted previously'the complex of histones,nonhistone proteins, and DNA constituteschromatin' which exists in various degreesof folding or compaction (seeFigure 6-1)' Chromatin, which is about half DNA and half protein by mass, is dispersedthroughout much of the nucleus in interphase cells (those that are not undergoing mitosis). Further folding and compaction of chromatin during mitosis producesthe visible metaphasechromosomes,whose morphology and staining characteristicswere detailed by early cytogeneticists.Although every eukaryotic chromosome includes millions of individual protein molecules,each chromosome contains iust one, extremely long, linear DNA molecule.The lonqest DNA molecules in human chromosomes,for inst"n.., are2.8 x 108 basepairs, or almost 10 cm, in length! The structuraI organization of chromatin allows this vast length of DNA to be compacted into the microscopic constraints of a cell nucleus.Yet chromatin is organized in such a way that specificDNA sequenceswithin the chromatin are

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< EXPERIMENTAL FIGURE 6-28 Theextended and condensedforms of extractedchromatin have very different appearancesin electron (a)Chromatin micrographs. isolated in low-ionic"beads-on-astrengthbufferhasan extended string"appearance The"beads"arenucleosomes (10-nmdiameter) andthe "string"isconnecting (linker) DNA (b)Chromatin isolated in bufferwith (0 15M KCI)appears a physiological ionicstrength asa condensed fiber30 nm in diameter. lpart(a) courtesy of 5 McKnight andO Miller, Jr.Part(b)courtesy o f B H a m k a l o a nBdRJa t t n e r l

readily availablefor cellularprocesses like the transcription, replication, repair, and recombination of DNA molecules.In this section,we consider the properties of chromatin and its organizationinto chromosomes.Important featuresof chromosomesin their entirety are covered in the next section.

dant proteins in chromarin, constitute a family of small, basic proteins. The five major types of histone proteinstermed H1, H2A, H2B, H3, and H4-are rich in positively charged basic amino acids, which interacr with the negatively charged phosphategroups in DNA. 'When chromatin is extracted from nuclei and examined in the electronmicroscope,its appearancedependson the salt concentration to which it is exposed.At low salt concentration in the absenceof divalent cations such as Mg*', isolated chromatin resembles"beadson a string" (Figure 6-28a).In this extendedform, the string is composedof free DNA called "linker" DNA connectingbeadlike structurestermed nucleosomes. Composed of DNA and histones, nucleosomesare

ChromatinExistsin Extended a n d C o n d e n s e dF o r m s lfhen the DNA from eukaryotic nuclei is isolated using a method that preservesnative protein-DNA interactions,it is associatedwith an equal mass of protein in the nucleoprotein complex known as chromatin.Histones,the most abun-

FIGURE 5-29 Structureof the nucleosome basedon x-ray (a)Nucleosome crystallography. with space-filling modelof the histones. Thesugar-phosphate backbones of the DNAstrands are represented asgraytubesto allowbettervjsualizatton of the histonesNucleosome shownfromthetop (/eft)andfromthe side (right',the sideviewis rotatedclockwise 90. f romthe rop vrew.,r (b)SpaceJilling modelof histones andDNA(white)viewedfromthe 248

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sideof the nucleosome. Thismodelshowsmoreclearly thatDNA "covers"muchof the proteinon the nucleosomes lateral surface. H2Asubunits aregold;H2Bsarered;H3sareblue;H4saregreen TheN-terminal tailsof the eighthistones andthetwo H2AandH2B C-terminal tailsinvolved in condensation of chromatin arenotvisible because theyaredisordered in the crystal. etal, 1997, [AfterK Luger Nature 389:251 l

G E N E SG , E N O M | C SA, N D C H R O M O S O M E S

about 10 nm in diameter and are the primary structural units of chromatin. If chromatin is isolated at physiological salt concentration, it assumesa more condensedfiberlike form that is 30 nm in diameter(Figure6-28b). Structure of Nucleosomes The DNA componentof nucleosomesis much lesssusceptible to nucleasedigestionthan is the linker DNA between them. If nucleasetreatment is carefully controlled,all the linker DNA can be digested,releasingindividual nucleosomeswith their DNA component. A nucleosome consistsof a protein core with DNA wound around its surface like thread around a spool. The core is an octamercontaining two copieseach of histonesHZA, H2B, H3, and H4. X-ray crystallography has shown that the octameric histone core is a roughly disk-shapedstructure made of interlocking histonesubunits (Figure6-29). Nucleosomesfrom all eukaryotescontain 147 base pairs of DNA wrapped one and nvo-thirds turns around the protein core. The length of the linker DNA is more variable among species,and even betweendifferent cells of one organism,ranging from about 10 to 90 basepairs. During cell replication,DNA is assembledinto nucleosomesshortly after the replicationfork passes(seeFigure 4-33). This processdependson specificchaperonesthat bind to histonesand assemble them together with newly replicated DNA into nucleosomes. Structure of the 30-nm Fiber When extracted from cells in isotonic buffers (i.e.. buffers with the samesalt concentration found in cells, :0.15 M KCl, 0.004 M MgCl2), most chromatin appearsas fibers :30 nm in diameter(seeFigure 6-28b). Current research,includingX-ray crystallographyof nucleosomesassembledfrom recombinant histones,indicate that the 30-nm fiber has a "zig-zagribbon" structurethat is wound into a "two-start" helix made from two "strands" of nucleosomesstacked on top of each other like coins (Figure 6-30). The two "strands" of stackednucleosomesare then wound into a double helix similarly to the two strands in a DNA double helix, except that the helix is left handed, rather than right handed as it is in DNA. The 30-nm fibers also includeH1, the fifth major histone.Hl is bound to the DNA as it entersand exits the nucleosomecore, but its structure in the 30-nm fiber is not known at atomic resolution. The chromatin in chromosomalregionsthat are not being transcribed or replicated exists predominantly in the condensed,30-nm fiber form and in higher-order folded structures whose detailed conformation is not currently understood. The regions of chromatin actively being transcribed are thought to assumethe extendedbeads-on-a-stringform. Conservation of Chromatin Structure The generalstructure of chromatin is remarkably similar in the cells of all eukaryotes, including fungi, plants, and animals, indicating that the structure of chromatin was optimized early in the evolution of eukaryotic cells.The amino acid sequencesfor four histones (H2A, IJ'ZB, H3, and H4) are highly conservedbetween distantly related species.For example,the sequencesof histone H3 from sea urchin tissue and calf thymus differ by only a single amino acid, and H3 from the garden pea and calf thymus differ only in four amino acids. Apparently significant deviations

from the histone amino acid sequenceswere selectedagainst strongly during evolution. The amino acid sequence'ofH1, however, varies more from organism to organism than do the sequencesof the other major histones.The similarity in sequence among histones from all eukaryotes suggeststhat they fold into very similar three-dimensionalconformations, which were optimized for histone function early in evolution in a common ancestorof all modern eukaryotes'

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6-30 Structureof the 30-nmchromatinfiber' (a)Model FIGURE ribbon" chainat top intoa "zig-zag for thefoldingof a nucleosomal " In each"strand"the two "strands containing of nucleosomes with eachotherlikea stackof coins.These arealigned nucleosomes "strands" arethenwoundintoa left-handed nucleosomes of two DNAisnot doublehelixcalleda "two-start"helixForsimplicity, helix(b)Modelof the 30-nmfiber in thetwo-start represented (a shortstretch of a tetranucleosome basedon x-raycrystallography iscolored nucleosomes on alternating DNA of four nucleosomes) (a) them [Part distinguishing to simplify lightanddarkblue,respectively, adapted from C L F.Woodcocket al , 1984, J CellBiol 99:42 Part(b) from T S c h a l c he t a l , 2 0 0 5 , N a t u r e4 3 5 : 13 8 l

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Minor histone variants encodedby genesthat differ from the highly conservedmajor types also exist, particularly in vertebrates.For example, a specialform of H2A, designated H2AX, is incorporated into nucleosomesin place of H2A in a small fraction of nucleosomesin all regionsof chromatin. At sites of DNA double-strandedbreaks in chromosomal DNA, H2AX becomesphosphorylatedand participaresin the chromosome-repairprocess,probably by functioning as a binding site for repair proteins. In the nucleosomesat centromeres,H3 is replacedby another variant histone called CENP-A, which participatesin the binding of spindle microtubules during mitosis. Most minor histone variants differ only slightly in sequencefrom the major histones.These slight changesin histonesequence may influencethe stability of the nucleosome as well as its rendency to fold into the 30-nm fiber and other hieher-orderstructures.

termini, called histone tails, are represented in the model shown in Figure 6-31.a.The histone tails are required for chromatin to condensefrom the beads-on-a-stringconformation into the 30-nm fiber. For example, recent experiments indicate that the N-terminal tails of histone H4, particularly lysine 16, are critical for forming the 30-nm fiber. This positively charged lysine interacts with a negative patch at the H2A-H2B intefiace of the nexr nucleosome in the stacked nucleosomesof the 30-nm fiber (see F i g u r e6 - 3 0 ) . Histone tails are subject to multiple post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. Figure 6-31b summarizesthe types of post-translationalmodifications observedin human histones.A particular histone protein never has all of these modifications simultaneously, but the histones in a single nucleosomemay collectivelycontain a severaldifferent types of modifications. The particular combinations of posttranscriptional modifications found in different regions of chromatin have been suggestedto constitute a bistone code that influenceschromatin function by creating or removing binding sitesfor chromatin-associatedproteins. Here we describethe most abundant kinds of modifications found in histone tails and how these modifications control chromatin condensation and function. We end with a discussionof a

Modificationsof HistoneTailsControl C h r o m a t i nC o n d e n s a t i o a nn d F u n c t i o n Each of the histone proteins making up the nucleosome core contains a flexible N-terminus of 19-39 residuesextending from the globular structureof the nucleosome;the H2A and H2B proteins also contain a flexible C-terminus extending from the globular histone octamericcore. These

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Ub U b i q u i t i n a t i o m n ark FIGURE6-31 Histone tails and their post-translational modifications. (a) Model of a nucleosome viewedfrom top with histonesdepictedas ribbondiagramsThismodeldepictsthe lengths of the histonetails(dottedlines),which are not visiblein the crystal structure(seeFigure6-29).The H2A N-terminaltailsare at the bottom, and C-terminaltails,at the top The H2B N-terminaltatls are on the right and left, and C-terminaltailsat the bottom center HistonesH3 and H4 haveshortC-terminaltailsthat are not modified

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(b)Summary of post-translational modifications observed in human histones Histone-tail sequences areshownin theone-letter aminoacid code(seeFigure2-14)Themainportionof eachhistoneisdepicted as an ovalThese modifications do notalloccursimultaneously on a single histonemoleculeRather, specific combinations of a few modifications of onehistone areobserved in anyparticular nucleosome [part(a)from K Luger andT.J Richmond, 1998, CurrOpinGenet. & Deret8:140part(b) adapted fromR Margueron etal,2005,Curr. OpinGenet& Devel. 15:1631

G E N E SG , E N O M t C SA,N D C H R O M O S O M E S

special case of chromatin condensation,the inactivation of X chromosomesin femalemammals. Histone Acetylation Histone-tail lysines undergo reversible acetylation and deacetylation by enzymesthat act on specific lysines in the N-termini. In the acetylatedform, the positive charge of the lysine e-amino group is neutralized. As mentioned above, lysine 16 in histone H4 is particularly important for the folding of the 30-nm fiber becauseit interacts with a negatively charged patch on the surface of the neighboring nucleosome in the fiber. Consequently, when H4 lysine 16 is acetylated,the chromatin tends to form the less condensed "beads-on-a-string" conformation conducive for transcription and replication. Histone acetylation at other sitesin H4 and in other histones (seeFigure6-31b) is correlatedwith increasedsensitivity of chromatin DNA to digestion by nucleases.This phenomenon can be demonstrated by digesting isolated nuclei with DNase L Following digestion, the DNA is completely separated from chromatin protein, digested to completion with a restriction enzyme, and analyzed by Southern blotting. An intact gene treated with a restriction enzyme yields fragments of characteristicsizes.If a gene is exposedfirst to DNase, it is cleavedat random siteswithin the boundariesof the restriction enzymecut sites.Consequently,any Southern blot bands normally seen with that gene will be lost. This method has been used to show that the transcriptionally inactive B-globingenein non-erythroidcells,where it is associated with relatively unacetylated histones, is much more resistant to DNase I than is the active, transcribed B-globin gene in erythroid precursor cells, where it is associatedwith acetylatedhistones(Figure6-32). Theseresultsindicatethat the chromatin structure of nontranscribedDNA is more condensed,and therefore more protected from DNase digestion, than that of transcribed DNA. In condensedchromatin, the DNA is largely inaccessibleto DNase I becauseof its close associationwith histones and other chromatinassociatedproteins that bind to unacetylatedhistone tails. In contrast, actively transcribed DNA is much more accessible to DNase I digestion becauseit is present in the extended, beads-on-a-stringform of chromatin. Genetic studies in yeast indicate that histone acetylases (HATs), which acetylatespecific lysine residuesin histones, are required for the full activation of transcription of a number of genes.Consequently,the control of acetylation of histone N-termini in specific chromosomal regions is thought to contribute to the transcriptional control of gene expression by mechanismsdescribedbelow and in the next chapter. Just as genesin condensed,folded regions of chromatin are less accessibleto exogenouslyadded DNase I than genesin decondensed, extended regions of chromatin, RNA polymeraseand other proteins required for transcription are also inhibited from interactins with DNA in condensed chromatin. Other Histone Modifications As shown in Figure6-31.b, histone tails in chromatin can undergo a variety of other covalent modificationsat specificamino acids.Lysinee-amino

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the C-terminal tails of H2A and H2B. Recall that addition of multiple linked ubiquitin moleculesto a protein can mark it for degradationby the proteasome(seeFigure3-Z9b).In this case, however, the addition of a single ubiquitin molecule does not affect the stability of a hisrone, although it does influence chromatin strucrure. As mentioned previouslS it is the precisecombination of modified amino acids in histone tails that helps control the condensation,or compaction,of chromatin and its ability to be transcribed,replicated,and repaired.This can be illustrated by comparing the specific modifications observed in highly condensedchromarin, known as heterochromatin, with those in less condensedchromatin, known as euchromatin (Figure6-33a).Heterochromatindoesnot fully decondensefollowing mitosis, remaining in a compactedstate during interphase.It is typically found at the cenrromeresand telomeresof chromosomes,as well as some other discretelocations.'Whencellsare subjectedto dyesthat bind DNA, regions of heterochromatinstain very darkly. In contrast, areas of euchromatin, which are in a less compacted state during interphase,stain lightly with DNA dyes.Typically,most transcribedregionsof DNA are found in euchromatin,while heterochromatin remains transcriptionally inactive. Reading the Histone Code The histonecodeis "read" by proteinsthat bind to the modified histonetails and in turn promote condensationor decondensationof chromatin, forming "closed" or "open" chromatin structures.Higher eukaryotes expressa number of proteins containing a so-calledchromodomain that binds to histone rails when they are methylated at specific lysines. One example is heterochromatin protein 1 (HP1).In addition to histones,HP1 is one of rhemajor proteins associatedwith heterochromatin. The HP1 chromodomain binds the H3 N-terminal tail only when it is tri-methylated at lysine9 (Figure6-33b).HP1 alsoconrainsa seconddomain called a chromosbadow domain becauseit is frequently found in proteins that contain a chromodomain. The chromoshadowdomain binds to other chromoshadow domains. Consequentlg chromatincontainingH3 tri-methylatedat lysine9 (H3K9Me3) is assembledinto a condensedchromatin structure by HP1, although the srructure of this chromatin is not well understood (Figure6-34a). In addition to binding to itself, the chromoshadow domain also binds the enzyme that methylatesH3 lysine 9, H3K9 histone methyl transferase (HMT). As a consequence,nucleosomesadjacentto a region of HP1-containing heterochromatin also become methylated at lysine 9 (Figure 6-34b). This createsa binding site for another Hp1 that can bind the H3K9 histone merhyl transferase, resulting in "spreading" of the heterochromatinstrucrure along the chromosome until a boundary element is encountered that blocks further spreading. Boundary elements so far characterizedare generally regions in chromatin where several nonhistone proteins bind to DNA. p o s s i b l yb l o c k i n gh i s t o n em e t h y l a i i o no n r h e o r h e r s i d e o f the boundary. Significantly,the model of heterochromatin formation in Figure 6-34b provides an explanation for how heterochro252

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FIGURE 6-33 Heterochromatin versuseuchromatin.(a)Inthis electron micrograph of a bonemarrowstemcell,thedark-staining areas (N)outside (n)areheterochromatin in thenucleus thenucleolus The (b)Themodifications light-staining, whitishareas areeuchromatin of histone N-terminal tailsin heterochromatin andeuchromatin differ, as illustrated herefor histone H3 Notein oarticular thathistone tarlsare generally muchmoreextensively acetylated in euchromatin compared with heterochromatin (thus Heterochromatin ismuchmorecondensed lessaccessible to proteins) andismuchlesstranscriptionally activethan iseuchromatin PC CrossandK L Mercer, 1993,Cell andTissue [Part(a) Ultrastructure, W H Freeman p 165 Part(b)adapted andCompany, fromT. Jenuwein andC D Allis, 2001, Science293:10741

matic regions of a chromosome are re-establishedfollowing DNA replication during the S phase of the cell cycle. \fhen DNA in heterochromatin is replicated,the histone octamers that are tri-methylated at H3 lysine 9 becomedistributed to both daughter chromosomesalong with an equal number of newly assembled histone octamers. The H3K9 histone methyl transferaseassociatedwith the H3K9 tri,methylated nucleosomesmethylate lysine 9 of the newly assembled nucleosomes,regenerating the heterochromatin in both daughterchromosomes.

c E N E S ,G E N O M t C SA, N D C H R O M O S O M E S

< FIGURE5-34 Model for the formation of heterochromatin by binding of HP1 to histone H3 tri-methylated at lysine 9. (a) by binding of heterochromatin HP1contributesto the condensation at lysine9 (H3K9Mer), to histoneH3 N-terminaltailstri-methylated with each of histone-boundHP'lmolecules followedby association can spreadalonga condensation other (b) Heterochromatin (HMT) chromosomebecauseHP1bindsa histonemethyltransferase that methylateslysine9 of histoneH3 Thiscreatesa bindingsitefor HPl on the neighboringnucleosomeThe spreadingprocess continuesuntil a "boundaryelement" is encounteredlPart(a)adapted from G Thielet al . 2004,Eur.J Biochem271:2855Part(b)adaptedfrom 410i120 et al . 2001,Nature A J Bannister I

I ui.,on"H 3 K 9 methvl transferase I

I a i n u i nosf H e t chromodomain to H3K9Me3 J

In summarS multiple types of covalent modifications of histone tails can influence chromatin structure by altering nucleosome-nucleosomeinteractions and interactions with additional proteins that participatein or regulateprocesses suchas transcriptionand DNA replication.The mechanisms and molecular processesgoverning chromatin modifications that regulatetranscriptionare discussedin greaterdetail in the next chapter.

Herorisorn"rization f

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S p r e a d i n go f s i l e n c e da n d n HP1-coatedheterochromati

Other protein domainsassociatewith histone-tailmodifications typical of euchromatin. For example, the bromodomain binds to acetylatedhistonetails and thereforeis associated with transcriptionally active chromatin. TFIID, a protein involved in transcription, contains two closely spacedbromodomains,which probably help TFIID to associate with transcriptionallyactive chromatin (i.e., euchromatin). This protein also has histone acetylaseactivity, which may maintain the chromatin in a hyperacetylated s t a t ec o n d u c i v et o t r a n s c r i o t i o n .

X-Chromosome lnactivation in Mammalian Females One important case of heterochromatin formation that correlateswith geneinactivation in mammals is the random inactivation and condensationof one of the two female sex chromosomes(the X chromosomes)in virtually all the diploid cells clf adult females.Inactivationof one X chromosomein females results in dosagecompensation,a processthat generatesequal expressionof genes on the sex chromosome in males and females.The inactive X appears as heterochromatin in interphasecells.It is visible as a dark-staining,peripheralnuclear structure called the Barr body, named after its discoverer' Each female mammal has two X chromosomes?one contributed by the egg from which they developed(X-) and one contributedby the sperm(Xo). Early during embryologicdevelopment, random inactivation of either the X* or the Xp chromosome occurs in each cell. In the female embryo, about half the cells have an inactive X*, and the other half have an inactiveXe. All subsequentdaughtercellsmaintain the same inactive X chromosomesas their parent cells. As a result,the adult femaleis a mosaic of clones,some expressing the genesfrom the X- and the rest expressingthe genes from the Xo. Histones associatedwith the inactive X chromosome have post-translationalmodifications characteristic of other regions of heterochromatin:hypoacetylation of lysines,di- and tri-methylation of histone H3 lysine 9, tri-methylation of H3 lysine27, and a lack of methylation a t h i s t o n eH 3 l y s i n e4 ( s e eF i g u r e 6 - 3 3 b ) . X - c h r o m o s o m e inactivation at an early stagein embryonic developmentis controlled by the X-inactivation center, a complex locus on the X chromosomethat determineswhich of the two X chromosomeswill be inactivated and in which cells. The X-inactivation center also contains the Xlsr gene, which encodesa remarkableRNA that coats only the X chromosome it was transcribedfrom, thereby triggering silencing of the chromosome.

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Although the mechanismof X-chromosome inactivation is not fully understood, it involves severalprocessesincluding the action of Polycomb protein complexesthat are discussedfurther in Chapter 7. One subunit of the Polycomb complex contains a chromodomain that binds to histone H3 tails when they are tri-methylated at lysine 27. The Polycomb complex also contains a histone methyl transferase specificfor H3 lysrne27. This finding helps to explain how the X-inactivationprocessspreadsalong largeregionsof the X chromosome and how it is maintained through DNA replication,similar to heterochromatizationby the binding of HPl to histone H3 tails methylaredat lysine 9 (seeFigure 6-34b). X-chromosomeinactivationis an epigeneticprocess:that is, a processthat affects the expressionof specificgenesand is inheritedby daughtercells,but is not the resultof a change in DNA sequence.Instead,the activity of geneson the X chromosomein femalemammalsis controlled by chromatin structurerather than the nucleotidesequenceof the underly, i n g D N A . A n d t h e i n a c t i v a t e dX c h r o m o s o m el e i r h e rX - o r Xu) is maintainedas the inactivechromosomein the progeny of all future cell divisions becausethe histones are modified in a specific, repressingmanner that is faithfully inherited through eachcell divisron.

Loops of DNA

protein scaffold

A EXPERIMENTAL FIGURE 6-35 An electronmicrograph of a histone-depleted metaphasechromosomerevealsan apparent scaffoldaroundwhich the DNAappearsto be organized.Long loopsof DNAarevisible extending fromthe nonhistone protein "scaffold" (thedarkstructure) thatreflects theshapeof the metaphase chromosome However, recent studies indicate thatthesenonntsrone proteins do notforma continuous structure responsible solely for determining theshapeof a metaphase chromosome asonemight expectfor a truescaffold structureThechromosome wasprepared f romHeLacellsbytreatment witha milddetergent I R paulson [From

N o n h i s t o n eP r o t e i n sP r o v i d ea S t r u c t u r a l Scaffoldfor Long ChromatinLoops Although histonesare the predominantproteinsin chromatin, lessabundant, nonhistonechromatin-associated proteins,and eventhe DNA moleculeitself, are also crucial to chromosome structure.Electronmicrographsof histone-depleted metaphase chromosomesfrom HeLa cellsreveallong loops of DNA anchored to what appearsto be a protein chromosomescaffold composedof nonhistoneproteins (Figure6-35). Although this chromosomescaffoldhas the shapeof the metaphasechromosome, recent results indicate that it is not protein alone that givesa metaphasechromosomelts structure. Micromechanical studies of large metaphasechromosomesfrom newts in the presenceof proteasesor nucleases indicate that DNA, nor protein, is responsiblefor the mechanical integrity of a metaphasechromosome when it is pulled from its ends. These results are inconsistent with a continuous protein scaffold at the chromosome axis. Rather, the integrity of chromosome strucrure requires the complete chromatin complex of DNA, histoneoctamers,and nonhistone chromatin-associated proteins. In situ hybridization experimenrswith several different fluorescent-labeled probesto the DNA of one chromosomein human interphasecellssupport a model in which chromatin is arrangedin large loops. In theseexperiments,some probe sequencesseparatedby millions of basepairs in linear DNA appeared reproducibly very close to one another in interphase nuclei from different cells of rhe same type (Figure 6-36). Thesecloselyspacedprobe sitesare postulatedto lie closeto regions of chromatin, called scaffoLd-associatedregions /SARs/ or matrix-attacbment regions (MARs), that are located at the basesof the DNA loops observed in histone254

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depleted metaphase chromosomes (see Figure 6-35). SARs/MARs have beenmapped by digestinghistone-depleted chromosomeswith restriction enzymesand then recovenng the fragments that remain associated with the histonedepletedpreparation.The measureddistancesbetweenprobes are consistentwith chromatin loops ranging in size from 1 million to 4 million basepairs in mammalian interphasecells. In general,SARs/MARs are found betweentranscription units, and genesare located primarily within the chromatin loops. As discussedbeloq the loops are tetheredat their bases by a mechanismthat does not break the duplex DNA molecule, which extendsthe entire length of the chromosome.Evidenceindicatesthat SARs/MARs may affect transcription of neighboringgenes.Experimentswith transgenicmice indicate that in somecasesSARs/MARs are requiredfor high-levelexpression of genes in the vicinity of SARs/MARs. And in Drosophila, some SARs/MARs function as insulators,that is, DNA sequences of tens to hundredsof basepairs that insulate transcription units from each other. Proteinsregulating transcription of one gene cannot influence the transcription of a neighboringgenethat is separatedfrom it by an insulator.

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probes EXPERIMENTAL FIGURE 5-36 Fluorescent-labeled hybridizedto interphasechromosomesdemonstratechromatin of loopsand permittheir measurement. In situhybridization interphase cellswascarried out with several different soecific orobes in linear, clonedDNA for sequences separated by knowndistances probes. Measurement of thedistances Lettered redcircles represent probes, between different hybridized whichcouldbe distinguished (e.g.,A, B,andC), bytheircolor,showedthatsomesequences separated fromoneanotherby millions of basepairs,appearlocated the nearoneanotherwithinnucleiForsomesetsof sequences, measured distances in nucleibetweenoneprobe(e g , C)and (eg , successively fartherawayinitially appearto increase sequences (e g , G andH).[Adapted D, E,andF)andthenappearto decrease fromH Yokota etal. 1995. J CellBiol130:1239 I

An SMC monomer contains two globular domains, a head domain and hinge domain, that are separatedby a very long coiled-coil domain. The head domain is formed from the N- and C-termini of the polypeptide, which are folded together in the native protein structure. The hinge domain forms where the polypeptide folds back on itself. The hinge domain of one monomer binds to the hinge domain of a second monomer, forming a roughly U-shapeddimeric complex (Figure 6-38a). The head domains of the monomers have ATPaseactivity and are linked by membersof another small protein family calledkleisins. Interphase Chromosome Structure Studieshave indicated that SMC proteins can link two circular DNA molecules by a mechanism that does not require direct protein-DNA binding. Rather, the two DNA molecules are topologically linked and can be separatedby either cleavageof the SMC complex with a protease,or cleavageof one of the circular DNA moleculesby a restriction enzyme.Theseresults,combined with the U-shaped structure of an SMC complex, suggestthat an SMC complex can link two 30-nm chromatin fibers by encircling both of them as depicted in Figure 6-38b. Using the technique of chromatin immunoprecipitation, discussedin the next chapter, researchershave demonstrated that SMC proteins in yeast interphasecells associatewith chromatin primarily at regions between genes.It is possible that ringlike SMC protein complexes are "pushed" into these regions by RNA polymerasestranscribing the regionsof chromatin betweenthem.

Individual interphasechromosomes,which are less condensedthan metaphasechromosomes,cannot be resolvedby standardmicroscopyor electronmicroscopy.Nonetheless,the chromatin of one chromosomein interphasecellsis not spread throughout the nucleus.Rather,interphasechromatin is organized into chromosometerritories.As illustratedin Figure 6-37, in situ hybridization of interphasenuclei with chromosomeprobes shows that the probes are specificfluorescent-labeled visualizedwithin restrictedregionsof the nucleusrather than appearingthroughout the nucleus.Use of probes specificfor different chromosomesshows that there is little overlap between chromosomesin interphasenuclei.However,the precise positionsof chromosomesare not reproduciblebetweencells. Ringlike Structure of SMC Protein Complexes Characterizationof proteinsassociatedwith metaphasechromosomes identified a small family of proteins called structwral maintenance of chromosome proteins, or SMC proteins. These nonhistoneproteinsare critical for maintainingthe morphological structureof chromosomes.In yeastwith mutations in certain SMC proteinschromosomecondensationduring the prophase period of mitosisdoesnot occur.Mutants with defectsin other SMC proteins fail to properly associatedaughter chromatids following DNA replicationin the S phase.As a result,chromoto daughtercellsduring mitosomesdo not properly segregate proteins required for proper segregation are Related SMC sis. of chromosomesin bacteriaand archaea,indicatingthat this is an ancientclassof proteinsvital to chromosomestructureand segregationin all kingdoms of life.

human FIGURE 6-37 Duringinterphase, a EXPERIMENTAL Fixed the nucleus. in territories remainin specific chromosomes in situto f luorescently werehybridized humanfibroblasts interphase alongthef ull lengthof human probes icfor sequences specif labeled bluewith and8 (purple)DNAwasstained 7 (cyan) chromosomes 7sandtwo DAPIInthisdiploidcell,eachof thetwo chromosome or domain to a territory 8scanbe seento be restricted chromosome the entire throughout ratherthanstretching withinthe nucleus, andT.Cremerl of DrsI Solovei nucleus. [Courtesy

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(a) Hinge domain

many more loops of chromatin, so that the length of each Ioop is greatly reduced compared with that in interphase cells. However, the folding of chromatin in metaphasechromosomes is not well understood. Microscopic analysis of mammalian chromosomesas they condenseduring prophase indicatesthat the 30-nm fiber folds into a 100- to 130-nm fiber called a chromonema fiber. As depicted in Figure 6-39, a chromonema fiber then folds into a structure with a diameter of 200-250-nm called a middle prophase chromatid, which then folds into the 500- to 750,nm diameter chromatids observedduring metaphase.

Head domain Kleisin

A d d i t i o n a lN o n h i s t o n eP r o t e i n sR e g u l a t e Transcriptionand Replication

C h r o m a t i nl o o p c o n t a i n i n g a transcriotionunit

The total mass of the histones associated with DNA in chromatin is about equal to that of the DNA. Interphase chromatin and metaphasechromosomesalso contain small amounts of a complex set of other proteins. For instance, hundreds to thousands of different transcription factors are associated with interphase chromatin. The strucrure and function of these critical nonhistone proteins, which help regulate transcription, are examined in Chapter 7. Other low-abundance nonhistone proteins associatedwith chromatin regulate DNA replication during the eukaryotic cell cycle (Chapter 20). A few other nonhistone DNA-binding proteins are present in much larger amounts than the transcription or replication factors. Some of these exhibit high mobility during electrophoretic separation and thus have been designated

FIGURE 6-38 Modelsof SMCcomplexesand their association with 30-nmchromatinfibersin interphasecells.(a)An SMCprotein complex consists of two monomers, SMC2(blue)andSMC4(red), whosehingedomains associate Theheaddomains, whichhaveATpase activity, areIinkedbya kleisin protein, forminga ringlike structure (b)Theringlike SMCcomplex topologically linkstwo chromatin fibers (graycylinders). Thecylinder diameter represents thediameter of a nucleosome andisto scalerelative to thedimensions of theSMC complex(c)Loopsof transcriptionally activechromatin maybetethered at theirbasebyseveral SMCcomplexes, forminga topological knot

30 nm

[Adapted from K Nasmythand C H Haering, ZOO5,Ann Rev_Biochem 74:595]

Basedon these various lines of evidence,a recent model proposesthat the long loops of chromatin detectedin interphase chromosomes(seeFigure 5-36) are tethered at the baseof eachloop by severalSMC complexes(Figure6-38c). These topological knots of SMC proteins and chromatin at the base of each loop are probably linked together in some way to produce the apparent protein scaffold shape visualized in histone-depletedmetaphasechromosomes (seeFigure 6-35). The linkage may require additional types of proteins, or may result from linkage of SMC complexes alone. In either case,the model in Figure 5-38c can explain why cleavageof the DNA at a relatively small number of sites leads to rapid dissolution of chromosome srructure, whereas proteasecleavagehas only a minor effect on chromosome structure until most of the protein is digested: When the DNA is cut anywhere in a chromatin loop, the broken ends can slip through the SMC protein rings, ,.untying" the topological knots that constrain the loops of chromatin. In contrast, most of the individual rings of SMC proteins must be broken before the topological constraints holding the baseof the loops together is released. Metaphase Chromosome Structure Condensation of chromosomesduring prophasemay involve the formation of 255

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100-130nm ( c h r o m o n e mf iab e r )

200-250nm ( m i d d l ep r o p h a sceh r o m a t i d )

500-750nm (metaphase chromatid)

FIGURE 6-39 Modelfor the folding of the 30-nmchromatin fiber in a metaphasechromosome. Thisdrawingdepicts the sequential foldingof a single30-nmfiberintoa singlechromatid of a metaphase chromosome fromN Kireeva et-al [Adapted ,2004,J Cell Biol.166:775l

G E N E 5G , E N O M I C SA, N D C H R O M O S O M E S

HMG (bigh-mobility group) proteins. !7hen genesencoding the most abundant HMG proteins are deleted from yeast cells, normal transcription is disturbed in most genesexamined. SomeHMG proteins have been found to bind to DNA cooperativelywith transcription factors that bind to specific DNA sequences,stabilizing multiprotein complexesthat regulate transcription of a neighboring gene.

Structural Organization of Eukaryotic Chromosomes r In eukaryoticcells,DNA is associatedwith about an equal massof histoneproteinsin a highly condensednucleoprotein complex called chromatin. The building block of chromatin is the nucleosome,consistingof a histone octamer around which is wrapped 1.47bp of DNA (seeFigure 6-29).

r Each eukaryotic chromosome contains a single DNA molecule packaged into nucleosomesand folded into a 3O-nm chromatin fiber, which is associatedwith a protein scaffold made up in part of structural maintenanceof chromosome (SMC) proteins at sites between transcription units (seeFigure 6-38c). Additional folding of the scaffold further compacts the structure into the highly condensed form of metaphasechromosomes(seeFigure 6-39).

Wl

andFunctional Morphology

Elementsof EukaryoticChromosomes

r The chromatin in transcriptionally active regionsof DNA within cells is thought to exist in an open, extended form (seeFigure6-28a).

Having examined the detailed structural organization of chromosomes in the previous section' we now view them from a more global perspective.Early microscopic observations on the number and sizeof chromosomesand their staining patterns led to the discovery of many important general characteristicsof chromosome structure. Researcherssubsequently identified specific chromosomal regions critical to their replication and segregationto daughter cells during cell division. In this sectionwe discussthesefunctional elements of chromosomes and consider how chromosomes evolved through rare rcarrangementsof ancestralchromosomes.

r The histone H4 tails, particularly H4 lysine 16, arc rcquired for beads-on-a-stringchromatin (the 10-nm chromatin fiber) to fold into a 30-nm fiber.

ChromosomeNumber,Size,and ShaPe at MetaphaseAre Species-Specific

r The chromatin in transcriptionally inactive regions of DNA within cells is thought to exist in a condensed,30-nm fiber form and higher-order structures built from it (see Figures6-30b and 6-39).

r Histone tails can be modified by acetylation, methylation, phosphorylation, and monoubiquitination (see Figure 6-31). Thesemodificationsinfluencechromatin structure by regulating the binding of histone tails to other less abundant chromatin-associatedproteins. r The reversible acetylation and deacetylation of Iysine residues in the N-termini of the core histones regulates chromatin condensation.Proteins involved in transcription, replication, and repair, and enzymeslike DNaseI can more easily accesschromatin with hyperacetylatedhistone tails (euchromatin)than chromatin with hypoacetylatedhistone tails (heterochromatin). r'When metaphasechromosomesdecondenseduring interphase, areas of heterochromatin remain much more condensedthan regions of euchromatin. r Heterochromatin protein 1 (HP1) usesa chromodomain to bind to histone H3 tri-methylated on lysine 9. The chromoshadow domain of HP1 also associateswith itself and with the histone methyl transferasethat methylatesH3 lysine 9. Theseinteractions causecondensationof the 30-nm chromatin fiber and spreading of the heterochromatic structure along the chromosome until a boundary element is encountered(seeFigure 6-34). r One X chromosome in nearly every cell of mammalian females is highly condensedheterochromatin, resulting in repressionof expressionof nearly all geneson the inactive chromosome. This inactivation results in dosagecompensation so that geneson the X chromosome are expressedat the same level in both males and females.

As noted previously in nondividing cells individual chromosomesare not visible,evenwith the aid of histologic stainsfor DNA (e.g.,Feulgenor Giemsastains)or electronmicroscopy. During mitosis and meiosis,however' the chromosomescondenseand becomevisible in the light microscope.Therefore, almost all cytogenetic work (i.e., studies of chromosome morphology) has beendone with condensedmetaphasechromosomes obtained from dividing cells-either somatic cells in mitosis or dividing gametesduring meiosis' The condensationof metaphasechromosomesprobably results from several orders of folding of 30-nm chromatin fibers (seeFigure 6-39\. At the time of mitosis, cells have already progressedthrough the S phase of the cell cycle and have replicated their DNA. Consequently,the chromosomes that becomevisible during metaphase are duplicated structures. Each metaphase chromosome consists of two sister chromatids,which are linked at a constrictedregion, the centromere (Figure 5-40). The number, sizes,and shapesof the metaphasechromosomesconstitute the karyotype, which is distinctive for each species.In most organisms' all cells have the same karyotype. However, species that appear quite similar can have very different karyotypes, indicating that similar genetic potential can be organized on chromosomes in very different ways. For example, two speciesof small deer-the Indian muntjac and Reeves muntjac-contain about the same total amount of genomic DNA. In one species,this DNA is organized into 22 pairs of homologous autosomesand two physically separatesex chromosomes.In contrast, the other speciescontains the smallest number of chromosomesin any mammal, only three pairs of autosomes;

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reagent produces R bands in a pattern that is approximately the reverseof the G-band pattern. The distinctive banding patterns of each chromosome permit cytologists to identify specific parts of a chromosome and to locate the sitesof chromosomal breaks and translocations(Figure 6-42a).In addition, cloned DNA probes that have hybridized to specificsequences in the chromosomescan be located in particular bands. The method of spectral karyotyping or chromosome painting greatly simplifies differentiating chromosomes of similar size and shape. This technique, a variation of fluorescence in situ hybridization (FISH), makes use of probes specific for sites scatteredalong the length of each chromosome. The probes are labeled with several different fluorescent dyes with distinct excitation and emissionwavelengths.Probesspecific for each chromosome are labeled with a predetermined fraction of each of the dyes.After the probes are hybridized to chromosomesand the excessremoved,the sampleis observed with a fluorescent microscope in which a detector determines the fraction of each dye present at each fluorescing position in the microscopic field. This information is conveyed to a computer, and a special program assigns a false color image to each type of chromosome. A related technique called mubicolor FISH can detectchromosomaltranslocations(Figure542b). The much more detailed analysispossible with these techniques permits detection of chromosomal translocations that banding analysis does not reveal. The photograph at the beginning of the chapter illustrates the use of multicolor FISH in preparing the karyotype of a human female.

FIGURE 6-40 Typicalmetaphasechromosome.As seenin this scanning electron micrograph, eachchromosome hasreplicated and comprises two chromatids, eachcontaining oneof two identical DNA molecules. Thecentromere, wherechromatids areattached at a constriction, isrequired for theirseparation latein mitosis. Special telomere sequences at theendsfunctionin preventing chromosome shortening[Andrew Syred/photo Researchers, Inc] one sex chromosome is physically separate,but the other is joined to the end of one autosome.

During Metaphase,ChromosomesCan Be Distinguished b y B a n d i n gp a t t e r n s and Chromosome Painting Certain dyes selectivelystain some regions of metaphasechromosomesmore intenselythan other regions,producing characteristic banding patterns that are specific for individual chromosomes.The regularity of chromosomal bands serveas useful visible landmarks along the length of each chromosome and can help to distinguishchromosomesof similar sizeand shaoe. G bands are produced when metaphasechromoso.., "ra subjected briefly to mild heat or proteolysis and then stained with Giemsareagenr,. p.r-un.ni DNA dye (Figure6-41). G bands correspond to large regions of the human genome that have an unusually low G + C content. Treatment of chromosomeswith a hot alkaline solution before staining with Giemsa 258

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EXPERIMENTAL FIGURE 6-41 G bandsproducedwith Giemsa stainsare useful markersfor identifying specificchromosomes. Shownherearethechromosomes froma humanmalethatwere subjected to briefproteolytic treatment andthenstaining with Giemsa reagent. Theresulting darkbandsat characteristic places are distinctive for eachchromosome. of Sabine Mal,phD , Manrtoba [Courtesy I n s t i t u t eo f C e l lB i o l o g yC , anadal

G E N E SG , E N O M t C SA, N D C H R O M O S O M E S

to the Iong arm of chromosome 10 (Figure 6-43b). Further' when multiple probes from the long arm of human chromoP h i l a d e l p h i a some 10 with different fluorescentdye labelswere hybridized chromosome to human chromosome 10 and tree shrew metaphasechromosomes,tree shrew sequenceshomologous to each of these probes were found along tree shrew chromosome 16 in the sameorder that they occur on human chromosome 10. (221 der These results indicate that during the evolution of humans and tree shrews from a common ancestor that lived -85 million yearsago, a long, continuous DNA sequenceon one of the ancestralchromosomesbecamechromosome 16 in tree shrews,but evolvedinto the long arm of chromosome 10 in humans. The phenomenon of genesoccurring in the same order on a chromosome in rwo different speciesis referred to as conservedsynteny (derived from Latin for "on the same ribbon"). The presenceof two or more genesin a common chromosomal region in two or more speciesindider (9) catesa conservedsyntenic segment. The relationships between the chromosomes of many primates have been determined by cross-specieshybridizations of chromosome paint probes as shown for human and tree shrew in Figure 6-43a and b. From these relationships and higher resolution analysesof regionsof synteny by DNA sequencingand other methods, it has been possible to propoie the karyotype of the common ancestor of all primates basedon the minimum number of chromosomal rearrangements necessaryto generatethe regions of synteny in chromosomesof contemporaryprimates. Human chromosomes are thought to have derived from a common primate ancestor with23 autosomesplus the X and Y sex chromosomes by several different mechanisms (Figure 6-43c). Somehuman chromosomeswere derivedwithout large scale rearrangementsof chromosome structure. Others arethought to have evolvedby breakageof an ancestralchromosome into two chromosomes or' conversely, by fusion of tvvo ancestral chromosomes' Still other human chromosomes translocations 5-42 Chromosomal FIGURE EXPERIMENTAL appear to have been generated by exchangesof parts of the can be analyzedusingbandingpatternsand multicolorFl5H. aims of distinct chromosomes,that is, by reciprocal translowith certain areassociated translocations chromosomal Characteristic two ancestralchromosomes.Analysis of rein nearly cation involving Forexample, ictypesof cancers. genetic andspecif disorders gions of conserved synteny between the chromosomes of cells the leukemic leukemia, myelogenous with chronic allpatients many mammals indicatesthat chromosomal rearrangements 22 chromosome a shortened chromosome, containthe Philadelphia such as breakage,fusion and translocationsoccurredrarely in 9 [der(9)]("der" longchromosome [der(22)],andan abnormally mammalian evolution, about once every five million years' between Theseresultfrom a translocation standsfor derivative). \fhen such chromosomal rearrangementsdid occur, they very canbe detected 9 and22 Thistranslocation normalchromosomes (b). (a) (a)andby multicolor FISH IPart likely contributed to the evolution of new speciesthat cannot bandinganalysis by classical p andCompany, 3d ed,W H Freeman 1997 interbreed with the speciesfrom which they evolved' fromJ Kuby, , lmmunology, (b) R Espinosa J Rowley and of 578 Part courtesy l Chromosomal rearrangementssimilar to those inferred for

P a i n t i n ga n d D N A S e q u e n c i n g Chromosome Revealthe Evolutionof Chromosomes Analysis of chromosomesfrom different specieshas provided considerableinsight about how chromosomesevolved. For example, hybridization of chromosome paint probes for chromosome 16 of the tree shrew (Tupia belangeri) to tree shrew metaphasechromosomesrevealed the two copies of chromosome 16, as expected(Figure 6-43a). However, when the same chromosome paint probes were hybridized to human metaphasechromosomes,most of the probeshybridized

ture (i.e., among mammals, among insectswith similar body organization, among similar plants, etc.) and the evolutionary reLtionships basedon the fossil record and on the extent of divergenceof DNA sequencesfor homologous genesis a strong argument for the validity of evolution as the processthat generated the diversity of contemporary organisms.

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EXPERIMENTAL FIGURE 543 Evolutionof primatechromosomes.(a)Chromosome paintprobes for chromosome 16of thetree shrew(7fbelanger|a primate-like animaldistantly relatedto humans) (yellow) werehybridized to treeshrewmetaphase (red). chromosomes probes"painted"theentirety These of bothcopies of chromosome 16 (b)Thesametreeshrewchromosome 16 paintprobeswerehybridized to humanmetaphase chromosomes Theseprobesweretargety localized to the longarmsof thetwo chromosome 10s (c)proposed evolutron (bottom)fromthe chromosomes of humanchromosomes of the commonancestor (top)Theproposed of all primates common primateancestor chromosomes arenumbered according to theirsizes, with eachchromosome represented by a differentcolor.Thehuman chromosomes arealsonumbered according to theirrelative sizes with colorstakenfromthe colorsof the proposed commonprimate ancestor chromosomes fromwhichtheywerederivedSmallnumbers to therightof thecolored regions of thehumanchromosomes indicate thenumberof theancestral chromosome fromwhichtheregron wasderived. Humanchromosomes werederived fromthe proposed chromosomes of the commonpnmateancestor in severar ways: withoutsignificant (e.g, humanchromosome rearrangements 1);by fusion(e.g, humanchromosome 2 byfusionof ancestral chromosomes (eg , humanchromosomes 9 and11);breakage 14and 15by breakage of ancestral chromosome 5),andchromosomar (e9.,humanchromosomes translocations 12 and22bya reciprocal translocation betweenancestral chromosomes (a)and 14 and2j) lparts (b)courtesy of Professor Dr.Johannes Weinberg, Institute forHuman Genetics

InterphasePolyteneChromosomes Arise b y D N AA m p l i f i c a t i o n The larval salivary glands of Drosopbila species and other dipteran insects contain enlarged interphase chromosomes that are visible in the light microscope. When fixed and stained, these polytene chromosomes are characterized by a large number of reproducible, well-demarcated bands that

and Anthropology,Universityof Munich part(c) courtesyof LutzFroenicke Ph D , Schoolof VeterinaryMedicine,Universityof California,Davisl

glands (Figure 6-44b). Chromosomal translocarionsand inversionsalso are readily detectablein polytenechromosomes, and specificchromosomalproteins can be localizedon interphase polytene chromosomesby immunostaining with specific antibodiesraised against them (seeFigure 7-11). Insect polytene chromosomes offer one of the only experimental 260

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FIGURE 644 Bandingon Drosophilapolytene > EXPERIMENTAT and in situ hybridizationare used salivaryglandchromosomes (a)Inthislightmicrograph of togetherto localizegenesequences. glandchromosomes, four larvalsalivary melanogaster Drosophila (X,2, 3, and4),with a totalof approxicanbe observed chromosomes from Thebandingpatternresults bands. mately5000distinguishable site packing of DNAandproteinwithineachamplified reproducible of morehighlycomDarkbandsareregions alongthechromosome. often of allfourchromosomes oactedchromatinThecentromeres 2 and3 are Thetipsof chromosomes appearfusedat thechromocenter (L : leftarm;R : rightarm),asisthetip of theX chromosome. labeled (b)A particular salivary canbe mappedon Drosophila DNAsequence Thisphotomicrograph glandchromosomes by in situhybridization. with a cloned thatwashybridized showsa portionof a chromosome Hybridization nucleotides. with biotin-derivatized labeled DNAsequence proteinavidinthatiscovalently with the biotin-binding isdetected phosphatase Onadditionof a soluble alkaline boundto theenzyme of thatresultsin formation catalyzes a reaction theenzyme substrate, (asterisk). at thesiteof hybridization coloredprecipitate an insoluble of each arecharacteristic bandingpatterns theveryreproducible Since canbe polytene sequence the hybridized chromosome, Drosophila majorbands indicate Thenumbers chromosome located on a particular andletters with numbers aredesrgnated thoseindicated Bandsbetween (a)courtesy (notshown). of F.Pignoni ofJ GallPart(b)courtesy ] [Part systemsin all of nature where such immuno-localizationstudies on decondensedinterphasechromosomesare possible. A generalizedamplification of DNA gives rise to the polytene chromosomesfound in the salivary glands of Drosophila. This process,termed polytenizatioin,occurs when the DNA repeatedly replicateseverywhereexcept at the telomeresand centromere, but the daughter chromosomesdo not separate.The result is an enlarged chromosome composed of many parallel copiesof itself (Figure6-45). The amplification of chromosomal DNA greatly increasesgenecopy number,presumablyto supply sufficient mRNA for protein synthesisin the massivesalivary gland cells. Although the bands seen in G-banded human metaphasechromosomesprobably representvery long folded or compacted stretchesof DNA containing about 10' base pairs, the bands in Drosophila polytene chromosomesrepresent much shorterstretchesof only 50,000-100,000basepairs.

Three FunctionalElementsAre Required f o r R e p l i c a t i o na n d S t a b l eI n h e r i t a n c e of Chromosomes Although chromosomes differ in length and number between species,cytogeneticstudieshave shown that they all behave similarly at the time of cell division. Moreover, any eukaryotic chromosomemust contain three functional elementsin order to replicate and segregatecorrectly: (1) replication origins at which DNA polymerasesand other proteins initiate synthesis of DNA (seeFigures 4-31 and a.33); (2) the centromere' the constricted region required for proper segregationof daughter chromosomes;and (3) the two ends, or telomeres.The yeast transformation studiesdepictedin Figure 6-45 demonstrated the functions of thesethree chromosomalelementsand establishedtheir importance for chromosomefunction. As discussedin Chapter 4, replication of DNA begins from sitesthat are scatteredthroughout eukaryotic chromo-

(a) Chromocenter

somes.The yeastgenomecontainsmany -100-bp sequences' caIIedautonomously replicating sequences(ARSs/, that act as replication origins. The observationthat insertion of an ARS into a circular plasmid allows the plasmid to replicatein yeast cells provided the first functional identification of origin sequencesin eukaryotic DNA (seeFigure 6-46a). Even though circular ARS-containingplasmidscan replicate in yeast cells, only about 5-20 percent of progeny cells contain the plasmid becausemitotic segregationof the plasmids is faulty. However, plasmids that also carry a CEN sequence,derived from the centromeresof yeastchromosomes'

645 The Patternof generalizedDNAamplificationof A FIGURE one polytenechromosomeduring five replications'Double-stranded and telomere bya singleline'Duringpolytenization, DNAisrepresented do chromosomes andthedaughter DNAarenotamplified centromere eachparental glandpolytene chromosomes, Insalivary notseparate. -10 replications (210: 1024strands)' [Adapted undergoes chromosome 1l 38:31 Biol' Symp Harbor Spring Quant' 1973, Cold al, et D Laird fromC 261

MoRPHoLoGYANDFUNcT|oNALELEMENTSoFEUKARYoT|CcHRoMo5oMES

Plasmid with Transfected sequencefrom leu- cell normal yeast

Progenyof transfected cell Growth without leucine

(a)

Conclusion

Mitotic segregation

ARS required for plasmid replication

No

No vL

\

Lo

') Yes

Poor $-20% of cells have plasmid)

In presence ofARS. olasmid replicationoccurs, but mitotic segregationis fa u ltv

Good (>90% of cells have plasmid)

Genomic fragment CEN required for good segregation

(b)

Yes

{\ 5J

(c) '?dL E U l -'ARS l

No

L i n e a rp l a s m i d lackingTEL is unstable

Yes

L i n e a rp l a s m i d s c o n t a i n i n gA R S and CEN behave l i k en o r m a l chromosomesif genomic fragment TEL is added to both ends

Restriction enzyme p r o d u c e sl i n e a r plasmid

TEL TEL ** ARs- rEU- cEN *-*

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A EXPERIMENTAL FIGURE 6-46 yeast transfection experimentsidentify the functionalchromosomalelements necessaryfor normal chromosomereplicationand segregation. Intheseexperiments, plasmids containing theIEU genefromnormalyeastcellsareconstructed andintroduced into /eu cellsbytransfection. lf the plasmid is maintained in the/eucells,theyaretransformed to LEU*by the LEUgeneon the plasmid andcanformcolonies on mediumlackinoleucrne. (a)Sequences thatallowautonomous replication inns)of . plasmid wereidentified because theirinsertion intoa plasmid vectorcontatning a clonedLEUgeneresulted in a highfrequency of transformation to IEU* However, evenplasmids with ARS exhibitpoorsegregation duringmitosis, andtherefore do not appearin eachof the daughter cells.(b)Whenrandomlv broken

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Yes

pieces yeastDNAareinserted of genomic intoplasmros containing ARSandLEU,someof thesubsequently transfected cellsproduce largecolonies, indicating thata highrateof mitotic segregation amongtheirplasmids isfacilitating the continuous growthof daughter cells. TheDNArecovered fromplasmids in theselargecolonies yeastcentromere (CEN) contains (c)When/eu- yeastcellsaretransfected sequences. with l i n e a r i z epdl a s m i dcso n t a i n i ntgE U ,A R Sa, n dC E Nn, o c o l o n i e s grow.Additionof telomere (TEL) sequences to the endsof the linearDNAgivesthe linearized plasmids the abilityto replicate as new chromosomes that behaveverymuchlikea normal chromosome in both mitosisand meiosis. A W Murray and [See J W,Szostak, 1983,Nature 305:89, andL Clarke andJ.Carbon. 1985. Ann Rev.Genet19i29l

YeastCEN

AAT GTCACGTG

78-86bp

6-47 Yeastcentromere(CEN)sequence.The A FIGURE of yeastCENsequence shownhere,basedon analysis consensus includes chromosomes, S.cerevisiae from 10 different centromeres is variable in sequence, ll,although regionsRegion threeconserved segregateequally or nearly so to both mother and daughter cellsduring mitosis (seeFigure 6-46b1. If circular plasmidscontaining an ARS and CEN sequence are cut once with a restriction enzyme) the resulting linear plasmids do not produce LEU, coloniesunlessthey contain special telomeric (TEL) sequencesligated to their ends (see Figure 6-46c). The first successfulexperiments involving transfectionof yeast cellswith linear plasmidswere achieved by usingthe endsof a DNA moleculethat was known to replicate as a linear molecule in the ciliated protozoan Tetrahymena. During part of the life cycle of Tetrahymena, much of the nuclear DNA is repeatedlycopied in short piecesto form a so-calledmacronucleus.One of these repeatedfragments was identified as a dimer of ribosomal DNA, the ends of which containeda repeatedsequence(G+Tzl".\fhen a section of this repeatedTEL sequencewas ligatedto the endsof linear yeast plasmids containing ARS and CEN, replication and good segregationof the linear plasmidsoccurred.

CentromereSequencesVary Greatlyin Length Once the yeast centromereregions that confer mitotic segregation were cloned, their sequencescould be determinedand compared, revealingthree regions(I, II, and III) that are conserved among different chromosomes(Figure 6-47). Short, fairly well conservednucleotide sequencesare presentin regionsI and III. Although region II seemsto have a fairly constant length, it contains no definite consensussequence;however,it is rich in A and T residues.RegionsI and III are bound by proteinsthat interact with a set of more than 30 other proteins, which in turn bind to microtubules.As a result of theseinteractions,each of the S. cereuisiaechromosomesbecomesattached to one microtubule of the spindleapparatusduring mitosis.RegionII is bound to a nucleosomethat hasa variant form of histoneH3 replacingthe usualH3. Centromeresfrom all eukaryotessimilarlyare bound by nucleosomeswith this specialized,centromere-specificform of histoneH3, called CENP-A in humans,that is essentialfor centromere function. S. cereuisiaehas by far the simplest cenknown in nature. tromeresequence In the fission yeastS. pombe, centromeresare :40 kb in simlength and are composedof repeatedcopiesof sequences ilar to those in S. cereuisiaecentromeres.Multiple copies of proteins homologous to those that interact with S. cereuisiae centromeres bind to these complex S. pombe centromeres and in turn bind the much longer S. pombe chromosomesto severalmicrotubules of the mitotic spindle apparatus.In plants and animals, centromeresare megabasesin length and DNA. are composed of multiple repeatsof simple-sequence In humans,centromerescontain 2t to 4-megabasearrays of a 771-bp simple-sequenceDNA called alphoid DNA that is

TGTTTCTGNTTTCCGAAA

Yeast in lengthandisrichin A andT residues. fairlyconstant areslmpler arequiteshortandtheirCENsequences chromosomes Ann 1985, andJ Carbon, L Clarke [See thanthosein othereukaryotes Genet19:291 Rev. bound by nucleosomescontaining the CENP-A histone H3 DNA. variant, as well as other repeatedsimple-sequence In higher eukaryotes,a complex protein structure called the kinetochoreassemblesat centromeresand associateswith multiple mitotic spindle fibers during mitosis. Homologs of most of the centromericproteins found in the yeastsoccur in humans and other higher eukaryotesand are thought to be componentsof kinetochores.The role of the centromereand prot;ins that bind to it in the segregationof sisterchromatids during mitosis is describedin Chapters 18 and 20.

Addition of TelomericSequencesby Telomerase PreventsShorteningof Chromosomes Sequencingof telomeresfrom multiple organisms,including humans, has shown that most are repetitive oligomerswith a high G content in the strand with its 3' end at the end of the chromosome. The telomere repeat sequencein humans and other vertebratesis TTAGGG. Thesesimple sequencesare repeatedat the very termini of chromosomesfor a total of a few hundred base pairs in yeastsand protozoans, and a few thousand base pairs in vertebrates.The 3' end of the G-rich strand extends 12-16 nucleotidesbeyond the 5' end of the complementary Crich strand.This region is bound by specificproteins that protect the endsof linear chromosomesfrom attack by exonucleases' The needfor a specializedregion at the endsof eukaryotic

would be shortenedat eachcell division. The problem of telomereshorteningis solved by an enzyme that addi telomeric (TEL) sequencesto the ends of each chromosome. The enzyme is a protein-RNA complex called telomere terminal transferase,ot telomerase.Becausethe sequence RNA, as we will see,servesas the of the telomerase-associated

263

MoRPHoLoGYANDFUNcT|oNALELEMENTSoFEUKARYoT|ccHRoMosoMEs

FocusAnimation:TelomereReplication flltt < FIGURE 6-48 StandardDNA replicationleadsto lossof DNA at the 5' end of eachstrandof a linearDNAmolecule. Replication of the rightendof a linearDNAisshown;thesame process occurs at the leftend(shownby inverting thefigure)As the replication forkapproaches theendof the parental DNAmolecule, the leading strandcanbe synthesized allthewayto theendof the parental template strandwithoutthe lossof deoxyribonucleotides. However, sincesynthesis of the lagging strandrequires RNAprimers, the rightendof the laggingdaughter DNAstrandwouldremainas ribonucleotides whichcannotserveasthetemplate for a replicative DNApolymerase Alternative mechanisms mustbe utilized by cells (andviruses with linearDNAgenomes) to prevent successive shortening of the laggingstrandwith eachroundof replication. Drosopbila speciesmaintain telomere lengths by the regulated insertion of non-LTR retrotransposonsinto telomeres. This is one of the few instancesin which a mobile element has a specificfunction in its host organism. I 5',

L a g g i n gs t r a n d

'11

Morphology and Functional Elements of Eukaryotic Chromosomes J

L a g g i n gs t r a n d

l-Shortenedl J

mutated RNA sequenceto the ends of telomeric primers. Thus telomeraseis a specializedform of a reversetranscriptasethat carriesits own internal RNA template to direcr DNA synthesis. Figure 6-49 depicts how telomerase,by reversetranscription of its associatedRNA, elongatesthe 3, end of the single-strandedDNA at the end of the G-rich srrand mentioned above. Cells from knockout mice that cannot produce the telomerase-associated RNA exhibit no telomeraseactivity, and their telomeres shorten successivelywith each cell generation.Such mice can breed and reproduce normally for three generations before the long telomere repeats become substantiallyeroded.Then, the absenceof telomereDNA results in adverseeffects,including fusion of chromosome termini and chromosomal loss. By the fourth generation, the reproductive potential of theseknockout mice declines,and they cannot produce offspring after the sixth generation. The human genes expressing the telomerase protein and the telomerase-associated RNA are active in germ cells and stem cells, but are turned off in most cells of adult tissuesthat replicate only a limited number of times, or will never replicate again (such cells are called postmitotic). However, these genes are activated in most human cancer cells, where telomeraseis required for the multiple cell divisions necessaryto form a tumor. This phenomenon has stimulated a search for inhibitors of human telomeraseas potential therapeutic agentsfor treatrng cancer. While telomerase prevents telomere shortening in most eukaryotes, some organisms use alternative strategies. 264

CHAPTER 6

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G E N E SG, E N O M t C SA, N D C H R O M O 5 O M E S

r During metaphase,eukaryotic chromosomes become sufficiently condensedthat they can be visualizedindividually in the light microscope. r The chromosomal karyotype is characteristic of each species.Closely related speciescan have dramatically different karyotypes, indicating that similar genetic information can be organized on chromosomesin different ways. r Banding analysis and chromosome painting are used to identify the different human metaphasechromosomesand to detect translocationsand deletions (seeFigure 6-42). r Analysis of chromosomal rearrangementsand regions of conservedsynteny betweenrelated speciesallows scientists to make predictions about the evolution of chromosomes (seeFigure 6-43c). The evolutionary relationshipsbetween organisms indicated by these studies are consistent with proposed evolutionary relationships based on the fossil record and DNA sequenceanalysis. The highly reproducible banding patterns of polytene romosomes make it possibleto localize cloned Drosophila DNA on a Drosophila chromosome by in situ hybridization (seeFigure 6-44) andto visualizechromosomal deletionsand rearrangementsas changesin the normal pattern of bands. r Three types of DNA sequencesare required for a long linear DNA moleculeto function as a chromosome:a reolication origin, called ARS in yeast; a centromere (CEN) sequence; and two telomere (TEL) sequencesat the ends of the DNA (seeFigure5-46). r Telomerase,a protein-RNA complex, has a special reverse transcriptase activity that completes replication of telomeresduring DNA synthesis(seeFigure 6-49).In the absenceof telomerase,the daughter DNA strand resulting from lagging-strandsynthesiswould be shortened at each cell division in mosr eukaryotes(seeFigure 6-48).

llll)

pe6usAnimation:TelomereReplication Catalyticsite for dNTP addition Telomerase protein Telomeraseassociated RNA template

G Q Q GT

Cccc44

Dissociationof 3' end of DNA

649 Mechanismof actionof telomerase.Thesingle< FIGURE bytelomerase, isextended of a telomere 3' terminus stranded to mechanism of the DNAreplication the inability counteracting elongates Telomerase DNA. linear of terminus the extreme synthesize reverse-transcription endby a reiterative thissingle-stranded fromthe protozoan Theactionof thetelomerase mechanism. other whichaddsa TaGarepeatunit,isdepicted; Oxytricha, contains The telomerase sequences. addslightlydifferent telomerases laggingthe of (red) 3' end to the base-pairs that template an RNA thenadds site(green) catalytic Thetelomerase strandtemplate. this asa template; (blue)usingtheRNAmolecule deoxyribonucleotides template RNA position of the 35 proceeds to reverse transcription duplexarethen DNA-RNA (steptr). Thestrands of the resulting of a singleto displacement leading other, to each relative slip to thought of part DNAstrandandto uncovering regionof the telomeric stranded telomeric (step of the RNAtemplatesequence E) Thelagging-strand andthe to position35 by telomerase, isagainextended sequence asbefore hybridization and translocation undergoes duplex DNA-RNA by repetition canaddmultiplerepeats (stepsB and 4). Telomerases of canprimesynthesis a-primase of stepsB and El. DNApolymerase net The strand template on thisextended fragments newOkazaki of the laggingstrandat eachcycleof DNA shortening resultprevents Nature 1990, andE H Blackburn, Shippen-Lentz from D replication. [Adapted 2471550 |

The human genome sequenceis a goldmine for new discoveries in molecular cell biology in identifying new proteins that

5',

I

nl Il I Translocationand hybridization I Y

GGGTTT u

T T T T N N N 5'

terization of cDNA copies of mRNAs isolated from the hundreds of human cell types, will likely lead to the discovery of new proteins, to a better understanding of biological processes, and may lead to applications in medicine and agriculture' We have seen that although most transposons do not function directly in cellular processes'they have helped to

-3', p

of reverse-transcription I Repetition translocation-hybridization and I I steps

Y

history. Large numbers of these interspersed repeats are P E R S P E C T I VF EO S RT H E F U T U R E

.

265

polymorphic within populations, occurring at a particular site in some individuals and not others. Individuals sharing an insertion at a particular site descendedfrom a common ancestorthat developedfrom an egg or sperm in which that insertion occurred. The time elapsedfrom the initial insertion can be estimated by the differencesin sequencesof the element that arose from the accumulation of random mutations. Analysis of retrotransposon polymorphisms will undoubtedly add immensely to our understanding both of human migrations sinceHomo sapiensfirst evolved, as well as the history of contemporarypopulations.

KeyTerms Barr body 253 bioinformatics 243

matrix-associatedregions (MARs)254

centromere 262 cnromatld li / chromatin 216

monocistronic245 nucleosome248 open reading frame (oRF) 244 polytene chromosome 25-1

cytoplasmic inheritance 2 3 7 DNA transposons227 eprgenetic 254 eachromatin 252 exon shuffling 235

protein famlly 220 pseudogene 220 retrotransposons 227

fluorescencein situ hybridization (FISH) 2'l8 gene famrly 220

repetitiousDNA 215 scaffold-associatedregrons (SARs)254

genomics216 heterochromatin 252

simple-sequence(satellite) DNA 224 SINEs230

histones 247

SMC proteins255 synteny 259

histone code250 insulator 254 | ^ -KaryotypezJ /

telomere 252 transcription unit 217 transposableDNA element216

LINEs 230 long terminal repeats (I:fP.s) 229

Review the Concepts 1. Cenescan be transcribedinto mRNA for protein-coding genes or RNA for genes such as ribosomal or transfei RNAs. Define a gene. Describehow a complex transcription unit can be alternativelyprocessedto geneiatea variety tf m R N A s a n d u l r i m a t e l yp r o t e i n s . 2. Sequencingof the human genome has revealedmuch about the organization of genes.Describe the differences between solitary genes, gene families, pseudogenes,and tandemlyrepeatedgenes. 3. Much of the human genomeconsistsof repetitiousDNA. Describethe differenceberweenmicrosatelliteand minisatel_ lite DNA. How is this repetitiousDNA useful for identifying individuals by the techniqueof DNA fingerprinting?

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4. Mobile DNA elementsthat can move or transposeto a new site directly as DNA are called DNA transposons.Describethe mechanismby which a bacterial DNA transposon, called an insertion sequence,can transpose. 5. Retrotransposons are a class of mobile elements that transposevia a RNA intermediate. Contrast the mechanism of transposition betweenretrotransposonsthat contain long terminal repeats(LTRs) and those that lack LTRs. 6. Discussthe role that transposonsmay have played in the evolution of modern organisms.!7hat is exon shuffling? What role do transposonsplay in the processof exon shuffling? 7. Mitochondria contain their own DNA molecules.Describe the typesof genesencodedin the mitochondrial genome.How do the mitochondrial genomesof plants, fungi, and animalsdiffer? 8. Mitochondria and chloroplasts are thought to have evolvedfrom symbiotic bacteriapresentin nucleatedcells.Review the experimental evidencethat supports this hypothesis. 9. Why is screeningof sequencedatabasesfor genesbasedon the presenceof ORFs (open reading frames)more useful for bacterialgenomesthan for eukaryoticgenomes?\Jfhatare paralogousand orthologousgenes?What are some of the explanations for the finding that humans are a much more complex organism than the roundworm C. elegans,yet have only fewer than one and a half as many genes(25,000 versus18,000)? 10. The DNA in a cell associateswith proteins to form chromatin. What is a nucleosome?What role do histonesplay in nucleosomes?How are nucleosomesarranged in condensed 30-nm fibers? 11. Vhat post-translation modifications of histones are associatedwith transcribed genes (euchromatin) and with repressedgenes(heterochromatin)?What protein is associated with heterochromatin in most eukaryotes?How doesthis affect heterochromatin formation over a region of a chromosome? 12. Describethe general organization of a eukaryotic chro, 'Sfhat mosome. structural role do scaffold-associated regions (SARs) or matrix artachment regions (MARs) play? rX/here are genesprimarily locatedrelative to chromosomestructure? 13. FISH is a powerful diagnostic tool readily used by cyto, geneticists.\fhat is FISH? Briefly describehow it is used to charaaerize chromosomal translocations associated with certain geneticdisordersand specifictypes of cancers. 14. Metaphasechromosomescan be identified by characteristic banding paterns. What are G bands and R bands? \fhat is chromosome painting, and how is this technique useful?How can chromosome paint probes be used to "rrulyze the evolution of mammalian chromosomes? 15. Certain organismscontain cells that possesspolytene chromosomes.ril/hat are polytene chromosomes,where are they found and what function do they serve? 16. Replication and segregationof eukaryotic chromosomes require three functional elements:replication origins, a centromere, and telomeres.Describe how these three elements function. What is the role of telomerasein maintainins chromosome structure?

Analyzethe Data To determineif genetransferfrom an organellegenometo the nucleus can be observed in the laboratory' a chloroplast transformation vector was constructed that contained two markerseachwith its own proselectableantibiotic-resistance geneand the kanamycinmoter: the spectinomycin-resistance resistancegene (seeS. Stegemannet al., 2003, Proc. Ndt'|. Acad. Sci. USA 100:8828-8833). The spectinomycinresistancegene was controlled by a chloroplast promoter, yielding a chloroplast-specificselectablemarker. Plants grown on spectinomycin are white unless they expressthe spectinomycin-resistancegene in the chloroplast. The kanamycin-resistancegene, inserted into the plasmid adlagene' was under the cent to the spectinomycin-resistance control of a strong nuclearpromoter.Tiansgenic,spectinomycinresistanttobacco plants were selectedfollowing transformation with this plasmid by identifying green plants grown on medium with spectinomycin. These plants contain the two antibiotic-resistancegenes inserted into the chloroplast genome by a recombination eventl however, kanamycin resistanceis not expressedbecauseit is under the control of plants a nuclear promoter. These spectinomycin-resistant in followthe generations and used for multiple grown were ing studies. a. Leavesfrom the spectinomycin-resistanttransgenrc plants were placed in a plant regenerationmedium containing kanamycin. Some of the leaf cells were resistant to kanamycin, and grew into kanamycin-resistantplants. Pollen (paternal) from kanamycin-resistantplants was used to pollinate wild-type (nontransgenic)plants. In tobacco, no chloroplasts are inherited from pollen. The resulting seeds were germinated on media with and without kanamycin. Half of the resulting seedlingswere kanamycin resistant. lfhen thesekanamycin-resistantplants were allowed to selfpollinate, the offspring exhibited a 3:1 ratio of kanamycinresistantto sensitivephenotypes.\[hat can be deducedfrom these data about the location of the kanamycin-resistance gene? b. To determineif transfer of the kanamycin-resistance gene to the nucleus was mediated via DNA or an RNA intermediate,DNA was extractedfrom 10 seedlingplants germinated from seedsproduced by a wild-type plant pollinated with a kanamycin-resistantplant. The 10 seedling plants, numbered1-10 in the correspondinggel lanesin the figure below, consist of 5 kanamycin-resistant(+) and 5 kanamycin-sensitive(-) plants. Each DNA sample was subjected to PCR analysis using primers to amplify the kanamycin-resistancegene (gel at left) or the spectinomycinresistancegene (gel at right). The lane marked M shows molecular weight markers. -What does the correspondence between the presenceor absenceof PCR products generated in the same plant with both sets of primers suggest about the mode of transfer of the kanamycin gene to the nucleus?

10

M

10

M

t-

-

Kan resistance I P C Rp r o d u c t su s i n g i -resistance k an a m y c n geneprimersl

Kan resistance I P C Rp r o d u c t su s i n g spectinomycin-resistance geneprimersl

c. V4ren the original transgenic plants, which were selected on spectinomycin but not on kanamycin, were used to pollinate wild-type plants,none of the offspring were kanamycin iesistant. !7hat can be deducedfrom theseobservations?

References EukaryoticGeneStructure Blencowe,B. J.2006. Alternative splicing:new insightsfrom

Trends Genet. 19:640-648' Suzuki,Y., and S. Sugano.2006. Transcriptomeanalysesof human genesand applicationsfor proteomeanalyses'Curr' Protein PeptideSci.7:147-163. Chromosomal Organization of Genes and Nontoding DNA InternationalHuman GenomeSequencingConsortium' 2004' Finishingthe euchromaticsequenceof the human genome'Nature 431:931- 945. Paterson,A.H. 2006. Leafing through the.genomesof our maior crop plants: strategiesfor capturing unique informatron' Nature Reu.Genet.7z174-184. Pearson.C. E, K. Nichol Edamura, and J' D' Cleary' 2005' Repeatinstability: mechanismsof dynamic mutations' Natwre Reu' Genet.6:729-742. Ranum, L. P.,and T. A. Cooper.2006' RNA-mediatedneuromusculardisorders'Annu. Reu.Neurosci.29:259-277' Transposable (Mobile) DNA Elements lilessler'2002'Plant transposFeschotte,C., N. Jiang, and S. R' Natwre Reu'Genet' genomics' meets geneiics where able elements, 3:329-341'. Gray, Y. H. 2000' It takestwo transposonsto tango: transposs' TrendsG enet' chromosomalrearrangement able-ele#ent-mediated 16:461-468. elements:the full Jones,R. N. 2005. McClintock's controlling ttotl. Cytogrnet. GenomeRes.109:90-103' Kazazian,H. H.' Jr. 1999. An estimatedfrequencyof endogenous insertionalmutations in humans.Nature Genet' 22:130' Kazazian,H.H., Jr. 2004. Mobile elements:drivers of genome evolution. Science 303z\ 626-1632'

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Kleckner,N., et al. 1996.TnI} and IS10 transpositionand . chromosomerearrangements:mechanismand requlation in vivo and in vitro. Curr. TopicsMicrohiol. Immunol. 204?49-92. Mahillon, J., and M. Chandler.1998. Insertion sequences. Ml_ crobiol. Mol. Biol. Reu.62:725-774. . Morgante,M.2006. Plantgenomeorganisationand diversity: the year of the;unk! Cur. Opin. Biotechiol. 17:169-173. E. M., and H.H.Kazazian, Jr. 2001. Biologyof mam_ ..Ost_errag, malian L1 rerrotransposons. Ann. Reu.Ginet.35:501-5jg. Steiniger-\?hiteM, L Rayment,and W. S. Reznikoff. 2004. Structu_relfunction insightsinto TnS transposition.Curr. Opin. Struc.Biol. 14:50-57.

Structural Organization of Eukaryotic Chromosomes Carroll, C. I7., and A. F. Straight.2006. Centromereformation: from epigeneticsto self-assemb\y. TrendsCell Biol. 16:70-78. Dehghani,H., G. Dellaire,and D. P.Bazen-Jones. 2005. Organization of chromatinin the inrerphasemammalianiell. Miuon 36:t5-10g. Dillon, N. 2004. Heterochromatinstructureand function. Blol. Cell.2004. 96:631-637. Henikoff, S., and Y. Dalal. 2005. Centromericchromatin: what makesit unique?Curr. Opin. Genet.Deu. 15:177-1,84. Horn, P.J., and C. L. Peterson.2002. Molecular biology. Chromatin higher order folding-wrapping up rranscription. Sience 29721.824-'1.827. Luger,K. 2003. Structureand dynamic behaviorof nucleosomes.Curr. Opin. Genet.Deu. 13:127-135. Luger,K., and T. J. Richmond. 1998. The histonetails of the nucleosome.Curr. Opin. Genet.Deu. 8:1.40-146. . Margueron, R., P. Trojer, and D. Reinberg.2005. The key to development:interpretingthe histone code?Curr. Opin. Genet.Deu. 15:163-176. McBryant,S.J., V. H. Adams,and J. C. Hansen.2006. Chromatin architecturalproteins. ChromosomeRes.l4:39-51.. Nasmyth,K., and C. H. Haering.2005. The strucrureand function of SMC and kleisincomplexes.Annu. Reu.Biochem.T4:595-64g. Sarma,K., and D. Reinberg.2005. Histone variants meertheir match.Nature Reu.Mol. Cell Biol. 6:139-149. Schalch,T., et al. 2005. X-ray strucure of a tetranucleosome and its implicarionsfor the chromatin fibre.Nature 436:13g-14I. 'Woodcock, C. L. 2006. Chromatin architecture.Curr. Onin. Struc. Biol. 16:2 | 3-220. 'Woodcock, C. L., A. I. Skoultchi,and y. Fan. 2006. Role of linker histonein chromatin structureand function: H1 stoichiometry and nucleosomerepeatlength. ChromosomeRes.14:17-25.

Organelle DNAs Bendich,A. J.2004. Circularchloroplastchromosomes: tne g r a n di l l u s i o nP. l a n tC e l l l 6 t l 6 6 l - 1 6 6 6 . Chan,D. C.2006. Mitochondria:dynamicorganelles in disease, aging, and development.Cell 125:1241-1252. . Clayton, D. A. 2000. Transcriptionand replication of mitochon_ drial DNA. Hum. Reprod.2(Suppi.):11-17. Daniell,H., M. S. Khan, and L. Allison.2002. Milestonesin chloroplastgenericengin_eering: an environmentallyfriendly era in brotechnology. Trcnd s Plant Sci. 7 zB4-91. W., G. Burger,and B. F. Lang.2001. The origin and early ,Glry,Y. evolutionof mitochondria.GenomeBft.,/. g.1-101g.5.' 2(Reviews):101 Shutt, T. E., and M. V. Gray. 2006. Baceriophageorigins of m^itochondrialreplication and transcriprionproteins.-Tren1s G enet. ( t t.gn-g

Sugiura,M., T. Hirose,and M. Sugita.199g. Evolutionand mech_ anismof translarionin chloroplasts.Ain. Reu.Genet.32:437459. Genomics: Genome-wide Analysis of Gene Structure and Expression BLAST Information can be found ar: hftp://www.ncbi.nlm.nih.gov/ Education/BLASTinfo/information3.htm Binnewies,T. T., et aL.2006.Ten yearsof bacterialgenomese_ quencing:c_omp_arative-genomics-based discoveries.Fuict. Integr. Genomics6:165-185. Celniker,S. E., and G. M. Rubin. 2003. The Drosoohila melanogastergenome.Annu. Reu.GenomicsHum. Geiet. 4:g9_177. ChimpanzeeSequencingand AnalysisConsorrium.2005. Initial sequenceof the chimpanzeegenomeand comparisonwith the human genome.Nature 437:69-87. Gebhardt,C., R. Schmidt,and K. Schneider. 2005. plant genomeanalysis:the stateof the art. Int'\. Reu.Cttot. 247:223-284. Gele Q119logyConsortium. 2006. The Gene Ontology (GO) projectin 2006. NucleicAcids Res.34:D322-D326. Guigo,R., et a|.2006.EGASp:the human ENCODE genome annotationassessmenr project. G enome B iot. 7(Suppl 1):S2:. 1_S2.3 1. Harris, T. \7., and L. D. Stein.2006. WormBase:methodsfor data mining and comparativegenomics.Methods Mol. Biol.351:31_50. InternationalHapMap Consortium. 2003. The international HapMap project. N ature 426:789-79 6. InternationalHuman GenomeSequencingConsortium.2004. Finishingthe euchromaticsequenceof the huiran genome.Nature 431:931.-945. Lander,E. S.,et al. 2001. Initial sequencing and analysisof rhe . human genome.Nature 409:860-927. Ness,S. A. 2006. Basicmicroarrayanalysis:strategies for suc_ cessfulexperiments.Methods Mol. Biol. 316:13_33. __ Shianna,K. V., and H. F. Willard. 2006.|n searchof normality. Nature 444:428-429. codingsequences of human _ Sioblom,T.2006. The consensus breastand colorectalcancers.Science3l4,i6g-274. . Wat€rston,R. H., et al.2002.Initial sequencingano compara_ tive analysisof the mousegenome.Nature AZO:SZi_SAZ. \7indsor,A. J., and T. Mitchell-Olds.2006.Comparatrvegenomrcs as a tool for genediscovery.Curr. Opin. Biotechnol.iZrrcttZZ.

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Morphology and Functional Elements of Eukaryotic Chromosomes Armanios, M., and C. $7. Greider.2005. Telomeraseano cancer stem celis.Cold Spring Harb. Symp. euant. Biol.70:205-20g. Belmont,A. S. 2002. Mitotic chromosomescaffold structure: New approachesto an old conrroversy.Proc. Nat'|. Acad. Sci.USA 9 9 : 1 5 8 5 5 -5 18 5 7 . Belmont, A. S., et al. 1999. Large-scalechromatin structureand function. Curr. Opin. Cell Biol. 11307-31I. Blackburn, E. H. 2005. Telomeresand telomerase:their mechanisms of acrion and the effectsof altering their functions. FEBSLett. 5792859-862. Carroll, C. W., and A. F. Straight.2006. Cenrromereformarion: from epigeneticsto self-assembly. TrendsCetl. Biol. 16:70-7g. \X/alter.2005. Eukaryotic origins of DNA C., and C. J. .Cvetic, replicafion:could you pleasebe more specific?Semin.Cell Deu. Biol. 16:343-353. de Lange,T.2006. Lasker laurelsfor telomerase.Cel/ 126:1.017-1.020. Froenicke,L. 2005. Origins of primate chromosomesas delin_ eatedby Zoo-FISH and alignmentsof human and mousedraft genomesequences. CytogenetGenome Res.l}8z122-I3g. Gassmann,R., et al. 2004. Mitotic chromosomeformation and the condensinparadox. Exp. Cell Res.296:35-42. MacAlpine, D. M, and S. P. Bell. 2005. A genomicview of eu_ karyotic DNA replication. ChromosomeRes.7l:SOg-SZe. Malik, H. S. 2006. A hitchhiker'sguide ro survival finally makes CENs. /. Cell Biol. 1742747-749. Marko, J. F., and M. G. Poirier.2003. Micromechanicsof chro_ matin and chromosomes.Biochem. Cell Biol. St:209-220. Robinson,N.. P.,and S._D.Bell. 2005. Origins of DNA replication in the three domains of life. FEBS/ . 272t3757-3766. \Tarburron, P. F,.2004. Chromosomaldynamicsof human neo_ centromereformation. ChromosomeRes.12617 -626.

G E N E SG , E N O M t C SA, N D C H R O M O S o M E S

CHAPTER

TRANSCRIPTIONAL OF GENE CONTROL EXPRESSION against withantibodies polytene stained chromosomes Drosophila (blue), RNA Kismet ATPase called remodeling chromatin a (red), andRNA polymerase ll wrthlowCTDphosphorylation (green) of polymerase ll withhighCTDphosphorylation [Courtesy 132:1623 et al, 2005,Development seeS Srinivasan I JohnTamkun;

I n previous chapters we have seenthat the properties and I functions of eachcell type are determined by the proteins it I contains. In this and the next chapter,we consider how the kinds and amounts of the various proteins produced by a particular cell type in a multicellular organism are regulated. This regulation of gene expression is the fundamental processthat controls the developmentof a multicellular orinto ganism such as ourselvesfrom a singlefertilized egg cell 'W'hen the thousands of cell types from which we are made. gene expressiongoes awr5 cellular properties are altered, a processthat all too often leadsto the developmentof cancer. As discussedfurther in Chapter 25, genesencoding proteins that restrain cell growth are abnormally repressedin cancer cells, whereas genesencoding proteins that promote cell growth and replication are inappropriately activated in cancer cells. Abnormalities in gene expressionalso result in developmental defectssuch as cleft pallet' tetrology of Fallot (a common, serious developmentaldefect of the heart that can be treated surgically), and many others. Regulation of gene expressionalso plays a vital role in bacteria and other singlecelled microorganisms, where it allows cells to adjust their enzymaticmachinery and structural componentsin response to their changing nutritional and physical environment. Consequently,to understand how microorganisms respond to their environment and how multicellular organisms normally develop, as well as how pathological abnormalities of gene expressionoccur, it is essentialto understand the molecular interactions that control protein production. The basic stepsin geneexpression,i.e.' the entire process whereby the information encodedin a particular gene is de-

coded into a particular protein, are reviewed in Chapter 4'

newly formed RNA transcripts, which function as mRNA

OUTLINE 7.1

Control of Gene Expressionin Bacteria

271

7.2

Overviewof EukaryoticGene Control and RNA Polymerases

276

7.3

Genes 282 in Protein-Coding RegulatorySequences

7.4

of Transcription Activatorsand Repressors

286

7.5

TranscriptionInitiation by RNA Polymerasell

295

7.6

MolecularMechanismsof Transcription and Activation Reoression

299

7.7

Activity Regulationof Transcription-Factor

311

7.8

RegulatedElongationand Terminationof Transcription

314

7.9

Other EukaryoticTranscriptionSystems

316

269

without further modification. In eukaryotes.however.the initial RNA transcript is subyected,o pio..rring that yields a functional mRNA (see Figure 4-15). The mRNA then is transported from its site of synthesisin the nucleusto the cytoplasm, where it is translatedinto protein with the aid of ribosomes,tRNAs, and translationfactors(seeFigure4-25). TheoreticallS regulation at any one of the various steps in gene expressionoutlined above could lead to differential production of proteins in different cell types or developmental stagesor in responseto external conditions. Although examples of regulation at each step in gene expressionhave been found, conrrol of transcription initiation-the first step-is the most important mechanism for determining whether most genesare expressedand how much of the encoded mRNAs and, consequently,proteins are produced. The molecular mechanismsthat regulate transcription initiation are critical to numerous biological phenomena,including the development of a multicellular organism from a single fertilized egg cell as mentioned above, the immune responsesthat protect us from pathogenic microorganisms, and neurological processessuch as learning and memory. Vhen theseregulatory mechanismscontrolliig transcription function improperly, pathological processesmay occur. For example, reduced activity of the pax6 gene causesaniridia, failure to develop an iris. Pax6 is a transcription factor that normally regulatestranscription of genesinvolved in eye development(seeFigure 1-26e).In other organisms,mutarions of transcription factors causean extra pair of wings to dev e l o pi n D r o s o p h i l a( s e eF i g u r e2 Z - 3 1 ) , t h " n g . t h e i t r u c t u r e of flowers in plants (seeFigure 22-36), and are responsible for multiple other developmentalabnormalities. Transcriptionis a complex processinvolving many lay_ ers of regulation. In this chapter, we focus on the molecular eventsthat determinewhen transcription of a gene occurs. First, we consider the relatively basic mechanismsof gene expressionin bacteria,where repressorand activator pro_

> FIGURE7-1 Overview of eukaryotic transcription control. Activatorproteinsbind to specificDNA controlelementsin chromatinand interactwith multiproteinco-activator machines, suchas mediator,to decondense chromatinand assembleRNApolymerase and general transcription factorson promotersInactivegenes are assembled into regionsof condensed chromatlnthat lnhibitRNApolymerases and their assocrated generaltranscription factors(GTFs) from interactingwith promotersAlternatively, repressor proteinsbind to other controlelements to inhibitrnitiationby RNAporymerase ano interactwith multiproteinco-repressor complexes to condensechromatin

teins recognize and bind to specific regions of DNA to control the transcription of a nearby gene.The remainder of the chapter focuseson eukaryotic transcription regulation and how the basic tenets of bacterial regulation are applied in more complex ways in higher organisms. Figure 7-1. provides an overview of eukaryotic gene regulation and the processesoutlined in this chapter. \Ve discusshow specific DNA sequencesfunction as transcription-control regions by serving as the binding sites for transcription factors (repressorsand activators)and how the RNA polvmerasesresponsiblefor transcription bind ,o pro-ot.. sequences initiate the synthesisof an RNA molecule complementary to template DNA. Next, we consider how activators and repressors influence transcription through interactions with large, multiprotein complexes. Some of these multiprotein complexes modify chromatin condensation, altering access of chromosomal DNA to transcription factors and RNA polymerases.Other complexes influence the rate at which RNA polymerase binds to DNA at the site of transcription initiation, as well as the frequency of initiation. Then we discusshow transcription of specific genescan be specified by particular combinationsof the -2,000 transcriptionfactors encoded in the human genome, giving rise to cell-typespecific gene expression.rWealso further consider the various ways in which the activities of transcription factors themselvesare controlled to ensuregenesare expressedonly at the right time and in the right place. Finally, we'll examine control of transcription elongation and termination and the transcription of nonprotein-coding RNAs. RNA processingand various post-transcriptional mechanismsfor controlling eukaryotic gene expression are covered in the

GENE "OFF"

,f *"Or"arora

f

Decondensed chromatin

GENE "ON" Activators

General transcription factors

270

c H A P T E R7

|

o",,u,.o,.,

TRANSCRtpTtoNC AL ONTROL O F G E N EE X p R E S S T O N

RNA polymerase

Jn

Controlof GeneExpression

in Bacteria Since the structure and function of a cell are determined by the proteins it contains, the control of gene expressionis a fundamental aspectof molecular cell biology. Most commonly, the "decision" to initiate transcription of the gene encoding a particular protein is the major mechanism for controlling production of the encoded protein in a cell. By controlling transcription initiation, a cell can regulate which proteins it 'When transcription of a gene is produces and how rapidly. mRNA and encodedprotein or the corresponding repressed, proteins are synthesizedat low rates. Conversely,when transcription of a geneis actiuated,both the mRNA and encoded protein or proteins are produced at much higher rates. In most bacteria and other single-celledorganisms,gene expressionis highly regulatedin order to adjust the cell'senzymatic machinery and structural componentsto changesin the nutritional and physical environment. Thus, at any given time, a bacterial cell normally synthesizesonly those proteins of its entire proteome required for survival under the particular conditions. Here we describethe basic featuresof transcription control in bacteria, using the lac operon and the glutamine synthetasegene in E. coli as our primary examples. Many of the same processes,as well as others, are involved in eukaryotic transcription control, which is the subject of the remainder of this chapter.

TranscriptionInitiation by BacterialRNA PolymeraseRequiresAssociationwith a SigmaFactor ln E. coli, about half the genesare clustered into operons, each of which encodesenzymesinvolved in a particular metabolic pathway or proteins that interact to form one multisubunit protein. For instance,the trp operon discussed in Chapter 4 encodesfive enzymesneededin the biosynthesis of tryptophan (seeFigure 4-13). Similarly the lac operon encodesthree enzymesrequired for the metabolism of lactose, a sugar presentin milk. Sincea bacterial operon is transcribed from one start site into a singlemRNA, all the genes within an operon are coordinately regulated;that is, they are all activated or repressedto the sameextent. Transcription of operons, as well as of isolated genes,is controlled by an interplay between RNA polymerase and specific repressorand activator proteins. In order to initiate transcription, however,E. coli RNA polymerasemust be associated with one of a small number of o (sigma) factors. The most common one in eubacterialcells is oto. oto bi.tds to RNA polymeraseand to promoter DNA sequences,bringing the RNA polymerase enzyme to a promot r. o70 recognizes and binds to both a six-basepair sequencecenteredat -1,0 and a seven-base pair sequencecenteredat -35 from the +1 transcriptionstart. Consequently,the -10 plus the -35 sequenceconstitute a promoter for E. coli RNA polymeraseassociatedwith o70 (seeFigure4-10b). Although the promoter sequencescontactedby o'o are located at -35 and

- 10, E. coll RNA polymerasebinds to the promoter region to:*20 through interactionswith DNA DNA from:-50 that do not dependon the sequencr. o'0 also assiststhe RNA polymerasein separatingthe DNA strands at the transcription start site and inserting the coding strand into the active site of the polymeraseso that transcription starts at +1 (see Figure4-11, step2).The optimal o7O-RNApolymerasepromoter sequence,determined as the consensussequenceof multiple strongpromoters,is: -35 region

a16trQ,\1-

-10 region

15 -17 bp-1a-t66t

The sizeof the font indicatesthe importance of that baseat this position. The sequenceshows the strand of DNA that has the same5'-+3' orientation as the transcribedRNA (i.e.,the nontemplate strand). However, the o7O-RNA polymerase initially binds to double-strandedDNA. After the polymerasetranscribesa few tensof basepairs,o70is released.Thus o7oactsas an initiation factor required for transcription initiation but not for RNA-strand elongation once initiation has taken place.

Initiation of lacOperonTranscriptionCan Be Repressedand Activated When E. coli is in an environment that lacks lactose' synthesis of lac mRNA is repressedso that cellular energy is not wasted synthesizingenzymesthe cellscannot use.In an environment containing both lactose and glucose, E- coli cells preferentially metabolize glucose, the central molecule of carbohydrate metabolism. Lactose is metabolized at a high rate only when lactose is present and glucose is largely depleted from the medium. This metabolic adjustment is achievedby repressingtranscription of the lac operon until lactoseis presentand allowing synthesisof only low levelsof /ac mRNA until the cytosolic concentration of glucosefalls to low levels.Transcription ofthe lac operon under different conditions is controlled by lac tepressor and catabolite activator protein (CAP) (also called CRP for catabolite /eceptor protein), each of which binds to a specificDNA sequencein the lac transcription-control region (Figure 7-2, top). -^ For transcription of the lacipeton to begin, the o70subunit of the RNA polymerasemust bind to the lac promoter 'When no Iactoseis at the -35 and - 10 promoter sequences. present, the lac repressor binds to a sequence called the lac operator, which overlaps the transcription start site' Therefore, lac repressorbound to the operator site blocks binding and hencetranscription initiation by RNA polymerase(Figure 7-2a\. -Whenlactoseis present, it binds to specific binding sitesin each subunit of the tetrametic lac repressor,causing a conformational change in the protein that makes it diisociate from the lac operatot.As a result, the polymerase

N BACTERIA C O N T R O LO F G E N EE X P R E S S I OIN

.

27'I

+1 (transcriptionstart site) PromoterV -_l

F

F

CAPsite

--

Ooerator E. coli lac transcription-control regions

(4,

- lactose + glucose (lowcAMP)

N o m R N At r a n s c r i p t i o n

(b) + lactose + glucose ( l o wc A M P )

Low transcription

cAMP

(c) + lactose - grucose ( h i s hc A M P )

A FIGURE 7-2 Regulationof transcription from the /acoperon ol E. coli.(Top)The transcription-control region, composed of -100 basepairs,rncludes threeprotein-binding regions: theCApsite,which bindscatabolite activator protein; the/acpromoter, whichbindsthe polymerase o70-RNA complex; andthe/acoperator, whichbinds/ac repressor The/acZgene, thefirstof threegenesin theoperon, is shownto the right (a)In theabsence of lactose, verylittle/acmRNA isproduced because the/acrepressor bindsto theoperator, inhibiting transcription initiation by,I7o-RNA polymerase. (b)Inthe presence of glucose andlactose, /acrepressor bindslactose anddissociates from theoperator, allowing o7o-RNA polymerase to initiate rranscnptton at a low rate (c)Maxlmal transcription of the/acoperon occurs in the presence of lactose andabsence of glucoseInthissrtuation, cAMp increases in response to the lowglucose concentration andformsthe CAP-cAMP complex, whichbindsto theCApsite,wherert interacts with RNApolymerase to stimulate the rateof transcription initiation the -35 and - 10 sequences in the lac promorer differ from the ideal o"'-binding sequences shown previously. Once glucoseis depletedfrom the media and the intracellular glucoseconcentrationfalls, E. coli cellsrespond by synthesizingcyclic AMP, or cAMp. As the concentration of cAMP increases,it binds to a sire in each subunit of the dimeric CAP protein, causinga conformarionalchangethat allows the protein to bind to the CAp site in the lac transcription-control region. The bound CAp-cAMp complex interacts with the polymerase bound to the promoter, greatly stimulating the rate of transcriptioninitiation. This activationleadsto synthesisof hieh levelsof /ac mRNA and subsequentlyof the enzymesencoded by the lac operon (Frgure7-2c1. 272

.

c H A p r E R7

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In fact, the lac operon is more complex than depicted in the sirnplified model of Figure 7-2. The tetrameric lac repressoractually binds to two sitessimultaneously,one at the primary operator (lacO1) that overlapsrhe region of DNA bound by RNA polymerase at the promoter and at one of two secondaryoperatorscenreredat +412 (lacO2) and -82 (lacO3) (Figure7-3). The /ac repressortetrameris a dimer of dimers. Each dimer binds to one operaror. Simultaneous binding of the tetram eric lac repressorto the prima ry lac operator O1 and one of the two secondaryoperators is possible becauseDNA is quite flexible, as we saw in the wrapping of DNA around the surfaceof a histone octomer in the nucleosomesof eukaryotes(Figure6-29l.These secondaryoperators function to increasethe local concentration of lac repressor in the micro-vicinity of the primary operator where repressorbinding blocks RNA polymerasebinding. Sincethe equilibrium of binding reactions dependson the concentrations of the binding partners, the resulting increasedlocal concentrationof ldc repressorin the vicinity of Ol increases repressorbinding to OL. There are approximatelyI0 lac repressor tetramers per E. coli cell. Becauseof binding to 02 and 03, there is nearly always a /ac repressortetramer much closerto 01 than would otherwisebe the caseif the 10 repressorswere diffusing randomly through the cell. If both 02 and 03 are mutated so that the lac repressorno longer binds to them with high affinity, repressionat the lac promoter is reduced by a factor of 70. Mutation of only 02 or only 03 reducesrepressiontwofold, indicating that either one of thesesecondaryoperators provides most of the stimulation of repression. Although the promoters for different E. coli genesexhibit considerablehomology, their exact sequencesdiffer. The promoter sequencedeterminesthe intrinsic rate at which an RNA polymerase-o complex initiates transcription of a gene in the absenceof a repressoror activator protein. Promoters that support a high rate of transcription initiation have -10 and -35 sequencessimilar to the ideal promoter shown previously and are calledstrong promoters. Those that support a low rate of transcription initiation differ from this ideal sequenceand are called weak promoters. The lac operon, for instance,has a weak promoter. Its sequencediffers from the consensusstrong promoter at severalpositions. This low intrinsic rate of initiation is further reducedby the lac repressor and substantiallyincreasedby the cAMP-CAP acuvaror. 02l+412]r

+

os (-821

t

01(+111 FIGURE 7-3 Lacrepressor-operator interactions.The tetrameric /acrepressor bindsto the primary (O/) and /acoperator oneof two secondary (O2or C.3) operators simultaneously. Thetwo structures arein equilibrrum fromB Muller-Hill, 1998, IAdapted Curr. Op Microbiol 1:1451

T R A N S c R t p I o N A Lc o N T R o L o F G E N EE X e R E S s t o N

nf M a n y S m a l lM o l e c u l e sR e g u l a t eE x p r e s s i o o Repressors B a c t e r i aG l e n e sv i a D N A - B I n d i n g and Activators

cellular needsby binding specificsmall moleculeligands (e.g., cAMP) or by post-translationalmodifications, such as phosphorylation, that alter the conformation of the activator.

Transcription of most E. coli genesis regulated by processes similar to those described for the lac operon, although the detailed interactions differ at each promoter. The general mechanisminvolves a specificrepressorthat binds to the operator region of a gene or operon, thereby blocking transcription initiation. A small molecule ligand (or ligands) binds to the repressor,controlling its DNA-binding activity and consequentlythe rate of transcription as appropriate for the needsof the cell. As for the lac operon,many eubacterial transcription-control regionscontain one or more secondary operators that contribute to the level of represslon. Specificactivator proteins, such as CAP in the lac operon, also control transcription of a subsetof bacterial genesthat have binding sitesfor the activator.Like CAP,other activators bind to DNA together with RNA polymerase, stimulating transcription from a specific promoter. The DNA-binding activity of an activator can be modulated in response to

TranscriptionInitiation from SomePromoters RequiresAlternative SigmaFactors Most E. coli promoters interact with o7O-RNA polymerase, the major initiating form of the bacterial enzyme. Transcription of certain groups of genes,however, is initiated by E. coli RNA polymerasescontaining one of severalalternative sigmafactorsthat recognizedifferent consensusprothan o70does(Table7-1). Thesealternative moter sequences for the transcription of sets of genes required are o-factors with related functions such as those involved in the response to heat shock or nutrient deprivation, motility, or sporulation in gram-positive eubacteria.In E. coli there are six alternativeo-factors in addition to the major "housekeeping" o-factor, o70. The genome of the gram-positive, sporulating bacterium Streptomycescoelicolor encodes 63 o-factors,the current record, basedon sequenceanalysisof

IONSENSUS PROMOTER

- 35REGION

- I OREGION

Housekeeping genes,most genesin exponentially replicating cells

TTGACA

TATAAT

Stationary-phasegenesand general stressresponse

TTGACA

TAIAAT

o't2

Induced by unfolded proteins in the cytoplasm; genesencoding chaperonesthat refold unfolded proteins and protease systems leading to the degradation of unfolded proteins in the cytoplasm

TCTCNCCCTTGAA

CCCCATNTA

F C'-

Activated by unfolded proteins in the periplasmic spaceand cell membrane;genesencodingproteins that restore integrity to the cellular envelope

GAACTT

TCTGA

Genes involved in flagellum assembly

CTAAA

CCGATAT

Genesrequired for iron uptake

TTGGAAA

GTAATG

FACTOR SIGMA 70

('s

o

F

FecI

RECt)GNIZED PROMOTERS

Genes for nitrogen metabolism and other functions eds', Cold Spring Souncr-s: C. A. Gross, M Lonetto, and R. Losick, 1992, rn Transcriptional Regulation, S L. McKnight and K' R, Yamamoto, 1 9 93,Proc'Nat'l' 1 . 9 8 9 , P r o c . N a t ' l . A c a d . S c l . U S A 8 6 : 8 3 0 ; K . T a n a k a e t a l . , HarborLaboratoryPress;D N.ArnostiandM. I.Chamberlin, 169:483' A c a d . S c i . ,{ / S A 9 0 : 3 5 1 1 ; C . D a r t i g a l o n g u e e t a l . , 2 0 0 1 , J . B i o l . C h e m . 2 7 6 2 2 0 8 6 6 ; A . A n g e r e r a n d V B r a u n , 1 9 9 8 , A r c h ' M i c r o b i o l ' N BACTERIA C O N T R O LO F G E N EE X P R E S S I OIN

.

273

tal

N t r Cd i m e r s P a i ro f p h o s p h o r y l a t e d NtrC dimers \ o54- RNA polymerase

( - 1 4 0a n d - 10 8 )

promoter

NtrC dimers

osa- RNA polymerase

EXPERIMENTAL FTGURE 7-4 DNAloopingpermitsinteraction of bound NtrCand oso-RNApolymerase.(a)Drawing (/eft)and (right)oI DNArestrrction electron mrcrograph fragment with phosphorylated NtrCdimersbindingto theenhancer regionnearone endandoso-RNA polymerase boundto theqtnApromoter nearthe

otherend (b)Drawing(/eff)andelectronmicrograph (flght)of the samefragmentpreparation showingNtrCdimersandosa-RNA polymerase bindingto eachotherwith the intervening DNAforming procNat':. a loopbetween them [Micrographs fromW Suetal, 1990, Acad.SciU5A87:5505; courtesv of S Kustu I

nearly 100 eubacterialgenomes.Most are structurally and functionally relatedto o70.But one classis unrelated,representedin E. coli by oto. Transcription initiation by RNA polymerasescontaining o70-likefactors is regulatedby repressorsand activatorsthat bind to DNA near the region where the polymerasebinds, similar to initiation by ot0R N A p o l y m e r a s ei t s e l f .

which synthesizesthe amino acid glutamine from glutamic acid and ammonia. The osa-RNA polymerase binds to the glnA promoter but does not melt the DNA strands and initiate transcription until it is activated by NtrC, a dimeric protein. NtrC, in rurn, is regulatedby a protein kinase called NtrB. In responseto low levels of glutamine, NtrB phosphorylaresdimeric NtrC, which then binds to an enhancer upstream of the glnA promoter. Enhancer-bound phosphorylated NtrC then stimulatesthe osa-polymerasebound at the promoter to separate the DNA strands and initiate transcrlptron. Electron microscopy studies have shown that phosphorylated NtrC bound ar enhancersand osa-polymerasebound at the promoter directly interact, forming a loop in the DNA between the binding sites (Figure 7-4). As discussedlater in this chapter, this activation mechanism resemblesthe predominant mechanism of transcriptional activation in eukaryotes. NtrC has MPase activirl, and ATP hydrolysis is required for activation of bound osa-polymeraseby phosphorylated NtrC. Evidence for this is that mutants with an NtrC defectivein ATP hydrolysis are invariably defectivein stimulating the osa-polymeraseto melt the DNA strands at the

Transcriptionby oto-RNApolymerasels Controlledby ActivatorsThat Bind Farfrom the Promoter The sequenceof one E. coli sigma factor, o54, is distinctly different from that of all the o7o-like facrors. Transcription of genesby RNA polymerasescontaining o5a is regulated solely by activators whose binding sitesin DNA, referred to as enhancers,generallyare located 80-160 basepairs upstream from the start site. Even when enhancers -ou.d "r. more than a kilobase away from a start site, o-5a-activators can activate transcription. The best-characterizedosa-activator-the NtrC protein (nitrogen regulatory protein C)-stimulates transcription of the glnA gene.glnA encodesthe enzymeglutamine synthetase, 274

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transcription start site. It is postulated that AIP hydrolysis supplies the energy required for melting the DNA strands. In contrast, the o70-polymerasedoes not require ATP hydrolysis to separatethe strands at a start site.

Are Controlledby Many BacterialResponses Two-ComponentRegulatorySystems As we have just seen,control of the E. coli glnA gene depends on two proteins, NtrC and NtrB. Such twocomponent regulatory systemscontrol many responsesof bacteria to changesin their environment. Another example involves the E. coli proteins PhoR and PhoB, which regulate transcription in responseto the concentration of free phosphate. PhoR is a transmembraneprotein, located in the inner (plasma)membrane, whose periplasmic domain binds phosphate with moderate affinity and whose cytosolic domain has protein kinase activity; PhoB is a cytosolic protein. Large protein pores in the E. coli outer membrane allow ions to diffuse freely between the external environment and the periplasmic space. Consequently,when the phosphate concentration in the environment falls, it also falls in the periplasmic space,causing phosphateto dissociatefrom the PhoR periplasmicdomain, as depictedin Figure 7-5. This causesa conformational changein the PhoR cytoplasmic domain that activatesits protein kinase activity. The activated PhoR initially transfers a 1-phosphate from ATP to a histidine (H) side chain in the PhoR kinase domain itself. The

samephosphateis then transferredto a specificaspartic acid (D) side chain in PhoB, converting PhoB from an inactive to an active transcriptional activator. Phosphorylated' active PhoB then inducestranscription from severalgenesthat help the cell cope with low phosphateconditions. Many other bacterial responsesare regulated by two proteins with homology to PhoR and PhoB. In each of these regulatory systems,one protein, called a sensor,contains a transmitter domain homologous to the PhoR protein kinase domain. The transmitter domain of the sensorprotein is regulatedby a secondunique protein domain (e.g.,the periplasmic domain of PhoR) that sensesenvironmental changes. The second protein, called a responseregulator, contains a receiver domain homologous to the region of PhoB that is phosphorylated by activated PhoR. The receiver domain of the responseregulator is associatedwith a second domain that determinesthe protein's function. The activity of this second functional domain is regulated by phosphorylation of the receiverdomain. Although all transmitter domains are homologous (as are receiverdomains),the transmitter domain of a specificsensorprotein will phosphorylate only the receiver domains of specific responseregulators, allowing specific responsesto different environmental changes.Note that NtrB and NtrC, discussedabove, function as sensor and response regulator proteins, respectively,in the twocomponent regulatory systemthat controls transcription of glnA. Simrlar two-component histidyl-aspartyl phosphorelay regulatory systemsare also found in plants.

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Control of Gene Expressionin Bacteria r Gene expressionin both prokaryotes and eukaryotes is regulated primarily by mechanismsthat control the initiation of transcription. r The first step in the initiation of transcription in E. coli is binding of the o subunit complexed with an RNA polymeraseto a promoter. r The nucleotide sequenceof a promoter determines its strength,that is, how frequently different RNA polymerase moleculescan bind and initiate transcription per minute. r Repressorsare proteins that bind to operator sequences, which overlap or lie adjacentro promorers.Binding of a repressorto an operator inhibits transcription initiation. r The DNA-binding activity of most bacterial repressorsis modulated by small molecule ligands.This allows bacterial cells to regulate transcription of specificgenesrn response to changesin the concentration of various nutrients in the environment and metabolitesin the cytoplasm. r The lac operon and some other bacterial genesalso are regulated by activator proteins that bind next ro promorers and increase the rate of transcription initiation by RNA polymerase. r The major sigma factor in E. coli is o70, but severalother less abundant sigma factors are also found, each recognizing different consensuspromoter sequences. r Transcription initiation by all E. coli RNA polymerases, except those containing osa, can be regulated by repressors and activators that bind near the transcriotion start site ( s e eF i g u r e7 - 2 ) . Genes transcribed by osa-RNA polymerase are reguted by activators that bind to enhancerslocated :100 basepairs upstream from the start site. When the activator and osa-RNA polymeraseinteracr, the DNA betweentheir binding sitesforms a loop (seeFigure 7-4). r In two-component regulatory systems,one prorern acts as a senso! monitoring the level of nutrients or other components in the environment. Under appropriate conditions, the "y-phosphateof an ATP is transferredfirst to a histidine in the sensorprotein and then to an aspartic acid in a second protein, the responseregulator. The phosphorylated response regulator then binds to DNA regulatory sequences,thereby stimulating or repressingtranscription of specificgenes(seeFigure 7-5).

JA Overviewof EukaryoticGene Controland RNAPolymerases In bacteria,geneconrrol servesmainly to allow a singlecell to adjust to changesin its environmentso that its growth and division can be optimized. In multicellular organisms,environmental changesalso induce changesin geneexpression.An ex276

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ample is the responseto low oxygen (hypoxia) in which a specific set of genesis rapidly induced that help the cell survive under the hypoxic conditions. These include secretedangiogenic proteins that stimulate the growth and penetration of new capillaries into the surrounding tissue.However, the most characteristic and biologically far-reachingpurpose ofgene control in multicellular organisms is execution of the genetic program that underlies embryological development. Generation of the many different cell types that collectively form a multicellular organism depends on the right genes being activated in the right cells at the right time during the developmentalperiod. In most cases,once a developmentalstep has been taken by a cell, it is not reversed. Thus these decisions are fundamentally different from the reversible activation and repression of bacterial genesin responseto environmental conditions. In executingtheir geneticprograms, many differentiated cells (e.g., skin cells, red blood cells, and antibodyproducing cells) march down a pathway to final cell death, leaving no progeny behind. The fixed patterns of gene control leading to differentiation serve the needs of the whole organism and not the survival of an individual cell. Despite the differencesin the purposes of gene control in bacteria and eukaryotes, two key features of transcription control first discoveredin bacteriaand describedin the previous section also apply to eukaryotic cells.First, protein-binding regulatory DNA sequences,or control elements,are associated with genes.Second, specific proteins that bind to a gene's regulatory sequencesdetermine where transcription will start and either activate or repressits transcription. As representedin Figure 7-1, in multicellular eukaryotes, inactive genes are assembledinto condensedchromatin, which inhibits the binding of RNA polymerasesand general transcription factors required for transcription initiation. Activator proteins bind to control elementsnear the transcription start site of a gene as well as kilobases away and promote chromatin decondensationand binding of RNA polymerase to the promoter. Repressorproteins bind to alternative control elements,causing condensation of chromatin and inhibition of polymerasebinding. In this section,we discussgeneral principles of eukaryotic genecontrol and point out some similarities and differencesbetweenprokaryotic and eukaryotic systems.Subsequentsectionsof this chapter will address specificaspectsof eukaryotic transcription in greater detail.

RegulatoryElementsin EukaryoticDNA Are Found Both Closeto and Many KilobasesAway from TranscriptionStart Sites Direct measurementsof the transcription rates of multiple genesin different cell types have shown that regulation of transcription initiation is the most widespread form of gene control in eukaryotes,as it is in bacteria.In eukaryotes,as in bacteria, a DNA sequencethat specifieswhere RNA polymerasebinds and initiates transcription of a gene is called a promoter. Transcription from a particular promoter is controlled by DNA-binding proteins that are functionally equivalent to bacterial repressorsand activators. Sincethesetranscriptional regulatory proteins can often function either to

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activateor to represstranscriptiondependingon their association with other proteins, they are more generallycalled transcriptionfactors.The DNA control elementsin eukaryotic genomesthat bind transcription factors often are located much farther from the promoter they regulatethan is the casein prokaryotic genomes.In some cases,transcrlption factors;that regulateexpressionof protein-codinggenes in higher eukaryotesbind at regulatory sitestens of thousandsof basepairs either upstream (oppositeto the direction of transcription)or downstream(in the samedirection a s t r a n s c r i p t i o n )f r o m t h e p r o m o t e r . A s a r e s u l t o f t h i s arrallgemernt,transcription of a single gene may be regulated by binding of multiple transcriptionfactorsto alternative control elements,directing expressionof the samegene in different types of cells and at different times during develclpment. F o r e x a m p l e , s e v e r a l s e p a r a t et r a n s c r i p t i o n - c o n t r o l regulateexpressionof the mammaliangene DNA sequ,:nces factor Pax6. Pax6 protein is retranscription the encoding quired for developmentof the eye, certain regions of the brain and spinal cord, and the cellsin the pancreasthat secrete horrnonessuch as insulin. Heterozygoushumanswith only one ftrnctional Pax6 geneare born with aniridia, a lack of irisesin the eyes(Figure1-26).The Pax6 geneis expressed from at leastthreealternativepromotersthat function in differentcell rrypesand at different timesduring embryogenesis ( F i g u r e7 - 6 a ) .W h e n t r a n s g e n im c i c ea r ep r e p a r e d( s e eF i g u r e

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regionsof the 7-6 Analysisof transcription-control < FIGURE Pax6 mousePax6genein transgenicmice.(a)Threealternative indifferent timesduringembryogenesis at distinct promoters areutilized regtons embryoTranscription-control of thedeveloping tissues specific by colored indicated are tissues Pax6 in different oI expression regulating controlregionin intron1 between Thetelencephalon-specific rectangles Theother to highresolution Oandt hasnotbeenmapped exons in length(b)Bshownare:200-500basepairs controlregions with a of a mouseembryo intissues galactosidase expressed The fertilization days after 1 0 5 transgene reporter B-galactosidase withB kbof DNA a transgene contained embryo genome of themouse codingregionLense fromexonOfusedto theB-galactosidase upstream of theeye Expression lense into the (LP) develop thatwill pit isthetissue (p) (c)Bintothepancreas thatwilldevelop intissue wasalsoobserved witha B-galactosidase embryo ina 13,5-day galactosidase expression exons4 in part(a)between geneundercontrolof thesequence reporter of the regions andtemporal to nasal Arrowpoints Retina, and5 marked been have also regions Pax6 transcription-control relina developing fromthe3' exonin an intronof the found:17 kbdownstream DeuBiol (a)adapted etal, 1999, fromB Kammendal geneIPart neighboring Grussl ofPete" patstbranotcr:Couflesv 205:79;

-5-43)containing a B-galactosiddserepotter gene fused to 8 is kb of DNA upstreamhom Pax6 exon 0, B-galactosidase pancreas of the and cornea' lense, developing in the observed embryo halfway through gestation(Figure7-6b).Analysisof transgenicmice with smallerfragmentsof DNA from this region allowed the mapping of separatetranscription-control regions regulatingtranscriptionin the pancreasand in the lenseand cornea.Transgenicmice with other reporter gene constructsrevealedadditional transcription-controlregions (Figure7-6a).Thesecontrolledtranscriptionin the developing retina and different regions of the brain (encephalon). Sorneof thesetranscription-controlregionsare in introns between exons4 and 5 and betweenexons 7 and 8. For example, a reporter geneunder control of the region labeledretina in Figure 7-6a betweenexons 4 and 5 led to reporter gene expressionspecificallyin the retina (Figure7-6c). Control regionsfor many genesare found severalhundreds of kilobasesaway from the coding exons of the gene. Or.remethodfor identifyingsuchdistantcontrol regionsis to

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compare the sequencesof distantly related organisms.Transcription control regions for a conservedgene are also often conservedand can be recognizedin the background of nonfunctional sequencethat divergesduring evolution. For example, there is a human DNA sequence:500 kilobases downstream of the SALLI gene thai is highly conservedin mice, frogs, and fish (Figure 7-7a).This geneencodesa transcription repressorrequired for normal development of the lower intestine, kidneys, limbs, and ears. Vhen transgenic mice were generated containing this conserved DNA sequence linked to a B-galactosidasereporter gene (Figure 77b), the transgenic embryos expresseda very high level of the B-galactosidasereporrer gene specificallyin the developing limb buds (Figure 7-7c).Human patients with deletions in this region of the genome develop with limb abnormalities. Theseresults indicate that this conservedregion directs transcription of the SALLl genein the developinglimb. presumably, other enhancerscontrol expressionof this gene in other types of cells where it functions in the normal divelooment of the lower intestine.kidneys and ears. 278

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< EXPERIMENTAL FIGURE 7-7 The human SALLIgeneenhanceractivates expressionof a reportergene in limb budsof the developingmouseembryo. (a)Graphicrepresentation of the conservation . ^., ^ of DNAsequence rna regionof the human (from50214-50220 genome 5 kb of the =500 kb chromosome 16sequence) downstream fromtheSAlt 7 geneencoding a zincfingertranscription repressor. A regionof :500 bp of non-coding sequence isconserved fromfishto human.900bp including this conserved regionwereinserted intoa plasmid nextto thecodingregionfor E.coli (b)Theplasmid was B-galactosidase microinjected intoa pronucleus of a fertilized mouseeggandimplanted in the uterus of a pseudo-pregnant mouseto generate a transgenic mouseembryowith the "reporter gene"on the injected plasmidincorporated intoitsgenome(seeFigure5-43).(c)After 11.5daysof development whenlimbbuds develop, thefixedandpermeabilized embryo wasincubated in X-galwhichisconverted into an insoluble intensely bluecompound by Bgalactosidase The= 900 bp regionof human DNAcontained an enhancer thatstimulated strongtranscription of the B-galactosidase genein limbbudsspecifically. reporter fFrom theVISTA Enhancer Browser http://enhancer. lblgov;

Three EukaryoticPolymerases Catalyze Formationof Different RNAs The nuclei of all eukaryotic cells examined so far (e.g.,vertebrate, Drosophila, yeast, and plant cells) contain three different RNA polymerases, designated I, II, and III. These enzymesare eluted at different salt concentrations during ionexchangechromatography, reflecting the polymerases'various net charges.The three polymerasesalso differ in their sensitivity to o'-amanitin, a poisonous cyclic octapeptide produced by somemushrooms (Figure7-8). RNA polymeraseI is very insensitiveto o-amanitin, but RNA polymeraseII is very sensitive-the drug binds near the active site of the enzyme and inhibits translocarion of the enzyme along the DNA template.RNA polymeraseIII has intermediatesensitivity. Each eukaryotic RNA polymerase catalyzestranscription of genesencodingdifferent classesof RNA (Table 7-2). RNA polymerase1, located in the nucleolus,transcribesgenesencoding precursor rRNA (pre-rRNA), which is processedinto 28S, 5.8S, and 18S rRNAs. RNA polymerase III transcribes genes encoding tRNAs, 55 rRNA, and an array of small,

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subunits and 1.0-14smallersubunits,some of which are common betweentwo or all three of the polymerases.The bestcharacterizedeukaryotic RNA polymerasesare from the yeast ,A Saccbaromycescereuisiae.Each of the yeast genes encoding II I the polymerasesubunits has been cloned and sequencedand O the effects of gene-knockout mutations have been characterc O ized. In addition, the three-dimensional structure of yeast o: A RNA polymeraseII has beendetermined(Figure7-9b, c).The aE I co I three nuclear RNA polymerasesfrom all eukaryotes so far exo amined are very similar to those of yeast. ACt o. t; TThe two large subunits (RPB1 and RPB2) of all three euEE tc -> karyotic RNA polymerasesare related to each other and are 9< z* similar to the E. coli B' and p subunits, respectively(Figure YZ 7-10). Each of the eukaryotic polymerasesalso contains an co-likeand two nonidentical a-like subunits. The extensive 10 20 30 40 50 similarity in the structures of these core subunits in RNA Fraction number polymerasesfrom various sourcesindicatesthat this enzyme FIGURE 7-8 Columnchromatography A EXPERIMENTAL arose early in evolution and was largely conserved. This separatesand identifiesthe three eukaryoticRNApolymerases, seemslogical for an enzyme catalyzing a process so basic as eachwith its own sensitivityto a-amanitin.A proteinextract copying RNA from DNA. cellsispassed througha DEAE fromthe nucleiof cultured eukaryotic In addition to their core subunits related to the E. coli proteineluted(black witha curve) Sephadex columnandadsorbed RNA polymerasesubunits, all three yeast RNA polymerases Threefractions increasing NaClconcentration of constantly solution contain four additional small subunits, common to them but activity(red fromthe eluatesubsequently showedRNApolymerase polymerase not to the bacterial RNA polymerase.FinallS each eukaryinhibits of 1 pglml,o-amanitin curve)At a concentration otic nuclear RNA polymerase has several enzyme-specific I andlll(greenshading) but hasno effecton polymerases ll activity subunits that are not present in the other two nuclear RNA whereas lll isinhibited by 10 pglmlof o-amanitin, Polymerase Gene-knockout experiments in yeast indicate polymerases. R polymerase I is unaffected evenat thishigherconcentration [See that most of these subunits are essentialfor cell viability. 1974. J BiolChen249:241 G Roeder. I Disruption of the few polymerasesubunit genesthat are not absolutely essentialfor viability (subunits4 andT) neverthestable RNAs, including one involved in RNA splicing (U6) lessresults in very poorly growing cells.Thus it seemslikely and the RNA component of the signal-recognitionparticle that all the subunits are necessaryfor eukaryotic RNA poly(SRP)involved in directing nascentproteins to the endoplasmerasesto function normally. mic reticulum (Chapter 13). RNA polymerase11 transcribes all protein-codinggenes;that is, it functions in production of The LargestSubunit in RNA Polymerasell Has mRNAs. RNA polymeraseII also produces four of the five Repeat an EssentialCarboxyl-Terminal small nuclearRNAs that take part in RNA splicing. The carboxyl end of the largest subunit of RNA polymerase II Each of the three eukaryotic RNA polymerasesis more (RPB1)contains a stretch of sevenamino acids that is nearly complex than E. coli RNA polymerase,although their strucprecisely repeated multiple times' Neither RNA polymerase I tures are similar (Figure 7-9a,b). All three contain two large

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A FIGURE 7-9 €omparisonof three-dimensional structures of bacterialand eukaryoticRNApolymerases. (a,b) TheseC. trace models arebasedon x-raycrystallographic analysis of RNApolymerase fromthe bacterium T.aquaticus andcoreRNApolymerase ll from (a)Thefivesubunits 5 cereyr''siae of the bacterial enzymeare distinguished bycolorOnlythe N-terminal domains of thecrsubunits areincluded in thismodel(b)Tenof the 12subunits constituting yeastRNApolymerase ll areshownin thismodelSubunits thatare similar in conformation to thosein the bacterral enzyme areshownin thesamecolorsTheC-terminal domainof the largesubunitRpBl wasnotobserved in thecrystal structure, but it isknownto extend fromtheposition marked witha redarrow(RpBistheabbreviation for "RNApolymerase 8," whichisan alternative wayof referring to nor III contains these repeating units. This heptapeptide repeat,with a consensussequenceof Tyr-Ser-Pro-Thr-Ser-proSer,is known asthe carboxyl-terminaldomain (CTD).yeast RNA polymeraseII contains 26 or more repears,vertebrate enzymes have 52 repeats, and an intermediate number of repeats occur in RNA polymerase II from nearly all other eukaryotes. The CTD is critical for viability, and at least 10 copiesof the repeatmust be presentfor yeastto survlve. In vitro experimentswith model promoters first showed that RNA polymerasell moleculesthat initiaretranscription have an unphosphorylated CTD. Once the polymerase initiatestranscription and beginsto move away from the promoter, many of the serineand some tyrosine residuesin the CTD are phosphorylated.Analysis of polytene chromosomes from Drosophila salivaryglandspreparedjust beforemolting of the larva, a time of active transcription, indicate that the CTD also is phosphorylatedduring in vivo transcription.The large chromosomal "puffs" induced at this time in development are regions where the genome is very actively transcribed.Staining with antibodiesspecificfor the phosphorylated or unphosphorylated CTD demonstrared thai RNR polymeraseII associatedwith the highly transcribedpuffed regions contains a phosphorylatedCTD (Figure 7-11). 280

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RNApolymerase ll ) (c)Space-filling modelof yeastRNApolymerase including subunits 4 andT These subunits extendfromthecore portionof theenzyme shownin (b)neartheregionof the C-terminal domaio n f t h el a r g es u b u n iC t l a m pi sa d o m a i o n f R P Btlh a ts w i n g s on a hingewhenRNArsin theexitchannel to closeoverupstream DNAsothatthe polymerase cannotrelease fromthetemplate until transcription hasterminated Wallisthedomainof RpB2 thatforces DNAenteringthejawsof the polymerase fromthe leftto bendbefore it exitsthe polymerase TheRNAexitchannel (a) isindicated Ipart based oncrystal structures fromG Zhang etal, 1999,Ce// 98:81 1 part(b) fromP Crameret al , 200'1,Science2g2:1863 Part(c)fromK J Armache et al , 2003,PNAS 100:6964, andD A Bushnell andR D Kornberg, 2003,?NAS 100:6969; coloredmodelcourtesv of Steven HahnI

RNA Polymerasell InitiatesTranscriptionat D N A S e q u e n c eC s o r r e s p o n d i ntgo t h e 5 ' C a p of mRNAs In vitro transcription experiments involving RNA polymeraseII, a protein extract prepared from the nuclei of cultured cells, and DNA templatescontaining sequencesencoding the 5' ends of mRNAs for a number of abundantly expressedgenes revealed that the transcripts produced always contained a cap structure at their 5' endsidentical with that present at the 5' end of nearly all eukaryotic mRNAs (seeFigure4-14).lnthese experimenrs,the 5'cap is addedto the 5' end of the nascentRNA by enzymesin the nuclear extract, which can only add a cap to an RNA that has a 5, trior diphosphate.Becausea 5' end generatedby cleavageof a longer RNA would have a 5' monophosphate,it would not be capped. Consequently,researchersconcluded that the cappednucleotidesgeneratedin the in vitro transcription reactions must have been the nucleotides with which transcription was initiated. Sequenceanalysisrevealedthat for a given gene,the sequenceat the 5' end of the RNA transcriprs produced in vitro is the same as that at the 5' end of the mRNAs isolated from cells, confirming that the capped

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Overview of Eukaryotic Gene Control and RNA Polymerases r The primary purpose of genecontrol in multicellular organismsis the execution of precisedevelopmentaldecisions so that the proper genesare expressedin the proper cells during developmentand cellular differentiation. r Transcriptional control is the primary means of regulating gene expressionin eukaryotes,as it is in bacteria. r In eukaryotic genomes,DNA transcription-control elements may be located many kilobasesaway from the promoter they regulate. Different control regions can control transcription of the samegene in different cell types.

r Eukaryotes contain three types of nuclear RNA polymerases.All three contain two large and three smaller core subunits with homology to the F" F, o, and to subunits of E. coli RNA polymerase, as well several additional small subunits(seeFigure 7-10). r RNA polymeraseI synthesizesonly pre-rRNA. RNA polymerase II synthesizesmRNAs and some of the small nuclear RNAs that participate in mRNA splicing' RNA polymerase III synthesizestRNAs' 55 rRNA, and several other relatively short, stable RNAs (seeTable 7-2). r The carboxyl-terminal domain (CTD) in the largest subunit of RNA polymeraseII becomesphosphorylatedduring transcription initiation and remains phosphorylated as the enzymetranscribesthe template. r RNA polymerase II initiates transcription of genes at the nucleotide in the DNA template that corresp o n d s t o t h e 5 ' n u c l e o t i d e t h a t i s c a p p e di n t h e e n c o d e d mRNA.

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A21

to

491

Base c23 100 frequency G 2 8 35 3 0 t%l T

09567975241 00 0

0

0

0

0

31240

ArAfn+ _3; to -25

RegulatorySequences in Protein-

CodingGenes As noted in the previous section,expressionof eukaryotic protein-codinggenesis regulatedby multiple protein-binding DNA sequences, genericallyreferredto as rranscriptioncontrol regions.Theseinclude promotersand other typesof control elementslocated near transcription start sites, as well as sequences locatedfar from the genesthey regulate.In this section,we take a closerlook at the propertiesof various control elementsfound in eukaryotic protein-codinggenes and sometechniquesusedto identify them.

The TATABox, Initiators,and CpGlslands F u n c t i o na s P r o m o t e r si n E u k a r y o t i cD N A The first genesto be sequencedand studiedthrough in vitro transcriptionsystemswere viral genesand cellular protein, coding genesthat are very activelytranscribedeither at particular times of the cell cycle or in specificdifferentiated cell types.In all thesehighly transcribedgenes,a conservedsequencecalledthe TAIA box was found :25-35 basepairs upstreamof the start site (Figure7-12). Mutagenesisstudies have shown that a single-base changein this nucleotidesequence drastically decreasesin vitro transcription by RNA polymerase II of genes adjacent to a TAIA box. In mosr cases,sequencechangesbetweenthe TAIA box and start site do not significantly affect the transcription rate. If the base pairs between the TAIA box and the normal start site are deleted,transcriptionof the altered,shortenedtemplate begins at a new site -25 base pairs downstream from the TAIA box. Consequently,the TATA box acts similarly to an E. coli promoter to position RNA polymerase II for transcriptioninitiation (seeFigure4-12). Instead of a TATA box, some eukaryotic genesconraln an alternative promoter element called an initiator. Most naturally occurringinitiator elementshave a cytosine(C) at the - 1 position and an adenine(A) residueat the transcription start site (+1). Directed muragenesisof mammalian geneswith an initiator-containingpromoter revealedthat the nucleotide sequenceimmediately surrounding the start .

c H A p r E R7

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9 35 37 38 30

A G

mRNAstarts A=50% G=25% C , U= 2 5 %

I

v

Transcription

rAAA-/9

Consensus sequence

FIGURE7-12 Determination of consensusTATAbox sequence.The nucleotidesequences upstreamof the startsite in 900 differenteukaryoticprotein-coding geneswere alrgneoro maximizehomologyin the regionfrom -35 to -25 Thetabulated numbersare the percentage frequencyof eachbaseat eachposition

282

0

2810 83 9100 5 33 0 36 10 11 9

5',

lE

16 24

Maximum homology occurs overan eight-base region,referred to as the TATA box,whoseconsensus sequence isshownat the bottom, Theinitialbasein mRNAs encoded by genescontaining a TATAbox mostfrequently isan A [SeeP Bucher, 1990, J Mol.Biol212:5631

site determinesthe strength of such promoters. Unlike the conservedTAIA box sequence,however, only an extremely degenerateinitiator consensussequencehas been defined: (5') Y-Y-A+ r-N-T/A-Y-Y-Y (3') where A*' is the baseat which transcriptionstarts,Y is a pyrimidine (C or T), N is any of the four bases,and T/A is T or A at position *3. Transcription of genes with promoters containing a TAIA box or initiator element beginsat a well-defined initiation site. However, transcription of many protein-coding geneshas been shown to begin at any one of multiple possible sitesover an extendedregion,often20-200 basepairs in length. As a result, such genesgive rise to mRNAs with multiple alternative 5' ends. These genes,which generally are transcribedat low rates(e.g.,genesencodingthe enzymesrequired for basic metabolic processesrequired in all cells, often called "housekeeping genes"), do not contain a TATA box or an initiator. Most genesof this type contain a CGrich stretchof 20-50 nucleotideswithin :100 basepairs upstream of the start-siteregion. The dinucleotide CG is statistically underrepresentedin vertebrate DNAs, and the presenceof a CG-rich region, or CpG island, just upstream from a start site is a distinctly nonrandom distribution. For this reason, the presenceof a CpG island in genomic DNA suggeststhat it may contain a transcription-initiation region.

Promoter-Proximal ElementsHelp Regulate E u k a r y o t i cG e n e s Recombinant DNA techniqueshave been used to systematically mutate the nucleotide sequencesof various eukaryotic genes in order to identify transcription-control regions. For example, alternative transcription-control elementsregulate expression of the mammalian gene that encodes transthyretin (TTR), which transports thyroid hormone in blood and the cerebrospinalfluid that surrounds the brain and spinal cord. Transthyretin is expressedin hepatocytes, which synthesizeand secretemost of the blood serum proteins, and in choroid plexus cells in the brain, which secrete cerebrospinalfluid and its constituent proteins. The control

T R A N s c R t p l o N AcLo N T R o Lo F G E N EE X p R E s s t o N

DNA techniques.Reporter genesexpressenzymesthat are easily assayedin cell extracts. Commonly used reporter genesinclude the E. coli lacZ geneencoding B-galactosidase; t NA the firefly gene encoding luciferase,which converts energy [ | neco-uinanD Reporter techniaues from ATP hydrolysis into light; and the jellyfish geneencodI ing green fluorescentprotein (GFP). 1 T--------------Tl By constructing and analyzing a S'-deletion series up.-a 2 l-----of the T?R gene,researchersidentified two control elstream T-----l 3 4 l-----;-1 ementsthat stimulate reporter-geneexpressionin cultured he5 f-;-l patocytes but not in other cell types. One region mapped 5'-deletionseries betweenthe transcription start site and -200 basepairs upLigate into vector streamof the start site; the other mapped between:1.85 and carrying reporter gene 2.01 kb upstream of the ?TR gene transcription start site. Further studiesdemonstratedthat alternativeDNA sequences Transform E. coli and isolateolasmid DNAs control TTR transcription in choroid plexus cells, demonstrating again that alternative control elementsoften regulate transcription of eukaryotic genesin different cell types. By now, hundreds of eukaryotic genes have been analyzed, and scoresof transcription-control regions have been identified. These control elements,together with the TATA 5'-deletion mutants box or initiator, often are referred to as the promoter of the gene they regulate. However, we prefer to reservethe term - | Transfecteach type of promoter for the TATA box or initiator sequencesthat E I nlasmio (1-5) separatelyinto 'We use the c u l t u r e dc e l l s determine the initiation site on the template. I regions lying for control elements term promoter-proximal Reporter In start site. of the pairs upstream within 100-200 base Reporter plasmid enzyme speare cell-type elements promoter-proximal some cases, cific; that is, they function only in specific differentiated cell types. In eukaryotes the term enhancer is used to refer to Reporter mRNA transcription-control regions greater than -200 bp from I Pr"o.r" cellextractand the transcriptionstart site. E | ...iy activityof reporter Once a transcription-control region has been identified, enzyme J analysis of linker scanning mutations can pinpoint the sequenceswithin the regulatory region that function to conPlasmidno. Reporter-geneexpression trol transcription. In this approach, a set of constructs with +++ 1 2 +++ contiguous overlapping mutations are assayedfor their ef3+ fect on expressionof a reporter gene or production of a spe4+ mRNA (Figure 7 -1.4a). One of the first usesof this type cific 5of analysis identified promoter-proximal elements of the FIGURE 7-13 S'-Deletion A EXPERIIVIENTAL analysiscan thymidine kinase (lk) genefrom herpessimplex virus (HSV). identify transcription-controlsequencesin DNA upstreamof a The results demonstratedthat the DNA region upstream DNAtechniques areused eukaryoti
of s'ffis'

r7F DNA

Start

r-->

z E

N P R O T E I N - C O D I NGGE N E S R E G U L A T O RS Y E Q U E N C EIS

283

(a)

fiePortergene

VectorDNA -

..

|

controlresion

|

//

fk mRNA i

+++

Mutant no. 1

+++

2

+ T

+++

4

f

+++ +++ I 9

+++ Controlelements

Control region of fk gene

A E X P E R I M E N TFA L U R7E- 1 4 L i n k e r s c a n n i nmgu t a t i o n s IG identify transcription-control elements.(a)A regionof eukaryotic DNA(tan)thatsupports high-level expression gene(light of a reporter purple) isclonedin a plasmid vectorasdiagrammed at thetop. (15)mutations Overlapping linkerscanning (crosshatch) areintroduced fromoneendof the regionbeinganalyzed to the other.These mutations resultfromscrambling the nucleotide sequence in a short stretchof theDNA Afterthe mutantplasmids aretransfected separately intoculturedcells,the activityof the reporter-gene productisassayed

Inthe hypothetical example shownhere,LSmutations 1,4, 6,7, and t havelittleor no effecton expression of the reportergene,indicating thatthe regions altered in thesemutants containno controlelements Reporter-gene expression issignificantly reducedin mutants2, 3, 5, (brown)liein the intervals and8, indicating thatcontrolelements shown at the bottom(b)Analysis of LSmutations in thetranscription-control (tk)genefromherpes regionof the thymidine kinase simplex virus(HSV) identified (PE-1 a TATAboxandtwo promoter-proximal elements and (b)seeS L McKnight PE-2)[Part andR Kingsbury 1982,Science217:316]

Distant EnhancersOften Stim u late Transcription by RNA Polymerasell

thousands of base pairs from the start site. An extensive linker scanningmutational analysisof rhe SV40 enhancerindicated that it is composed of multiple individual elements, each of which contributes to the total activity of the enhancer.As discussedlater, each of theseregulatory elements is a protein-binding site. Soon after discovery of the SV40 enhancer, enhancers were identified in other viral genomesand in eukaryotic cellular DNA. Some of these control elementsare located 50 or more kilobasesfrom the promoter they control. Analyses of many different eukaryotic cellular enhancershave shown that they can occur upstream from a promoter, downstream from a promoter within an intron, or even downstream from the final exon of a gene,as in the caseof the Pax6 gene (see Figure 7-6). Llke promoter-proximal elements, many enhancersare cell-type specific. For example, an enhancer controlling Pax6 expressionin the retina has been characterized in the intron between exons 4 and 5 (seeFigureT-6). Analysesof the effects of deletions and linker scanningmutations in cellular enhancershave shown that. like the SV40

As noted earlier, transcription from many eukaryotic promoters can be stimulated by control elementslocated thousands of base pairs away from the start site. Such longdistance transcription-control elements, referred to as enhancers, are common in eukaryotic genomes but fairly rare in bacterial genomes.The first enhancer to be discovered that stimulatestranscription of eukaryotic geneswas in a 366-bp fragment of the simian virus 40 (SV40) genome (Figure 7-15). Further analysisof this region of SV40 DNA revealedthat an :100-bp sequencelying -1gg basepairs upstream of the SV40 early transcription start site was responsible for its ability to enhance transcription. In SV40, this enhancer sequencefunctions ro stimulate transcription from viral promoters. The SV40 enhancer,however, ,ii-ulates transcription from all mammalian promoters that have beentestedwhen it is insertedin either oiientation anywhere on a plasmid carrying the test promoter, even when it is 284

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enhancer,they generally are composed of multiple elements that contribute to the overall activity of the enhancer. p - gl o b i n

A.3' p - g l o b i nm R N A Treat with S1 nuclease and then denature

Perform gel electrophoresis and autoradiography

c12

527 404

**t 309* 2'r? 238

IG A E X P E R I M E N TFA L U R7E- 1 5 P l a s m i dcso n t a i n i n a g particular5V40DNAfragment showed markedincreasein mRNAproductioncomparedwith plasmidslackingthis genewith or withouta Plasmids containing the B-globin enhancer. of SV40DNAwereconstructed. Theseplasmids 366-bpfragment RNAwas weretransfected intoculturedcells,andanyresulting ( s t e p s p r o b e T h ea m o u n t h y b r i d i z et od a B - g l o b iD N A a n d B Z ) n by cellstransfected with oneor the of B-globinmRNAsynthesized method wasassayed by the S1nuclease-protection otherplasmid (stepB) Therestriction fragmentprobe,generated froma Bg l o b i nc D N Ac l o n ew , a sc o m p l e m e n t at or yt h e 5 ' e n do f B - g l o b i n with 32P(reddot) mRNAThe5' endof the probewaslabeled an -340of B-globinmRNAto the probeprotected Hybridization probe nuclease, fragmentof the fromdigestion by S1 nucleotide w h i c hd i g e s tssi n g l e - s t r a n dDeNdAb u t n o t D N Ai n a n R N A - D N A hybridAutoradiography of electrophoresed 51-protected (step4) revealed with plasmid1 fragments that cellstransfected ( l a n e1 ) p r o d u c em d u c hm o r eB - g l o b im n R N At h a nt h o s e of Bwith plasmid2 (lane2) LaneC is a controlassay transfected globinmRNAisolated from reticulocytes, whichactively synthesize p-globinTheseresults showthatthe SV40DNAfragmentin plasmi1 d c o n t a i nasn e l e m e nt h, ee n h a n c et rh,a tg r e a t l y et al, fromJ Banerji synthesis of B-globinmRNA[Adapted stimulates 1981, Cell 27:299 l

Most EukaryoticGenesAre Regulatedby Elements Multiple Transcription-Control Initially, enhancers and promoter-proximal elements were thought to be distinct types of transcription-controlelements. Howeve! as more enhancers and promoter-proximal elements were analyzed, the distinctions between them became less clear. For example, both types of element generallycan stimulate transcription even when inverted, and both types often are cell-typespecific.The generalconsensusnow is that a spectrum of control elements regulates transcription by RNA polymeraseII. At one extremeare enhancers,which can stimulate transcription from a promoter tens of thousandsof base pairs away (e.g., the SV40 enhancer).At the other extreme are promoter-proximal elements,such as the upstream elementscontrolling the HSV tk gene,which lose their influence when moved an additional 30-50 base pairs farther from the promoter. Researchershave identified a large number of transcription-control elementsthat can stimulate transcription from distancesbetweenthesetwo extremes. Figure 7-16a summarizesthe locations of transcriptioncontrol sequencesfor a hypothetical mammalian gene. The start site at which transcription initiates encodesthe first (5') nucleotide of the first exon of an mRNA, the nucleotidethat is capped. For many genes,especiallythose encoding abundantly expressedproteins, a TATA box located approximately 25-35 basepairs upstream from the start site directs RNA polymeraseII to begin transcription at the proper nucleotide. Promoter-proximal elements,which are relatively short (:lQ base pairs), are located within the first -200 basepairs upstream of the start site. Enhancers,in contrast, usually are about 50-200 basepairs long and are composed of multiple elementsof :10 base pairs. Enhancersmay be located up to 50 kilobasesor more upstream or downstream from the start site or within an intron. Many mammalian genesare controlled by more than one enhancerregion. The S. cereuisiaegenome contains regulatory elements called upstream activating sequences(UASs),which function similarly to enhancersand promoter-proximal elementsin higher eukaryotes.Most yeast genescontain only one UAS, which generally lies within a few hundred base pairs of the start site. In addition, S. cereuisiaegenescontain a TATA box :90 base pairs upstream from the transcription start site (Figure7-1.6b).

Regulatory Sequencesin Protein-CodingGenes r Expressionof eukaryotic protein-coding genesgenerally is regulated through multiple protein-binding control regions that are located close to or distant from the start site ( F i g u r e7 - 1 6 ) . r Promotersdirect binding of RNA polymeraseII to DNA, determine the site of transcription initiation, and influence transcrlptlon rate. N P R O T E I N - C O D I NGGE N E S R E G U L A T O RS Y E Q U E N C EIS

285

(a) Mammalian gene

up to -50 kb or more

+ 1 0t o +50 kb or more

(b) S. cerevrblaegene

Exon fl Intron l-l rnrn uo* Promoter-proximal Enhancer; l-l - yeast element UAS

=-90 A FIGURE 7-16 Generalorganizationof control elementsthat regulategene expressionin multicellulareukaryotesand yeast.(a)Genes of multicellular organisms containbothpromoterproximal elements andenhancers, aswellasa TATAboxor other promoter elementThepromoter position elements RNApolymerase ll to initiatetranscription at thestartsiteandinfluence the rateof transcription Enhancers maybe eitherupstream or downstream and

asfar awayas 50 kb or morefromthe transcription startsite In somecases, enhancers liewithinintronsForsomegenes,promoterproximalelements occurdownstream fromthe startsiteaswellas genescontainonlyone regulatory upstream(b) MostS.cerevisiae region,calledan upstreamactivatingsequence(UAS)and a TATA box,whichis :90 basepairsupstream fromthe startsite.

r Three principal types of promorer sequenceshave been identified in eukaryotic DNA. The TAIA box, the most common, is prevalent in highly transcribed genes.Initiator promoters are found in some genes,and CpG islands are characteristicof genestranscribed at a low rate.

analyses like those described in Chapter 5. However, in mammals and other vertebrates,which are lessamenableto such genetic analysis,most transcription factors have been detected initially and subsequentlypurified by biochemical techniques.In this approach, a DNA regulatory elementthat has been identified by the kinds of mutational analysesdescribed in the previous section is used to identify cognate proteins that bind specificallyto it. Two common techniques for detectingsuch cognateproteins are DNase I footprinting and the electrophoreticmobility shift assay. DNase I footprinting takes advantage of the fact that when a protein is bound to a region of DNA, it protects that DNA sequencefrom digestion by nucleases.As illustrated in Figure 7-17, when samples of a DNA fragmenr that is labeled at one end are digestedunder carefully controlled conditions in the presenceand absenceof a DNA-binding protein and then denatured, electrophoresed,and the resulting gel subjectedto autoradiographg the region protected by the bound protein appearsas a gap, or "footprint," in the array of bands resulting from digestion in the absenceof protein. When footprinting is performed with a DNA fragment containing a known DNA control element,the appearanceof a footprint indicatesthe presenceof a transcription factor that bincis that control element in the protein sample being assayed. Footprinting also identifies the specific DNA sequenceto which the transcription factor binds. The electrophoretic mobility shift assay (EMSA), also called the gel-shift or band-shift assay,is more useful than the footprinting assay for quantitative analysis of DNAbinding proteins. In general,the electrophoreticmobility of a DNA fragment is reducedwhen it is complexed to protein, causing a shift in the location of the fragment band. This assay can be used to detect a transcription factor in protein fractions incubated with a radiolabeled DNA fragment containing a known control element (Figure 7-18). In the biochemical isolation of a transcription factor, an extract of cell nuclei commonly is subjectedsequentiallyto severaltypes of column chromatography (Chapter 3). Fractions eluted from the columns are assayedby DNase I footprinting or EMSA using DNA fragmentscontaining an identified regulatory element (see Figures 7-"1.7 and 7-18).

r Promoter-proximal elements occur within -200 base pairs upstream of a start site. Severalsuch elements.containing -10 basepairs, may help regulatea particular gene. r Enhancers, which contain multiple short control elements, may be located from 200 basepairs to tens of kilobasesupstream or downstream from a promoter, within an intron, or downstream from the final exon of a gene. r Promoter-proximal elements and enhancers often are cell-type specific,functioning only in specificdifferentiated cell types.

Activatorsand Repressors of lA Transcription The various transcription-control elementsfound in eukaryotic DNA are binding sitesfor regulatory proteins. The simplest eukaryotic cells encode hundreds of transcription factors, and the human genome encodes over 2000. The transcription of each gene in the genome is independently regulated by combinations of specific transcription factors that bind to its transcription-control regions.The number of possible combinations of this many transcription factors is astronomical, sufficient to generate unique controls for every geneencodedin the genome.In this section,we discuss the identification, purification, and structures of these transcription factors, which function to activate or repressexpressionof eukaryotic protein-coding genes.

Footprintingand Gel-5hiftAssaysDetect Protein-DNAInteractions In yeast,Drosophila, and other geneticallytractable eukaryotes, numerous genes encoding transcriptional activators and repressors have been identified by classical genetic 286

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T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X P R E S S | O N

SampleA (DNA-binding protein absentl

Sample B (DNA-binding protein present) Sequ e nce-specific b i n d i n gp r o t e i n

Protein-binding sequence

J

(b)

Fraction MNEOFTl

Fraction o N 1

Bound p r o b e*

Free PrObe+

8 9 1 0 1 11 2 1 3 1 4 1 51 6 1 8 2 02 2

2 3 4 5 6 7 8 9 1011121416182022

;l'

7-'17DNaselfootprinting reveals FIGURE < EXPERIMENTAL control-elementsequencesand can be usedas an assayin transcriptionfactor purification.(a)DNaseI footprintingcan A DNAfragmentknownto sequences control-element identify at oneendwith 12P(reddot) containthecontrolelementislabeled I in with DNase thenaredigested DNAsample of the labeled Portions thoughtto containa of proteinsamples andabsence the presence the phosphodiester hydrolyzes protein. I randomly DNase cognate of one bondsof DNAbetweenthe 3' oxygenon the deoxyribose A low of the nextnucleotide andthe 5' phosphate nucleotide eachDNA I isusedsothaton average, of DNase concentration justonce(vertical lf the proteinsample arrows). iscleaved molecule protein, the DNAfragment doesnot containa cognateDNA-binding positions andunlabeled betweenthe labeled at multiple iscleaved A on the left lf the asin sample fragment, endsof theoriginal B on the protein, asin sample a cognate proteinsample contains portionof protecting a DNA, thereby protein to the binds right,the the DNAis treatment, DNase fromdigestionFollowing thefragment and thestrands, to separate denatured fromprotein, separated geldetects only of the resulting Autoradiography electrophoresed fromthe labeled extending fragments andreveals strands labeled fragments L Cleavage by DNase endto thesiteof cleavage A showup on thegelfor sample thecontrolsequence containing the boundcognateprotein B because in sample but aremissing of the andthusproduction withinthatsequence cleavages blocked gel constitute on the bands The missing fragments corresponding a sequence-specific thefootprint(b)A proteinfractioncontaining proteincanbe purifiedby columnchromatography. DNA-binding whichof the elutedfractions canthenidentify I footprinting DNase of addedprotein(NE,no thecognateproteinInthe absence contarn multiple sites, at fragment the DNA I cleaves DNase extract), bandson the gelshownhereA cognateprotein producing multiple to the column(O,onput) extractapplied present in the nuclear generated a footprintThisproteinwasboundto the column,since protein in theflow-through wasnot detected activity footprinting to the column,mostof wasapplied fraction(FT)Aftera saltgradient by the 9-12,asevidenced the cognateproteinelutedin fractions of the protein-binding Thesequence bands(footprints). missing with markerDNA bycomparison regioncanbe determined (b) the samegel(M).Ipart on length analyzed known of fragments J BiolChem264:105291 et al, 1989, fromS Yoshinaga

mobilityshift 7-18 Electrophoretic FIGURE < EXPERIMENTAL assaycan be usedto detecttranscriptionfactorsduring bycolumn proteinfractions separated purification.Inthisexample, to bindto a radiolabeled for theirability wereassayed chromatography elementAfteran a knownregulatory probecontaining DNA-fragment ontothecolumn(ON)and wasloaded aliquotof theproteinsample (numbers) withthelabeled wereincubated columnfractions successive thatdo not conditions under probe,the samples wereelectrophoresed probe to protein protein-DNA not bound free The interactions disrupt applied to the bottomof the gel A proteinin the preparation migrated 7 and8 boundto theprobe,forminga to thecolumnandin fractions that migratedmoreslowlythanthefreeprobe complex DNA-protein proteinbeing likelycontainthe regulatory therefore Thesefractions etal, 1989,,/Biol.Chem264:10529] 5 Yoshinaga souqht[From OS F TRANSCRIPTION ACTIVATORA SND REPRESSOR

287

Adenovirus DNA

*t

SV40 DNA

rt

SP1:-+-+ A EXPERIMENTAL FIGURE 7-19 Transcription factorscan be identified by in vitro assayfor transcriptionactivity.Sp1was identified basedon itsabilityto bindto a regionof the SV40genome thatcontains sixcopies promoter-proximal of a GC-rich element and waspurified by columnchromatography Totestthetranscriptionactivating ability of purified 5P1,it wasincubated in vitrowithtemplate DNA,a proteinfractron containing RNApolymerase ll andassociated general transcription factors, andlabeled ribonucleoside triphosphates Thelabeled RNAproducts weresubjected to electrophoresis and autoradiography Shownhereareautoradiograms fromassays with adenovirus (-) andpresence andSV40DNAin the absence (+) of SP1SP1hadno significant effecton transcription fromtheadenovirus promoter, whichcontains no SP1-binding sitesln contrast, Sp1 stimulated transcription fromthe SV40promoter abouttenfold [Adaptedfrom M R Briggset al , 1986, Science234:4jI

Fractionscontaining protein that binds to the regulatory element in these assaysprobably contain a putative transcription factor. A powerful technique commonly used for the final stepin purifying transcriptionfacrorsis sequence-specific DNA affinity cbromatography, a particular type of affinity chromatography in which long DNA strands containing multiple copies of the transcription factor-binding site are coupled to a column matrix. As a final test that an isolated protein is in fact a transcription factor, its ability to modulate transcription of a template containing the corresponding protein-binding sites is assayedin an in vitro transcription reaction. Figure 7-19 shows the results of such an assayfor SP1, a transcription factor that binds to GC-rich sequences, thereby activating transcription from nearby promoters. Once a transcription factor is isolated and purified, its partial amino acid sequencecan be determined and used to clone the geneor cDNA encodingit, as outlined in Chapter 5. The isolated gene can then be used to test the ability of the encodedprotein to activate or represstranscription in an in vivo transfection assay(Figure 7-20).

ActivatorsAre Modular ProteinsComposedof D i s t i n c tF u n c t i o n aD l o m a i n sa n d p r o m o t e Transcription Studieswith a yeast transcription activator called GAL4 provided early insight into the domain structure of transcripiion factors. The gene encoding the GAL4 protein, whichpromotes expression of enzymes needed to metabolize galac288

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Reporter-gene tra nscri pts

A EXPERIMENTAL FIGURE 7-20 ln vivo transfectionassay measurestranscriptionactivity to evaluateproteinsbelieved to be transcriptionfactors.Theassaysystemrequires two plasmids Oneplasmid contains the geneencoding the putative transcription factor(protein X) Thesecondplasmid contains a gene(eg,lac\ andoneor morebindingsitesfor proteinX. reporter Bothplasmids aresimultaneously introduced intocellsthat lackthe geneencoding proteinX. Theproduction of reporter-gene RNA transcripts ismeasured; alternatively, theactivity of theencoded proteincanbe assayedlf reporter-gene transcription isgreaterin the presence plasmid of theX-encoding thanin itsabsence, thenthe proteinisan activator; if transcription isless,thenit isa repressor. By useof plasmids encoding a mutatedor rearranged transcription factor,important domains of the proteincanbe identified

tose, was identified by complementation analysis of gal4 mutants (Chapter 5). Directed mutagenesisstudieslike those describedpreviously identified UASs for the genesactivated by GAL4. Each of these UASs was found to contain one or more copies of a related 17-bp sequencecalled UAS6AL. DNase I footprinting assayswith recombinant GAL4 protein produced in E. coli from the yeast GAL4 gene showed that GAL4 protein binds to UAS6al sequences.rWhen a copy of UAS541 w4s cloned upstream of a TAIA box followed by a lacZ reporter gene, expression of lacZ was activated in galactosemedia in wild-type cells but not in gal4 mutants. These results showed that UAS6a1 is a transcription-control element activated by the GAL4 protein in galactosemedia. A remarkable set of experiments with gal4 deletion mutants demonstrated that the GAL4 transcription factor is composedof separablefunctional domains: an N-terminal DNA-binding domain, which binds to specific DNA sequences,and a C-terminal actiuation domain, which interacts with other proteins to stimulate transcription from a nearby promoter (Figure 7-21). Sfhen the N-terminal DNA-binding domain of GAL4 was fused directly ro various portions of its own C-terminal region, the resulting truncated proteins retained the ability to stimulate expression of a reporter gene in an in vivo assay like that

T R A N S C R T P T T O NCAOLN T R O LO F G E N E E X P R E S S t O N

(a) Reporter-gene construct

UASGAT

TATA oox

(b) Wild-type and mutant GAL4 proteins 1 Wild-type

74

Binding to UASor, 738 823

N DNA-binding domain

Activation domain 881

50

Fe+e N-and C-terminal deletion m u t an t s

f-l,o 74 I n t e r na I deletion murants

f_l zq f-],0

oeol-----l

eer

zeel--_-] esr zoaf-l

ear

7-21 Deletion FIGURE < EXPERIMENTAL mutantsol the GAL4gene in yeastwith a UASGAT constructdemonstratethe seParate reporter-gene (a)Diagram of functionaldomainsin an activator. geneand a laczreporler containing DNAconstruct elementthat a regulatory TATAboxligatedto UASCAL B - g a l a c t o s i d a s e contains sitesThereporter-gene GAL4-binding several activity or mutant wild-type andDNAencoding construct into (deleted) introduced GAL4weresimultaneously +++ mutant(9al4)yeastcells,andthe activityof BActivity fromlacZwasassayed galactosidase expressed a DNA encodes GAL4 if the introduced high willbe of wild-type protein(b)Schematic diagrams f unctional refer mutantformsSmallnumbers GAL4andvarious +++ of 50 Deletion sequence in thewild-type to positions the enddestroyed aminoacidsfromthe N-terminal +++ andto stimulate of GAL4to bindto UAS6a1 ability gene fromthe reporter of B-galactosidase expression fromtheC-terminal deletions withextensive Proteins the localize These results endstillboundto UASGAT. of GAL4 end the N-termrnal to domain DNA-binding was expression Theabilityto activate F-galactosidase 126 between somewhere unless eliminated notentirely '1 fromthe and 89 or moreaminoacidsweredeleted domainliesin the end Thustheactivation C-terminal +++ with internal regionof GAL4Proteins C-terminal (bottom)alsowereableto stimulate deletions +++ thatthe indicating of B-galactosidase, expression for itsfunctionin regionof GAL4isnotcrucial central ++ 1987, Cell48:847 'I J MaandM Ptashne, thisassay. [See A H o o ea n d K S t r u h l .1 9 8 6 ,C e / / 4 6 : 8 8 5a; n d R B r e n ta n d M Ptashne,1985. Cell43:729I

depicted in Figure 7-20. Thus the internal portion of the protein is not required for functioning of GAL4 as a transcription factor. Similar experiments with another yeast transcription factor, GCN4, which regulatesgenesrequired for synthesisof many amino acids,indicated that it contains an -60-aa DNA-binding domain at its C-terminusand an :20-aa activation domain near the middle of its sequence. Further evidencefor the existenceof distinct activation domains in GAL4 and GCN4 came from experimentsin which their activation domains were fused to a DNAbinding domain from an entirely unrelated E. cctli DNAbinding protein. \7hen thesefusion proteins were assayedin vivo, they activated transcription of a reporter genecontaining the cognate site for the E. coli protein. Thus functional transcriptionfactorscan be constructedfrom entirely novel combinationsof prokaryotic and eukaryoticelements. Studiessuchas thesehavenow beencarried out with many eukaryotic activators.The structural model of eukaryotic activatorsthat has emergedfrom thesestudiesis a modular one in which one or more activationdomainsare connected DNA-binding domain through flexito a sequence-specific ble protein domains (Figure 7-22). In some cases,amlno acids included in the DNA-binding domain also contribute to transcriptionalactivation.As discussedin a later section, activationdomainsare thought to function by binding other proteins involved in transcription.The presenceof flexible domainsconnectingthe DNA-binding domainsto activation

Examples N

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GCN4

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DNA-binding domain Activation domain F l e x i b l ep r o t e i n domain FfGURE7-22 Schematicdiagrams illustrating the modular structure of eukaryotic transcription activators. These factorsmay containmore than one activationdomain transcription b u t r a r e l yc o n t a i nm o r et h a n o n e D N A - b i n d i ndgo m a i n G A L 4a n d receptor The glucocorticord activators. GCN4are yeasttranscription of target geneswhen certainhormones (GR)promotestranscription a r eb o u n dt o t h e C - t e r m i n aalc t i v a t i o dn o m a i n S P 1b i n d st o G C - r i c h p r o m o t ee r l e m e n t si n a l a r g en u m b e ro f m a m m a l r agne n e s OS F TRANSCRIPTION AND REPRESSOR ACTIVATORS

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domains may explain why alterationsin the spacingbetween control elementsare so well tolerated in eukaryotic control regions. Thus even when the positions of transcription factors bound to DNA are shifted relative to each other, their activation domains may still be able to interact becausethey are attachedto their DNA,binding domains through flexible protein regions.

Repressors Inhibit Transcriptionand Are the FunctionalConverseof Activators Eukaryotic transcription is regulated by repressorsas well as activators. For example, geneticistshave identified mutations in yeast that result in continuously high expressionof certain genes.This type of unregulated,abnormally high expression is called constitutive expression and results from the inactivation of a repressorthat normally inhibits the transcription of these genes. Similarly, mutants of Drosophila and Caenorhabditis eleganshave been isolated that are defective in embryonic development becausethey expressgenesin embryonic cells where those genesare normally repressed.The mutations in these mutanrs inactivate repressors,leading to abnormal development. Repressor-bindingsites in DNA have been identified by systematiclinker scanning mutation analysis similar to that depictedin Figure 7-14.In this type of analysis,mutation of an activator-binding site leadsto decreasedexpressionof the linked reporter gene, whereas mutation of a repressorbinding site leads to increasedexpressionof a reporter gene. Repressorproteins that bind such sites can be purified and assayedusing the same biochemicaltechniquesdescribed earlier for activator proteins. Eukaryotic transcriptionrepressorsare the functional converseof activators.They can inhibit transcription from a gene they do not normally regulatewhen their cognatebinding sites are placedwithin a few hundred basepairs of the gene'sstart site. Like activators,most eukaryotic repressorsare modular proteins that have two functional domains: a DNA-binding domain and a repressiondomain. Similar to activation domains,repressiondomainscontinue to function when fusedto another type of DNA-binding domain. If binding sitesfor this secondDNA-binding domain are insertedwithin a few hundred basepairs of a promoter,expressionof the fusion protein inhibits transcription from the promoter. Also like activation domains, repression domains function by interacting with other proteins, as discussedlater in this chapter. The absence of appropriate repressor activity can have devastatingconsequences. For instance,the pro-Wilms' tein encodedby the tumor (WT1)gene rs a repressor that is expressedpreferentially in the developingkidney. Children who inherit mutations in both the maternal and paternal WT1 genes,so that they produce no functional WT1 prorein, invariably develop kidney tumors early in life. The WT1 protein binds to the control region of the gene encoding EGR1, which itself is a transcripiion factor as well (Figure7-23).This gene,like many other eukaryotic genes,is subjectto both repressionand activation.

290

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A FIGURE 7-23 Diagramof the controlregionof the gene encodingEGR-1. a transcriptionactivator.Thebindingsitesfor WT1, protein, a eukaryotic repressor do notoverlap the bindingsitesfor theactivator AP1or thecomposite bindingsitefor the activators SRF andTCF. Thusrepression byWT1doesnot involve directinterference with bindingof otherproteins asin thecaseof bacterial repressors. Experiments have shown that binding by \7T1 represses transcription of the EGRi genewithout inhibiting binding of the activatorsthat normally stimulateexpressionof this gene. These experiments demonstrate that WT1 can function as a transcriptional repressor.It is likely that WT1 functions to represstranscription of multiple other genesin addition to EGRl. Consequently,tumor formation in patients with homozygous mutations of the'WT1 gene may result in part from abnormal activation of multiple genes such as the EGRI gene.I

DNA-B|nding D o m a i n sC a n B e C l a s s i f i e d into NumerousStructuralTypes The DNA-binding domains of eukaryotic activarors and repressorscontain a variety of structural motifs that bind specific DNA sequences.The ability of DNA-binding proteins to bind to specific DNA sequencescommonly results from noncovalent interactions betweenatoms in an ct helix in the DNA-binding domain and atoms on the edgesof the bases within a major groove in the DNA. Interactions with sugarphosphate backbone atoms and, in some cases,with atoms in a DNA minor groove also contribute to binding. The principles of specificprotein-DNA interactionswere first discoveredduring the study of bacterialrepressors.Many bacterial repressorsare dimeric proteins in which an cr helix from each monomer insertsinto a major groove in the DNA helix (Figure 7-24).This ct helix is referredto as the recognition helix or sequence-readinghelix becausemost of the amino acid sidechainsthat contact DNA extend from this heIix. The recognition helix that protrudes from the surfaceof bacterialrepressorsto enter the DNA major groove and make multiple, specificinteractionswith atoms in the DNA is usually supported in the protein structure in part by hydrophobic interactions with a secondct helix just N-terminal to it. This structural element, which is present in many bacterial repressors,is called a helix-turn-helix motif. Many additional motifs that can presentan cr helix to the major groove of DNA are found in eukaryotic transcription factors, which often are classifiedaccording to the type of DNA-binding domain they contain. Becausemost of these motifs have characteristicconsensusamino acid sequences, newly characterizedtranscription factors frequently can be classifiedonce the correspondinggenesor cDNAs are cloned and sequenced.The genomes of higher eukaryotes encode

T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X p R E S S t O N

(a)

7-24 lnteractionof bacteriophage434 repressor FIGURE boundto itsspecific of 434 repressor diagram with DNA.(a)Ribbon arein yellowandgreenThe monomers DNA,Repressor operator modelof A space-filling by asterisks. helices areindicated recognition (b) protein interacts how the shows complex the repressor-operator overa lengthof 1 5 with onesideof the DNAmolecule intimately Science242:8991 fromA K Aggarwal etal, 1988, turns [Adapted

dozensof classesof DNA-binding domains and hundredsto thousands of transcription factors. The human genome' for instance,encodes-2000 transcription factors. Here we introduce several common classesof DNAbinding proteins whose three-dimensional structures have been determined.In all theseexamplesand many other transcription factors, at least one ct helix is insertedinto a major groove of DNA. However. some transcription factors contain alternative structural motifs (e.g., F strands and loops) that interact with DNA. Homeodomain Proteins Many eukaryotic transcription factors that function during developmentcontain a conserved 60-residue DNA-binding motif, called a homeodomain that is similar to the helix-turn-helixmotif of bacterial repressors.These transcription factors were first identified in Drosophila mutants in which one body part was transformed into another during development(Chapter 22). The conserved homeodomain sequence has also been found in vertebrate transcription factors' including those that have similar master-controlfunctions in human development. Zinc-Finger Proteins A number of different eukaryotic proteins have regions that fold around a central Zn"- ion, producing a compact domain from a relatively short length of the polypeptide chain (Figure 7-25a). Termed a zir.c finger, this structural motif was first recognizedin DNA-binding domains but now is known to occur also in proteins that do not bind to DNA. Here we describe two of the several classesof zinc-finger motifs that have been identified in eukarvotic transcriptionfactors.

The C2H2 zinc finger is the most common DNA-binding motif encoded in the human genome and the genomes of most other multicellular animals. It is also common in multicellular plants but is not the dominant type of DNAbinding domain in plants as it is in animals. This motif has a 23- to 26-residue consensussequencecontarnrng two conservedcysteine(C) and two conservedhistidine (H) residues, whose side chains bind one Znz* ion (Figure 3-9c). The name "zinc finger" was coined becausea two-dimensional diagram of the structure resemblesa finger. Sfhen the threedirnensional structure was solved, it became clear that the binding of the Znz* ion by the two cysteineand two histidine residuesfolds the relatively short polypeptide sequence into a compact domain, which can insert its ct helix into the

zinc finger (becauseit has four conservedcysteinesin contact with the Zn2*\. is found in -50 human transcription factors. The first members of this class were identified as specific intracellular high-affinity binding proteins, or "receptors," for steroid hormones, leading to the name steroid receptor swperfamily.Becausesimilar intracellular receptors for nonsteroid hormones subsequently were found, these transcription factors are now commonly called nuclear re."ptott. The characteristicfeature of Ca zinc fingers is the p..r..t.. of two groups of four critical cysteines'one toward each end of the 55- or 56-residuedomain' Although the Ca

three or more repeating finger units and bind as monomers' whereas Ca zinc-finger proteins generally contain only two finger units and generally bind to DNA as homodimers or heterodimers.Homodimers of Ca zinc-finger DNA-binding domains have twofold rotational symmetry (Figure 7-25c)' ConsequentlS homodimeric nuclear receptors bind to consensusDNA sequencesthat are inverted repeats' Leucine-Zipper Proteins Another structuralmotif present in the DNA-binding domains of alarge classof transcription factors contains the hydrophobic amino acid leucine at every seventh position in the sequence'These proteins bind to DNA as dimers, and mutagenesis of the leucines showed that they were required for dimerization' Consequently, the name leucine zipper was coined to denote this structural motif. The DNA-binding domain of the yeast GCN4 transcription factor mentioned earlier is a leucine-zipperdomain' Xray crystallographicanalysisof complexesbetweenDNA and the GCN4 DNA-binding domain has shown that the dimeric protein contains two extendeda helicesthat "grip" the DNA holecule, much like a pair of scissors,at two adjacentmajor grooves separatedby about half a turn of the double helix OS F TRANSCRIPTION O AND REPRESSOR ACTIVATORS

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> FfGURE 7-25 Zinc-Fingerproteins.(a)This two-dimensional depiction oI C2H2 zincfingers illustrates the shapethatgavethismotifits n a m el n d i v i d uaam l i n oa c i d a s r ei n p u r p l et ;h e aminoacidsthatcontacttheZn*2ionarein blue (b)GL1isa monomeric proteinthat contains fiveC2H2 zincf ingersa-Helices are shownascylinders, Zn*2ionsasspheresFinger '1 doesnot interact with DNA,whereas the otherfourfingersdo (c)Theglucocorticoid receptor isa homodimeric Cozinc-finger proteino-Helices areshownaspurpleribbons, asgreenarrows, Zn*2ionsasspheres B-strands (darker Twoo helices shade). onerneach monomer, interact with the DNA LikeallCa zinc-finger homodimers, thistranscription factor hastwofoldrotational symmetry; the centerof symmetry isshownby theyellowellipse, In contrast, heterodimeric nuclear receptors do not (b) pavletich p exhibitrotational symmetry N [See

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5' 7;26a). The portions of the o helicescontacting the !{qure DNA include positively charged (basic)residuesthat interact with phosphates in the DNA backbone and additional residuesthat interact with specificbasesin the major groove. GCN4 forms dimers via hydrophobic interactions be-

3'

subsequentlywere identified. Like leucine-zipper proreins, they form dimers containing a C-terminal coiLi-coil dimerizationregion and an N-terminal DNA-binding domain. The term basi zipper (bZIP)now is frequently ,ri.d to refer to all proteins with these common structural features. Many basic-zipper transcription factors are heterodimers of two different polypeptide chains, each containing one basic_ zipper domain. Basic Helix-Loop-Helix (bHLH) proteins The DNA_binding domain of another classof dimeric transcription factors con-

coiled-coildimer (seeFigure 3-9a). Although the first leucine-zippertranscription factors to be analyzedcontainedleucineresidues seventhposition "t.u..y in the dimerization region, additional DNA-binding proteins containing other hydrophobic amino acids in thesepositions 292

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contain an N-terminal a helix with basicresiduesthat interact

T R A N s c R t p l o N AcLo N T R o Lo F G E N EE X p R E S s t o N

tion domains marked by an unusually high percentageof particular amino acids. GAL4, GCN4, and most other yeast transcription factors, for instance'have activation domains that are rich in acidic amino acids (aspartic and glutamic acids).These so-called acidic actiuation domains generally are capable of stimulating transcription in nearly all types of eukaryotic cells-fungal, animal, and plant cells.Activation domains from some Drosophila and mammalian transcription factors are glutamine-rich, and some are proline-rich; still others are rich in the closely related amino acids serineand threonine, both of which have hydroxyl groups. However, some strong activation domains are not particularly rich in any specificamino acid' -. I , Biophysical studies indicate that acidic activation domains have an unstructured, random-coil conformation' Thesedomains stimulate transcription when they are bound I to a protein co-actiuator.The interaction with a co-activator 7-26 Interactionof homodimericleucine-zipperand A FIGURE ."nr.t the activation domain to assumea more structured ctbasichelix-loop-helix(bHLH)proteinswith DNA.(a)In leucinehelical conformation in the activation domain-co-activator regions of cr-helical in the extended basicresidues zipperproteins, complex. A well-studied example of a transcription factor at adjacentmajor interactwith the DNAbackbone the monomers with an acidic activation domain is the mammalian CREB by domainisstabilized groovesThecoiled-coil dimerization protein, which is phosphorylated in responseto increased (b)In bHLH the monomers. interactions between hydrophobic ievels of cAMP. This regulated phosphorylation is required of the at the bottom(N-termini proteins, helices the DNA-binding (CREB binding loopsfroma leucine-zipper- for CREB to bind to its co-activator CBP by nonhelical monomers) areseparated whose congenes protein), resulting in the transcription of (a)see domainlPart dimerization a coiled-coil likeregioncontaining (see 1'6-31')' Figure part(b)seeA R Ferre-D'Amare trol regionscontain a CREB-binding site elal,1992,Cell71:1223; T.E Ellenberger of domain activation 353:38 \Thenihe phosphorylatedrandom coil etal , 1993,Nature l a conformational CREB interacts with CBR it undergoes to form two ct helices linked by a short loop that change with DNA, a middle loop region, and a C-terminal region wrap around the interacting domain of CBP. with hydrophobic amino acids spacedat intervals characterisSomeactivation domains arelarget and more highly structic of an amphipathic a helix. As with basic-zipper proteins' tured than acidic activation domains. For example' the liganddifferent bHLH proteins can form heterodimers. binding domains of nuclear receptors function as activation domains when they bind their specific ligand (Figure 7-27)' StructurallyDiverseActivation and Repression Binding of ligand induces alarge conformational change that DomainsRegulateTranscription allows the ligand-binding domain with bound hormone to inof the GAL4 teract with a short ct helix in nuclear-receptor co-activators; with fusionproteinscomposed Experiments the resulting complex then can activate transcription of genes DNA-binding domain and random segmentsof E. coli prowhose control regions bind the nuclear receptor. teins demonstrated that a diverse group of amino acid se-1 Thus the acidic activation domain in CREB and the ligpercent of quencescan function as activation domains, activation domains in nuclear receptors repreand-binding all E. coli sequences,even though they evolved to perform extremes. The CREB acidic activation structural two sent other functions. Many transcription factors contain activa(a)

(b)

7-27 Eftectof ligand binding on conformation > FIGURE domarnof of the estrogenreceptor.Onlythe ligand-binding the receptorisshown.(a)Whenestrogenis boundto the with the ligand,generating the greeno helixinteracts domain, domain(darkbrown a hydrophobic aroovein theligand-binding cthelixin a co-activator thatbindsan amphipathic helices) of the estrogenreceptorin subunit(blue).(b)Theconformation by binding of hormoneisthoughtto bestabilized theabsence the Inthisconformation, tamoxifen. antagonist of theestrogen that greenhelixof the receptorfoldsinto a conformation bindinggrooveof the active with the co-activator interacts AK blockingbindingof co-activators. [From sterically receptor, et al, 1998,Cell95:927 Shrau )

(a)

o-helixfrom interacting co-activator Estrogen (agonist)

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> FIGURE 7-28 Combinatorial possibilities due to formationof (a) heterodimerictranscriptionfactors.(a)In someheterodimeric Factor Factor Factor transcrrption factors, A eachmonomer C z Activation recognizes thesameDNA (D/ domain sequence In the hypothetical example O shown,transcription factors \\ A, B,andC canallinteract E with oneanother; RDNA-binding creating sixdifferent alternative combinations of activation domains thatcanallbindat domain thesamesite Eachcomposite bindingsiteisdivided intotwo halfsites,andeachheterodimeric factorcontains theactivation domains .)r) YY of itstwo constituent monomers(b)Whentranscription factor -r---fl monomers recognize differentDNAsequences, alternative combinations of the threefactorsbindto sixdifferentDNAsequences (sites1-6),eachwith a uniquecombination of activation domarns(c) ( b ) Expression of an inhibitory factor(red)thatinteracts onlywith factor Factor Factor Factor A inhibits binding;hencetranscriptional activation at sites1,4, and5 A B C -, Activation isinhibited, but activation at sites2, 3. and6 is unaffected a) Inhibitory domain

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tf

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domain is a random coil that folds into two a-heliceswhen it binds to the surfaceof a globular domain in a co-activaror. In contrast, the nuclear-receptorligand-binding activation domain is a structured globular domain that interactswith a short cr helix in a co-activator, which probably is a random coil before it is bound. In both cases, however, specific protein-protein interactions between co-activators and the activation domains permit the transcription factors to stimulate gene expression. CurrentlS lessis known about the structure of repression domains. The globular ligand-binding domains of some nuclear receptorsfunction as repressiondomains in the absence of their specific hormone ligand. Like activation domains, repressiondomains may be relatively short, comprising 15 or fewer amino acids. Biochemical and genetic st;dies indicate that repression domains also mediate protein-protein interactions and bind to co-repressorproteins, forming a complex that inhibits transcription initiation by mechanisms that are discussedlater in the chaoter.

TranscriptionFactorInteractionsIncreaseGeneControl Options Two typesof DNA-binding proteins discussedpreviouslybasic-zipper proteins and bHLH proteins-oiten exisi in alternative heterodimeric combinations of monomers. Other classesof transcription factors not discussedhere also form heterodimeric proteins. In some heterodimeric transcription factors, each monomer recognizes the same sequence.In these proteins, the formation of alternative heterodimers does not increase the number of different sites on which the monomers can act but rather allows the activation domains associatedwith each monomer to be brought together in alternative combinations that bind to

transcriptional responsesdepending on which 6ZIp or bHLH monomers that bind to that ,it. expressedin a ".. 294

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particular cell at a particular time and how their activities are regulated. In some heterodimeric transcription factors, however, each monomer has a different DNA-binding specificity.The resulting combinatorial possibilities increasethe number of potential DNA sequencesthat a family of transcription factors can bind. Three different factor monomers theoretically could combine to form six homo- and heterodimericfactors, as illustrated in Figure 7-28b. Four different facor monomers could form a total of 10 dimeric factors; five monomers, 15 dimeric factors; and so forth. In addition, inhibitory factors are known that bind to some basic-zipper and bHLH monomers, thereby blocking their binding to DNA. Ifhen these inhibitory factors are expressed,they repress transcriptional activation by the factors with which they interact (Figure 7-28c). The rules governing the interactions of members of a heterodimeric transcription factor class are complex. This combinatorial complexity expands both the number of DNA sites from which these factors can activate transcription and the ways in which they can be regulated. Similar combinatorial transcriptional regulation is achieved through the interaction of structurally unrelated transcription factors bound to closely spaced binding sites in DNA. An example is the interaction of two rranscription factors, NFAT and AP1, which bind to neighboring

T R A N S C R | p T t o N ACLO N T R O LO F G E N E E X P R E S S | O N

7-29 Cooperativebinding of < FIGURE two unrelatedtranscriptionfactorsto neighboringsitesin a compositecontrol NFAT both monomeric element.Bythemselves, factors AP1transcription andheterodimeric binding havelow affinityfor theirrespective region. sitesin the /L-2promoter-proximal betweenNFATand n interactions n-protei Protei AP1addto the overallstabilityof the NFATsothatthe two proteins complex, AP1-DNA sitecooperatively. [SeeL bindto the composite etal.1998,Nature392:421 Chen

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Weak NFAT b i n d i n gs i t e

Weak AP1 b i n d i n gs i t e

Cooperativebinding of NFATand AP1

sitesin a compositepromoter-proximal elementregulating the gene encoding interleukon-Z (lL-Z). Expressionof the IL-2 geneis critical to the immune response,but abnormal expressionof IL-2 can lead to autoimmune diseasessuch as rheumatoid arthritis. Neither NFAT nor AP1 binds to its site in the lL-2 control region in the absence of the other. The affinities of the factors for these particular DNA sequencesare too low for the individual factors to form a stable complex with DNA. However, when both NFAT and AP1 are present, protein-protein interactions between them stabilize the DNA ternary complex composed of NFAT, AP1, and DNA (Figure 7-29). Such cooperatiue DNA binding of various transcription factors results in considerablecombinatorial complexity of transcription control. As a result, the approximately 2000 transcription factors encoded in the human genome can bind to DNA through a much larger number of cooperative interactions, resulting in unique transcriptional control for each of the several tens of thousands of human genes.In the caseof IL-2, transcription occurs only when both NFAT is activated,resulting in its transport from the cytoplasm to the nucleus,and the two subunits of API' are synthesized.These eventsare controlled by distinct signal transductionpathways (Chapters15 and 16)' allowing stringent control of IL-2 expression. Cooperative binding by NFAT and AP1 occurs only when their weak binding sites are positioned quite close to each other in DNA. The sites must be located at a precise distancefrom each other for effectivebinding. Recentstudies have shown that the requirementsfor cooperativebinding are not so stringent in the case of some other transcription factors and control regions.For example, the EGR-1 control region contains a composite binding site to which the SRF and TCF transcription factors bind cooperatively(see Figure 7-23). Becausea TCF has a long' flexible domain that interactswith SRF.the two proteins can bind cooper-

atively when their individual sites in DNA are separated by any distanceup to 10 basepairs or are inverted relative to each other.

Multiprotein ComplexesForm on Enhancers As noted previouslS enhancers generally range in length from aboui 50 to 200 basepairs and include binding sitesfor severaltranscription factors. The multiple transcription factors that bind to a single enhancer are thought to interact' Analysis of the :70-bp enhancer that regulatesexpression of B-lnterferon, an important protein in defenseagainstviral infections in humans, provides a good example of such transcription-factor interactions.The B-interferonenhancercontains four control elements that bind four different transcription factors simultaneously.In the presenceof a small, abundant protein associatedwith chromatin called HMGI, binding of the transcription factors is highly cooperative' similai to the binding of NFAT and AP1 to the composite promoter-proximal site inthe IL-2 control region (FigureT-29)' Thi, .oop.."tive binding produces a multiprotein complex on the B-interferon enhancerDNA (FigureT-30)' The term enhancesomehas been coined to describesuch large nucleoprotein complexes that assemblefrom transcription factors a. th.y bind cooperatively to their multiple binding sites in an enhancer. HMGI binds to the minor groove of DNA regardlessof the sequence and, as a result, bends the DNA molecule sharply. This bending of the enhancerDNA permits bound tr"nr.riptio.t factors to interact properly' The inherently weak, noncovalent protein-protein interactions between transcription factors are strengthened by their binding to neighboring DNA sites, which keeps the proteins at very high relative concentrations. Becauseof the presenceof flexible regions connectlng the DNA-binding domains and activation or represston

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more a helicesthat inreract cognatesite in DNA.

malor grooves in their

r Activation and repressiondomains in transcription factors exhibit a variety of amino acid sequencesand threedimensional structures. In general, these functional domains interact with co-activators or co-repressors, which are critical to the ability of transcription factors to m o du l a r eg e n ee x p r e s s i o n .

IRF.7 F

^\FIGURE7-30 Model of the enhancesomethat forms on the p-interferon enhancer.Two monomerictranscription factors,IRF_3 and IRF-7,and two heterodimeric factors,)un/AfF-2and p50/ p65 (NF-rB),bind to the four controlelementsin this enhancer. Cooperative bindingof thesetranscription factorsis facilitatedby HMGI,which bindsto the minor grooveof DNA and alsointeracts directlywith the dimericfactors.Bendingof the enhancersequence resultingfrom HMGI bindingis critrcalto formationof an enhancesome. DifferentDNA-bendingproteinsact similarlyat other enhancers. fromD.Thanos [Adapted andT.Maniatis, 1995,Ce//g3:1091. andM A Wathelet al , 1998,Mot Cett1:5OlI

r The transcription,control regions of most genescontain binding sites for multiple transcription factors. Transcription of such genesvaries dependingon the particular ..p.rtoire of transcription factors that are expressedand activated in a particular cell at a particular time. r Combinatorial complexity in transcription control results from alternative combination, of -ono-ers that form heterodimeric transcription facors (seeFigure 7-2g) and from cooperative binding of transcription factors to composite control sites (seeFigure 7-29). r Cooperative binding of multiple activators to nearby sitesin an enhancerforms a multiprotein complex called an enhancesome(seeFigure 7-30). Assemblyof enhancesomesoften requiressmall proteins that bind to the DNA minor groove and bend the DNA sharply, allowing bound proteins on either side of the bend to rnteract more readily.

lE

Transcription Initiationby RNA

Polymerasell

r Transcription factors, which stimulate or repress transcription, bind to promoter-proximal regulatory elements and enhancersin eukaryotic DNA.

In previous sections,many of the eukaryotic proteins and DNA sequencesthat participate in transcription and its control have beenintroduced. In this section,we focus on assembly of transuiption preinitiation complexe.s.The preinitiation complex is an associationof RNA polymerase II and several protein initiation factors that assembletogether at the start site and begin to unwind the DNA in preparation for transcription of the gene. Recall that this eukaryotic RNA polymeraseII catalyzessynthesisof mRNAs and a few small nuclear RNAs (snRNAs). Specific activator and repressorproteins regulate the mechanismsthat control the assembly of Pol II transcription preinitiation complexesand hencethe rate of transcription of protein-coding genesand are consideredin the nexr section.

r Transcription activators and repressorsare generally modular proteins containing a singleDNA-binding domain and one or a few activation domains (for activators) or re_ pression domains (for repressors).The different domains frequently are linked through flexible polypeptide regions (seeFigure 7-22).

GeneralTranscriptionFactorspositionRNA Polymerasell at Start Sitesand Assistin Initiation In vitro transcriprion by purifiedRNA polymerase II requires

subjectedto this evolutionaryexperimentationthan would be the caseif constraintson the spacingbetweenregulatory elementswere strict, as for most genesin bacteria.

Activatorsand Repressors of Transcription

r Among the most common structural motifs found in the DNA-binding domains of eukaryotic transcription factors are the C2H2 zinc finger,homeodomain, basichelix_ Ioop-helix (bHLH), and basic zipper (leucinezipper). All these and many other DNA-binding motifs conrain one or 296

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the addition of several initiation factors that are seoaiated from the polymerase during purification. These initiation factors, which position polymerase molecules at transcription start sites and help to melt the DNA strands so rhar the tem_ plate strand can enter the active site of the enzyme, are called general transuiption factors. In contrast to the transcription

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Complex ffi eod."rt: Assemblyof the Pol ll Preinitiation ll 7-31 In vitro assemblyof RNApolymerase > FIGURE factors general transcriptton preinitiationcomplex.Theindicated to TATA-box ll (Polll)bindsequentially andpurifiedRNApolymerase thenprovides complexATPhydrolysis DNAto forma preinitiation subunit by a TFIIH start site DNA at the of for unwinding energy the the opencomplex, in the resulting transcrtption As Polll initiates polymerase movesawayfromthe promoterand itsCTDbecomes for factors(except transcription phosphorylated Invitro,thegeneral yet it is not but complex, from the TBP-promoter TBP)dissociate regions with promoter knownwhichfactorsremainassociated in vivo initiation followingeachroundof transcription factors discussedin the previous section' which bind to specific sitesin a limited number of genes,generaltranscription factors are required for synthesisof RNA from most genes. The generaltranscription factors that assistPol II in initiation of transcription from most TATA-box promoters in vitro have been isolated and characterized.Theseproteins are designated TFIIA, TFIIB, etc., and most are multimeric proteins. The largest is TFIID, which consistsof a single 38-kDa TATAbox-binding protein (TBP) and 13 TBP-associatedfactors (TAFs). General transcription factors with similar activities have been isolated from cultured human cells' rat liver' Drosophila embryos, and yeast.The genesencoding theseproteins in yeasthave beensequencedas part of the completeyeast genome sequence,and many of the cDNAs encoding human and DrosophilaPol II general transcription factors have been cloned and sequenced.In all cases,equivalentgeneraltranscription factors from different eukaryotesare highly conserved.

TFIIB GL

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SequentialAssemblyof ProteinsFormsthe Pol ll TranscriptionPreinitiationComplexin Vitro The complex of RNA polymeraseII and its general transcription factors bound to a promoter and ready to initiate transcription is called a preinitiation complex. Understanding how a preinitiation complex is assembledon a promoter is important because,in principle, each step in the processcan be controlled, allowing complex regulation of the overall processof transcription initiation. Detailed biochemical studies using DNase I footprinting and electrophoreticmobility shift assays were used to determine the order in which Pol II and general transcription factors bound to TAIA-box promoters. Because the complete, multisubunit TFIID is difficult to purifS researchersused only the isolatedTBP component of this general transcription factor in these experiments.Pol II can initiate transcription in vitro in the absenceof the other TFIID subunits. Figure 7-31 summarizesour current understandingof the stepwiseassemblyof the Pol II transcription preinitiation complex in vitro. TBP is the first protein to bind to a TATAbox promoter. All eukaryotic TBPs analyzed to date have very similar C-terminal domains of 180 residues'The sequenceof this region is 80 percent identical in the yeast and hu-"n proteins, and most differencesare conservativesubstitutions. This conserved C-terminal domain functions as well as the full-length protein does for in vitro transcription' (The N-terminal domain of TBP, which varies greatly in se-

Preinitiation complex

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Nascent RNA Releaseof generalfactors, exceptTBP

Elongating Pol ll with phosphorylated CTD

quence and length among different eukaryotes, functions in the Pol ll-catalyzed transcription of genes encodlng snRNAs.) TBP is a monomer that folds into a saddle-shape structure; the two halves of the molecule exhibit an overall dyad symmetry but are not identical' Like the HMGI and T R A N S C R I P T I OI N I T I A T I O NB Y R N A P O L Y M E R A S IEI

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TFIIF

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template strand is bound at the polymerase active site. If the remaining ribonucleosidetriphosphatesare present,pol II begins transcribingthe templatestrand. As the polymerasetranscribes away from the promoter region, another subunit of TFIIH phosphorylates the Pol II CTD at multiple sites (see Figure 7-31).In the minimal in vitro transcription assaycontaining only these general transcription factors and purified RNA polymerase II, TBP remains bound to the TATA box as the polymerase transcribes away from the promoter region, but the other general transcription factors dissociate.

The first subunitsof TFIIH to be cloned from hu-

A FIGURE 7-32 Model for the structureof an RNApolymerase ll preinitiationcomplex.yeastRNApolymerase ll issnownasa space-filling modelwith the direction of transcription to the left.The templatestrandof DNAisshownin darkblueandthe nontemolate strandin red.Thestartsiteof transcription isshownasa space-filling redanddarkbluebasepair.TBpandTFIIB areshownasgreenand yellowwormtraces of thepolypeptide backbone. Structures for TFllE, F, andH havenot beendetermined to highresolution Theirapproximate positions lyingoverthe DNAin the preinitiation complex areshownby (lightblue),TFilF ellipses for TFIIE (red),andTFI|H (orange). [Adapted fromG MillerandS Hahn,2006,Nature Strua.Biol..13:6031

mans were identified becausemutations in the genes El encoding them cause defects in the repair of damaged DNA. In normal individuals, when a transcribing RNA polymerase becomes stalled at a region of damaged template DNA, a subcomplex of TFIIH is thought to recognize the stalled polymerase and then recruit other proteins that function with TFIIH in repairing the damaged DNA region. In patients with mutant forms of TFIIH subunits, such repair of damaged DNA in transcriptionally active genesis impaired. As a result, affected individuals have extreme skin sensitivity to sunlight (a common cause of DNA dama.ge)and exhibit a high incidence of cancer.Depending on the severity of the defect in TFIIH function, theie individuals may suffer from diseasessuch as xerode a pigmentosum and Cockayne'ssyndrome (Chapter 25

In Vivo TranscriptionInitiation by pol ll Requires Additional Proteins Although the general transcription factors discussedabove allow Pol II to initiate transcription in vitro, another general transcription factor, TFIIA, is required for initiation by pol II in vivo. Purified TFIIA forms a complex with TBp and TATA-box DNA. X-ray crystallography of this complex shows that TFIIA inreracts with the side of TBp that is upstream from the direction of transcription. Biochemical experimentssuggestthat in cells of higher eukaryotes,TFIIA and TFIID, with its multiple TAF subunits, bind first to TATA-box DNA and then the orher general transcription factors subsequentlybind as indicated in Figure 7-31. The TAF subunits of TFIID appear to play a role in initi_ ating transcription from promoters that lack a TAIA box. For instance, some TAF subunits contact the initiator element in promoters where it occurs,probably explaining how such sequencescan replace a TATA box. Additional TFIID TAF subunits can bind to a consensussequenceA/G-G-A/ T-C-G-T-G centered =30 base pairs downstream from the transcription start site in many genesthat lack a TATA_box promoter. Becauseof its position, this regulatory sequenceis called the downstream promoter element (DpE). tt. Opn facilitatestranscription of TAIA-less genesthat contain it by increasingTFIID binding. In addition to general transcription factors, specific activators and repressorsregulate transcription of genesby Pol II. In the next sectionwe will examine how thesi regulatory proteins influence Pol II transcription initiation.

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TranscriptionInitiation by RNA Polymerasell r Transcription of protein-coding genes by Pol II can be initiated in vitro by sequentialbinding of the following in the indicated order: TBP, which binds to TMA-box DNA; TFIIB; a complex of Pol II and TFIIF; TFIIE; and finally TFIIH (seeFigure 7-31). r The helicase activity of a TFIIH subunit separatesthe template strands at the start site in most promoters' a process that requires hydrolysis of ATP. As Pol II begins transcribing away from the start site, its CTD is phosphorylated by another TFIIH subunrt. r In vivo transcription initiation by Pol II also requires TFIIA and, in metazoans,a completeTFIID protein, including its multiple TAF subunits as well as the TBP subunit.

of MolecularMechanisms lB and Activation Repression Transcription The repressorsand activators that bind to specificsitesin DNA and regulate expressionof the associatedprotein-coding genes do so by trno generalmechanisms.First, theseregulatoryproteins act in concert with other proteins to modulate chromatin structure, inhibiting or stimulating the ability of general transcription factors to bind to promoters.Recall from Chapter 6 thai the DNA in eukaryotic cells is not free but is associated with a roughly equal massof protein in the form of chromatin. The basic structural unit of chromatin is the nucleosome, which is composed of :147 base pairs of DNA wrapped tightly around a disk-shapedcore of histoneproteins.Residues within the N-terminal region of each histone, and the Cterminal regionsof histonesH2A and H2B, calledhistonetails, extend from the surface of the nucleosomeand can be reversiblymodified (seeFigure 6-31b). Suchmodifications'especially the acetylation of histone H3 and H4 tails, influence the relative condensation of chromatin and thus its accessibilityto proteins required for transcription initiation. In addition to their role in such chromatin-mediatedtranscriptionalcontrol, activators and repressorsinteract with a large multiprotein complex called the mediator of transcription complex, or simply mediator. This complex in turn binds to Pol II and directly regulatesassemblyof transcriptionpreinitiation complexes. In this section,we review current understandingof how repressorsand activators control chromatin structure and preiniiiation complex assembly.In the next section of the chapter,we discusshow the concentrations and activities of activators and repressorsthemselvesare controlled,so that geneexpressionis preciselyaftunedto the needsof the cell and organism.

Formationof HeterochromatinSilencesGene Expressionat Telomeres,Near Centromeres,and in Other Regions For manl' years it has been clear that inactive genes in eukaryotic cells are often associatedwith heterochromatin,

regions of chromatin that are more highly condensed and stain more darkly with DNA dyes than euchromatin, where most transcribed genes are located (see Figure 6-33a)' Regions of chromosomes near the centromeresand telomer.i a.td additional specificregionsthat vary in different cell types are organized into heterochromatin. The DNA in hetertchromatin is less accessibleto externally added proteins than DNA in euchromatin and consequentlyis often referred to as "closed" chromatin. For instance, in an experiment describedin Chapter 6, the DNA of inactive genes was found to be far more resistantto digestion by DNase I than the DNA of transcribed genes(seeFigure 6-32)' Chromatin-Mediated Repression in Yeast Study of DNA regions in S. cereuisiaeth^t behavelike the heterochromatin oi high.t eukaryotes provided early insight into the chroiatin-mediated repressionof transcription' This yeast can grow either as haploid or diploid cells' Haploid cells exhibit one of two possiblemating types, called a and ct' Cells of different mating type can "mate," or fuse, to generatea diploid cell (seeFigure 1-6). Vhen a haploid cell dividesby buddlng, the larger "mother" cell switches its mating type (seeFigure 21-27). Genetic and molecular analyseshave revealedlhat three genetic loci on yeast chromosome III control the mating type of yeast cells (Figure 7-33)' Only the central mating-type locus, termed MAT, is actively transcribed. How the proteins encoded at the MAT locws determine whether a cell has the a or ct phenotype is explained in Chapter 21. The two additional loci, termed HML and HMR, near the left and right telomere, respectively,contain "silent" (nontranscribed)copies of the a or ct genes'These seouencesare transferred alternately from HMLa or HMRa into the MAT locus by a type of nonreciprocal recombination between sister chromatids during cell division' \fhen the MAT locus contains the DNA sequencefrom HMLa, the cells behave as ct cells. \fhen the MAT locus contains the DNA sequencefrom HMRa, the cells behavelike a cells' Our interest here is how transcription of the silent mating-type loci at HML and HMR is repressed'If the genesat theseloci are expressed'as they are in yeastmutants with defects in the repressingmechanism,both a and ct proteins are expressed,causing the cells to behave like diploid cells, which cannot mate. The promoters and UASs controlling transcription of the a and a geneslie near the center of the DNA sequencethat is transferred and are identical whether the sequencesare at the MAT locus or at one of the silent loci. Tilis indicatesthat the function of the transcription factors that interact with these sequencesmust somehow be blocked ^t HML and HMR but not at the MAT locus' This repressionof the silent loci dependson silencersequenceslocaied next to the region of transferred DNA at HML and HMR (Figure 7-33).If the silenceris deleted,the adjacent silent locris is transcribed.Remarkably' any geneplaced near the yeast mating-type silencer sequence-by recombinant DNA techniques is repressed,or "silenced"' even a tRNA gene transcrited by RNA polymeraseIII, which usesa difierent set of general transcription factors than RNA polymeraseII uses.

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a sequences at MATlocus -..l-----------L -1-l-

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FIGURE 7-33 Arrangementof mating-typelocion chromosomelll in the yeastS. cerevisiae. Silent(unexpresseo,t mating-type genes(eithera or ct,depending on the strain) are located at theHMLlocus. Theopposite mating-type genesare present at the silentHMRlocusWhentheo or a sequences are present at the MAI locus, theycanbetranscribed intomRNAs whose

proteins encoded specify the mating-type phenotype of thecell.The silencer sequences nearHML andHMRbindproteinsthat arecritical for repression of thesesilentloci.Haploid cellscanswitchmating typesrn a process that transfers the DNAsequence fromHMLor HMRIo the transcriptionally activeMATlocus

Severallines of evidenceindicate that repressionof the HML and HMR loci results from a condensedchromatin

silencersequences. For instance,when a geneis placedwithin a few kilobases of any yeast telomere, its expression is repressed.In addition, this repressionis relievedby the same mutations in the H3 and H4 histone tails that interfere with repressionat the silent mating-typeloci.

Theseresultsindicate that the DNA of the silent loci is inac_

peated multiple times at each yeast chromosome telomere. Further biochemical studiesshowed that the SIR2 protein is a,histone deacetylase;it removes acetyl groups on lysinesof the histone tails. Also, the RAP1, and SIR2, 3, and 4 proteins bind to one another, and SIR3 and SIR4 bind to the N_ terminal tails of histonesH3 and H4 that are maintained in a-largely unacetylated state by the deacetylaseactivity of SIR2. Several experiments using fluorescenceconfocal

( a ) N u c l e ia n d t e l o m e r e s

(b)Telomeres

yeast nuclei. (a)Confocalmicrograph0.3 pm thick throughthree diploidyeastcells,eachcontaining6g telomeres.Telomeres were labeledby hybridization to a fluorescenttelomere_specific probe (yellow).DNA was stainedred to revealthe nuclei. The 6g telomeres coalesceinto a much smallernumberof regionsnearthe nuclear 300

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periphery. (b, c) Confocalmicrographs of yeastcellsIabeledwith a telomere-specific hybridization probe(b) and a fruorescent-rabered antibodyspecificfor SlR3(c) Note that SlR3is locailzedin the repressed telomericheterochromatin. similarexperiments with RAp1, SlR2,and SlR4haveshown that theseproteinsalsocolocalize with the repressed telomericheterochromatin[Fromlvt cottaet al , 1996, J CellBiol 134:1349;courtesyof M Gotta.T.Laroche, andS M Gasserl

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microscopy of yeast cells either stained with fluorescentlabeled antibody to any one of the SIR proteins or RAP1 or hybridized to a labeled telomere-specificDNA probe revealed that these proteins form large, condensedtelomeric nucleoprotein structures resembling the heterochromatin found in higher eukaryotes (Figure 7-34). Figure 7-35 depicts a model for the chromatin-mediated silencing at yeast telomeres based on these and other studies. Formation of heterochromatin at telomeresis nucleatedby multiple RAPl proteins bound to repeated sequencesin a nucleosomefree region at the extreme end of a telomere. A network of protein-protein interactions involving telomere-bound RAP1, three SIR proteins (2, 3, and 4), and hypoacetylated histones H3 and H4 createsa stable, higher-order nucleoprotein complex that includes several telomeres and in which the DNA is largely inaccessibleto external proteins. One additional protein, SIR1, is also required for silencing of the silent mating-type loci. It binds to the silencerregions associatedwith HML and HMR together with RAP1 and other proteins to initiate assemblyof a similar multiprotein silencingcomplex that encompassesHML and HMR.

Si12 Si14 Si13

Chapter 6 that acetylation of lysines neutralizes their positive chargeand eliminatestheir interaction with DNA phosphate groups, reducing chromatin condensation' Repressionat ielome.es and at the silent mating-type loci was defectivein the mutants with glycine substitutions but not in mutants with arginine substitutions. Further, acetylation of H3 and H4 lysinesinterfereswith binding by SIR3 and SIR4 and consequentlypreventsrepressionat the silent loci and telomeres' Chromatin Condensation in Higher Eukaryotes In higher eukaryotes, a similar processleads to the formation oicond.ns.d heterochromatinat centromeresand telomeres, as well as repressionof genesat internal chromosome positions that differ depending on the cell type' But in

Hypoacetylatedhistone N - t e r m i n atla i l s

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Nucleosomescondense a n d m u l t i p l et e l o m e r e s associate

modelof 7-35 Schematic < FIGURE yeast telomeres. at mechanism silencing (Iop)Multiplecopies of RAPlbindto a simple region at eachtelomere sequence repeated SlR3andSlR4bindto thatlacksnucleosomes R A P 1a,n dS l R 2b i n d st o S l R 4S l R 2i sa h i s t o n e thetailson the thatdeacetylates deacetylase RAPlbinding therepeated neighboring histones histonetails stte(Middle)Thehypoacetylated which arealsobindingsitesfor SlR3andSlR4, deacetylating SlR2, additional in turn binds of this histonesRepetition neighboring of the regionof in spreading process results SlR2, with associated histones hypoacetylated (Bottom) between Interactions SlR3,and SlR4 the andSlR4cause SlR3, of SlR2, comolexes telomeres andseveral to condense chromatin 7-34.fhe asshownin Figure to associate, generated structure chromatin higher-order interacting proteins from other blocks sterically fromM DNA.[Adapted with the underlying OpinCellBiol9:383| Curr. 1997, Grunstein,

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multicellular organisms,methylation of specificlysineresidues in histone H3 contribute to chromatin condensationin addition to the deacetylation of histone lysines. We learned in Chapter 5 how HP1 (heterochromatinprotein 1) promotes chromatin condensationby binding to nucleosomesthat are methylated at lysine 9 of histone H3. BecauseHp1 also binds to a bistone methyl transferasethat methylatesH3 lysine 9 on neighboring nucleosomes,additional Hpl is recruited to the area. Further associationsbetweenthe individual HP1 moleculesthemselvesremodel chromatin into a more compact structure(seeFigure6-34).

sential for maintaining the repression of genes in specific types of cells and in all of the subsequentcells that develop from them throughout the life of an organism. Importani genes regulated by Polycomb proteins include the Hox genes,encoding master regulatory transcription factors. As discussed in Chapter 22, different combinations of Hox transcription factors help to direct the development of spe_ cific tissuesand organs in a developingembryo. Early during embryogenesis,expressionof Hox genesis controlled by typical activator and repressor proteins. However, the ex_

quent descendentsof that cell.

Remarkably, virtually all cells in the developing embryo and adult expressa similar set of Polycomb and Trithorax proteins and all cells contain the same set of Hox genes.yet only the Hox genes in cells where they were initially repressed in early embryogenesis remain repressed, even though the same Hox genesin other cells remain active in the presenceof the same Polycomb proteins. Consequentl5 as in the caseof the yeast silent-mating-typeloci, the expression of Hox genes is regulated by a process that involves more than simply specific DNA sequencesinteracting with proteins that diffuse through the nucleoplasm.The reason is the samecollection of Polycomb and Trithorax proteins and the same Hox DNA sequencesin all cells result in the expression of specific Hox genesin cells comprising the ante, rior of an embryo and their repressionin cells located in the posterior of the embryo. A current model for repressionby polycomb proteins is depictedin Figure 7-36. Most Polycomb proteins are subunits of one of two multiprotein complexes, pRCl and pRC2. PRC2 is thought to act initially by associatingwith specificrepressorsbound to their cognateDNA sequencesearly in embryogenesis.The PRC2 complex contains a subunit with a SET domain, the enzymaticallyactive domain of severalhistone methyl transferases.This SET domain methylates histone H3 on lysine 27. The PRC1 complex then binds the methylated nucleosomesthrough dimeric Pc subunits eachcontaining a binding domain (called a chromodomain\ soecjhc for methylated H3 lysine 27. Binding of the dimeric pi to neighboring nucleosomesis proposed to condensethe chromatin into a structure rhat inhibits transcription. PRC2 or another methyltransferase is postulatedto associatewith pRC1, maintaining methylation of H3 lysine 27 in nucleosomesin the region. This resultsin associationof the chromatin with pRCl and PRC2 complexeseven after expressionof the initial repressorproteins in Figure 7-36ahas ceased. A key feature of Polycomb repressionis its maintenance in daughter cells through successivecell divisions for the life

PRCl complex

H3.

H3.

H3.

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K27tMeK27tMeK27+Me K

A FIGURE 7-36 Model for repressionby polycombcomplexes. (a)Duringearlyembryogenesis repressors associate with the pRC2 (b)Thisresults complex. (Me)of neiqhborinq in methylation nucleosomes on histone H3 lysine 27 \K27)Oytfreiff-Oomiin_ containing (c)ThepRClcomplexes subunitE(z). bindnucreosomes methylated at H3 lysine 27 througha dimeric, chromodomain_ containing subunitPc ThepRClcomplex condenses the chromatin 302

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pRC2complexes Intoa repressed chromatin structure. or another histone methyltransferase associates with pRClcomptexes to maintain H3lysine 27 methylation of neighboring nucreosomes (not shown)As a consequence, PRC 1 association with the regionrs maintained whenexpression of the repressor proteins in (a)ceases [Modified from A H Lund and M van Lohuizen,2OO4,Curr Op CettBiol. 16:239 l

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7-37ThC FIGURE < EXPERIMENTAL chromatinimmunoprecipitationmethod can revealthe acetylationstate of histones arelightlycross-linked in chromatin.Histones to DNAin vivousinga cell-permeable, agent. cross-linking chemical reversible, l s o t a t ea n d s h e a rc h r o m a t i nm e c h a n i c a l l y tailsare histone with acetylated Nucleosomes N-terminal shownin green.StepE: Cross-linked for acetvlated specific ffl*i:tibodv to an andsheared isthenisolated chromatin Antibody against lengthof two to threenucleosomes average acetylatedhistone a particular against StepZ: An antibody N-terminaltail isadded,and tailsequence histone acetylated (step are nucleosomes bound E) N u c l e o s o m ew i t h acetylatedhistone immunoprecipitated Step4: DNAin the tails is fragments chromatin immunoprecipitated andthenis thecross-link by reversing released quantitated PCRmethod.The usinga sensitive the in vivo methodcanbe usedto analyze of anyproteinwith a specific association trnrnunoprecipitate against of DNAby usingan antibody sequence SE the proteinof interestin stepE. [See 392:831 Nature et al. 1998, l Rundlett

+

of an organism (-199 years for some vertebrates,2'000 years for a sugar cone pine!). This stability of Hox gene expression state is thought to result from the distribution of nucleosomeswith methylated H3 lysine 27 to both daughter DNA molecules immediately following DNA replication. Association of PRC1 complexes with these H3 lysine 27methylated nucleosomes and methylation of new nucleosomes assembledon the replicated DNA on H3 lysine 27 would allow the complete PRC1 complex to be reestablishedover the sameregion of chromatin in both replicated daughter chromosomes. Although Polycomb repression may involve additional mechanismsas well, this model can explain how repressionof specificgenescan be maintained in all daughter cells derived from an initial cell where the gene was repressedby a transientlyexpressedset of repressors. Alternatively, a complex of Trithorax proteins includes a histone methyl transferasethat methylateshistone H3 lysine 4, a histone methylation associatedwith the promoters of actively transcribed genes. This histone modification is thought to create a binding site for histone acetylaseand chromatin remodeling complexesthat promote transcription and prevent methylation of histone H3 at lysine 9, preventing the binding of HP1, and at Iysine 27, preventing the binding of the PRC1 repressingcomplex. Nucleosomes marked with histone H3 lysine 4 methylation also are

thought to be distributed to both daughter DNA molecules during DNA replication. Binding of Trithorax complexesto nocleosomeswith the histone H3 lysine 4 methylation mark may causethe same methylation at unmodified histones incorporated into the daughter chromatin, leading to perpetuation of the chromatin mark in this region' In this way, inheritance of the expression status of Hox genes and other genes regulated by the Polycomb/Irithorax system is templat.d through chromatin replication by post-translational modifications on histones rather than DNA sequence'This type of inheritance through modifications of chromatin structure rather than modification of DNA sequenceis referred to as epigeneticinheritance.

Can DirectHistoneDeacetylation Repressors and Methylation at SPecificGenes The importance of bistone deacetylation and methylation in chro-atitt-tttediated gene repression has been further supported by studiesof eukaryotic repressorsthat regulategenes at internal chromosomal positions. These proteins are now known to act in part by causingdeacetylationof histonetails in nucleosomesthat bind to the TATA box and promoterproximal region of the genesthey repress.In vitro studies have shown that when promoter DNA is assembledonto a

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nucleosomewith unacetylatedhistones,the generaltranscription factors cannor bind to the TATA box and initiation region. In unacetylatedhistones,the N-terminal lysinesare positively charged and inreract strongly with DNA phosphates. The unacetylatedhistonetails also inreractwith neighboring histone octamers,favoring the folding of chromatin into condensed,higher-order structureswhose preciseconformation is not well understood. The net effect is that general transcription factors cannot assembleinto a preinitiation complex on a promoter associatedwith hypoacetylatedhistones. In contrast, binding of general transcription factors is repressedmuch less by histones with hyperacetylatedtails in which the positively charged lysinesare neutralizedand electrostatic interactionswith DNA phosphatesare eliminated. The connection between histone deacetylationand repressionof transcriptionat specificyeastpromotersbecame clearerwhen the cDNA encodinga human histonedeacetylase was found to have high homology to the yeast RpD3 gene,known to be required for the normal repressionof a number of yeast genes.Further work showed that RpD3 protein has histonedeacetylase activity.The ability of RpD3 to deacetylate histonesat a number of promotersdependson two other proteins:UME6, a repressorthat binds to a specific upstreamregulatorysequence(URS1),and SIN3, which is part of a large, multiprotein complex that also contains RPD3. SIN3 also binds to the repressiondomain of UME6, thus positioning the RPD3 histonedeacetylasein the complex

so it can interact with nearby promoter-associatednucleosomesand remove acetylgroups from histonetail lysines.Additional experiments,using the chromatin immunoprecipitation technique outlined in Figure 7-37, demonstrated that in wild-type yeast, one or two nucleosomesin the immediate vicinity of UME6-binding sites are hypoacetylated. These DNA regions include the promoters of genesrepressedby UME6. In sin3 and rpd3 deletion mutants, not only were these promoters derepressed,but the nucleosomesnear the UME6-binding siteswere hyperacetylated. All these findings provide considerable support for the model of repressor-directeddeacetylationshown in FigureT-38a.In this model, the SIN3-RPD3complex funcions as a co-repressor,Co-repressor complexes containing histone deacetylasesalso have been found associatedwith many repressorsfrom mammalian cells. Some of thesecomplexes contain the mammalian homolog of SIN3 (mSin3), which interacts with the repressiondomain of the repressor, as in yeast.Other histone deacetylasecomplexesidentified in mammalian cells appear to contain additional or different repressor-bindingproteins. These various repressorand corepressor combinations are thought to mediate histone deacetylationat specificpromoters by a mechanism similar to the yeast mechanism (Figure 7-38a). In higher eukaryotes,some co-repressorcomplexesalso contain histone methyl transferasesubunits that methylate histone H3 at lysine 9, generatinga binding site for Hp1 protein,

> FIGURE7-38 Proposedmechanismof (a) Repressor-directed histonedeacetvlation histone deacetylation and hyperacetylation in yeast transcription control. (a) RepressorDeacetvlationof histone d i r e c t e dd e a c e t y l a t i oonf h i s t o n eN - t e r m i n a l t a i l s .T h e D N A - b i n d i n gd o m a i n( D B D )o f t h e repressorUfvlE6interactswith a specific u p s t r e a mc o n t r o le l e m e n t( U R S 1o) f t h e g e n e s i t r e g u l a t e sT h e U M E 6r e p r e s s i odno m a i n ( R D )b i n d sS l N 3 ,a s u b u n i to f a m u l t i p r o t e i n c o m p l e xt h a t i n c l u d e sR p D 3 ,a h i s t o n e deacetylaseDeacetylation of histone N - t e r m i n atla i l so n n u c l e o s o m ei sn t h e r e g i o n o f t h e U M E 6 - b i n d r nsgi t ei n h i b r t sb i n d i n go f generaltranscriptionfactorsat the TATA box, therebyrepressinggene expression (b) Activator-directed hyperacetylation of h i s t o n eN - t e r m i n atla i l s T h e D N A - b i n d i n g (b) Activator-di rectedh istone hyperacetylation d o m a i no f t h e a c t i v a t o G r C N 4i n t e r a c t sw i t h specificupstreamactivatingsequences(UAS) o f t h e g e n e si t r e g u l a t e sT h e G C N 4a c t i v a t i o n Hyperacetylation of histone d o m a i n( A D )t h e n i n t e r a c t sw i t h a m u l t i p r o t e r n GCNS tails \ N t e r m i n a l h i s t o n ea c e t y l a sceo m p l e xt h a t i n c l u d e st h e \ \ G C N 5c a t a l y t i cs u b u n i t S u b s e q u e n t h y p e r a c e t y l a t i oonf h i s t o n eN - t e r m i n atla i l so n n u c l e o s o m eisn t h e v i c i n i t yo f t h e G C N 4 b i n d i n gs i t ef a c i l i t a t e as c c e s so f t h e g e n e r a l t r a n s c r i p t i ofna c t o r sr e q u i r e df o r i n i t i a t i o n R e p r e s s i oann d a c t i v a t i o no f m a n yg e n e si n h i g h e re u k a r y o t eosc c u r sb Vs i m i l a m r echanisms

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7-39 Associationof a repressedtransgenewith FIGURE with a werestablytransformed Mousefibroblasts heterochromatin. repressor. The with bindingsitesfor an engineered transgene domain,a repression wasa fusionbetween a DNA-binding repressor complex, andthe with the KAP1co-repressor domainthatinteracts receptor thatallowsthe nuclear ligand-binding domainof a nuclear (see experimentally importof thefusionproteinto be controlled dye DAPI with the intercalating 7-49)DNAwasstained blue Figure wherethe regions areregions of heterochromatin, Brighter-staining was Thetransgene ishigherthanin euchromatin DNAconcentration

complementary labeled of a fluorescently by hybridization detected in the wasretained repressor probe(green)Whenthe recombinant (/eft)andwasassociated wastranscribed thetransgene cytoplasm, wasaddedsothat in mostcells.Whenhormone with euchromatin was thetransgene the nucleus, entered repressor the recombinant Chromatin (right) heterochromatinwith associated and repressed (seeFigure 7-37)showedthatthe assays immunoprecipitation at lysine H3 methylated with histone genewasassociated repressed of Frank theactivegenewasnot.[Courtesy 9 andHP1,whereas andDevl7:1855l et al, 2003Genes fromAyyanathan Rauscher

as discussedearlier. For example, the KAP1 co-repressor complex functions with a class of more than 200 zinc-finger transcription factors encodedin the human genome.This corepressorcomplex includes an H3 lysine 9 methyl transferasethat methylatesnucleosomesover the promoter region of repressedgenes,leading to HP1 binding and repressionof transcription. An integrated transgenein cultured mouse fibroblaststhat was repressedthrough the action of the KAP1 co-repressorassociatedwith heterochromatin in most cells, whereas the active form of the same transgene associated with euchromatin (Figure 7-39). Chromatin immunoprecipitation assays(seeFigure 7-37) showedthat the repressedgene was associatedwith histone H3 methylated at lysine 9 and HP1, whereasthe active genewas not. Interestingly,in addition to methylation of histone proteins, methylation of the DNA sequenceitself can also be a trigger for chromatin condensation.The discoveryof mSin3containing histone deacetylasecomplexes provided an explanation for earlier observations that in vertebratestranscriptionally inactive DNA regions often contain the modified cytidine residue 5-metbylcytidine (mC) followed immediately by a G, whereas transcriptionally active DNA regions contain fewer mC residues.DNA containing 5methylcytidine has beenfound to bind a specificprotein that

in turn interacts specifically with mSin3' This finding suggests that association of mSin3-containing co-repressors with methylated sites in DNA leads to deacetylationof histones in neighboring nucleosomes,making these regions inaccessibleto general transcription factors and Pol II and hencetranscriptionallyinactive.

ActivatorsCan DirectHistoneAcetylationand Methylation at SPecificGenes bind Just as repressorsfunction through co-repressorsthat DNAof domains activation the domains' repression to their binding activators function by binding multisubunit coactiuator complexes.One of the first co-activator complexes to be characterized was the yeast SAGA complex, which functions with the GCN4 activator protein describedin Section 7.4. Early genetic studies indicated that full activity of the GCN4 activator required a protein called GCNS. The clue to GCNS's function came from biochemical studiesof a histone acetylasepurified from the protozoan Tetrahymena, the first histone acetylaseto be purified. Sequenceanalysis revealed homology between the Tetrahymena protein and yeast GCN5, which was soon shown to have histone acetylase activity as well. Further geneticand biochemical studies

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revealedthat GCN5 is one subunit of a multiprotein co-activator complex, named the SAGA complex after genes encoding someof the subunits.Another subunit of this histone acetylasecomplex binds to acrivation domains in multiple yeast activator proteins, including GCN4. The model shown in Figure 7-38b is consistenrwith the observationthat nucleosomesnear the promoter region of a gene regulated by the GCN4 activator are specifically hyperacetylated compared to most histonesin the cell. This activator-directedhyperacetylationof nucleosomesnear a promoter region opens the chromatin strucure so as to facilitate the binding of other proteins required for transcription initiation. The chromatin structure is less condensedcompared to most chromatin, as indicated by its sensitivity to digestion with nucleasesin isolated nuclei. Also, the acetylation of specific histone lysinesgeneratesbinding sitesfor proreins wiih bromodomains that bind them. For example.a subunit of the general transcription factor TFIID contains two bromodomains that bind to acetylatednucleosomeswith high affinity. Recall that TFIID binding ro a promoter initiates assembly of an RNA polymeraseII preinitiation complex (see Figure 7-31). Nucleosomesar promoter regionsof virtually all active genesare hyperacetylated. A similar activation mechanismoperatesin hieher euk a r y o t e s .M a m m a l i a n c e l l sc o n r a i n m u l r i s u b u n i th i s r o n e acetylaseco-activatorcomplexeshomologous to the yeast SAGA complex. They also expresstwo related -400-kDa, multidomain proteins called CBP and P300, which are thought to function similarly. As noted earlier, one domain of CBP bindsthe phosphorylatedacidicactivariondomain in the CREB transcription factor. Other domains of CBp interact with different activation domains in other activators. yet another domain of CBP has histone acetylaseactivity, and another CBP domain associateswith additional multisubunit histone acetylasecomplexes.CREB and many other mammalian activators are thought to function in part by directing CBP and the associatedhistone acetylasecomplex to specific nucleosomes,where they acetylatehistone tails, facilitating the interaction of generaltranscription factors with promoter DNA. In addition, the largest TFIID subunit also has histone acetylaseactivity and may function to maintain histone tail hyperacetylationin promoter regions. Methylation of histone H3 lysines9 or 27 resultsin rranscriptional repression mediated by binding proteins of the HP1 or Polycombclass,respectivelgas discussedabove.In contrast, H3 lysine 4 methylation is observed in the promoter regionsof active genesthrough the rargetingof methyl transferasesspecificfor H3 lysine 4. One example of this involves the Trithorax complex proteins. As discussedearlier, Trithorax complex proteins maintain expressionof Hox g e n e si n a p p r o p r i a t ec e l l s ,j u s t a s p o l y c o m bp r o t e i n sm a i n tain repressionof the same Hox genesin other cells. Trithorax proteins, including one with a SET domain that merhylates lysines, assembleinto a multiprotein complex that tri-methylatesH3 lysine 4. H3 tails tri-methylated at lysine 4 then serveas a binding site for another subunit of the tithorax complex so that the methyl transferasesubunit of the Trithorax complex can maintain H3 lysine 4 in the methy305

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lated state in chromatin associatedwith the complex. This is a similar mechanismto the maintenanceof histone H3 lysine 27 methylation by Polycomb complexes (seeFigure 7-36). The H3 amino-terminal tail tri-methylated on lysine 4 also servesas a binding site for co-activator complexes.For example, SAGA-like histone acetylasecomplexes also contain a domain that binds specificallyto tri-methylated H3 lysine 4. This results in acetylation of histone tail lysines,thereby generatinga chromatin structure conducive to transcription. Many genesin multicellular organisms in addition to Hox genesare expressedin lineage-specificexpressionprograms regulated by Trithorax and Polycomb proteins. This is most clearly revealedby staining Drosophila salivary gland polytene chromosomeswith antibodies to Polycomb and Trithorax proteins.This experimentrevealsbinding of theseproteins to more than 100 siteson fly chromosomesin thesecells.

C h r o m a t i n - R e m o d e l i nFga c t o r sH e l pA c t i v a t eo r RepressTranscription In addition to histone acetylase complexes, multiprotein chromatin-remodelingcomplexesalso are required for activation at many promoters. The first of thesecharacterizedwas the yeastSWVSNFchromatin-remodelingcomplex.One of the SWVSNF subunitshas homology to DNA helicases,enzymes that use energy from AIP hydrolysis to disrupt interactions between base-pairednucleic acids or between nucleic acids and proteins. In vitro, the SVIiSNF complex is thought to pump or push DNA into the nucleosomeso rhat DNA bound to the surface of the histone octomer transiently dissociates from the surfaceand translocates,causingthe nucleosomesto "slide" along the DNA. The net result of such chromatin remodelingis to facilitate the binding of transcription factors to specific DNA sequencesin chromatin. Many activation domains bind to chromatin-remodelingcomplexes,and this binding stimulates in vitro transcription from chromatin templates(DNA bound ro nucleosomes).Thus the SWSNF complex representsanothertype of co-activatorcomplex.The experiment shown in Figure 7-40 dramatically demonstrates how an activation domain can cause decondensationof a region of chromatin. This is thought to result from the interaction of the activation domain with chromatin-remodeling and histoneacetylasecomplexes. Chromatin-remodelingcomplexesare required for many processesinvolving DNA in eukaryotic cells, including transcription control, DNA replication, recombination, and repair. Severaltypes of chromatin-remodeling complexes are found in eukaryotic cells,all with homologous DNA helicase domains. S\fVSNF complexes and related chromatinremodeling complexes in multicellular organisms contain subunits with bromodomains that bind to acetylatedhistone tails. Consequently,SI7VSNF complexes remain associated with activated, acetylatedregions of chromatin, presumably maintaining them in a decondensedconformation. Some chromatin-remodelingcomplexescontain subunits that bind to histone H3 methylated on lysine 4, contributing to rranscriptional activation by Trithorax proteins. Surprisingly, chromatin-remodeling complexes can also participate in

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EXPERIMENTAL FIGURE 7-40 Expression of fusionproteins demonstrateschromatindecondensationin responseto an to activationdomain.A cultured hamster cellIinewasengineered containmultiple copies of a tandemarrayof E.colilacoperator sequences integrated intoa chromosome in a regionof (a)Whenan expression heterochromatin vectorfor the/acrepressor intothesecells,/acrepressors wastransfected boundto the /ac operator sitescouldbevisualized in a regionof condensed chromatin (red)DNAwasvisualized usingan antibody against the /acrepressor (b)Whenan by staining with DAPI(blue), revealing the nucleus. vectorfor the /acrepressor fusedto an activation domain expression intothesecells,staining wastransfected asin (a)revealed thatthe to decondense into activation domaincauses thisregionof chromatin fiberthatfillsa muchlarger volume a thinnerchromatin of thenucleus Bar: 1 pm [Courtesy of Andrew S Belmont, 1999, J CellBiol145:1341 ] transcriptionalrepression.Thesechromatin-remodelingcomplexesbind to transcription repressiondomains of repressors and contribute to repression,presumably, by folding chromatin into condensedstructures.Much remainsto be learned about how this important classof proteins alters chromatin structure to influencegeneexpressionand other processes.

HistoneModificationsVary Greatlyin T h e i rS t a b i l i t i e s radiolabelingexperimentshave shown that acetyl Pulse-chase groups on histone lysinesturn over rapidly, whereasmethyl groups are much more stable.The acetylationstate at a specific histone lysine on a particular nucleosomeresultsfrom a dynamic equilibrium between acetylation and deacetylation by histone acetylasesand histone deacetylases,respectively. Acetylation of histonesin a localizedregion of chromatin predominateswhen local DNA-bound activatorstransientlybind histone acetylasecomplexes. De-acetylation predominates when repressorstransiently bind histone deacetylasecomplexes. In addition to these processes,localized to relatively short lengths of chromatin, which include promoters and other transcription-control regions, histone acetylasesand also function globally on all euchromatin, condeacetylases stantly removing and replacinghistonelysine acetylgroups. In contrast to acetyl groups, methyl groups on histone lysinesare much more stableand turn over much lessrapidly than acetyl groups. Histone lysine methyl groups can be

removed by recently discovered histone lysine demethylases. But the resulting turnover of histone lysine methyl groups is much slower than the turnover of histonelysine acetylgroups. Multiple other post-translationalmodifications have been characterizedon histones, many of them summarized in Figure 6-31b. Theseall have the potential to positively or negatively regulate the binding of proteins that interact with the chromatin fiber to regulatetranscription and other processes. A picture of chromatin is emergingin which histone tails extending as random coils from the chromatin fiber are posttranslationally modified to generate one of many possible combinations of modifications that regulate transcription and other processesby regulating the binding of a large number of different protein complexes.Some of these modifications, like histone lysine acetylation, are rapidly reversible, whereas others, like histone lysine methylation, can be templated through chromatin replication, generating epigenetic inheritancein addition to inheritanceof DNA sequence.

T h e M e d i a t o rC o m p l e xF o r m sa M o l e c u l a r Bridge BetweenActivation Domainsand Pol ll Now let's shift our attention from how activators and repressorscontrol chromatin structure to the other mechanism of gene regulation outlined in the introduction to this section-regulation of the assemblyof transcription preinitiation complexes. Interaction of activators with the multiprotein mediator complex (Figure 7-41.1directly assistsin assemblyof Pol II preinitiation complexes.Someof the -30 mediator subunits bind to RNA polymerase II, and other mediator subunits bind to activation domains in various activator proteins. Thus mediator can form a molecular bridge between an activator bound to its cognate site in DNA and Pol II at a promoter. In addition, one of the mediator subunits has histone acetylaseactivity and may function to maintain a promoter region in a hyperacetylatedstate. yeast mutants inExperimentswith temperature-sensitive dicate that some mediator subunits are required for transcription of virtually all yeastgenes.Thesesubunits most likely help maintain the overall structure of the mediator complex or bind to Pol II and therefore are required for activation by all activators. In contrast, other mediator subunits are required for normal activation or repressionof specific subsetsof genes.DNA microarray analysis of yeast gene expression in mutants with defectsin these mediator subunits indicates that each such subunit influencestranscription of -3-10 percent of all genes to the extent that its deletion either increasesor decreases mRNA expression by a factor of rwofold or more (seeFigure 5-29 for DNA microarray technique).Thesemediator subunits are thought to interact with specific activation domains; thus when one subunit is defective,transcription of genesregulated by activators that bind to that subunit is severely depressed, but transcription of other genesis unaffected. Consistent with this explanation are binding studies showing that some activation domains do indeed interact with specific mediator subunits. However, recent studies suggestthat most activation domains may interact with more than one mediator subunit.

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< FIGURE7-41 Structureof yeast and human mediator complexes. (a) Reconstructed imageof mediatorfrom 5 cereyrsiae boundto Poill N/ultipleelectronmicroscopy imageswerealignedand computer-processed to producethis averageimagein whichthe threedimensional Polll structure(lightorange)is shownassociated with the yeastmediatorcomplex(darkblue) (b) Diagrammatic representation of mediatorsubunitsfrom 5 cereyrsr'ae Subunitsshownin the samecolor arethoughtto form a module Mutationsin one subunitof a module may inhibitassociatron of othersubunitsin the samemodulewith the restof the complex(c)Diagrammatic representation of human mediatorsubunitsThe relativepositionof eachhumanmediator subunitis arbitraryexceptfor the subunitsthat are homologousto 5 cerevisiae mediatorsubunits IPart(a)fromS Hahn,2004,Nat.Struct.Mol B i o l1 1 : 3 9 4b,a s e d o n D J a v i s e t a , 2 O O 2 , MCoel l1l 0 : 4 0 9P a r t ( bf r)o m B Gugliemi el al ,2004,Nuc AcidsRes32:5379Part(c)adapted fromS Malik andR G Roeder, 2005,Trends BtochemScl 30:256l or promoter-proximal elements can interact with mediator associatedwith a promoter becausechromatin, like DNA, is flexible and can form a loop bringing the regulatory regions and the promoter close together, as observed for the E. coli NtrC activator and oso-RNA polymerase (see Figure 7-4). The multiprotein nucleoprotein complexes that form on eukaryotic promoters may comprrse as many as 100 polypeptides with a total mass of :3 megadaltons (MDa), as large as a ribosome.

MED19

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T r a n s c r i p t i o on f M a n y G e n e sR e q u i r e s O r d e r e dB i n d i n ga n d F u n c t i o no f A c t i v a t o r s and Co-activators 'We can now extend the model of Pol II transcr:iotioninitiation in Figure 7-31 to take into accountthe role of activarors and co-activators.These accessoryproteins function not oniy to make geneswithin nucleosomalDNA accessible to generaltranscriptionfactors and Pol II but also directly recruit Pol II to promoter regions.

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Largemediatorcomplexes,isolatedfrom yeastand from culturedmammaliancells,are requiredfor mammalian activators to stimulate transcription by Pol II in vitro. Since genesencodinghomologsof mediator subunitsare found in the genomesol C. elegans,Drosophila, and plants, it appears that most multicellular organismshave homologous mediator complexes.About half of the merazoan(multicellular animals)mediator subunitsare clearly homologousto yeastmediatorsubunits(Figure7-411r,c). But the remaining subunits,which appear to be distinct from any yeasrprot e l n s , m a y i n t e r a c t w i t h a c t i v a t i o nd o m a i n s t h a t a r e n o t found in yeast. The various experimentalresultsindicatingthat individuai mediator subunits bind to specificactivation domains suggestthat multiple acrivatorsinfluencetranscriptionfrom a single promoter by interactingwith a mediator complex simultaneously(Figure7-42). Activatorsbound at enhancers

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FIGURE7-42 Model of several DNA-bound activators interacting with a single mediator complex. The abilityof differentmediatorsubunitsto interactwith specific activation domainsmay contributeto the integrationof signalsfrom several activatorsat a singlepromoter Seethe text for drscussron

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7-43 Orderedbinding and interactionof activators < FIGURE and co-activatorsleadingto transcriptionof the yeast HO intocondensed the HOgeneis packaged gene.Stepn: Initially, bindsto whenthe SWl5activator begins chromatinActivation of the startsiteand sites1200-1400 basepairsupstream enhancer complexStep[: chromatin-remodeling with the SWI/SNF interacts thereby the chromatin, actsto decondense TheSWI/SNF complex histone acetylase histone tails StepS: A GCN5-containing exposing histone tailsin with boundSWl5andacetylates associates complex adlacent to decondense continues the HOlocusasSWI/SNF fromthe DNA,buttheSWI/SNF chromatinStep4:SWl5 is released withthe HOcontrolregion remainassociated andGCN5complexes histone thatbindacetylated of bothcomplexes of subunits because to Theiractionallowsthe SBFactivator tailsthroughbromodomains then SBF promoter-proximal region Step in the sites E: bindseveral bindingof Polll Step@: Subsequent complex. bindsthe mediator of a transcription in assembly factorsresults transcription andgeneral 7-42in Figure aredetailed whosecomponents preinitiation complex [ A d a p t e df r o m C J F r ya n d C L P e t e r s o n2, 0 0 1 , C u r r . B i o l1 1 : R 1 8 5S e ea l s o M P C o s m a e t a l, 1 9 9 9 . C e l l9 7 : 2 9 9 a, n d M P C o s m a e t a l, 2 O O l , M o l C e l l 7 : 1 2 1 3l

binding of the SS7I5 activator to an upstream enhancer. Bound S\7I5 then interacts with the S\7VSNF chromatinremodeling complex and the GCNS-containing SAGA histone acetylasecomplex. Once the chromatin in the HO control region is decondensedand hyperacetylated' a second activator,SBE,can bind to severalsitesin the promoter-proximal region. Subsequentbinding of the mediator complex by SBF then leads to assemblyof the transcription preinitiation complex containing Pol II and the generaltranscription factors shown in Figure 7-31. N7e can now see that the assembly of a preinitiation complex and stimulation of transcription at a promoter results from the interaction of severalactivators with various Preinitiation multiprotein co-activator complexes.Theseinclude chromatincomplex remodeling complexes, histone acetylasecomplexes, and a mediator complex. Although much remains to be learned about these processes,it is clear that the net result of these multiple molecular eventsis that activation of transcription at a promoter depends on highly cooperative interactions initiated by severalactivators. This allows genesto be regulated in a cell-type-specificmanner by specificcombinations of transcription factors. The TTR gene,which encodestransthyretin in mammals, is a good example of this. As noted earlier, transthyretin is expressedin hepatocytesand in choroid plexus cells' TranRecentstudieshave analyzedthe order in which activascription of the TTR gene in hepatocytesis controlled by at tors bind to a transcription-control region and interact with least five different transcriptional activators (Figute 7-441. co-activatorsas a gene is induced. Such studiesshow that Even though three of these activators-HNF4, C/EBP,and assemblyof preinitiation complexesdependson multiple AP1-are also expressedin cells of the intestine and kidney, protein-DNA and protein-protein interactions, as illusTTR transcription does not occur in these cells, becauseall trated in Figure 7-43,which depictsactivation of the yeast nuclease five activators are required and HNF1 and HNF3 are missHO gene. This gene encodesa sequence-specific ing in kidney and intestine cells. Other hepatocyte-specific that initiates mating-type switching in haploid yeast cells (seeFigure 7-33). Activation of the HO gene begins with enhancers and promoter-proximal regions that regulate M O L E C U L A RM E C H A N I S M sO F T R A N S C R I P T I ORNE P R E S S I OANN D A C T I V A T I O N

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A FIGURE 7-44 Transcription-control region of the mouse transthyretin(rlfl gene.Binding sitesfor thefiveactivators required for transcription of IIR in hepatocytes areindicated. Thecomplete set of activators isexpressed at the required concentrations to stimulate transcription onlyin hepatorytes A different setof activators stimulates transcription in choroidplexus cells[See R Costa etal, 1989, Mot.Cett Biol

A two-step selectionprocessis used (Figure 7-45c).The bait vector also expressesa wild-type TRP gene, and the hybrid vector expressesa wild-type LEU gene.Transfected cells are first grown in a medium that lacks tryptophan and leucine but contains histidine. Only cells that have taken up the bait vector and one of the fish plasmids will survive in this medium. The cells that survive then are plated on a medium that lacks histidine. Those cells expressinga fish hybrid that does not bind to the bait hybrid cannot transcribe the HIS geneand consequentlywill not form a colony on medium lacking histidine. The few cells that express a bait-binding fish hybrid will grow and form colonies in the absenceof histidine. Recoveryof the fish vectors from these colonies yields cDNAs encoding protein domains that interact with the bait domain.

9:1415, and K Xanthopouluset al , 1989,Proc Nat'l.Acad. Sci USA86:4iil l

additional genesexpressedonly in hepatocytescontain binding sitesfor other specificcombinations of transcription factors found only in thesecells, together with those expressed ubiquitously.

The YeastTwo-HybridSystemExploitsActivator Flexibilityto DetectcDNAsThat Encode InteractingProteins A powerful molecular genetic method called the yeast twohybrid systemexploits the flexibility in activator structuresto identify geneswhose products bind to a specific protein of interest. Becauseof the importance of protein-protein interactions in virtually every biological process,the yeast twohybrid systemis usedwidely in biological research. This method employs a yeast vector for expressing a DNA-binding domain and flexible linker region without the associated activation domain, such as the deleted GAL4 containing amino acids 1-592 (seeFigure 7-21). A cDNA sequenceencoding a protein or protein domain of interest, called the bait domain, is fused in frame to the flexible linker region so that the vector will expressa hybrid protein composed of the DNA-binding domain, linker region, and bait domain (Figure 7-45a, Ieft). A cDNA library is cloned into multiple copiesof a secondyeastvector that encodesa strong activation domain and flexible linker to produce a vector library expressingmultiple hybrid proteins, eachcontaining a different fish domain (Figure 7-45a,rigbt). The bait vector and library of fish vectors are then rransfected into engineeredyeastcells in which the only copy of a generequired for histidine synthesis(HIS) is under control of a UAS with binding sitesfor the DNA-binding domain of the hybrid bait protein. Transcription of the HlS gene requires activation by proteins bound to the UAS. Transformed cells that expressthe bait hybrid and an interacting fish hybrid will be able to activate transcription of the H1S gene (Figure 7 -45b). This systemworks becauseof the flexibility in the spacingbetweenthe DNA-binding and activation domains of eukaryotic activators.

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Molecular Mechanismsof TranscriptionRepression and Activation r Eukaryotic transcription activators and repressorsexert their effects largely by binding to multisubunit co-activators or co-repressorsthat influence assemblyof Pol II transcription preinitiation complexes either by modulating chromatin structure (indirect effect) or by interacting with Pol II and generaltranscription factors (direct effect). r The DNA in condensed regions of chromatin (heterochromatin) is relatively inaccessibleto transcription factors and other proteins, so that geneexpressionis repressed. r The interactions of severalproteins with each other and with the hypoacetylated N-terminal tails of histones H3 and H4 are responsiblefor the chromatin-mediatedrepression of transcription that occurs in the telomeres and the silent mating-type loci in S. cereuisiae(seeFigure 7-35). r Some repression domains function by interacting with co-repressorsthat are histone deacetylasecomplexes.The subsequentdeacetylationof histone N-terminal tails in nucleosomesnear the repressor-bindingsite inhibits interaction betweenthe promoter DNA and generaltranscriprion factors, thereby repressingtranscription initiation (seeFigure 7-38a). r Someactivation domains function by binding multiprotein co-activator complexes such as histone acetylasecomplexes. The subsequent hyperacetylation of histone N-terminal tails in nucleosomesnear the activator-binding site facilitates interactions between the promoter DNA and general transcription factors, thereby stimulating transcription initiation (seeFigure 7-38b). r SWI/SNF chromatin-remodeling factors constitute another type of co-activator. These multisubunit complexes can transiently dissociateDNA from histone cores in an AT?-dependentreaction and may also decondenseregions of chromatin, thereby promoting the binding of DNA-binding proteins neededfor initiation to occur at some promoters. r Mediator, another type of co-activator, is an :30subunit complex that forms a molecular bridge between

T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X P R E S S T O N

llll+ Technique Animation:YeastTwo-HybridSystem > EXPERIMENTAL FIGURE 7-45 The yeasttwo-hybrid systemprovidesa way of screeninga cDNAlibrary for clonesencodingproteinsthat interactwith a specific proteinof interest,Thisisa commontechnique for screening a proteins with a for clonesencoding thatinteract cDNAlibrary specificproteinof interest(a)Twovectorsareconstructed proteinsIn one genesthatencodehybrid(chimeric) containing for the DNA-binding domainof vector(/eft),the codingsequence for a known factorisfusedto the sequences a transcription protein,referred to asthe "bait"domain(lightblue)Thesecond (right) vector expresses an activation domainfusedto a "fish" thatinteracts with the baitdomain.(b)lf yeast domain(green) with vectors expressing bothhybrids, the cellsaretransformed proteins interact to produce of the chimeric baitandfishportions theactivator transcriptional activator. In thisexample, a functional promotes of a HISgene.Oneendof thisprotein transcription (UAS) of the bindsto the upstream activating sequence complex domain, Hi53gene;theotherend,consisting of theactivation preinitiation complex stimulates assembly of thetranscription (orange) (yellow). (c)Toscreen a cDNAlibrary for at the promoter proteins thatinteract with a particular bait clones encoding proteinof interest, the libraryisclonedintothevectorencoding The areexpressed. the activation domainsothathybridproteins genes containwild-type selectable baitvectorandfishvectors (e g , a TRPor IEUgene)Theonlytransformed cellsthatsurvive the bait the indicated selection scheme arethosethatexpress with it Seethetextfor hybridanda fishhybridthatinteracts Nature 34O:2451 S Fields andO Song,1989, discussion [See

(al Hybridproteins

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(b) Transcriptional activation by hybrid proteins in yeast

r-> Transfect yeast cells with genes encoding bait and fish hybrids

and Co-activators pretranscription initiationcomplex

^€ ^^^ /\^} H/S mRNA

(c) Fishing for proteins that interact with bait domain FishcDNA from library

LEU Fish vector

activation domains and RNA polymerase II by binding directly to the polymeraseand activation domains.By binding to severaldifferent activators simultaneouslgmediator probably helpsintegratethe effectsof multiple activatorson a singlepromoter (seeFigure 7-42).

1. Transfect into trp, leu, his mutant yeast cells 2 . S e l e c tf o r c e l l s t h a t g r o w i n a b s e n c eo f t r y p t o p h a n and leucine 3 . P l a t e s e l e c t e dc e l l s o n m e d i u m lackinghistidine

r Activators bound to a distant enhancer can interact with transcription factors bound to a promoter because DNA is flexible and the intervenins DNA can form a large loop. r The highly cooperative assembly of preinitiation complexesin vivo generallyrequiresseveralactivators.A cell must produce the specificset of activators required for transcription of a particular genein order to expressthat gene. r The yeast two-hybrid system is widely used to detect cDNAs encoding protein domains that bind to a specific protein of interest(seeFigure 7-45).

I

*

Colony formation

ption-Factor Regulation of Transcri lE Activity We have seenin the precedingdiscussionhow combinations of activators and repressorsthat bind to specificDNA regulatory sequencescontrol transcription of eukaryotic genes. Whether or not a specificgenein a multicellular organism is expressedin a particular cell at a particular time is largely a

I

v

No colony formation

consequenceof the nuclear concentrations and activities of the transcription factors that interact with the regulatory sequences of that gene. Which transcription factors are expressedin a particular cell type, and the amounts produced, are determined by multiple regulatory interactions between transcription-factor genes that occur during the development and differentiation of a particular cell type' In O F T R A N S C R I P T I O N . F A C TAOCRT I V I T Y REGULATION

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> FIGURE 7-46 Examples of hormonesthat bind to nuclearreceptors. These andrelated lipid-soluble hormones bindto receptors located in thecytosol or nucleusTheligand-receptor complex f unctions asa transcription activator Retinoicacid .\-

NH

o cH,-cH c''oH ' "-14-) ,Y-/ Thyroxine

Chapters76 and22,we presentexamplesof suchregulatory interactions during development and discussthe principles of development and differentiation that have emergedfrom theseexamples. In addition to controlling the expressionof hundredsto thousandsof specifictranscriptionfactors,cellsalso regulate the activities of many of the transcription factors expressed in a particular cell type. For example,transcriptionfactors are often regulatedin responseto extracellularsignals.Interactions between the extracellular domains of transmembrane receptor proteins on the surfaceof the cell and specific protein ligandsfor thesereceptorsacrivateprotein domains associated with the intracellulardomainsof theserransmembrane proteins,transducingthe signal receivedon the outside of the cell to a signalon the insideof the cell that eventually reaches transcription factors in the nucleus. In Chapter 16, we describethe major types of cell-surfacereceptors and intracellular signalingpathways that regulate t r a n s c r i p t i o n - f a c r oarc t i v i r y . In this section,we discussthe second major group of extracellular signals, the small, lipid-soluble hormonesincluding many different steroid hormones, retinoids, and thyroid hormones-that can diffuse throueh plasma and

nuclear membranesand interact directly with the transcription factors they control (Figure 7-46). As noted earlier,the intracellular receptors for most of these lipid-soluble hormones, which constitute the nuclear-receptorsuperfamily, function as transcription activators when bound to their ligands.

A l l N u c l e a rR e € e p t o r S s h a r ea C o m m o n Domain Structure Cloning and sequencingof the genesencoding various nuclear receptors revealed a remarkable conservation in their amino acid sequencesand three functional regions (Figure 7-47). All the nuclearreceptorshave a unique N-terminal region of variablelength (100-500 amino acids).Portionsof this variable region function as activation domains in some nuclear receptors.The DNA-binding domain maps near the center of the primary sequenceand has a repeat of the Ca zinc-finger motif. The hormone-binding domain, located near the C-terminal end, contains a hormone-dependent activation domain. In some nuclear receptors,the hormonebinding domain functions as a repressiondomain in the absenceof lieand.

Estrogenreceptor(ER) Progesteronereceptor(PR) Glucocorticoidreceptor(GR) Thyroxinereceptor(TR) Retinoicacid receptor(RAR) Ng

Generalprimary structure V a r i a b l er e g i o n ( 1 0 0 - 5 0 0a a )

Amino acid identity:

O

DNA-binding d o m a i n( 6 8a a ) 42-94o/o

FIGURE7-47 General design of transcription factors in the nuclear-receptorsuperfamily.The centrally locatedDNA-binding domainexhibitsconsiderable sequencehomologyamong different receptorsand containstwo copiesof the Ca zinc-fingermotif The 312

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Ligand-binding domain 1225-285aal 15-57o/o C-terminalhormone-binding domainexhibitssomewhatless homologyThe N-terminalregionsin variousreceptorsvary in length, haveuniquesequences, and may containone or more activation d o m a i n s[ S e e RM E v a n s1,9 8 8 , S c i e n c e 2 4 0 : 8 8 9 ]

T R A N s c R t p l o N AcLo N r R o L o F G E N EE X P R E S S I O N

ResponseElementsContain Nuclear-Receptor Invertedor DirectRepeats

trast, the monomers in homodimeric nuclear receptors(e.g., GRE and ERE) have an inverted orientation.

The characteristicnucleotide sequencesof the DNA sites, called response elements, that bind several nuclear recepof the consensus tors have beendetermined.The sequences responseelementsfor the glucocorticoid and estrogenreceptors are 6-bp inverted repeats separatedby any three basepairs (Figure7-48a, b). This finding suggestedthat the cognatesteroid hormone receptorswould bind to DNA as symmetrical dimers, as was later shown from the x-ray crystallographic analysis of the homodimeric glucocorticoid receptor's Ca zinc-finger DNA-binding domain (see Figure 7-25c). Some nuclear-receptorresponseelements,such as those for the receptors that bind vitamin D3, thyroid hormone, and retinoic acid, are direct repeatsof the same sequencerecognized by the estrogenreceptor, separatedby three to five base pairs (Figure 7-48c-e). The specificity for responding to these different hormones by binding distinct receptorsis determined by the spacingbetweenthe repeats.The receptorsthat bind to such direct-repeatresponseelementsdo so as heterodimers with a common nuclear-receptormonomer called RXR. The vitamin D3 responseelement, for example, is bound by the RXR-VDR heterodimer,and the retinoic acid responseelement is bound by RXR-RAR. The monomers composing theseheterodimersinteractwith eachother in such a way that the two DNA-binding domains lie in the samerather than inverted orientation, allowing the RXR heterodimersto bind to direct repeatsof the binding site for each monomer. In con-

H o r m o n eB i n d i n gt o a N u c l e a rR e c e p t o r Regulateslts Activity as a TranscriptionFactor

(a) GRE

5'AGAACA(N)3TGTTC3 T' , 3 ' T C T T G T ( N } 3 A C A A G5A'

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ERE

(c)VDRE 5' AGZ;;A(N).nccr& s'

The mechanism whereby hormone binding controls the activity of nuclear receptors differs for heterodimeric and homodimeric receptors.Heterodimericnuclearreceptors(e.g.,RXRVDR, RXR-TR, and RXR-RAR) are located exclusively in the nucleus.In the absenceof their hormone ligand' they repress transcriptionwhen bound to their cognatesitesin DNA. They do so by directing histone deacetylation at nearby nucleosomesby the mechanismdescribedearlier (seeFigure 7-38a). In the ligand-bound conformation' heterodimericnuclear receptors containing RXR can direct hyperacetylation of histones in nearby nucleosomes,therebyreversingthe repressing effectsof the free ligand-binding domain. In the presenceof ligand, ligand-bindingdomains of nuclearreceptorsalso bind mediator,stimulating preinitiation complex assembly. In contrast to heterodimeric nuclear receptors' homodimeric receptorsare found in the cytoplasm in the absence of their ligands.Hormone binding to thesereceptorsleadsto their translocation to the nucleus. The hormone-dependent translocation of the homodimeric glucocorticoid receptor (GR) was demonstrated in the transfection experiments shown in Figure 7-49. The GR hormone-binding domain alone mediates this transport. Subsequentstudies showed that, in the absenceof hormone, GR is anchored in the cytoplasm as a large protein aggregatecomplexed with inhibitor proteins, including Hsp90, a protein related to Hsp70, the major heat-shockchaperone in eukaryotic cells. As long as the receptor is confined to the cytoplasm, it cannot interact with target genes and hence cannot activate transcription. Hormone binding to a homodimeric nuclear receptor releasesthe inhibitor proteins, allowing the receptor to enter the nucleus, where it can bind to responseelementsassociated with target genes(Figure 7-50)' Once the receptor with bound hormone binds to a response element, it activates transcription by interacting with chromatin-remodeling and histone acetylasecomplexesand mediator.

3 ' T C C A G T ( N ) . T C C A G5T'

(d) TRE

5' AGGTCA(N}4AGG3 T 'C A ,' taaoottr)4TccAGT 5'

(e) RARE ;:+::Hlill,+::f+;; elements of DNAresPonse 7-48 Consensus sequences A FIGURE for the elements that bind three nuclearreceptors.Theresponse (ERE) (GRE) receptor contain glucocorticoid receptor andestrogen proteinsTheresponse repeats that bindthesehomodimeric inverted receptors containa commondirectrepeat for heterodimeric elements bythreeto fivebasepairsfor thevitaminD3receptor separated (VDRE), and retinoicacidreceptor thyroidhormonereceptor(TRE), (RARE) K by redarrows[See areindicated Therepeatsequences , n d A M N a a re t a l , 1 9 9 1 ,C e l l6 5 : 1 7 6 7I U m e s o n oe t a l , 19 9 1 , C e l l6 5 : 1 2 5 5 a

Regulation of Transcription-Factor Activity r The activitiesof many transcription factors are indirectly regulated by binding of extracellular proteins and peptides to cell-surfacereceptors.These receptors activate intracellular signal-transduction pathways that regulate specific transcription factors through a variety of mechanismsdiscussedin Chapter 16. r Nuclear receptorsconstitute a superfamily of dimeric Ca ztnc-fnger transcription factors that bind lipid-soluble hormones and interact with specificresponseelementsin DNA (seeFigure 7-47). r Hormone binding to nuclear receptors induces conformational changesthat modify their interactions with other protelns. O F T R A N S C R I P T I O N - F A C TAOCRT I V I T Y REGULATION

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Video: Hormone-Regulated Nuclear Translocationof the GlucocorticoidReceptor (b)

Proteins t-I-a expressed:

/ B-Galactosidase

NC \-r

Glucocorticoid receptor

EXPERIMENTAL FTGURE 7-49 Fusionproteinsfrom expression vectorcdemonstratethat the hormone-binding domainof the glucocorticoidreceptor(GR)mediatestranslocationto the nucleusin the presenceof hormone.Cultured animalcellswere transfected with expression vectors encoding the proteins diagrammed at thebottom.lmmunofluorescence with a labeled antibody specific for p-galactosidase wasusedto detectthe exoressed oroteinsin transfected cells(a)In cellsthatexpressed alone,the B-galactosidase enzyme waslocalized to the cytoplasm in the presence andabsence

r Heterodimeric nuclear receptors (e.g., those for retinoids, vitamin D, and thyroid hormone) are found only in the nucleus. In the absence of hormone, they repress transcription of target genes with the corresponding responseelement.When bound to their ligands, they activate transcription. r Steroid hormone receptors are homodimeric nuclear receptors.In the absenceof hormone, they are trapped in the cytoplasm by inhibitor proteins. When bound to their ligands, they can translocateto the nucleusand activate transcription of target genes(seeFigure 7-50).

GR ligand-binding domain

of the glucocorticoid (Dex).(b)ln cellsthat hormonedexamethasone expressed a fusionproteinconsisting of B-galactosidase andthe (GR),the fusionproteinwaspresentin entireglucocorticoid receptor the cytoplasm in the absence of hormonebut wastransported to the nucleus in the presence of hormone.(c)Cellsthat expressed a fusionproteincomposed of B-galactosidase andjusttheGRligandbindingdomain(lightpurple) alsoexhibited hormone-dependent transport of the fusionproteinto the nucleus. D picard andK R [From Yamamoto, 1987, EMBO J 5:3333; courtesy of theauthors I

RegulatedElongationand lf, Termi nation of Transcri ption In eukaryotes,the mechanismsfor terminating transcription differ for each of the three RNA polymerases.Transcription of pre-rRNA genesby RNA polymeraseI is terminated by a mechanism that requires a polymerase-specifictermination factor. This DNA-binding protein binds to a specific DNA sequencedownstream of the transcription unit. Efficient termination requires that the termination factor bind to the template DNA in the correct orientation. Purified RNA

Hormone

Resoonse element

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< FIGURE 7-50 Model of hormone-dependentgene activation by a homodimericnuclearreceptor.In the absence of hormone, the receptoris keptin the cytoplasm by interaction betweenits ligand-binding domain(LBD) andinhibitor proteins. Whenhormone ispresent, it diffuses throughthe plasma membrane andbindsto the ligand-binding domain,causing a conformational change that releases the receptor fromthe inhibitorproteins. Thereceotorwith boundligandisthentranslocated intothe nucleus, whereitsDNAbindingdomain(DBD) bindsto response elements, allowing the ligand-binding domainandan additional activation domain(AD)at the N-terminus to stimulate transcription of tarqetqenes.

TRANSCR|PT|ONA CLO N T R O LO F G E N EE X P R E S S I O N

polymerase III terminates after polymerizing a series of U residues.The deoxy(A),-ribo(U),DNA-RNA hybrid that results when a stretch of U's are synthesizedis particularly unstable compared with all other base-pairedsequences.The ease with which this hybrid can be melted probably contributes to the mechanism of termination by RNA polymeraseIII. In most mammalian protein-coding genestranscribed by RNA polymeraseII, once the polymerasehas transcribed beyond about 50 bases,further elongation is highly processive and does not terminate until after a sequenceis transcribed that directs cleavageand polyadenylation of the RNA at the sequencethat forms the 3' end of the encodedmRNA. RNA polymerase II then can terminate at multiple sites located over a distanceof 0.5-2 kb beyond this poly(A) addition site. Experiments with mutant genesshow that termination is coupled to the processthat cleavesand polyadenylatesthe 3' end of a transcript, which is discussedin the next chapter. Biochemical and chromatin immunoprecipitation experiments suggestthat the protein complex that cleavesand polyadenylatesthe nascentmRNA transcript at specific sequences associateswith the phosphorylated carboxylterminal domain (CTD) of RNA polymeraseII following initiation (see Figure 7-31). This cleavage/polyadenylation complex may suppresstermination by RNA polymerase II until the sequencesignaling cleavageand polyadenylation is transcribed by the polymerase. Although transcription termination is unregulated for most genes, for some specific genes, a choice is made between elongation and termination or pausing within a few tens of basesfrom the transcription start site. This choice between elongationand termination or pausing can be regulated; thus expression of the encoded protein is controlled not only by transcription initiation but also by control of transcription elongation early in the transcription unit. We discusstwo examplesof such regulation next.

RNA-binding protein. It binds Tat is a sequence-specific to the RNA copy of a sequencecalled TAR, which is located near the 5' end of the HIV transcript' The TAR sequence folds into an RNA hairpin with a bulge in the middle of the stem (Figure 7-51). TAR contains two binding sites:one that interacts with Tat and one that interacts with a cellular protein called cyclin T. As depicted in Figure 7-51', the HIV Tat protein and cellular cyclin T each bind to TAR RNA and also interact directly with each other so that they bind cooperatively,much like the cooperativebinding of DNA-binding transcription factors (seeFigure 7-29).lnteruction of cyclin T with a protein kinase called CDKS activates the kinase, whose substrateis the CTD of RNA polymeraseII. In vitro transcription studies using a specificinhibitor of CDK9 suggest that RNA polymerase II molecules that initiate transcription on the HIV promoter terminate after transcribing -50 bases unless the CTD is hyperphosphorylatedby CDK9. Cooperative binding of cyclin T and Tat to the TAR sequenceat the 5' end of the HIV transcript positions CDK9 so that it can phosphorylate the CTD, thereby preventing termination and permitting the polymerase to continue chain elongation. Severaladditional cellular proteins, including Spt4 and Spt5 and the NELF complex' participate in the processby which HIV Tat controls elongation versustermination (Figure 7-51). Experiments with the specific inhibitor of CDK9 mentioned above and with spt4 and sptS yeastmutants indicatethat thesecellular proteins are required for transcription elongation beyond -50 basesfor most cellular genes.But for most genes,these proteins appear to function constitutively, that is, without being regulated.As discussedin Chapter 8, RNA polymeraseII pausing instigated by Spt4/5 and NELF is thought to delay elongation until mRNA processing factors associatewith the phosphorylated CTD. Further phosphorylation of the CTD by cyclin T-CDK9 (alsoknown

Transcriptionof the HIV Genomels Regulated b y a n A n t i t e r m i n a t i o nM e c h a n i s m Currentln transcription of the human immunodeficiency virus (HIV) genome by RNA polymerase II provides the best-understoodexample of regulatedtranscription termination in eukaryotes. Efficient expression of HIV genes requires a small viral protein encoded atthe tat locus. Cells infected with tat- mutants produce short viral transcripts that hybridize to restriction fragments containing promoterproximal regions of the HIV DNA but not to restriction fragments farther downstream from the promoter. In contrast, cells infected with wild-type HIV synthesizelong viral transcripts that hybridize to restriction fragments throughout the singleHIV transcription unit. Thus Tat protein functions as an antitermination factor, permitting RNA polymerase II to read through a transcriptional block. Since antitermination by Tat protein is required for HIV replication, further understanding of this gene-control mechanism may offer possibilities for designing effective therapies for acquired immunodeficiencysyndrome (AIDS).

CTD 7-51 Modelof antiterminationcomplexcomposed A FIGURE of HIVTat protein and severalcellularproteins.TheTARelement byTatandthe recognized sequences contains in the HIVtranscript position the helps and T activates T. Cyclin protein cyclin cellular the CTDof RNApolymerase CDKgnearitssubstrate, oroteinkinase andallowstranscription prevents termination ll CTDphosphorylation complex proteins Spt4andSpt5andthe NELF Cellular to continue. P weiet termination transcript HIV [See regulating in arealsoinvolved al , 1998, Cell92:451,T. Wada et al , 1998, GenesDev 12:357; and Y Yamaguchiet al , 1999, Cell97:41 l

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as pTEFb) appears to reversethis pause and allow elongation to continue. Currently, it is not clear why this processis not constitutive for the HIV promoter, where cooperative binding of HIV Tat and cyclin T to the TAR RNA sequence is required for CDK9 activation and efficient elongation.

Promoter-Proximal Pausingof RNA polymerasell O c c u r si n S o m eR a p i d l yI n d u c e dG e n e s The heat-shockgenes(e.g.,hsp70) illustrate another mechanism for regulating RNA chain elongation in eukaryotes. During transcription of these genes,RNA polymerase II pausesafter transcribing :25 nucleotidesbut does not terminate transcription (as it does when transcribing the HIV genome in the absenceof Tat protein). The paused polymeraseremains associatedwith the nascent RNA and template DNA and then, after a few minutes, continues transcription of the gene. As the first polymerase transcribes away from the promoter region, another RNA polymeraseII binds to the promoter, initiates transcription, and pausesafter transcribing:25 nucleotides,waiting severalminutes be'S7hen fore the process is repeated. heat shock occurs, the heat-shock transcription factor (HSTF) is activated. Subsequent binding of activated HSTF to specificsitesin the promoter-proximal region of heat-shock genes stimulates the paused polymerase to continue chain elongation and promotes rapid re-initiation by additional RNA polymerase II molecules, leading to many transcription initiations per mtnute. The pausing during transcription of heat-shockgenesinitially was discovered in Drosophila, but a similar mechanism has been shown to occur in human cells. Heat-shock genesare induced by intracellular conditions that denature proteins(suchas elevatedtemperature,"heat shock"). Some encode proteins that are relatively resistant to denaturing conditions and act to protect other proteins from denaturation; others are chaperonins that refold denatured proteins (Chapter 3). The mechanism of transcriptional control that evolved to regulate expressionof thesegenespermits a rapid response:these genes are always paused in a state of suspended transcription and therefore, when an emergency anses, require no time to remodel and acetylatechromatin over the promoter and assemblea transcription preinitiation comolex.

Regulated Elongation and Termination of Transcription r Different mechanisms of transcription termination are employed by each of the eukaryotic nuclear RNA polymerases.Transcription of most protein-coding genesis not terminated until an RNA sequenceis synthesizedthat specifies a site of RNA cleavageand polyadenylation. r Transcription of the HIV genome by RNA polymeraseII is regulated by an antitermination mechanismthat requires cooperative binding by the virus-encodedTat protein and '114R cyclin T to the sequencenear the 5, end of the HIV RNA. 316

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r During transcription of Drosophila heat-shock genes, RNA polymerase II pauses within the downstream promoter-proximal region; this interruption in transcription is releasedwhen the HSTF transcription factor is activated, resultingin very rapid transcription of the heat-shockgenes in responseto the accumulation of denatured proteins.

Other Eukaryotic Transcription W Systems We concludethis chapterwith a brief discussionof transcription initiation by the other two eukaryotic nuclear RNA polymerases,Pol I and Pol III, and by the distinct polymerasesthat transcribe mitochondrial and chloroplast DNA. Although thesesystems,particularly their regulation,are lessthoroughly understoodthan transcription by RNA polymeraseII, they are equally as fundamentalto the life of eukaryotic cells.

T r a n s c r i p t i o Inn i t i a t i o nb y P o l I a n d P o l l l l l s A n a l o g o u st o T h a t b y P o l l l The formation of transcription-initiation complexesinvolving Pol I and Pol III is similar in somerespecrsto assemblyof Pol II initiation complexes (seeFigure 7-31). However, each of the three eukaryotic nuclear RNA polymerasesrequires its own polymerase-specificgeneraltranscription factors and recognizesdifferent DNA control elements.Moreover, neither Pol I nor Pol III requiresATP hydrolysis to initiate transcription, whereasPol II does. Transcription initiation by Pol I, which synthesizesprerRNA, and by Pol III, which synthesizestRNAs, 55 rRNA, and other short, stable RNAs (seeTabIe 7-2), has beencharacterrzedmost extensively in S. cereuisiaeusing both biochemical and geneticapproaches.It is clear that synthesisof tRNAs and of rRNAs, which are incorporated into ribosomes,is tightly coupled to the rate of cell growth and proliferation. However, much remains to be learned about how transcription initiation by Pol I and Pol III is regulated so that synthesisof pre-rRNA, 55 rRNA, and tRNAs is coordinated with the growth and replication of cells. Initiation by Pol I The regulatory elementsdirecting pol I initiation are similarly located relative to the transcription start site in both yeast and mammals. A core element spanning the transcription start site from -40 to *5 is essential for Pol I transcription. An additional upstream element extending from roughly -155 to -60 stimulatesin vitro pol I transcription tenfold. Assemblyof a fully active Pol I initiation complex begins with binding of a multimeric upstream activating factor (UAF) to the upstreamelement(Figure7-52).Two of the six subunits composing UAF are histones,which probably participate in DNA binding. Next, a trimeric core factor binds to the core element together with TBP, which makes contact with both the bound UAF and the core factor. Finally a preformed complex of Pol I and Rrn3p associateswith the bound proteins, positioning Pol I near the start site. In human cells.TBP is stablv

T R A N S C R | p T t O N ACLO N T R O LO F G E N EE X P R E S S | O N

Pre-rRNApromoter DNA

I

Upstreamactivating factor (UAF)

tRNA gene

Pollll

r

Corefactor (CF),TPB, and other factors

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P o ll l l

Initiation complex

7-52 ln vitro assemblyof the yeastPolI A FfGURE transcriptioninitiationcomplex.UAFandCF,bothmultimeric (UE)and general factors, bindto the upstream element transcription DNA TBPanda respectively, in the promoter coreelement, | (Poll) factor(Rrn3p) associated with RNApolymerase monomeric fromN in formingthe initiation complex[Adapted alsoparticrpate by Transcription of Ribosomal RNAGenes Nomura, 1998,in R M Paule, pp 157-1721 RNA Polymerase Bioscience, Eukaryotic 1 Landes bound to three other polypeptides,forming an initiation factor called SLI that binds to the core promoter elementand is functionally equivalentto yeastcore factor plus TBP. lnitiation by Pol lll Unlike protein-codinggenesand prerRNA genes,the promoter regions of IRNA and SS-rRNA geneslie entirelywithin the transcribedsequence(Figure7-53). Two such internal promoter elements,termed the A box and B box, are presentin all IRNA genes.Thesehighly conserved sequencesnot only function as promoters but also encode two invariant portions of eukaryotic tRNAs that are required for protein synthesis.In SS-rRNA genes,a singleinternal control region, the C box, acts as a promoter. Three general transcription factors are required for Pol III to initiate transcription of IRNA and SS-rRNA genesin vitro. Two multimeric factors, TFIIIC and TFIIIB, participate in initiation at both IRNA and 5S-rRNA promoters;a third factor, TFIIIA, is required for initiation at SS-rRNA promoters. As with assemblyof Pol I and Pol II initiation complexes, the Pol III general transcription factors bind to promoter DNA in a definedsequence. The N-terminal half of one TFIIIB subunit, called BRF (for TFIIB-related /actor), is similar in sequenceto TFIIB

elementsin genes 7-53 TranscriPtion-control A FIGURE genes ll1.BothIRNAandSS-rRNA transcribedby RNApolymerase (yellow) downstream promoter located elements containinternal asindicated fromthestartsiteandnamedA, B,andC boxes, on thesegenesbegins complexes initiation of transcription Assembly general factorsTFlllA, transcription with the bindingof Pol-lll-specific to thesecontrolelementsGreenarrowsindicate andTFlllC TFlllB, Bluearrows protein-DNA interactions strong,sequence-specifrc factorsPurple transcription betweengeneral interactions indicate factors general transcription between interactions arrowsindicate Dev16:2593 Genes I andN Hernandez,2002, L Schramm andPollll.lFrom (a Pol II factor). This similarity suggeststhat BRF and TFIIB perform a similar function in initiation, namely' to direct the polymeraseto the correct start site. Once TFIIIB has bound to either a tRNA or 5S-rRNA gene,Pol III can bind and initiate transcription in the presenceof ribonucleosidetriphosphates.The BRF subunit of TFIIIB interacts specifically with one of the polymerase subunits unique to Pol III, accounting for initiation by this specific nuclear RNA polymerase. Another of the three subunits composing TFIIIB is TBP' which we can now see is a component of a general transcription factor for all three eukaryotic nuclear RNA polymerases.The finding that TBP participates in transcription initiation by Pol I and Pol III was surprising, since the promoters recognized by these enzymes often do not contain TATA boxes. Nonetheless,recent studies indicate that the TBP subunit of TFIIIB interacts with DNA similarly to the way it interacts with TATA boxes.

t N A sA r e M i t o c h o n d r i aal n d C h l o r o p l a sD T r a n s c r i b e bd y O r g a n e l l e - S p e c i fRi cN A Polymerases As discussedin Chapter 6, mitochondria and chloroplasts probably evolved from bacteria that were endocytosedinto ancestralcells containing a eukaryotic nucleus. In modernday eukaryotes, both organellescontain distinct DNAs that encode some of the proteins essentialto their specific functions. Interestingly, the RNA polymerases that transcribe T R A N S C R I P T I OSNY S T E M S O T H E RE U K A R Y O T I C

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mitochondrial (mt) DNA and chloroplast DNA are similar to polymerasesfrom bacteria and bacteriophages,reflecting their evolutionaryorigins. Mitochondrial Transcription The RNA polymerasethat transcribesmtDNA is encodedin nuclear DNA. After synthesisof the enzymein the cytosol, it is imported into the mitochondrial matrix by mechanismsdescribedin Chapter 13. The mitochondrial RNA polymerases from S. cereuisiae and the frog Xenopus laeuis both consist of a large subunit with ribonucleotide-polymerizingactivity and a small B subunit (TFBM). Another matrix protein, mitochondrial transcription factor A (TFAM), binds to mtDNA promoters and is essentialfor initiating transcription at the start sitesused in the cell. The large subunit of yeastmitochondrial RNA polymerase clearly is related to the monomeric RNA polymerasesof bacteriophageT7 and similar bacteriophages. However, the mitochondrial enzyme is functionally distinct from the bacteriophage enzyme in its dependenceon two other polypeptides for transcription from rhe proper start sites. The promoter sequencesrecognizedby mitochondrial RNA polymerases include the transcription start sire. These promoter sequences,which are rich in A residues. have been characterizedin the mtDNA from yeast, plants, and animals. The circular, human mitochondrial genome contains two related 15-bp promoter sequences,one for the transcription of each strand. Each strand is transcribed in its entirery; the long primary transcripts are then processedto yield mitochondrial mRNAs, rRNAs, and tRNAs. A secondpromoter appearsto be responsiblefor transcribing additional copies of the rRNAs. Currently, there is relatively little understandingof how transcription of the mitochondrial genomeis regulatedto coordinatethe production of the few mitochondrial proteins it encodes with synthesis and import of the thousands of nuclear DNA-encoded proteins that comprisethe mitochondria. Chloroplast Transcription ChloroplastDNA is transcribed by two types of RNA polymerases,one multisubunit protein similar to bacterial RNA polymerasesand one similar to the single subunit enzymesof bacteriophageand mitochondria. The core subunits of the bacterial-typeenzyme,o., B, B,, and o subunits, are encoded in the chloroplast DNAs of higher plants, whereassix o7o-like o factors are encodedin the nuclear DNA of higher plants. This is another example of the transfer of genes from organellar genomes to nuclear genomesduring evolution. In this case,genesencoding the regulatory transcription initiation factors have been transferred to the nucleus,where the control of their transcription by nuclear RNA polymeraseII likely indirectly controls the expression of sets of chloroplast genes. The bacterial-like chloroplast RNA polymeraseis called the plastid polymerase sinceits catalytic core is encodedby the chloroplast genome. Most chloroplast genesare transcribed by theseenzymesand have -35 and -10 control regionssimilar to promoters in cyanobacteria, from which they evolved. The chloroplast T7-like RNA polymerase is also encoded in the nuclear 318

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genome of higher plants. It transcribes a different set of chloroplast genes. Curiously, this includes genes encoding subunits of the bacterial-like multisubunit plastid polymerase. As for mitochondrial transcription, currently relatively little is known about how transcription of chloroplast DNA is regulated.

Other Eukaryotic TranscriptionSystems r The processof transcription initiation by Pol I and Pol III is similar to that by Pol II but requires different general transcription factors, is directed by different promoter elements, and does not require AIP hydrolysis. r Mitochondrial DNA is transcribed by a nuclearencoded RNA polymerase composed of two subunits. One subunit is homologous to the monomeric RNA polymerase from bacteriophage T7; the other resembles bacterial o factors. r Chloroplast DNA is transcribed by a chloroplastencoded RNA polymerase homologous to bacterial RNA polymerases,except that it lacks a o factor.

A great deal has been learned in recent years about transcription control in eukaryotes. Genes encoding about 2000 activatorsand repressorscan be recognizedin the human genome. We now have a glimpse of how the astronomical number of possible combinations of these transcription factors can generate the complexity of gene control required to produce organisms as remarkable as those we seearound us. But very much remains to be un, derstood. Although we now have some understandingof what processesturn a gene on and off, we have very little understanding of how the frequency of transcription is controlled in order to provide a cell with the appropriate amounts of its various proteins. In a red blood cell precursor, for example, the globin genes are transcribed at a far greater rate than the genesencoding the enzymes of intermediary metabolism (the so-called housekeepinggenes). How are the vast differencesin the frequency of transcrip'S(hat tion initiation at various genesachieved? happens to the multiple interactions between activation domains, coactivator complexes, general transcription factors, and RNA polymerase II when the polymerase iniriares transcription and transcribes away from the promoter region? Do thesecompletelydissociateat promoters that are transcribed infrequently, so that the combination of multiple factors required for transcription must be reassembled anew for each round of transcription? Do complexes of activators with their multiple interacting co-activators remain assembledat promoters from which re-initiation takes place at a high rate, so that the entire assemblydoes not have to be reconstructed each time a polymerase initiates?

T R A N S C R | p T t o N ACLO N T R O LO F G E N EE X P R E S S | O N

Much remains to be learned about the structure of chromatin and how that structure influencestranscription. What additional components besidesHP1 and methylated histone H3 lysine 9 are required to direct certain regions of chromatin to form heterochromatin, where transcription is repressed?Preciselyhow is the structure of chromatin changed by activators and repressors,and how does this promote or inhibit transcription? Once chromatin-remodeling complexes and histone acetylasecomplexes become associated with a promoter region, how do they remain associated? Current models suggestthat certain subunits of these complexes associatewith modified histone tails so that the combination of binding to a specific histone tail modification plus modification of neighboring histone tails in the same way resultsin retention of the modifying complex at an activated promoter region. In some cases,this type of assembly mechanismcausesthe complexesto spread along the length 'What of a chromatin fiber. controls when such complexes spread and how far they will spread? Singleactivation domains have been discoveredto interact with several co-activator complexes. Are these interactions transient, so that the same activation domain can interact with several co-activators sequentially?Is a specific order of co-activator interaction required?How does the interaction of activation domains with mediator stimulate transcription? Do theseinteractions simply stimulate the assembly of a preinitiation complex, or do they also influence the rate at which RNA polymeraseII initiates transcription from an assembledpreinitiation complex? Transcriptional activation is a highly cooperative processso that genesexpressedin a specifictype of cell are expressedonly when the complete set of activators that control that gene are expressedand activated. As mentioned earlier,someof the transcriptionfactors that control expressionof the TTR gene in the liver are also expressed in intestinal and kidney cells. Yet the TTR gene is not exp r e s s e di n t h e s e o t h e r t i s s u e s ,s i n c e i t s t r a n s c r i p t i o n r e quires two additional transcription factors expressedonly in the liver. \7hat mechanisms account for this highly cooperative action of transcription factors that is critical to geneexpression? cell-type-specific A thorough understanding of normal development and of abnormal processesassociatedwith diseasewill require answersto theseand many related questions.As further understandingof the principles of transcription control are discovered, applications of the knowledge will likely be made. This understandingmay allow fine control of the expression of therapeutic genesintroduced by gene therapy vectors as they are developed.Detailedunderstandingof the molecular interactionsthat regulatetranscription may provide new targets for the developmentof therapeutic drugs that inhibit or stimulate the expressionof specificgenes.A more complete understanding of the mechanismsof transcriptional control may allow improved engineeringof crops with desirable characteristics. Certainly, further advances in the area of transcription control will help to satisfy our desireto understand how complex organismssuch as ourselvesdevelop and function.

KeyTerms activationdomain 288

nuclear rcceptors291

activators 271

promoter 276

antitermination factor 31 5

promoter-proximal elements283

carboxyl-terminal d o m a i n( C T D ) 2 8 0 chromatin-mediated repression299 co-activator 293 co-repressor294 DNase I footprintrng 286 enhancers274 295 enhancesome generaltranscription factors 296 heat-shockgenes315 histone deacetylation300 leucine zipper 291 MAT locus (inyeastl 299

repressiondomain 290 repressors271 RNA polymerasell279 silencersequences299 specifictranscription factors 286 TATAbox 282 TAIA box-binding protein (TBP) 297 upstream actrvatlng (UASs)285 sequences yeast two-hybrid system 3 10 zinc finger 291

mediator 299

Review the Concepts 1. Describe the molecular events that occur at the lac operon when E. coli cellsare shifted from a glucose-containing medium to a lactose-containingmedium. 2. The concentration of free phosphate affects transcription of some E. coli genes.Describethe mechanismfor this. '$7hat types of genes are transcribed by RNA poly3. merasesI, II, and III? Design an experiment to determine whether a specificgeneis transcribedby RNA polymeraseII. II 4. The CTD of the largest subunit of RNA polymerase 'What are can be phosphorylated at multiple serineresidues. the conditions that lead to the phosphorylated versus unphosphorylated RNA polymeraseII CTD? 5. \What do TAIA boxes, initiators, and CpG islands have in common? Which was the first of these to be identified?

whv? 6. Describe the methods used to identify the location of DNA-control elements in promoter proximal regions of genes. 7. lfhat is the difference between a promoter-proximal element and a distal enhancer? 8. Describe the methods used to identify the location of DNA-binding proteins in the regulatory regions of genes. 9. Describe the structural features of transcriptional activator and repressorproteins. 10. What happensto transcription of the EGR-I genein patients with Wilm's tumor? Sfhy? 11. Using CREB and nuclear receptors as examples' compare and contrast the structural changesthat take place when thesetranscription factors bind to their co-activators. R E V I E WT H E C O N C E P T S

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12. \lhat general transcription factors associate with an RNA polymeraseII promoter in addition to the polymerase? '$fhat In what order do they bind in vitro? structural change occurs in the DNA when an "open" transcription-initiation complex is formed? L3. Expressionof recombinantproteins in yeast is an important tool for biotechnology companiesthat produce new drugs for human use. In an attempt to get a new geneX expressedin yeast, a researcherhas integrated gene X into the yeast genome near a telomere. Vill this strategy result in good expression of gene X? Why or why not? \fould the outcome of this experiment differ if the experiment had been performed in a yeast line conraining murations in the H3 or H4 histonetails? 14. You have isolated a new protein called STICKY. You can predict from comparisons with other known proteins that STICKY contains a bHLH domain and a Sin3-interacting domain. Predict the function of STICKY and rationale for the importance of thesedomains in STICKY function. 15. The yeast two-hybrid method is a powerful molecular genetic method to identify a protein(s) that interacts with a known protein or protein domain. You have isolatedthe glucocorticoid receptor (GR) and have evidencethat it is a modular protein containing an activation domain, a DNAbinding domain, and a second ligand-binding activation domain. Further analysisrevealsthat in pituitary cells,the protein is anchored in the cytoplasm in the absenceof its hormone ligand, a result leading you to speculatethat it binds to other inhibitory proteins. Describehow a two-hybrid analysis could be used to identify the protein(s) GR interactswith. How would you specifically identify the domain in the GR that binds the inhibitor(s)? 1,6. Some heat-shockgenesencodeproteins that act rapidly to protect other proteins from harsh conditions. Describethe mechanism that has evolved to regulate the expression of such genes.

Analyzethe Data In eukaryotes, the rhree RNA polymerases,pol I, II and III, each transcribe unique genesrequired for the synthesisof rib o s o m e s : 2 5 Sa n d 1 8 S r R N A s ( P o l I ) , 5 5 r R N A ( p o l I I I ) , and mRNAs for ribosomal proteins (Pol II). Researchers have long speculated that the activities of the three RNA polymerasesare coordinately regulated according to the demand for ribosome synthesis:high in replicating cells in rich nutrient conditions and low when nutrients are scarce.To determine if the activities of the three polymerasesare coordinated, Laferte and colleaguesengineereda strain of yeast to be partially resistant to the inhibition of cell growth by the drug rapamycin (2006, GenesDeu.2022030-2040).As discussedin Chapter 8, rapamycininhibits a protein kinase (called TOR for target of rapamycin) that regulates the overall rate of protein synthesisand ribosome synthesis. When TOR is inhibited by rapamycin, the transcription of rRNAs by Pol I and Pol III and ribosomal protein mRNAs by RNA polymeraseII are all rapidly repressed.part of the C H A P T E R7

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inhibition of Pol I rRNA synthesisresults from the dissociation of the Pol I transcription factor Rrn3 from Pol I (see Figure 7-52). ln the strain constructed by Laferte and colleagues,the wild-type Rrn3 gene and the wild-type A43 gene, encoding the Pol I subunit to which Rrn3 binds, were replaced with a gene encoding a fusion protein of the A43 Pol I subunit with Rrn3. The idea was that the covalent fusion of the two proteins would prevent the Rrn3 dissociation from Pol I otherwise caused by rapamycin rreatment. The resulting CARA (constitutive association of Rrn3 and A43) strain was found to be partially resistant to rapamycin. In the absenceof rapamycin, the CARA strain grew at the same rate and had equal numbers of ribosomes as do wild-type cells. a. To analyzerRNA transcription by Pol I, total RNA was isolated from rapidly growing wild-type (I7T) and CARA cells at various times following the addition of rapamycin. The concentration of the 35S rRNA precursor transcribedby Pol I (seeFigure 8-35) was assayedby the primer-extensionmethod. Sincethe 5' end of the 355 rRNA precursoris degradedduring the processingof 25S and 18S rRNA, this method measuresthe relatively short-lived prerRNA precursor. This is an indirect measure of the rate of rRNA transcription by Pol I. The results of this primer extension assayare shown below. How does the CARA Pol I-Rrn3 fusion affect the responseof Pol I transcriotion to rapamycin? Minutes after rapamycln

0 20 40 60 80 100

0 20 40 60 80 100

35S rRNA

b. The concentrations of four mRNAs encoding ribosomal proteins,RPL30, RPS6a,RPL7a, and RPLS, and the mRNA for actin (ACTL), a protein presentin the cytoskeleton, were assessed in wild-type and CARA cells by Northern blotting at various times after addition of rapamycin to rapidly growing cells (upper autoradiograms). 55 rRNA transcription was assayedby pulse labeling rapidly growing S7T and CARA cellswith 3H uracil (for 20 minutes)at varioustimes Minutesafter rapamycrn

CARA

0 20 40 60 80 100

0 20 40 60 80 100

WT 0 20 40 60 80 100

0 20 40 60 80100

RPL3O RPS6a RPLTa RPLS ACTl

Minutes after rapamycrn

T R A N S C R T P T T O NCAOLN T R O LO F G E N EE X P R E S S | O N

CJ

CARA

after addition of rapamycin to the media. Total cellular RNA was isolatedand subjectedto gel electrophoresisand autoradiography. The lower autoradiogram shows the region of the gel containing 55 rRNA. Basedon these data, what can be concluded about the influence of Pol I transcription on the transcription of ribosomal protein genesby Pol II and 5S rRNA by Pol III? c. To determine if the difference in behavior of wildtype and CARA cellscan be observedunder normal physiological conditions (i.e., without drug treatment), cells were subjectedto a shift in their food source,from nutrient-rich media to nutrient-poor media. Under these conditions, in wild-type cells, the TOR protein kinase becomes inactive. ConsequentlS shifting cells from nutrient-rich media to nutrient-poor media should result in a normal physiological responsethat is equivalent to treating cells with rapamycin, which inhibits TOR. To determine how the CARA fusion protein affected the responseto this media shift, RNA was extracted from wild-type and CARA cells and used to probe microarrays containing all yeast open reading frames. The extent of RNA hybridization with the arrays was quantified and is expressedin the graphs as log2 of the ratio of CARA-cell RNA concentration to wild-type-cell RNA concentration for each open reading frame. A value of zero indicates that the two strains of yeast exhibit the same level of expression for those specific RNAs. A value of 1 indicates that the CARA cells contain twice as much of that particular RNA as do wild-type cells. The graphs below show the number of open reading frames (y axis) that have values for log2 of this ratio, indicated by the x axis. The results of hybridization to open reading frames encoding mRNAs for ribosomal proteins are shown by black bars, those for all other mRNAs by white bars.The graph on the left givesresults for cells grown in nutrient-rich medium, the graph on the right for cells shifted to nutrient-poor medium for 90 minutes. \fhat do thesedata suggestabout the regulation of ribosomal protein genetranscription by Pol II?

Cells grown in rich media

Cells grown in poor media

1

q

o o)

6

E z

-2-1 0 1 2 3 Expression ratio (logzCARA/WT)

-3-2-1 0 1 2 3 ratio Expression (logzCARA/WT)

References Control of Gene Expression in Bacteria Borukhov,S., and J. Lee. 2005. RNA polymerasestructureand function at lac operon. C. R. Biol.328:576-587. Halford, S. 8., and J. F. Marko. 2004. How do site-specific DNA-binding proteins find their targets?Nucleic Acids Res. 32:3040-3052. Lawson, C. L., et a\.2004. Cataboliteactivator protein: DNA binding and transcription activation. Cwrr. Opin. Struct. Biol. 14:1.0-20. Lewis, M. 2005. The lac repressor.C. R. Biol.328:521'-548. Muller-Hill, B. 1,998.Somerepressorsof bacterialtranscription. Curr. C)pin.Microbiol. 1:145-151. Murakami, K. S., and S. A. Darst. 2003. BacterialRNA polymerases:the whole story.Curr. Opin. Struct. Biol. 13:31'-39. Wigneshweraraj,S. R., et al. 2005. The secondparadigm for activation of transcription.Prog. Nucleic Acid Res.Mol. Biol. 79:339-369. Overview of Eukaryotic Gene Control and RNA Polymerases Boeger,H., et al.. 2005. Structural basisof eukaryoticgenetranscription. FEBSLett. 579:899-903. Regulatory Sequences in Protein-Coding Genes Brenner,S., et al. 2002. Conservedregulation of the lymphocyte-specificexpressionof lck in the Fugu and mammals.Proc. Natl. Acad. Sci.USA 99:2936-2941. Dean, A. 2005. On a chromosomeIar, far away:LCRs and gene expression.TrendsGenet.22:3845. Gaszner,M., and G. Felsenfeld.2005. Insulators:exploiting transcriptionaland epigeneticmechanisms.Nat. Reu.Genet.T:703-713. Kleinian, D. A., and V. van Heyningen.2005. Long-range control of geneexpression:emergingmechanismsand disruption in Am. J. Hum. Genet.76:8-32. disease. Nobrega, M. A., L Ovcharenko,Y. AfzaI,and E' M' Rubin. 2003. Scanninghuman genedesertsfor long-rangeenhancers.Sclence 302:473. Smale,S. T., and J. T. Kadonaga'2003. The RNA polymeraseII core promoter.Annu. Reu.Biochem. T2:449479. Activators and Repressors of Transcription Brivanlou, A. H., and J. E. Darnell, Jr.2002. Signaltransduction and the control of geneexpression.Science295:813-818' Broun, P. 2004. Transcription factors as tools for metabolic engineeringin plants. Curr. Opin. Plant Biol.7:202:209Garvie, C. W., and C. Wolberger.2001. Recognitionof specific Mol. Cell. 8:937-946. DNA sequences. Kadonaga,I.T. 2004. Regulation of RNA polyrneraseII transcripDNA binding factors. Cell ll6:247J57. tion by sequence-specific Luscombe.N. M.. et al. 2000. An overview of the structuresof protein-DNA complexes.Genome Biol. t:I-37. Marmorstein, R., and M. X. Fitzgerald.2003. Modulation of DNA recognition. DNA-binding domains for sequence-specific Gene 304:I-'12. Riechmann,J. L., et al. 2000. Arabidopsistranscription factors: genome-widecomparativeanalysisamong eukaryotes.Science 290:2105J1'1.0. Tupler,R., G. Perini, and M. R' Green.2001. Expressingthe human genome. Nature 409:832-833. Transcription lnitiation by RNA Polymerase ll Hahn, S. 2004. Structureand mechanismof the RNA polymerase II transcription machinery. Nat. Struct. MoL Biol' 11:394403' REFERENCES

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Molecular Mechanisms of Transcription Repression and Activation Courey,A. J., and S.Jia. 2001. Transcriptionalrepression:rhe long and the short of it. GenesDeu. 15:2786-2796. Green,M. R. 2005. Eukaryotic transcription activation:right on target. Mol. Cell 18:399402. Horn, P.J., and C. L. Peterson.2005. Heterochromatrnassembly: a new twist on an old model. ChromosomeRes.14:83-94. Klose, R. J., and A. P. Bird. 2006. GenomicDNA methylation: the mark and its mediators.TrendsBiochem. Sci.3l:89-97. Kornberg, R. D. 2005. Mediator and the mechanismof transcriptionalactivation. TrendsBiochem. Sci.30:235-239. Levine,S. S., I. F. King, and R. E. Kingston.2004. Division of labor in polycomb group repression.Trends Biochem. Sci. 29:478-485. Lonard, D. M., and B. W. O'Malley.2006. The expandingcosmos of nuclearreceptorcoactivators.Cell 125:41,1414. Lund, A. H., and M. van Lohuizen.2004. Polycombcomplexes and silencingmechanisms.Curr. Opin. Cell Biol. 16:239-246. Malik, S., and R. G. Roeder.2005. Dynamic regulationof pol II transcription by the mammalian Mediator complex. Trends Biochem.Sci.3O:256-263. Margueron, R., P. Trojer, and D. Reinberg.2005. The key to development:interpretingthe histone code?Curr. Opin. Genet.Deu. 15:1,63-176. Martin, C., and Y. Zhang.2005. The diversefunctions of histone lysinemethylation. Nat. Reu.Mol. Cell Biol.6:838-849. Mohrmann, L., and C. P. Verriizer.2005. Composition and functional specificityof S'Wi2/SNF2classchromatin remodeling complexes.B io chim. B iophys. Acta 168l :5 9-7 3. Roeder,R. G. 2005. Transcriptionalregulationand the role of diversecoactivatorsin animal cells. FEBSLett.579:909-91.5. Smith, C. L., and C. L. Pererson.2005. ATP-dependentchromatin remodeling.Curr. Top. Deu. Biol. 65:115-i,48. . Spiegelman,B. M., and R. Heinrich.2004. Biologicalcontrol through regulatedtranscriptionalcoactivators.Cetl llgir 57-167. Struhl, K. 2005. Transcriptionalactivation:mediator can act after preinitiation complex formation. Mol. Cell 17:752-754. Taatjes,D.J., M. T. Marr, and R. Tjian. 2004. Regulatorydiversrty among metazoanco-activatorcomplexes.Nat. Reu.Mol. Cell Biol.5:403410. Thiel, G., M. Lietz, and M. Hohl. 2004. How mammalian transcriptionalrepressorswork. Eur. J. Biocbem. 27 l:28 5 5-2862. Trojer, P.,and D. Reinberg.2005. Histone lysinedemethylases and their impact on epigenetics.Cell 125:273-217. Villard, J.2004. Transcriptionregulationand human diseases. 9uiss Med. l(rkly. 134:571,-579. Wang, W. 2003. The SITVSNFfamily of ATP-dependentchromatin remodelers: similar mechanismsfor diverse functions. Curr. Top. Microbiol. Immunol. 274:143-169.

Regulation of Transcription-Factor Activity Boisvert,F. M., C. A. Chenard,and S. Richard. 2005. protein interfacesin signalingregulatedby argininemethylation.Sci. STKE2Tl:re2.

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Brahimi-Horn, C., N. Mazt;:e, and J. Pouyssegur. 2005. Signalling via the hypoxia-induciblefactor-lalpha requiresmultiple posttranslationalmodifications. Cell. Signal.17:1-9. Gardner,K. H., and M. Montminy. 2005. Can you hear me now? Regulatingtranscriptionalactivatorsby phosphorylation.Sci. STKE 301:pe44. Glozak,M. A., N. Sengupta,X. Zhang, and E. Seto.2005. Acerylation and deacetylationof non-histoneproteins. Gene 363:1.5-23. Khidekel,N., and L. C. Hsieh-I7ilson.2004. A "molecular switchboard"-covalent modificationsto proteins and their impact on transcription. Org. Biomol. Chem. 7:1-7. Moehren, U., M. Eckey,and A. Baniahmad.2004. Generepression by nuclearhormone receptors.EssaysBiochem. 40:89-104. Picard,D. 2005. Chaperoningsteroidhormone action.Trends Endocrinol. Metab. 17:229-235. Rosenfeld,M. G., V. V. Lunyak, and C. K. Glass.2005. Sensors and signals:a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptionalresponse.GenesDeu. 2006 2U1405-1,428. Regulated Elongation and Termination of Transcription Peterlin,B. M., and D. H. Price.2005. Controlling the elongation phaseof transcriptionwith P-TEFb.Mol. Cell 23:297-305. Saunders,A., L.J. Core, and J. T. Lis. 2005. Breakingbarriers to transcriptionelongation.Nat. Reu.MoL Cell Biol. 7:557-567 . Winston, F. 2001. Control of eukaryotic transcriptionelongation. GenomeBiol. 2:reviews1005. Yamada,T., et al. 2005. P-TEFb-mediatedphosphorylationof hSpt5 C-terminal repeatsis critical for processivetranscription elongation. Mol. Cell 2I:227-237. Other Eukaryotic Transcription Systems Gaspari,M., N. G. Larsson,and C. M. Gustafsson.2004.The transcriptionmachineryin mammalian mitochondria. Biochim. Biophys. Acta 1659:148-'152. Grummt, I. 2003. Life on a planet of its own: regulationof RNA polymeraseI transcriptionin the nucleolus.GenesDeu. 17:1691-1.702. Kanamaru,K., and K. Tanaka.2004. Rolesof chloroplastRNA polymerasesigma factors in chloroplastdevelopmentand st.essresponsein higher plants. Biosci. Biotechnol.Biochem. 68:2215-2223. Nomura, M.2001. RibosomalRNA genes,RNA polymerases, nucleolar structures,and synthesisof rRNA in the yeastSaccharomyces cereuisiae.Cold Spring Harb. Symp. Quant. Biol.66:555-565. Raska,I., et a\.2004. The nucleolusand transcriorionof ribosomal genes.Biol. Cell.96:579-594. Schramm,L., and N. Hernandez.2002. Recruitmenrof RNA polymeraseIII to its rargetpromorers.GenesDeu. 16:2593-2620. Shiina,T., Y. Tsunoyama,Y. Nakahira, and M. S. Khan. 2005. PlastidRNA polymerases,promoters,and transcription regulators in higher plants.Int. Reu.Cytol.244:L-68. '$7. Shutt, T. E., and M. Gray. 2005. Bacteriophageorigins of mitochondrial replication and transcriptionproteins. TrendsGenet. 22:90-95. 'White, R. J. 2004. RNA polymerasesI and III, growth control and cancer.Nat. Reu.Mol. Cell Biol.6:69-78.

T R A N S C R T p T T O NCAOLN T R O LO F G E N EE X P R E S S T O N

CHAPTER

POSTTRANSCRIPTIONAL GENECONTROL Portionof a "lampbrushchromosome"from an oocyteof the with hnRNPproteinassociated newt Nophtha/musviridescens; red afterstarningwith a fluoresces nascentRNAtranscripts of M RothandJ Gall] monoclonalantibody[Courtesty

I n the previous chapter, we saw that most genesare reguI lated at the first stepin geneexpression,namely,the initiaI tion of transcription. However, once transcription has been initiated, synthesisof the encoded RNA requires that RNA polymerasetranscribe the entire geneand not terminate prematurely. Moreover, the initial primary transcripts produced from eukaryotic genes must undergo various processing reactions to yield the corresponding functional RNAs. For mRNAs, the 5' cap structure necessaryfor translation must be added (seeFigure 4-14), introns must be spliced out of pre-mRNAs, and the 3' end must be polyadenylated (see Figure 4-15). Once formed in the nucleus, mature, functional RNAs are exported to the cytoplasm as components of ribonucleoproteins. Both processingof RNAs and their export from the nucleusoffer opportunities for further regulating geneexpressionafter the initiation of transcription. Recently, the vast amount of sequencedata on human cDNAs has revealedthat -60 percent of human genesgive rise to alternatively spliced mRNAs. These alternatively spliced mRNAs encode related proteins with differencesin sequenceslimited to specific functional domains. In many cases,alternative RNA splicing is regulatedto meet the need for a specificprotein isoform in a specificcell type. Given the complexity of pre-mRNA splicing, it is not surprising that mistakes are occasionally made, giving rise to mRNA precursors with improperly spliced exons. However, eukaryotic cells have evolved RNA surueillance mechanismsthat prevent the transport of incorrectly processedRNAs to the cytoplasm or lead to their degradation if they are transported. Additional control of gene expressioncan occur in the cytoplasm. In the caseof protein-coding genes,for instance,

the amount of protein produced depends on the stability of the corresponding mRNAs in the cytoplasm and the rate of their translation. For example, during an immune response' lymphocytes communicate by secretingpolypeptide hormones called cytokines that signal neighboring lymphocytes through cytokine receptors that span their plasma membranes (Chapter 24). It is important for lymphocytes to synthesize and secretecytokines in short bursts. This is possible because cytokine mRNAs are extremely unstable. ConsequentlS the concentration of the mRNA in the cytoplasm falls rapidly once its synthesis is stopped. In contrast' mRNAs encoding proteins required in large amounts that function over long periods, such as ribosomal proteins, are extremely stable so ihat multiple polypeptides are transcribed from each mRNA. In addition to regulation of pre-mRNA processing'nuclear export, and translation, the cellular locations of some mRNAs are regulated so that newly synthesizedprotein is concentrated

OUTLINE 8.1

of EukaryoticPre-mRNA Processing

325

8.2

Regulationof Pre-mRNAProcessing

337

8.3

Transportof mRNAAcrossthe Nuclear Envelope

8.4

I ic Mechanismsof Post-transcriptiona Cytoplasm 347 control

8.5

of rRNAand IRNA Processing

358

323

Nucleolus

DNArlr

polll 'lrrrrr

o

tt

pollll -t lll

polI

ll

lll

'--' Pre-rRNA t r an s c r p i tion

cap

Ribosomal subunit o s y n t h e s i si n nucleolus pre-rRNA

@

Correctly processeo mRNA

lmproperly processeo mRNA

mRNA export

o

Exosome

Excised pre-tRNA

tRNA export

Nucleus

Ribosome export

Cytoplasm AAAAA

Cytoplasmic exosome

Cytoplasmic polyadenylation

AAAAA

miRNA t r a n s l a t i o ni n h i b i t i o n

Translation initiation

FIGURE 8-1 Overviewof RNAprocessing and posttranscriptional genecontrol.Nearly allcytoplasmic RNAs are processed fromprimary transcripts in the nucleus beforetheyare exported to the cytoplasmForproteincodinggenestranscribed by RNApolymerase ll,genecontrol canbeexertedthroughI thechoice of alternative exonsduringpre-mRNA splicing andthe Z choice of alternative poly(A) siteslmproperly processed mRNAs areblocked from exportto thecytoplasm p bya largecomplex anddegraded called the exosome thatcontains multiple ribonucleases Onceexported to the cytoplasm, initiation factors @ translation bindto themRNA5,_cap cooperatively withpoly(A)-binding proteinI boundto thepoly(A) tail andinitiate (seeFigure4-29)E mRNAisdegraded translation in the cytoplasm byde-adenylation anddecapping followedbydegradation

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P o S T - T R A N S C R t p T t o NG AE L N EC O N T R O L

Cytoplasmic deadenylation

bycytoplasmic exosomes Thedegradation rateof eachmRNAis controlled, thereby regulating themRNAconcentration and, consequently, the amountof proteintranslated SomemRNAs are synthesized withoutlongpoly(A) tailsTheirtranslation isregulated by thesynthesis of a longpoly(A) @ controlling tailbya cytoplasmic poly(A) polymerase isalsoregulated byother Z Translatron mechanisms including miRNAs. Whenexpressed, these-22 nucleotide RNAs inhibittranslation of mRNAs to whichtheyhybridize, usually in the3'-untranslated region. tRNAs andrRNAs arealsosynthesized as precursor RNAsthat mustbe S processed beforetheyarefunctional Regions of precursors cleaved fromthe matureRNAsaredegraded by nuclear exosomes I QOO6\7:529 I

lAdaptedfrom Houseleyet.al. Nat RevMot CettBiot.

where it is needed.Particularlystriking examplesof this occur in the nervoussystemsof multicellularanimals.Someneurons in the human brain make more than 1000 separatesynapses with other neurons.During the processof learning, synapses that fire more frequently than others increase in size many times, even though other synapsesmade by the sameneuron do not. This can occur becausemRNAs encoding proteins critical for formation of an enlarged synapseare stored at all synapses.Then the translation of these localized, stored mRNAs is regulatedat eachsynapseindependentlyby the frequency at which it initiates translation. In this way, synthesis proteinscan be regulatedindependently of synapse-associated made by the sameneuron. many synapses each of the at Another type of generegulation that has recentlycome to light involvesmicro RNAs (miRNAs), which regulatethe stability and translation of specifictarget mRNAs in multicellular animals and plants. Analysesof these short miRNAs in various human tissuesindicatethat there are -1000 miRNAs expressedin the multiple typesof human cells.Although some have been recently discoveredto function through inhibition of target geneexpressionin the appropriate tissueand at the appropriatetime in development,the functionsof the vast majority of human miRNAs are unknown and are the subiectof a growing new area of research.If most miRNAs do indeed have significant functions, miRNA genesconstitute a significant fraction of the :25,000 human genes.A closelyrelated processcalled RNA interference(RNAi) leadsto the degradation of viral RNAs in infected cells and the degradation of transposon-encodedRNAs in most eukaryotes. This is of tremendoussignificanceto biological researchersbecauseit is possibleto design short interfering RNAs (siRNA) to inhibit the translationof specificmRNAs experimentallyby a process called RNA knockdown. This makesit possibleto inhibit the function of any desiredgene,even in organismsthat are not amenableto classicgeneticmethodsfor isolating mutants. \7e refer to all the mechanismsthat regulategeneexpression following transcription as post-transcriptionalgene control (Figure 8-1). Sincethe stability and translation rate of an mRNA contribute to the amount of protein expressedfrom a gene,thesepost-transcriptionalprocessesare important components of genecontrol. Indeed,the protein output of a gene is regulatedat every step in the life of an mRNA from the initiation of its synthesisto its degradation.Thus geneticregulatory processesact on RNA as well as DNA. In this chapter, we considerthe eventsthat occur in the processingof mRNA following transcription initiation and the various mechanisms that are known to regulatetheseevents.In the last section, we briefly discussthe processingof primary transcripts produced from genesencodingrRNAs and tRNAs.

E[

of EukaryoticPre-mRNA Processing

In this section,we take a closer look at how eukaryotic cells convert the initial primary transcript synthesizedby RNA oolvmerase II into a functional mRNA. Three maior events

occur during the process:5' capping,3' cleauage/polyadenyla' tion, and RNA splicing (Figure 8-2). Adding these specific modifications to the 5' and 3' ends of the pre-mRNA is important to protect it from enzymes that quickly digest uniapped RNAs generated by RNA processing' such as spliced-out introns and RNA transcribed downstream from a polyadenylationsite.The 5'-cap and 3'-poly(A) tail distinguish pre-mRNA molecules from the many other kinds of RNAs in ihe nucleus. Pre-mRNA molecules are bound by nuclear proteins that function in mRNA export to the cytoplasm. After mRNAs are exported to the cytoplasm, they are bound by another set of cytoplasmic proteins that stimulate translation and are critical for mRNA stability in the cytoplasm. Furthermore, introns must be removed to generatethe correct coding region of the mRNA. In higher eukaryotes, alternative splicing is intricately regulated in order to substitute different functional domains into proteins, producing a considerableexpansion of the proteome of theseorganisms,which include ourselves. The pre-mRNA processingeventsof capping, splicing, and polyadenylation occur in the nucleus as the nascent mRNA precursoris being transcribed.Thus pre-mRNA processingis co-transcriptional. As the RNA emergesfrom the surface of RNA polymeraseII, its 5' end is immediately modified by the addition of the S'-cap structure found on all mRNAs (seeFigwe 4-14).As the nascentpre-mRNA continuesto emergefrom the surfaceof the polymerase,it is immediately bound by members of a complex group of RNA-binding proteins that assistin RNA splicing and export of the fully processed mRNA through nuclear pore complexes into the cytoplasm. Some of these proteins remain associatedwith the mRNA in the cytoplasm, but most either remain in the nucleus or shuttle back into the nucleus shortly after the mRNA is exported to the cytoplasm. Cytoplasmic RNA-binding proteins are exchanged for the nuclear ones.Consequently,mRNAs never occur as free RNA moleculesin the cell but are always associatedwith protein as ribonucleoprotein (RNP) complexes, first as nascent pre-mRNPs that are capped and spliced as they ate transcribed. Then, following cleavage and polyadenylation, they are referred to as nuclear zzRNPs. Following the exchangeof proteins that accompaniesexport to the cytoplasm' they are called cytoplasmic mRNPs. Although we frequently refer to pre-mRNAs and mRNAs, it is important to remember that always associatedwith proteins as RNP complexes' ih.y "r.

The 5' Cap ls Added to NascentRNAsShortly After TranscriptionInitiation As the nascentRNA transcript emergesfrom the RNA chan-25 nunel of RNA polymeraseII and reachesa length of cleotides, a protective cap composed of 7-methylguanosine and methylated riboses is added to the 5' end of eukaryotic mRNAs (seeFigure 4-14).The 5' cap marks RNA molecules as mRNA precursorsand servesto protect them from RNAdigesting ..try-.t (5'-exoribonucleases)in the nucleus and cyiopl"s-. This initial step in RNA processingis catalyzed by a dimeric capping enzyme, which associateswith the

PRE-mRNA P R O C E S S I NOGF E U K A R Y O T I C

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phosphorylated carboxyl-terminal domain (CTD) of RNA

merasesI or III that do not have a CTD. This is important becausepre-mRNA synthesisaccountsfor less than 10 percent of the total RNA synthesizedin replicating cells.About 80 percent of RNA synthesis is preribosomal RNA transcribed by RNA polymeraseI, and about 15 percent is transcribed by RNA polymeraseIII, including 5S rRNA, tRNAs, and other stable small RNAs. The two mechanisms of (1) capping enzyme binding to initiated RNA polymerase II specificallythrough its unique, phosphorylated CTD and (2) activation of capping enzyme activity by binding to the phosphorylated CTD result in specificcapping of the minor fraction of RNAs transcribed by RNA polymeraseII.

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(stepB). Thepoly(A) -250 A residues tailcontains in mammals, -150 in insects, and-100 in yeastsForshortprimary transcripts (step4) usually withfew introns, splicing followscleavage and polyadenylation, asshownForlargegenes with multiple introns, introns oftenarespliced out of the nascent RNAduringits transcrrption, i e.,beforetranscription of thegeneiscomplete. Notethatthe 5' capandsequence adjacent to the poly(A) tailare retained in maturemRNAs.

initial phase of transcription the polymerase elongates the nascent transcript very slowly due to the action of elongation factors NELF (negative elongation factor) and Spt4/5, as occursat the HIV LTR promoter (seeFigure 7-51). Once the 5'-end of the nascentRNA is capped,phosphorylation of the RNA polymerase CTD and Spt5 by the cyclin TCdk9 protein kinase is stimulated, causing the release of NELF. This allows RNA polymerase II to enter into a faster mode of elongation that rapidly transcribes away from the promoter. The net effect of this mechanism is that the polymerase waits for the nascent RNA to be capped before elongating at a rapid rate. HIV appears to have exploited this mechanism to add an additional level of transcription control through the Tat protein and the TAR element, which are required to bring the cyclin T-Cdk9 kinase to the elongation complex that forms at the HIV LTR promoter (seeFigure 7-51). Currently, ir is not understoodwhat distinguishes transcription from the HIV LTR promoter from other promoters where Tat protein is not required to bring in the cyclin T-Cdk9 protein kinase.

A DiverseSet of Proteinswith ConservedRNABinding DomainsAssociatewith pre-mRNAs

Considerable evidence indicates that capping of the nascent transcript is coupled to elongation by RNA poly_ meraseII so that all of its transcriptsare cappedduring the earliest phase of elongation. In one -oJ.l, during the 326

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As noted earlier,nascentRNA transcripts from protein-coding genesand mRNA processingintermediates,collectively referred to as pre-mRNA, do not exist as free RNA molecules in the nuclei of eukaryotic cells. From the time nascentrranscripts first emerge from RNA polymerase II until mature mRNAs are transported into rhe cytoplasm, the RNA moleculesare associatedwith an abundant set of nuclear proteins.

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been identified by constructing deletionsof hnRNP proteins and testing their ability to bind RNA. Functions of hnRNP Proteins The associationof pre-mRNAs with hnRNP proteins preventsformation of short secondary structures dependenton base pairing of complementary regions, thereby making the pre-mRNAs accessiblefor interaction with other RNA molecules or proteins. Pre-mRNAs associatedwith hnRNP proteins present a more uniform substrate for subsequentprocessingsteps than would free' unbound pre-mRNAs' where each mRNA forms a unique secondarystructure due to its specificsequence. Binding studies with purified hnRNP proteins indicate that different hnRNP proteins associatewith different regions of a newly made pre-mRNA molecule. For example' ih. httRNp proteins A1, C, and D bind preferentially to the at the 3' endsof introns (seeFigure pyrimidine-rich sequences interact with the RNA sequences proteins hnRNP 8-7). Some cleavage/polyadenylationand or splicing RNA that specify by RNA-processing recognized structure the to contribute have shown that experiments cell-fusion Finally, factors. in the nucleus, localized remain proteins hnRNP some whereasothers cycle in and out of the cytoplasm, suggesting that they function in the transport of mRNA (Figure 8-4)' Conserved RNA'Binding Motifs The RNA recognition motif (RRM| also called the RNP motif and the RNA-binding domain (RBD), is the most common RNA-binding domain

Venkatesanand B Moss, 1982, Proc Natl Acad 'ci USA79:304)

These are the major protein components of heterogeneows ribonucleoprotein particles (hnRNPs), which conrain heterogeneous nuclear RNA /EnRNAl, a collective term referring to pre-mRNA and other nuclear RNAs of various sizes.These hnRNP proteins contribute to further steps in RNA processing,including splicing and polyadenylation and export through nuclear pore complexesto the cytoplasm' Researchersidentified hnRNP proteins by first exposing cultured cells to high-doseUV irradiation, which causescovalent cross-linksto form betweenRNA basesand closely associatedproteins. Chromatography of nuclear extracts from treated cells on an oligo-dT cellulose column, which binds RNAs with a poly(A) tail, was usedto recoverthe proteins that had become cross-linkedto nuclear polyadenylated RNA. Subsequenttreatment of cell extracts from unirradiated cells with monoclonal antibodies specific for the major proteins identified by this cross-linking technique revealed a complex set of abundant hnRNP proteins ranging in sizefrom -30 to -120 kDa. Like transcription factors, most hnRNP proteins have a modular structure. They contain one or more RNA-binding domains and at least one other domain that interacts with other proteins. Severaldifferent RNA-binding motifs have

eukaryotic evolution. Structural analyseshave shown that the RRM domain consists of a four-stranded B sheet flanked on one side by two cr helices.To interact with the negatively charged RNA phosphates,the B sheet forms a positively.chargedsurface' th. ionr.tu.d RNP1 and RNP2 sequenceslie side by side on the two central p strands, and their side chains make multiple contacts with a single-strandedregion of RNA that lies acrossthe surfaceof the B sheet (Figure 8-5)' The 45-residueKH motif is found in the hnRNP K protein and several other RNA-binding proteins' The threedimensionalstructureof representativeKH domains is similar to that of the RRM domain but smaller, consisting of a three-strandedp sheetsupported from one side by two o helices.Nonetheless,the KH domain interactswith RNA much differently than does the RRM domain' RNA binds to the KH domain by interacting with a hydrophobic surface formed by the a helices and one B strand' The RGG bor, another RNA-binding motif found in hnRNP proteins, contains five Arg-Gly-Gly (RGG) repeatswith severalinterspersedaromatic amino acids.Although the structure of this motif has not yet been determined,its arginine-rich nature is similar to the RNA-binding domains of the HIV Tat protein' KH domains and RGG repeatsare often interspersedin two or more setsin a single RNA-binding protein' PRE-mRNA P R O C E S S I NOGF E U K A R Y O T I C

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A FIGURE 8-4 HumanhnRNpA1 proteincancyclein and out of the cytoplasm,but humanhnRNpC proteincannot. CulturedHeLacellsandXenopus cellswerefusedbvtreatment with polyethylene glycol,producing heterokaryons containing nuclei fromeachcelltype Thehybridcellsweretreatedwith cycloheximide immediately protern afterfusionto prevent synthesis After2 hours,the cellswerefixedandstained with fluorescent-labeled antibodies specific for humanhnRNp C and A1 proteinsThese antibodies do not bindto the homologous Xenopus proteins(a)A fixedpreparation viewedby phasecontrast microscopy includes unfused HeLacells(arrowhead) and Xenopuscells(dottedarrow),aswellasfusedheterokaryons ( s o l i da r r o w )l n t h e h e t e r o k a r yionnt h i sm i c r o g r a pthh,e r o u n d

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Hela-cell nucleus isto therightof theoval-shaped Xenopus nucleus(b,c)Whenthesamepreparation wasvieweooy fluorescence microscopy, thestained hnRNpC proteinappeared greenandthestained hnRNP A1 proteinappeared red Notethat the unfused Xenopus cellon theleftisunstained, confirming that theantibodies arespecific for the humanproteinsln the heterokaryon, hnRNP C proteinappears onlyin the Hela-cell (b),whereas nucleus the41 protein (c) appears in bothnucleus proteinsynthesis Since wasblocked aftercellf usion,someof the humanhnRNP A'1proteinmusthaveleftthe Hela-cell nucleus, movedthroughthecytoplasm, andenteredlheXenopus nucleus in the heterokaryon S pinol-Roma andG Dreyfuss, [See 1992, Nature 355:730; courtesy of G Dreyfuss l

( b ) S e x - l e t h a(lS x l )R R Md o m a r n s

FIGURE8-5 Structure of the RRM domain and its interaction with RNA. (a) Diagramof the RRMdomainshowinqthe two cr helices(green)and four B strands(red)that characterrze this motif The conservedRNPl and RNp2regionsare locatedin the two central B strands (b) Surfacerepresentation of the two RRMdomainsin DrosophilaSex-lethal (Sxl)protein,which bind a nine_base sequence in transformerpre-mRNA(yellow).The two RRMsare orientedlike the two partsof an open pairof castanets, with the B sheetof RRMl facingupwardand the B sheetof RRM2facinqdownward.positivelv chargedregionsin Sxlproteinareshown in shadesof blue;negativeli chargedregions,in shadesof red.The pre-mRNAis bound to the posT-TRANSCR|pTtoNA GLE N EC O N T R O L

c Shu

(c) Polypyrimidinetract binding protein (pTB)

surfaces of the positively chargedB sheets, makingmostof its contacts with the RNPlandRNp2regions of eachRRM.(c)Strikingly d i f f e r e notr i e n t a t i oonf R R Md o m a i nisn a d i f f e r e nht n R N B the polypyrimidine tractbinding(PTB) protein, illustrating that RRMs are oriented in different relative positions in different hnRNps; colorsare asin (b) Polypyrimidine (p(y))singlestranded RNAis boundto the upward(RRM3) (RRM4) anddownward facingB-sheets RNAis (a)adapted shownin yellow[Part fromK Nagai et al, 1gg5,Trends Biochem Sci 20:735 Part(b) after N Haradaet al , 1999, Nature 399;579 Part(c) after F C Oberstrasset al , 2006, Science309:2054 l

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SplicingOccursat Short, Conserved via Two Sequencesin Pre-mRNAs Transesterification Reactions During formation of a mature, functional mRNA, the introns are removed and exons are spliced together.For short transcription units, RNA splicing usually follows cleavageand polyadenylationof the 3' end of the primary transcript' as depicted in Figure 8-2. However, for long transcription units containing multiple exons, splicing of exons in the nascent RNA beginsbefore transcription of the geneis complete. Early evidencethat introns are removed during splicing came from electron microscopy of RNA-DNA hybrids between adenovirusDNA and the mRNA encoding hexon, a major virion capsid protein (Figure 8-6). Other studiesrevealed nuclear viral RNAs that were colinear with the viral DNA (primary transcripts) and RNAs with one or r\rvoof the introns removed (processingintermediates).These results, together

8-5 EIECTTON FIGURE < EXPERIMENTAL microscopyof mRNA-templateDNA hybrids showsthat introns are splicedout during (a)Diagram of the processing. pre-mRNA DNA,which of adenovirus A fragment EcoRl extendsfromthe left endof the genometo just beforethe endof the finalexonof the hexon of threeshortexons geneThegeneconsists bythree andonelong(-3.5 kb)exonseparated (b) -1, Electron kb. 9 2 5, and intronsof (/eft)andschematic drawing(nght) micrograph A fragmentand of hybridbetweenan EcoRl TheloopsmarkedA, B,andC hexonmRNA. in (a).Since to the intronsindicated correspond genomtc viral in the sequences theseintron in maturehexonmRNA, DNAarenot present that theyloopout betweenthe exonsequences in sequences to theircomplementary hybridize al et M Berget 5 ' from ,19]7 the mRNA.[Micrograph ProcNat'lAcadSciuSA74:3171;courtesyofPA Sharp l

with the findingsthat the 5' cap and 3' poly(A)tail at eachend of long mRNA precursorsare retained in shorter mature cytoplasmic mRNAs, led to the realization that introns are removed from primary transcripts as exons are spliced together' The location of splice sites-that is, exon-intron iunc-

revealedmoderatelyconserved,short consensussequencesat the splicesitesflanking introns in eukaryotic pre-mRNAs; in higher organisms, a pyrimidine-rich region just upstream oflhe 3' splice site also is common (Figure 8-7)' Studiesof mutant genes with deletions introduced into introns have shown that much of the center portion of introns can be removed without affecting splicing; generally only 30-40

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fromthe 3' splice is20-50bases usually alsoinvariant, adenosine, site,Thecentralregionof the intron,whichmayrangefrom40 bases to occur. for splicing isunnecessary in length,generally to 50 kilobases t t a l , 1 9 8 6 , A n n R e vB i o c h e m 5 5 : 1 ' 1 9 , a n d E B K e l l e r [ S e eR A P a d g e t e a n d W A N o o n , 1 9 8 4 ,P r o c N a t ' l A c a d S c i U S A 8 1 : 7 4 1 71

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A FIGURE 8-8 Two transesterificationreactionsthat result in splicingof exonsin pre-mRNA. In thefirstreaction, the esterbond between the 5' phosphorus of the intronandthe 3, oxygen(dark red)of exon1 isexchanged for an esterbondwith the 2, oxygen (blue)of the branch-site A residueInthesecondreacrton, tneester bondbetween the 5' phosphorus of exon2 andthe 3, oxygen (orange) of the intronisexchanged for an esterbondwiththe 3, oxygenof exon1, releasing the intronasa lariatstructure andjoining the two exons.Arrowsshowwhereactivated hydroxyl oxygensreact with phosphorus atoms.

nucleotidesat each end of an intron are necessaryfor splic_ ing to occur at normal rates. Analysis of the intermediatesformed during splicing of pre-mRNAs in vitro led to the discovery that splicing of exons proceedsvia two sequential transesterification reac_ tions (Figure 8-8). Inrrons are removed as a lariat_like structure in which the 5' G of the inrron is joined in an u n u s u a l 2 ' , 5 ' - p h o s p h o d i e s t ebr o n d t o a n a d e n o s i n en e a r t h e 3 ' e n d o f t h e i n t r o n . T h i s A r e s i d u ei s c a l l e d b r a n c h point A because it forms an RNA branch in the lariat

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structure. In each transesterificationreaction) one phosphoesterbond is exchangedfor another.Sincethe number of phosphoesrerbonds in the molecule is not changed in either reaction, no energy is consumed. The net result of these two reactions is that two exons are ligated and the intervening intron is released as a branched lariat structure.

p o s T - T R A N S C R | p T t O NG AL E N EC O N T R O L

Splicing requires the presenceof both small nuclear RNAs (snRNAs), important for base pairing with the pre-mRNA, and associatedproteins. Five U-rich snRNAs, designated UI,U2, U4, U5, and U6, participate in pre-mRNA splic, ing. Ranging in length from 107 to 210 nucleotides,these snRNAs are associatedwith 6 to 10 proteins in the many small nuclear ribonucleoprotein particles (snRNps) in rhe nuclei of eukaryotic cells. Definitive evidencefor the role of U1 snRNA in splicing came from experiments indicating that base pairing between the 5' splicesite of a pre-mRNA and the 5, region of U1 snRNA is required for RNA splicing (Figure 8-9a). In vitro experiments showed that a synthetic oligonucleotide that hybridizeswith the 5'-end region of U1 snRNA blocks RNA splicing.In vivo experimentsshowed that base-pairingdisrupting mutations in the pre-mRNA 5' splice block RNA splicing. However, splicing can be restored by expressionof a mutant U1 snRNA with a compensating mutation that restoresbasepairing to the mutant pre-mRNA 5, splice site (Figure 8-9b). Involvement of U2 snRNA in splicing initially was suspectedwhen it was found to have an internal sequencethat is largely complementary to the consensus sequenceflanking the branch point in pre-mRNAs (see Figure 8-7). Compensatingmutation experiments,similar to those conducted with U1 snRNA and 5, splice sites, demonstratedthat basepairing betweenU2 snRNA and the branch-point sequencein pre-mRNA also is critical to splicing. Figure 8-9a illustrates the general strucrures of the U1 and U2 snRNAs and how they base-pair with pre-mRNA during splicing. Significantly,branch-point A itself, which is not base-pairedto U2 snRNA, "bulgesout" (Figure8-10a), allowing its 2' hydroxyl to participate in the firsr transesterification reaction of RNA splicing (seeFigure 8-8). Similar studies with other snRNAs demonstrated that base pairing between them also occurs during splicing. Moreover, rearrangementsin these RNA-RNA interactions are critical in the splicing pathway, as we describenext.

S p l i c e o s o m eA s ,s s e m b l e df r o m s n R N p s a n d a P r e - m R N AC, a r r yO u t S p l i c i n g The five splicing snRNPsand other proteins involved in splicing assembleon a pre-mRNA, forming a large ribonucleoprotein

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5' sPlicesite Mutationin pre-mRNA blockssplicing FIGURE 8-9 Basepairing between pre-mRNA,Ul snRNA, (a)Inthisdiagram, and U2 snRNAearlyin the splicingprocess. that arenot alteredduring in the snRNAs structures secondary Theyeastbranch-point schematically. aredepicted splicing with a isshownhere.Notethat U2 snRNAbase-pairs sequence is thisresidue A, although the branch-point thatincludes sequence that bind sequences represent Thepurplerectangles not base-paired Forunknown proteins antibodies. recognized by anti-Sm snRNP systemic disease from patients with the autoimmune reasons, antisera proteins, (SLE) to snRNP antibodies contain lupuserythematosus

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mutationin U1 ComPensatorY sPlicing restores of the components whichhavebeenusefulin characterizing and5' splice (b)Onlythe 5' endsof U1snRNAs reaction. splicing areshown.(Left)A mutation(A)in a pre-mRNA sitesin pre-mRNAs to the 5' endof U1 with basepairing sitethatinterferes splice witha U1 snRNA of a Expression splicing blocks snRNA basepairingalsorestores mutation(U)that restores compensating fromM J Moore lPart(a)adapted of the mutantpre-mRNA. splicing andJ Atkins,eds, Ihe RNAWorld,ColdSpring et al,,1993,in R Gesteland pp Part(b)seeY ZhuangandA M Weiner, 303-357 HarborPress, 1986.Cell45:827|

FactorMovementin LivingCells 8-10 Structuresof a bulged A in an RNA-RNA < FIGURE (a)The helix and an intermediatein the splicingProcess. containing shown, the sequence of an RNAduplexwith structure was helix, RNA (red) position in the 5 at bulgedA residues (b)ThebulgedA restdues by x-raycrystallography. determined helix.Thephosphate extendfromthe sideof an A-formRNA-RNA green; otherstrandin the of onestrandis shownin backbone for a view degrees 90 turned is right on the blue Thestructure of a (c) structure resolution 40 A helix the of axis downthe o n t a i n i nUg2 ,U 4 ,U 5 ,a n d s p l i c e o s o msapl i c i nign t e r m e d i act e andimage mtcroscopy by cryoelectron determined U6 snRNPs, has a similar complex tri-snRNP TheU4/U6/U5 reconstruction that suggesting complex, of this body triangular the to structure shownhereand areat the bottomof the structure thesesnRNPs (a)and(b) largelyof U2snRNP lParts that the headis composed Boehringer f r o m D P a r t ( c ) , NAT:682 f r o m J . AB e r g l u n d e,t2a0l 0 1R R Luhrmann, and H Stark also See 11:463 Biot. Mot. etal, 2004,Nat.Struct. BiomolStruct35:435l Biophys 2006,Annu Rev.

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complex called a spliceosome(Figure 8-11). The spliceosome is a large ribonuclear protein complex with a masssimilar to that of a ribosome.Assemblyof a spliceosomebeginswith the basepairing of the snRNAs of the U1 and U2 snRNps to the

After formation of the spliceosome,extensiverearrangements in the pairing of snRNAs and the pre-mRNA lead

332

C H A P T E R8

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< FIGURE 8-11 Modelof spliceosome-mediated splicingof pre-mRNA. Step[: AfterU1andU2snRNps associate withthe oremRNA, viabase-pairing interactions shownin Flgure 8-9,a trimeric snRNP complex of U4,U5,andU6joinsthe initialcomplex to form thespliceosome StepE: Rearrangements of base-pairing interactions between snRNAs converts thespliceosome intoa catalytically active conformation anddestabilizes the U1andU4snRNps, whichare released. StepB: Thecatalytic core,thoughtto beformedby U6 andU2,thencatalyzes thefirsttransesterif icationreaction, forming the intermediate containing a 2',5'-phosphodiester bondshownin Figure 8-8.StepZl: Following furtherrearrangements between the snRNPs, thesecond joinsthetwo exonsby transesterification reaction a standard 3',5'-phosphodiester bondandreleases the intronasa lariatstructure andthe remaining snRNPs. Step5: Theexcised Iariat intronisconverted intoa linearRNAby a debranching enzyme

P o S T - T R A N S C R | p T t O NGAELN EC O N T R O L

to releaseof first the U1 and then the U4 snRNPs.Figure8-10b shows a cryoelectron-microscopy structure of an intermediate in the splicing processlacking UlsnRNP. A further rearrangementof spliceosomalcomponents occurs with the loss of U4snRNP.This generatesa complex that catalyzesthe first transesterificationreaction that forms the 2,,5,-phosphodiesterbond between the 2' hydroxyl on branch point A and the phosphateat the 5' end of the intron (Figure8-11). Following another rearrangementof the snRNps, the second transesterificationreaction ligates the two exons in a standard 3',5'-phosphodiesterbond, releasingthe intron as a lariat structure associatedwith the snRNPs. This final intronsnRNP complex rapidly dissociates, and the individual snRNPs releasedcan participate in a new cycle of splicing. The excisedintron is then rapidly degradedby a debranching enzymeand other nuclear RNasesdiscussedlater. As mentioned above, a spliceosomeis roughly the size of a ribosome and is composed of about 100 proteins, collectively referred to as splicing factors, in addition to the five snRNPs.This makesRNA splicingcomparablein complexity to initiation of transcription and protein synthesis.Some of the splicing factors are associatedwith snRNPs, but others are not. For instance,the 65-kD subunit of the U2-associated factor (U2AF) binds to the pyrimidine-rich region near rhe 3' end of introns and to the U2 snRNP. The 35-kD subunit of U2AF binds to the AG dinucleotideat the 3, end of the intron and also interacts with the larger U2AF subunit bound nearby.Thesetwo U2AF subunitsact togetherto help specify the 3' splice site by promoting interaction of U2 snRNp with the branch point (seeFigure 8-9). Some splicing facors also exhibit sequencehomologiesto known RNA helicases;these are probably necessaryfor the base-pairingrearrangements that occur in snRNAs during the spliceosomalsplicing cycle. Following RNA splicing, a specific set of hnRNp proteins remain bound to the spliced RNA approximately 20 nucleotides 5' to each exon-exon junction, thus forming an exon-junction complex. One of the hnRNp proteins associated with the exon junction complex is the RNA export factor (REF), which functions in the export of fully processed

R N A p o l y m e r a s el l

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I I I I I t I I l$

ei* s $ s * & $ 6 $ & : q * { * $ $ $ r ' e $ a F !s x f s s s $ * 6 s l * * * * R S $ 6 * * 6 S * & & S * e

ll with diagramof RNApolymerase 8-12 Schematic A FIGURE yeastRNA the CTDextended.Thelengthof the fullyextended andthe linkerregion polymerase domain(CTD) ll carboxyl-terminal polymerase to theglobular isshownrelative it to the thatconnects

RNApolymerase TheCTDof mammalian domainof the polymerase. with associate CTD can the form, In its extended long ll istwiceas DA P Cramer, factorssimultaneously lFrom multipleRNA-processing 292:1863 Science 2001, ) andR D Kornberg, Bushnell,

mRNPs from the nucleus to the cytoplasm, as discussedin Section8.3. Other proteins associatedwith the exon junction complex function in a quality-control mechanism that leads to the degradation of improperly spliced mRNAs, decay(Section8.4). known as nonsense-mediated A small fraction of pre-mRNAs (-1o7' in humans) contain introns whose splicesitesdo not conform to the standard consensussequence.This classof introns beginswith AU and ends with AC rather than following the usual "GU-AG rule" (seeFigure 8-7). Splicing of this special class of introns appearsto occur via a splicingcycle analogousto that shown in Figure 8-11, exceptthat four novel,low-abundancesnRNPs, together with the standardU5 snRNP,are involved. Nearly all functional mRNAs in vertebrate, insect, and plant cellsare derivedfrom a singlemoleculeof the corresponding pre-mRNA by removal of internal introns and splicingof exons. However, in two types of protozoans-trypanosomes and euglenoids-mRNAs are constructedby splicingtogether separateRNA molecules.This process,referred to as transsplicing, is also used in the synthesisof 10-15 percentof the mRNAs in the nematode (roundworm\ Caenorhabditiselegdns, an important model organism for studying embryonic development.Trans-splicingis carried out by snRNPs by a processsimilar to the splicingof exonsin a singlepre-mRNA.

Recentresultsindicate that the associationof RNA splicing factors with phosphorylated CTD also stimulates transcription elongation. Thus chain elongation is coupled to the binding of RNA-processing factors to the phosphorylated CTD. This mechanismmay ensurethat a pre-mRNA is not synthesizedunlessthe machinery for processingit is properly positioned.

ll ls Coupled ChainElongationby RNAPolymerase Factors to the Presenceof RNA-Processing How is RNA processingefficiently coupled with the transcription of a pre-mRNA? The key lies in the long carboxylterminal domain (CTD) of RNA polymeraseII, which, as discussed in Chapter 7, is composed of multiple repeats of a seven-residue(heptapeptide)sequence.\X/henfully extended, the CTD domain in the yeast enzyme is about 65 nm long (Figure8-12); the CTD in human RNA polymeraseII is about twice as long. The remarkablelength of the CTD apparently allows multiple proteins to associatesimultaneouslywith a single RNA polymerase II molecule. For instance' as mentioned earlier,the enzymesthat add the 5' cap to nascenttranscripts associatewith the phosphorylatedCTD shortly after transcription initiation. In addition, RNA splicing and polyadenylationfactors have beenfound to associatewith the theseprocessingfacphosphorylatedCTD. As a consequence, tors are presentat high local concentrationswhen splicesites and poly(A) signals are transcribed by the polymerase,enhancing the rate and specificityof RNA processing.

S RP r o t e i n sC o n t r i b u t et o E x o nD e f i n i t i o n i n L o n gP r e - m R N A s The averagelength of an exon in the human genomeis :150 bases,whereasthe averagelength of an intron is much longer (:3500 bases).The longestintrons contain upward of 500 kb! Becausethe sequencesof 5' and 3' splice sites and branch points are so degenerate,multiple copies are likely to occur iandomly in long introns. Consequently,additional sequence information is required to define the exons that should be splicedtogether in higher organismswith long introns. The information for defining the splice sitesthat demarcate exons is encodedwithin the sequencesof exons' A family of RNA-binding proteins, the SR proteins, interact with sequenceswithin exons called exonic splicing enhancers.SR proteins are a subsetof the hnRNP proteins discussedearlier and so contain one or more RRM RNA-binding domains' They also contain several protein-protein interaction domains rich in serine(S) and arginine (R) residues'\fhen bound to exonic splicing enhancers,SR proteins mediate the cooperativebinding of U1 snRNP to a true 5' splice site and U2 snRNP to a branch point through a network of proteinprotein interactionsthat span acrossan exon (Figure8-13)' The comple* of SR proteins, snRNPs,and other splicing factors (e.g.,U2AF) that assembleacrossan exon' which has been called a cross-exonrecognition complex, permits precise specificationof exons in long pre-mRNAs. In the transcription units of higher organismswith long introns, exons not only encodethe amino acid sequencesof different portions of a protein but also contain bindi.rg sites for SR proteins. Mutations that interfere with the binding of an SR protein to an exonic splicing enhancer, evenif they do not changethe encodedamino acid sequence, would prevent formation of the cross-exonrecognition complex. Ai a result, the affectedexon is "skipped" during splicis not included in the final processedmRNA' The ing "nd t.Jncat.d mRNA produced in this caseis either degradedor PRE-mRNA P R O C E S S I NOGF E U K A R Y O T I C

o

333

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U2AF65

U1 GU

u2 //

5 ' s p l i c es i t e

Cross-exon recognition complex A FIGURE 8-13 Exonrecognitionthroughcooperativebinding of SRproteinsand splicingfactorsto pre-mRNA. Thecorrect5, GUand3'AG splice sitesarerecognized by splicing factorson the basisof theirproximity to exonsTheexonscontainexonicsplicing (ESEs) enhancers thatarebindingsitesfor SRproteinsWhenbound to ESEs, the SRproteins interact with oneanotherandpromotethe c o o p e r a t i vbei n d i n go f t h e U 1 s n R N tpo t h e 5 , s p l i c es i t eo f t h e d o w n s t r e aim n t r o nt,h e U 2s n R N tpo t h e b r a n c hp o i n to f t h e upstream i n t r o nt,h e 6 5 -a n d3 5 - k Ds u b u n i tosf U 2 A Ft o t h e pyrimidine-rich regionandAG 3, splicesiteof the upstream Intron, andothersplicing factors(notshown)Theresulting RNA-protein translatedinto a mutant, abnormally functioning protein. Recent studieshave implicated this type of mutation in human geneticdiseases.For example, spinal muscle atrophy is one of the most common genetic causesof childhood mortality. This diseaseresultsfrom mutations in a region of the genome containing two closelyrelated genes,SMNI and SMN2, that arose by geneduplication. SMN2 encodesa prorein identical with SMNI. SMN2 is expressedat much lower level because a silent mutation in one exon interfereswith the binding of an SR protein. This leadsro exon skipping in mosr of the SMN2 mRNAs. The homologous SMN gene in the mouse, where there is only a singlecopy, is essentialfor cell viability. Spinal muscle atrophy in humans results from homozygous murations that inactivate SMN1. The low level of protein translated from the small fraction of SMN2 mRNAs rhar are correctly spliced is sufficient to maintain cell viability during embryogenesisand fetal development,but it is not sufficient to maintain viability of spinal cord motor neurons in childhood, resulting in their death and the associateddisease. Approximately 15 percentof the single-base-pair mutations that causehuman geneticdiseasesinterfere with proper exon definition. Some of thesemutations occur in 5, or 3, splice sites, often resulting in the use of nearby alternative "cryptic" splice sitespresentin the normal genesequence.In the absenceof the normal splice site, the cross-exonrecognition complex recognizesthesealternative sites.Other mutations that causeabnormal splicing result in a new consensus splice site sequencethat becomesrecognizedin place of the normal splice site. Finally, some mutations can interferewith the binding of specific SR proteins to pre-mRNAs. These mutations inhibit splicing at normal splice sites, as in the caseof the SMN2 gene,and thus lead ro exon skipping. I

S e l f - S p l i c i nG g r o u pl l I n t r o n sp r o v i d eC l u e s t o t h e E v o l u t i o no f s n R N A s Under certain nonphysiological in vitro conditions, pure preparationsof some RNA transcriptsslowly spliceout in_ 334

o

c H A p r E8R |

p o s r - T R A N S c R t p I oG NE AN L cEo N T R o L

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B r a n c hp o i n t

3 ' s p l i c es i t e

5 ' s p l i c es i t e

Cross-exon recognition complex cross-exon recognition complex spansan exonandactivates the correct splice sitesfor RNAsplicingNotethatthe U1andU2snRNps in thisunitarenot partof thesamespliceosome TheU2snRNp on the rightformsa spliceosome with the U1snRNp boundto the 5, endof the sameintronTheU1snRNP shownon the rightformsa spliceosome with the U2snRNP boundto the branchpointof the downstream intron(notshown), andthe U2snRNp on the leftforms a spliceosome with a U1snRNP boundto the 5, splice siteof the upstream intron(notshown).Double-headed arrowsindicate protein-protein interactrons fromI Maniatis,2OO2, [Adapted Nature 418:236; seealsoS M Berget,1995,J Biol.Chen 27O:24111 trons in the absenceof any protein. This observationled to the recognition that some introns are self-splicing. Two types of self-splicingintrons have beendiscovered:group I introns, presentin nuclearrRNA genesof protozoans,and group II introns, present in protein-coding genesand some rRNA and IRNA genesin mitochondria and chloroplasts of plants and fungi. Discovery of the catalytic activity of selfsplicing introns revolutionized conceptsabout the functions of RNA. As discussedin Chapter 4, RNA is now thought to catalyzepeptide-bond formation during protein synthesisin ribosomes. Here we discussthe probable role of group II introns, now found only in mitochondrial and chloroplast DNA, in the evolution of snRNAs; the functioning of group I introns is consideredin the later sectionon rRNA processing. Even though their precisesequencesare not highly conserved, all group II introns fold into a conserved,complex secondarystructure containing numerous stem loops (Figure 8-t4a). Self-splicing by a group II intron occurs vra two transesterificationreactions, involving intermediatesand products analogous to those found in nuclear pre-mRNA splicing. The mechanistic similarities between group II intron self-splicingand spliceosomalsplicing led to the hypothesis that snRNAs function analogously to the stem loops in the secondarystructure of group II introns. According to this hypothesis,snRNAs interact with 5, and 3, solice sitesof pre-mRNAs and with each other to produce a threedimensional RNA structure functionally analogous to that of group II self-splicingintrons (Figure8-14b). An extension of this hypothesisis that introns in ancient pre-mRNAs evolved from group II self-splicing introns through the progressive loss of internal RNA structures. which concurrently evolved into trans-acting snRNAs that perform the same functions. Support for this type of evolutionary model comesfrom experimentswith group II intron mutants in which domain V and part of domain I are d e l e t e dR . N A t r a n s c r i p t cs o n t a i n i n gs u c hm u t a n ti n t r o n sa r e defectivein self-splicing,but when RNA moleculesequivalent

( a )G r o u p l l i n t r o n

5', 3',

( b ) U s n R N A si n s p l i c e o s o m e

-.Pre-mRNA

introns of group ll self'splicing 8-14 Comparison FIGURE the comparing diagrams Theschematic and the spliceosome. intronsand(b)U of (a)groupll self-splicing structures secondary present Thefirsttransesterification rnthe spliceosome snRNAs reaction, by thesecond by lightgreenarrows; reaction is indicated in these Thesimilarity A isboldfaced Thebranch-point bluearrows. f romgroup snRNAs evolved suggests thatthe spliceosomal structures analogous beingfunctionally snRNAs with thetrans-acting ll introns, bars group The colored ll introns in domarns to thecorresponding fromPA exons.[Adapted flankingthe intronsin (a)and(b)represent 254:663 1991,Science Sharp, I

to the deletedregions are added to the in vitro reaction, selfsplicing occurs. This finding demonstratesthat these domains in group II introns can be trans-acting,like snRNAs. The similarity in the mechanismsof group II intron selfsplicingand spliceosomalsplicingof pre-mRNAs also suggests that the splicing reaction is catalyzedby the snRNA, not the protein, components of spliceosomes.Although group II introns can self-splicein vitro at elevated temperatures and Mg2* concentrations, under in vivo conditions proteins called matura.ses,which bind to group II intron RNA, are required for rapid splicing. Maturases are thought to stabilize the precisethree-dimensionalinteractionsof the intron RNA required to catalyzethe two splicing transesterificationreacare thought tions. By analogg snRNP proteinsin spliceosomes intron nuand geometry snRNAs precise of to stabilize the pre-mRNA splicing. to catalyze required cleotides The evolution of snRNAs may have been an important step in the rapid evolution of higher eukaryotes'As internal rntron sequenceswere lost and their functions in RNA splicing supplantedby trans-actingsnRNAs, the remaining intron sequenceswould be free to diverge.This in turn likely facilitated the evolution of new genes through exon shuffling sincethere are few constraintson the sequenceof new introns generatedin the process(seeFigures6-18 and 6-19). It also permitted the increasein protein diversity that results from alternativeRNA splicing and an additional level of genecontrol resulting from regulatedRNA splicing.

during the S phase. They undergo a special form of 3'-end processingthat involves cleavagebut not polyadenylation. SpecializedRNA-binding proteins that help to regulate histone mRNA translationbind to the 3'end generatedby this specializedsystem.) Early studies of pulse-labeledadenovirus and SV40 RNA demonstratedthat the viral primary transcripts extend beyond the site from which the poly(A) tail extends. These results suggestedthat A residuesare added to a 3' hydroxyl generatedby endonucleolytic cleavage of a longer transcript, but the predicted downstream RNA fragments never were detectedin vivo, presumably becauseof their rapid degradation.However' detection of both predicted cleavageproducts was observed in in vitro processing reactions performed with nuclear extracts of cultured human cells. The cleavage/polyadenylation process and degradation of the RNA downstream of the cleavage site occurs much more slowly in thesein vitro reactions' simproduct. -plifying detection of the downstream cleavage animal cells from clones cDNA of Early sequencing sequence the contain mRNAs all showed that nearly poly(A) the from upstream nucleotides AAUAAA 10-35 is virtranscripts RNA of (Figure Polyadenylation 8-15). tail in the sequence the corresponding when tually eliminated except sequence other any to mutated is DNA template one encoding a closely related sequence(AUUAAA). The

animal cells.This downstreamsignalis not a specificsequence :50 but rather a GU-rich or simply a U-rich region within site' n u c l e o t i d eos f t h e c l e a v a g e Identification and purification of the proteins required for cleavageand polyadenylation of pre-mRNA have led to the modelihown in Figure 8-15. According to this model' a 360kDa cleavageand polyadenylation specificity factor (CPSF)' composed of four different polypeptides,first forms an unstabie complex with the upstream AAUAAA poly(A) signal' Then at leastthree additional proteins bind to the CPSF-RNA complex: a 200-kDa heterotrimer calledcleauagestimulatory sequence;a factor (CSIF),which interactswith the G/lJ-rich (CFI); and a I 150-kDa heterotrimer called cleauagefactor

3' Cleavageand Polyadenylationof Pre'mRNAs Are Tightly Coupled In eukaryotic cells,all mRNAs, excepthistone mRNAs, have a 3', poly(A) tail. (The major histone mRNAs are transcribed from repeatedgenesat prodigious levels in replicating cells PRE-mRNA P R O C E S S I NoGF E U K A R Y O T I C

'

335

> FIGURE 8-15 Modelfor cleavageand polyadenylation of pre-mRNAs in mammaliancells.Cleavage andpolyadenylation specificity factor(CPSF) bindsto the upstream AAUAAApoly(A) signalCSIFinteracts with a downstream GU-or U-richsequence a n dw i t h b o u n dC P S F f o, r m i n ga l o o pi n t h e R N Ab; i n d i n g of CFI andCFllhelpstabilize the complexBindingof poly(A)polymerase (PAP) thenstimulates cleavage at a poly(A) site,whichusually is 10-35nucleotides 3' of the upstream poly(A) signal.Thecleavage factorsarereleased, as isthe downstream RNAcleavage product, w h i c hi s r a p i d ldy e g r a d e dB.o u n dp A pt h e na d d s: 1 2 A r e s i d u east a slowrateto the 3'-hydroxyl groupgenerated by the creavage reactionBindingof poly(A)-binding proteinil (pABpil) to the initial shortpoly(A) tailaccelerates the rateof additionby pApAfter 200-250A residues pApto stoo havebeenadded,pABpll siqnals polVmenzation

Poly(A) signal [I

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1

residuesoccurs slowly, followed by rapid addition of up to 200-250 more A residues.The rapid phase requires the binding of multiple copies of a poty(A)-binding protein containing the RRM motif. This protein is designatedpABpII ro distinguish it from the poly(A)-binding protein presentin the cytoplasm. PABPII binds to the short A tail initially added by PAP,stimulating the rate of polymerization of additional A residuesby PAP,resulting in rhe fast phase of polyadenylation (Figure 8-15). PABPII is also responsiblefor signaling poly(A) polymerase to terminate polymerization when the poly(A) tail reachesa length of 200-250 residues,although the mechanismfor controlling the length of the tail is not yit understood. Binding of PABP to the poly(A) tail is essential for mRNA export into the cytoplasm.

N u c l e a rE x o n u c l e a s eDse g r a d eR N AT h a t l s Processed Out of Pre-mRNAs

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Becausethe human genome contains long introns, only :5 percent of the nucleotides that are polymerized by RNA polymerase II during transcription are retained in mature, processedmRNAs. Although this process appears inefficient, it probably evolved in multicellular organismsbecause the processof exon shuffling facilitated the evolution of new

As mentionedearlier,the 2,,5,-phosphodiesterbond in excised introns is hydrolyzed by a debranching enzyme,yielding a linear molecule with unprotected ends that can be attacked by exonucleases(seeFigure 8-11). The predominant nuclear decaypathway is 3' -+ 5' hydrolysis by 11 exonucleases that associatewith one another in a large protein complex called the exosome.Other proteins in the complex include RNR helicasesthat disrupt base pairing and RNA-protein interactions that would otherwiseimpedethe exonucleases. Exosomesalso function in the cytoplasm, as discussedlater. In addition, the exosome appears to degrade pre-mRNAs that have not been properly spliced or polyadenylated. It is not yet clear how the

336

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exosomerecognizesimproperly processedpre-mRNAs. But in yeast cells with temperature-sensitivemutant poly(A) polymerase(Figure8-15), pre-mRNAs are retainedat their sitesof transcription at the nonpermissivetemperature.Theseabnormally processedpre-mRNAs are releasedin cellswith a second mutation in a subunit of the exosomefound only in nuclear and not cytoplasmicexosomes(PM-Scl; 100 kD in humans). Also, exosomesare found concentratedat sitesof transcription in Drosophila polytene chromosomes,where they are associated with RNA polymeraseII elongation factors. Theseresults suggestthat the exosomeparticipatesin an as yet poorly understood quality-control mechanismthat recognizesaberrantly processed pre-mRNAs, preventing their export to the cytoplasm and ultimately leadingto their degradation. To avoid being degraded by nuclear exonucleases,nascent transcripts, pre-mRNA processing intermediates, and mature mRNAs in the nucleus must have their ends protected.As discussedabove,the 5' end of a nascenttranscript is protectedby addition ofthe 5'cap structureas soon as the 5' end emergesfrom the polymerase.The 5' cap is protected becauseit is bound by a nuclear cap-binding complex, which protects it from 5' exonucleasesand also functions in export of mRNA to the cytoplasm. The 3' end of a nascent transcript lies within the RNA polymeraseand thus is inaccessible to exonucleases(seeFigure4-12). As discussedpreviously, the free 3' end generatedby cleavageof a pre-mRNA downstream from the poly(A) signal is rapidly polyadenylatedby the poly(A) polymeraseassociatedwith the other 3' processing factors, and the resulting poly(A) tail is bound by PABPII (Figure 8-15). This tight coupling of cleavageand polyadenylation protects the 3' end from exonucleaseattack.

pre-mRNAs of higher organisms. A network of interactions between SR proteins, snRNPs' and splicing factors forms a cross-exonrecognition complex that specifiescorrect splicesites(seeFigure 8-13)' r The snRNAs in the spliceosomeare thought to have an overall tertiary structure similar to that of group II selfsplicing introns. r For long transcription units in higher organisms,splicing of exons usually begins as the pre-mRNA is still being formed. Cleavageand polyadenylation to form the 3' end of the mRNA occur after the poly(A) site is transcribed. r In most protein-coding genes' a conserved AAUAAA poly(A) signal lies slightly upstream from a poly(A) site where cleavageand polyadenylation occur. A GU- or Urich sequence downstream from the poly(A) site contributes to the efficiency of cleavageand polyadenylation. r A multiprotein complex that includes poly(A) polymerase(PAP)carries out the cleavageand polyadenylation of a pre-mRNA. A nuclear poly(A)-binding protein, PABPII, stimulatesaddition of A residuesby PAP and stops addition once the poly(A) tail reaches 200-250 residues ( s e eF i g u r e8 - 1 5 ) . r Excised introns and RNA downstream from the cleavagel poly(A) site are degradedprimarily by exosomes,multiprotein complexesthat contain eleven3' -> 5' exonucleasesas well as RNA helicases.Exosomesalso degradeimproperly processedpre-mRNAs.

fp| Regulationof Pre-mRNA Processing Processingof Eukaryotic Pre-mRNA r In the nucleus of eukaryotic cells, pre-mRNAs are associated with hnRNP proteins and processedby 5' capping, 3' cleavageand polyadenylation, and splicing before being transported to the cytoplasm (seeFigure 8-2). r Shortly after transcriptioninitiation, a cappingenzymeassociatedwith the phosphorylatedCTD of RNA polymerase II addsthe 5' cap to the nascenttranscript. Other RNA processingfactors involved in RNA splicing, 3' cleavage,and polyadenylation also associate with the phosphorylated CTD, increasingthe rate of transcriptionelongation.Consequently, transcription does not proceed at a high rate until RNA processingfactors become associatedwith the CTD, where they are poised to interact with the nascent premRNA as it emergesfrom the surface of the polymerase. r Five different snRNPs interact via basepairing with one another and with pre-mRNA to form the spliceosome(see Figure 8-11). This very large ribonucleoproteincomplex catalyzestwo transesterificationreactions that join two exons and remove the intron as a lariat structure, which is subsequentlydegraded(seeFigure 8-8). r SR proteins that bind to exonic splicing enhancer sequencesin exons are critical in defining exons in the large

Now that we've seenhow pre-mRNAs are processedinto mature, functional mRNAs' we consider how regulation of this processcan contribute to genecontrol. Recall from Chapter 5 that higher eukaryotescontain both simple and complex tran-

transcription units 1-69o7oof all human transcription units) can be processedin alternative ways to yield different mRNAs that encodedistinct proteins (seeFigure 6-3).

A l t e r n a t i v eS p l i c i n gl s t h e P r i m a r yM e c h a n i s m for RegulatingmRNAProcessing The discovery that a large fraction of transcription units in higher organisms encode alternatively spliced mRNAs and that differently splicedmRNAs are expressedin different cell types revealedthat regulation of RNA splicing is an importun, g.n"-.ontrol mechanismin higher eukaryotes.Although many examples of cleavageat alternative poly(A) sites in pre-mRNAs are known, alternative splicing of different exons is the more common mechanismfor expressingdifferent O F P R E - m R N AP R O C E S S I N G REGULATION

337

Pre-mRNAs

(a) sxl

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(b) tra

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gsxt'rotein

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(c) dsx

A FIGURE 8-16 Cascade of regulatedsplicingthat controlssex determinationin Drosophilaembryos.Forclarity,onlythe exons (boxes) andintrons(blacklines) whereregulated splicing occursare shownSplicing isindicated by reddashed linesabove(female) and bluedashed linesbelow(male) the pre-mRNAs Vertical redlinesin exonsindicate ln-frame stopcodons, whrchprevent synthesis of functional proteinOnlyfemaleembryos produce functional Sxl protein, whichrepresses splicing betweenexons2 and3 in sxlpremRNA(a)andbetweenexons1 and2 in tra pre-mRNA (b) (c)In contrast, the cooperative bindingof Traproteinandtwo SRproteins,

Rbpl andfra2,activates splicing betweenexons3 and4 and cleavage/polyadenylation Anat the 3' endof exon4 in dsxprem R N Ai n f e m a l e m b r y o sI n m a l ee m b r y o w s ,h i c hl a c kf u n c t i o n a l Tra,the SRproteins do not bindto exon4, andconsequently exon3 isspliced to exon5 ThedistinctDsxproteins produced in female andmaleembryos asthe resultof thiscascade of regulated splicing repress transcription of genesrequired for sexual differentiation of the opposite sex.[Adapted fromM J Mooreet al, 1993,in R Gesteland press, andJ Atkrns, eds,IheRNAWorld, ColdSpring pp 303-357 Harbor l

proteins from one complex transcription unit. In Chapter 4, for example, we mentioned that fibroblasts produce one type of the extracellular protein fibronectin, whereas hepatocytes produce another type. Both fibronectin isoforms are encoded by the same transcription unit, which is spliced differendy in the two cell types to yield two different mRNAs (seeFigure4-16).ln other cases,alternativeprocessingmay occur simultaneouslyin the samecell type in responseto different developmentalor environmental signals.First we discussone of the best-undersrood examplesof regulatedRNA processingand then briefly consider the consequencesof RNA splicing in the developmentof rhe nervous sysrem.

The Sxl protein, encodedby the sex-lethalgene,isthe first protein to act in the cascade(Figure 8-16). The Sxl protein is present only in female embryos. Early in development,the gene is transcribed from a promoter that functions only in female embryos. Later in development,this female-specific promoter is shut off and another promoter for sex-letbal becomesactive in both male and female embryos. However, in the absenceof early Sxl protein, the sex-lethalpre-mRNA in male embryos is spliced to produce an mRNA that contains a stop codon early in the sequence.The net result is that male embryos produce no functional Sxl protein either early or later in development. In contrast, the Sxl protein expressedin early female embryos directs splicing of the sex-lerEal pre-mRNA so that a functional sex-lethalmRNA is produced (Figure 8-16a). Sxl accomplishesthis by binding to a sequencein the pre-mRNA near the 3' end of the intron between exon 2 and exon 3, thereby blocking the proper association of U2AF and tJ2 snRNP.As a consequence, the U1 snRNP bound to the 3, end of exon 2 assembles into a spliceosomewith U2 snRNp bound to the branch point at the 3' end of the intron betweenexons 3 and 4,leading to splicing of exon 2 to 4 and skipping of exon 3. The resulting female-specificsex-lethal mRNA is translated into functional Sxl protein, which reinforces its own expressionin female embryos by continuing to cause skipping of exon 3. The absenceof Sxl protein in male embryos allows the inclusion of exon 3 and, consequently,of the stop codon that preventstranslation of functional Sxl protein.

A Cascade o f R e g u l a t e dR N AS p l i c i n gC o n t r o l s D r o s o p h i l aS e x u a lD i f f e r e n t i a t i o n One of the earliestexamplesof regulatedalternarivesplicing of pre-mRNA came from studiesof sexual differentiaiion in

splicing in Drosophila embryos. More recent researchhas provided insight into how theseproteins regulate RNA processingand ultimately lead to the creation of two different sex-specifictranscriptionalrepressorsthat suppressthe development of characteristicsof the oppositesex. 338

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posr-TRANscRtploN GA ELN E coNTRoL

U2AF and U2 snRNP to the 3' end of the intron between exons 3 and 4, just as other SR proteins do for constitutively spliced exons (see Figure 8-13). The TralTra2lRbpl complexes may also enhancebinding of the cleavage/polyadenylation complex to the 3' end of exon 4.

and ActivatorsControl SplicingRepressors Splicingat Alternative Sites

d

A FIGURE 8-17 Modelof splicingactivationby Traproteinand embryos, the 5RproteinsRbpl and Tra2.In femaleDrosophila isactivated by bindingof splicing of exons3 and4 in dsxpre-mRNA Rbpl andTra2 fra/Tra2lRbp1 complexes to sixsitesin exon4 Because in theabsence of Tra,exon4 isskipped cannotbindto the pre-mRNA A, : polyadenylation in maleembryos. Seethetextfor discussion 2002,Nature 418:236] fromT.Maniatis andB Tasic, lAdapted Sxl protein also regulatesalternative RNA splicing of the transformergenepre-mRNA (Figure8-16b).In male embryos, where no Sxl is expressed,exon 1 is spliced to exon 2, which contains a stop codon that prevents synthesisof a functional transformer protein. In female embryos, however, binding of Sxl protein to the 3' end of the intron between exons 1 and 2 blocks binding of U2AF at this site. The interaction of Sxl with transformer pre-mRNA is mediated by two RRM domains in the protein (seeFigure 8-5).Vhen Sxl is bound, U2AF binds to a lower-affiniry site farther 3' in the pre-mRNA; as a result exon 1 is spliced to this alternative 3' splice site, eliminating exon2 with its stop codon. The resulting female-specifictransformer nRNA, which contains additional constitutively spliced exons, is translated into functional Transformer (Tra) protein. Finally, Tra protein regulatesthe alternativeprocessingof pre-mRNA transcribed from the double-sex gene (Figure 8-16c). In female embryos,a complex of Tra and two constitutively expressedproteins,Rbpl and Tra2, directssplicingof exon 3 to exon 4 and also promotescleavage/polyadenylation at the alternativepoly(A) site at the 3' end of exon 4-leading to a short, female-specificversion of the Dsx protein. In male embryos,which produce no Tra protein, exon 4 is skipped,so that exon 3 is spliced to exon 5. Exon 5 is constitutively splicedto exon 6, which is polyadenylatedat its 3' end-leading to a longer,male-specificversion of the Dsx protein. As a result of the cascadeof regulatedRNA processingdepictedin Figure 8-16, different Dsx proteins are expressedin male and femaleembryos.The male Dsx protein is a transcriptionalrepressor that inhibits the expression of genes required for female development.Conversely,the female Dsx protein repressestranscription of genesrequired for male development. complex Figure 8-17 illustrates how theTrafta2iRbpl is thought to interact with double-sex(dsx)pre-mRNA. Rbpl andTra2 are SR proteins, but they do not interact with exon 4 in the absenceof the Tra protein. Tra protein interactswith Rbpl and Tra2, resulting in the cooperative binding of all three proteins to six exonic splicing enhancersin exon 4. The bound Tra2 and Rbpl proteins then promote the binding of

As is evident from Figure 8-15, the Drosophila Sxl protein and Tra protein have opposite effects:Sxl prevents splicing' causingexons to be skipped, whereasTra promotes splicing. The action of similar proteins may explain the cell-typespecificexpressionof fibronectin isoforms in humans. For instance,an Sxl-like splicingrepressorexpressedin hepatocytes might bind to splicesitesfor the EIIIA and EIIIB exons in the fibronectin pre-mRNA, causing them to be skipped during RNA splicing (seeFigure4-16). Alternatively,a Tralike splicing activator expressedin fibroblastsmight activatethe splice sites associatedwith the fibronectin EIIIA and EIIIB exons' leading to inclusion of these exons in the mature mRNA. Experimental examination in some systemshas revealedthat inclusion of an exon in some cell types versusskipping of the same exon in other cell types results from the combined influenceof severalsplicing repressorsand enhancers. Alternative splicing of exons is especially common in the nervous system,generatingmultiple isoforms of many proteins required for neuronal developmentand function in both vertebratesand invertebrates.The primary transcripts from thesegenesoften show complex splicing patterns that can generate several different mRNAs' with different spliced forms expressedin different anatomical locationswithin the central nervous system.We consider two remarkable examplesthat illustrate the critical role of this processin neural function. Expression of K*-Channel Proteins in Vertebrate Hair Cells In the inner ear of vertebrates, individual "hair cells," which are ciliated neurons' respond most strongly to a specific frequency of sound. Cells tuned to low frequency (:50 Hz) are found at one end of the tubular cochlea that makes up the inner ear; cells responding to high frequency (:5000 Hz\ arefound at the other end (Figure8-18a).Cells in between the ends respond to a gradient of frequenciesbetween theseextremes.One component in the tuning of hair cells in reptiles and birds is the opening of K*ion channelsin resDonseio increasedintracellular Ca2*concentrations.The Ca2* concentration at which the channel opens determines the frequency with which the membrane potential oscillates and hencethe frequency to which the cell is tuned' The gene encoding this Ca2+-activatedK+ channel is expressedas multiple, alternatively spliced mRNAs. The various proteins encodedby thesealternative mRNAs open at different Ca2* concentrations. Hair cells with different response frequenciesexpressdifferent isoforms of the channel protein depending on their position along the length of the cochlea (see Figure 23-30). The sequencevariation in the protein is very complex: there are at leasteight regions in the mRNA where alternative exons are utilized, permitting the expression of 576 possibleisoforms(Figure8-18b).PCR analysisof mRNAs from O F P R E - m R N AP R O C E S S I N G REGULATION

339

(a)

through post-translational modifications of splicing factors playsa significantrole in modularingneuron function.

Apical h a i rc e l l ( 5 0H z )

Auditory nerve cell body

Basal h a i rc e l l (5000Hz)

Cytosol

<7

FIGURE 8-18 Roleof alternativesplicingin the perception of soundsof differentfrequency.(a)Thechicken cochlea, a 5mm-longtube,contains an epithelium of auditory haircellsthatare tunedto a gradient of vibrational frequencies from50 Hzat the apicalend(/eft)to 5000Hzat the basalend(nght).(b)TheCa2*activated K* channel contains (50-56), seven transmembrane o helices whichassociate to formthechannelThecytosolic domain,which includes fourhydrophobic (S7-S10), regions regulates openingof the channel in response to Ca2* lsoforms of thechannel, encoded by alternatively spliced produced mRNAs fromthe sameprimary transcript, openat different Ca2*concentrations andthusrespond to different frequencies Rednumbers referto regions wherealternative splicing produces different aminoacidsequences in thevarious isoforms [ A d a p t e df r o m K P R o s e n b l a tet l a l , 1 9 9 7 , N e u r o n1 9 : 1 0 6 1]

individual hair cellshas shown that each hair cell expressesa mixture of different alternative Ca2*-activated K+-channel mRNAs, with different forms predominating in different cells according to their position along the cochlea.This remarkable arrangementsuggeststhat splicing of the Ca2*-activatedK+channel pre-mRNA is regulatedin responseto extracellular signals that inform the cell of its position along the cochlea. Other studiesdemonstratedthat splicing ar one of the alternative splice sites in the Ca2+-activatedK+-channel premRNA in the rat is suppressedwhen a specificprotein kinase is activatedby neuron depolarizationin responseto synaptic activity from interactingneurons.This observationraisesthe possibilitythat a splicingrepressorspecificfor this site may be activated when it is phosphorylated by this protein kinase, whose activity in turn is regulatedby synaptic activity. Since hnRNP and SR proteinsare extensivelymodified by phosphorylation and other post-translationalmodificarions, it seems likely that complex regulation of alternative RNA splicing

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Many examples of genessimilar to the cochlear K*channel neurons have been observed in vertebrate neurons; alternatively spliced mRNAs co-expressedfrom a specific gene in one type of neuron are expressedat different relativeconcentrationsin different regionsof the central nervous system.Expansions in the number of microsatellite repeatswithin the transcribedregionsof genesexpressedin neurons can causean alteration in the relative concentrationsof alternativelysplicedmRNAs transcribedfrom multiple genes. In Chapter 6, we discussedhow backward slippage during DNA replication can lead to expansionof a microsatelliterepeat (seeFigure 6-5). At least 14 different types of neurological diseaseresult from expansion of microsatellite regions within transcription units expressedin neurons.The resulting long regionsof repeatedsimple sequences in nuclearRNAs of theseneurons resultsin the abnormalitiesin the relative concentrationsof alternativelysplicedmRNAs. For example,the most common of thesetypes of diseases, myotonic dystrophy, is characterizedby paralysis,cognitive impairment, and personality and behavior disorders.Myotonic dystrophy results from increasedcopiesof either CUG repeatsin one transcripr in some patients or CCUG repeatsin another transcript in other patients.When the number of theserepeatsincreasesto 10 or more times the normal number of repeatsin thesegenes, abnormalitiesare observedin the levelof two hnRNP proteins that bind to these repeatedsequences.This probably results becausethesehnRNPs are bound by the abnormally high concentration of this RNA sequencein the nuclei of neurons in such patients.The abnormal concentrationsof thesehnRNP proteins are thought to lead to alterationsin the rate of splicing of different alternative splice sites in multiple pre-mRNAs normally regulatedby thesehnRNP proteins.I Expression of Dscam lsoforms in Drosophila Retinal Neurons The most extreme example of regulated alternative RNA processingyet uncovered occurs in expressionof the Dscam gene in Drosophila. Mutations in this gene interfere with the normal synaptic connectionsmade befweenaxons and dendrites during fly development.Analysis of the Dscam gene showedthat it contains 95 alternativelysplicedexonsthat could be splicedto generateover 38,000 possibleisoforms!Recentresults have shown that Drosopbila matantswith a version of the gene that can be spliced in only about 22,000 different ways have specificdefectsin connectivity betweenneurons.Theseresults indicate that expressionof most of the possibleDscam tsoforms through regulatedRNA splicing helps to specify the tens of millions of different specific synaptic connections between neurons in the Drosophila brain. In other words, the correct wiring of neurons in the brain requiresregulatedRNA splicing.

R N AE d i t i n gA l t e r st h e S e q u e n c e s of SomePre-mRNAs In the mid-1980s,sequencing of numerouscDNA clonesand corresponding genomic DNAs from multiple organisms led

CAA

-

t

apoB gene

apoB mRNA

5',

CAA+UAA v

UAA

.t,

A

2152 Proteins

cooH

NH,

cooH

NH,

APoB-48

ApoB-100 FfGURE8-19 RNAeditingof apo-Bpre-mRNA. TheapoB mRNAoroduced in the liverhasthesameseouence asthe exonsin t h ep r i m a rtyr a n s c r i pTth i sm R N Ai st r a n s l a t ei ndt oa p o B - 1 0w 0 ,h i c h ao l m a i n( g r e e nt )h a t h a st w o f u n c t i o n adlo m a i n sa:n N - t e r m i n d with lipidsanda C-terminal thatbindsto associates domain(orange)

in In theapo-BmRNAproduced on cellmembranes LDLreceptors stop to a UAA 26 is edited in exon CAA codon the the intestine. whichcorresponds apoB-48, intestinal cellsproduce codonAsa result, and fromP Hodges domainof apoB-100lAdapted to the N-terminal

to the unexpected discovery of another type of pre-mRNA processing.In this type of processing,called RNA editing, the sequenceof a pre-mRNA is altered;as a result, the sequenceof the correspondingmature mRNA differs from the exons encoding it in genomic DNA. RNA editingis widespreadin the mitochondriaof protozoansand plants and also in chloroplasts.In the mitochondria of certainpathogenictrypanosomes,more than half the sequenceof some mRNAs is altered from the sequenceof the corresponding primary transcripts. Additions and deletions of specificnumbers of U's follows templatesprovided by base-pairedshort "guide" RNAs. TheseRNAs are encoded by thousandsof mini-mitochondrialDNA circlescatenated to many fewer large mitochondrial DNA molecules.The reason for this baroquemechanismfor encodingmitochondrial proteinsin suchprotozoansis not clear.But this systemdoes rcpresenta potential target for drugs to inhibit the complex processingenzymesessentialto the microbe that do not exist in the cellsof their human or other vertebratehosts. In higher eukaryotes,RNA editing is much rarer,and thus far, only single-basechangeshave beenobserved.Such minor editing, however,turns out to have significantfunctional conin some cases.An important exampleof RNA editsequences ing in mammals involves the apoB gene. This gene encodes two alternativeforms of a serumprotein central to the uptake and transport of cholesterol.Consequently,it is important in the artethe pathogenicprocesses that lead to atherosclerosls, rial diseasethat is the major causeof death in the developed world. The apoB gene expressesboth the serum protein the major apolipoproteinB-100 (apoB-100)in hepatocytes, cell type in the liver, and apoB-48, expressedin intestinalepithelial cells.The :240-kDa apoB-48 correspondsto the Nterminal region of the :500-kDa apoB-100. As we detail in Chapter 10, both apoB proteins are componentsof large lipoprotein complexesthat transport lipids in the serum.However, only low-density lipoprotein (LDL) complexes,which contain apoB-100on their surface,delivercholesterolto body tissuesby binding to the LDL receptorpresenton all cells. The cell-type-specific expressionof the two forms of apoB results from editing of apoB pre-mRNA so as to changethe

nucleotideat position 6666 in the sequencefrom a C to a U. This alteration, which occurs only in intestinalcells,converts a CAA codon for glutamine to a UAA stop codon, leadingto synthesisof the shorter apoB-48 (Figure 8-19)' Studieswith the partially purified enzymethat performs the post-transcriptional deaminationof C6666to U shows that it can recognize and edit an RNA as short as 26 nucleotideswith the sequence surrounding C5566in the apoB primary transcript.

J Scott, 1992, TrendsBiochem Sci 17:77 l

Regulation of Pre-mRNAProcessing r Becauseof alternativesplicing of primary transcripts,the use of alternative promoters, and cleavageat different poly(A) sites,different mRNAs may be expressedfrom the same gene in different cell types or at different developmental stages(seeFigure 6-3 and Figure 8-16). r Alternative splicing can be regulated by RNA-binding proteins that bind to specific sequencesnear regulated splice sites. Splicing repressorsmay sterically block the binding of splicing factors to specific sites in pre-mRNAs or inhibit their function. Splicing activators enhancesplicing by interacting with splicing factors, thus promoting their associationwith a regulated splice site. r In RNA editing the nucleotide sequenceof a pre-mRNA is altered in the nucleus.In vertebrates,this processis fairly rare and entails deamination of a single basein the mRNA sequence,resulting in a change in the amino acid specified by the correspondingcodon and production of a functionally differentprotein (seeFigure 8-19).

Ifil

of mRNAAcross Transport

the NuclearEnvelope Fully processedmRNAs in the nucleus remain bound by hnRNP proteins in complexes now referred to as nuclear zRNPs. Before an mRNA can be translated into its encoded protein. it must be exported out of the nucleusinto ENVELOPE THENUCLEAR T R A N S P O ROTF m R N A A C R O S S

341

the cytoplasm. The nuclear envelopeis a double membrane that separatesthe nucleusfrom the cytoplasm(seeFigure9-1). Like the plasma membrane surrounding cells, each nuclear membrane consistsof a water-impermeablephospholipid bilayer and multiple associatedproteins. mRNPs and other

macromolecules including tRNAs and ribosomal subunits traverse the nuclear envelope through nwclear pores. This section will focus on the export of mRNPs through the nuclear pore and the mechanismsthat allow some level of regulation of this step.

(al

NuclearPoreComplexesControl lmport a n d E x p o r tf r o m t h e N u c l e u s

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rlir:a:i.t:rjr ,

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(d)

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A FIGURE8-20 Model of transporter passagethrough an NpC. (a) Diagramof NPCstructure(b)The FG-domarns of FG-nucleoporrns arethoughtto havean extendedconformationwith long hydrophilic regionsof polypeptide separatedby shortFG-hydrophobic domains (c) FG-repeats arethoughtto associate with eachother reversibly and rapidly,forminga constantlyre-arranging molecularsievethat allows diffusionof smallwater-soluble molecules throughit However, macromolecules aretoo big to fit throughthe channelsin the molecularsieve (d) Nucleartransporters havehydrophobicregionson their surface(darkbluedots)that bind reversibly to the FG-domains in the FG-nucleoporins As a consequence, they can penetratethe molecularsievein the NPCcentralchanneland diffusein and out of the nucleus[FromK Ribbeck andD Gorlich, zOOj,EMBO I 20:1320 ] 342

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c H A p r E R8

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p o s r - T R A N s c R t p l o N AGLE N Ec o N T R o L

Nuclear pore complexes (NPCs) are large, complicated structurescomposedof multiple copiesof approximately 30 different proteins called nucleoporins. Embedded in the nuclear envelope, NPCs are roughly octagonal in shape with eight filaments extending into the nucleoplasm and another eight filaments extending into the cytoplasm. (Figure 8-20a; seealso Figure 13-32 for a more detailed discussionof NPC structure). The filaments extending from the nuclear face of the NPC are joined at their ends by a terminal ring forming a structure called the nuclear basket. A special class of nucleoporins called FG-nucleoporins line the central channel through the NPC. FG-nucleoporins contain long stretches of hydrophilic amino acid sequencespunctuated by hydrophobic FG-repeats,short sequencesrich in hydrophobic phenylalanine(F) and glycine(G). 'Water, ions, metabolites,and small globular proteins up to =40 kDa can diffuse through the water-filled channel in the nuclear pore complex. However, FG-nucleoporinsin the central channel form a barrier restricting the diffusion of macromoleculesbetweenthe cytoplasm and nucleus. These larger molecules must be selectivelytransported across the nuclear envelope with the assistanceof soluble transporter proteins that bind them and also interact with FG-repeatsof FG-nucleoporins. In one current model of the nuclear pore complex central channel, FG-repeat domains of FG-nucleoporins form random coil regions of polypeptide that extend into the channel (Figure 8-20b). The FG-repeat domains of different FGnucleoporin molecules are proposed to associatewith each other reversibly,forming a constantly rearranging molecular meshwork that acts like a sieve(Figure8-20c). Small molecules can diffuse through the spaces between FG-repeat domains. But large proteins and ribonucleoproteins, including mRNPs, are too large to pass betweenthe protein filaments that form the molecular sieve and consequently cannot diffuse through the nuclear pore complex. Nuclear transport proteins bind reversibly to the hydrophobic FG-regionsof FG-nucleoporins.These interactions are thought to involve multiple surfacesof the transporters, allowing the proteins to diffuse through the central channel(Figure8-20d). mRNPs are transported through the NPC by the zRNP exporter. The mRNP exporter is a heterodimer consisting of a large subunit, called nuclearexport factot | (NXF1) or TAP,and a small subunit, ntclear export transporter1 (Nxt1). TAP binds nuclear mRNPs through associationswith both RNA and other proteins in the mRNP complex. One of the most important of these is REF (RNA export factor), a component of the exon junction complexes discussed

of mRNPs 8-21 Remodeling < FIGURE proteins during nuclearexport.SomemRNP (rectangles) mRNP fromnuclear dissociate beforeexportthroughan NPC complexes throughthe NPC areexported Some(ovals) in the but dissociate with the mRNP associated andareshuttledbackintothe cytoplasm n u c l e utsh r o u g ha n N P Cl n t h ec y t o p l a s m , CBC replaces factorelF4E initiation translation replaces boundto the 5' capandPABPI PABPII. r4*1g*!s:ii!llttl!irlb4*e*'.**,,,r,,uuu*,r,.,r,rt,*.r**u,*,r,urril

Gytoplasm

earlier, bound approximately 20 nucleotides 5' to each exon-exon junction (Figure 8-21). The TAP/Nxt1 nRNP exporter also associateswith SR proteins bound to exonic splicingenhancers.Thus SR proteins associatedwith exons function to direct both the splicing of pre-mRNAs and the export of fully processedmRNAs through NPCs to the cytoplasm. mRNPs are probably bound along their length by several TAPiNxtl mRNP exporters, which both interact with the FG-domains of FG-nucleoporins to facilitate export of mRNPs through the NPC central channel (Figure 8-20). The filamentsthat extend from the nuclearand cytoplasmic faces of the NPC also assist in mRNP export. Gle2, an adapter protein that reversibly binds both TAP and a nucleoporinin the nuclear basket,brings nuclearmRNPs to the pore in preparationfor export. A nucleoporin in the cytoplasmicfilaments of the NPC binds an RNA helicase that is proposed to function in the dissociationof TAPA{xt1 and other hnRNP proteins from the mRNP as it reachesthe cytoplasm. In a processcalled zRNP remodeling,the proteins associated with an mRNA in the nuclear mRNP complex are exchanged for a different set of proteins as the mRNP is transported through the NPC. Some nuclear mRNP proteins dissociateearly in tranport, remaining in the nucleus to bind to newly synthesizednascentpre-mRNA. Other nuclear mRNP proteins remain with the mRNP complex as it traversesthe pore and do not dissociatefrom the mRNP until the complex reachesthe cytoplasm.Proteinsin this category include the TAP/Nxt1 mRNP exporter, CBC bound to the 5' cap, and PABPII bound to the poly(A) tail. They dissociate from the mRNP on the cytoplasmic side of the NPC throush the action of the RNA helicasethat associateswith

cytoplasmicNPC filaments,as discussedabove.Theseproteins are then imported back into the nucleusas discussed for other nuclear proteins in Chapter 13' where they can function in the export of another mRNP. In the cytoplasm, the cap-binding translation initiation factor eIF4E replaces CBC bound to the 5' cap of nuclearmRNPs. In vertebrates, the nuclearpoly(A)-bindingprotein PABPIIis replacedwith the cytoplasmicpoly(A)-binding protein PABPI (so named becauseit was discoveredbefore PABPII). (Only a single PABP is found in budding yeast' in both the nucleus and the cytoplasm.) Yeast SR Protein Recentresultssuggestthat the direction of mRNP export from the nucleus into the cytoplasm is controlled by phosphorylation and dephosphorylation of mRNP adapterproteins suchas REF that assistin the binding of the TAPNxtl mRNP exporter to mRNPs. In one case,a yeast SR protein (Npl3) functions as an adapter protein that promotes the binding of the yeastmRNP exporter (Figure 822).The SR-protein initially binds to nascentpre-mRNA in its phosphorylatedform.'When 3'-cleavageand polyadenylation are completed,the adapter protein is dephosphorylated by a specificnuclear protein phosphataseessentialfor mRNP export. Only the dephosphorylatedadapter protein can bind the mRNP exporter, thereby coupling mRNP export to correct polyadenylation. This is one form of mRNA "qualify control. " If the nascentmRNP is not correctly processed,it is not recognized by the phosphatasethat dephosphorylates Npl3. Consequently,it is not bound by the mRNA exporter and not exported from the nucleus.Insteadit is degradedby exosomes,the multiprotein complexesthat degrade unprotected RNAs in the nucleusand cytoplasm (seeFigure 8-1)' ' T R A N S P O RO T F m R N A A C R O S ST H E N U C L E A RE N V E L O P E

343

RNApol ll R N Ap o l l l

R N Ap o l l l

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z

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A FIGURE 8-22 Reversible phosphorylation and directionof mRNPnuclearexport.StepIl: TheyeastSRproteinNpl3binds nascent pre-mRNAs in itsphosphorylated form StepA When p o l y a d e n y l a thi oanso c c u r r esdu c c e susl fl yt,h e G l c Tn u c l e a r p h o s p h a t aesses e n t ifaolr m R N pe x p o rdt e p h o s p h o r y l aNt e p sl 3 , promoting the bindingof the yeastmRNpexporter, TAp/Nxt1. Step B. the mRNP e x p o r t earl l o w sd i f f u s i oonf t h e m R N pc o m p l e x t h r o u g ht h ec e n t r acl h a n n eolf t h e n u c l e acro r ec o m p l e(xN p C )

Step4 Thecytoplasmic proteinkinase Skyl phosphorylates Npl3in the cytoplasm, causing E dissociation of the mRNP exporter, and phosphorylated Npl3probably throughtheactionof an RNAhelicase associated with NPCcytoplasmic filaments6 ThemRNAtransporter andphosphorylated Npl3aretransported backintothe nucleus throughNPCsZ Transported mRNAisavailable for translation in thecytoplasm E lzaurralde,2004,Nat StructMol Biol11:Z1O-Z\2 fFrom seew Gilbert andc Guthrie, 2004,Mol Cell13:201-2121

Following exporr to the cytoplasm, the Npl3 SR protein is phosphorylated by a specific protein kinase resrricted to the cytoplasm.This causesit to dissociatefrom the mRNp, along with the mRNP exporter. In this wa5 dephosphorylation of adapter mRNP proteins in the nucleus once RNA processingis complete and their phosphorylation in the cytoplasm results in a higher concentration of mRNp exporter-mRNP complexesin the nucleus,where they form, and a lower concentration of these complexes in the cyto-

plasm, where they dissociate.As a result, the direction of mRNP export may be driven by simple diffusion down a concentration gradient of the transport-competent mRNP exporter-mRNP complex across the NPC from high in the nucleusto low in the cytoplasm.

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CHAPTER 8

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P o S T - T R A N S C R t p T t OG NE AN L EC O N T R O L

Nuclear Export of Balbiani Ring mRNPs The larval salivary glands of the insect Chironomows tentans provide a good model systemfor electron microscopy studiesof the

8-23 Formationof heterogeneousribonucleoprotein < FIGURE from the nucleus' and exportof mRNPs particles(hnRNPs) of loopandassembly (a)Modelof a singlechromatin transcription RNA tentansNascent in Chironomous ring(BR)mRNP Balbiani with produced associate DNArapidly fromthetemplate transcripts in sizeof the hnRNPs increase Thegradual proteins, forminghnRNPs distances at greater lengthof RNAtranscripts the increasing reflects from startsite.Themodelwasreconstructed fromthetranscription glandcells. of salivary of serialthinsections micrographs electron s .o l l o w i n g ( b )S c h e m a tdi ci a g r a m o f t h e b i o g e n e soi sf h n R N P F particle ribonucleoprotein the resulting processing of the pre-mRNA, (c) of BR mRNPs transport for the Model mRNP as an referred to is (NPC) microscopic on electron porecomplex based throughthe nuclear appearto uncoilastheypass Notethatthe curvedmRNPs studres, it rapidly pores. Asthe mRNAentersthecytoplasm, throughnuclear passes through 5' end that the indicating ribosomes, with associates (a)fromC Erricson courtesy of Ceil55:631; etal, 1989, the NPCfirst [Part

\ Template DNA

(c)

mRNP

B Daneholt Parts(b) and (c) adaptedfrom B Daneholt,1997, Ce//88:585 Seealso B Daneholt,2001, Proc Nat'l Acad Sci USA 98:7012 l

N u c l e a re n v e l o P e

Nucleoplasm i :i Cytoplasm

formation of hnRNPs and their export through NPCs. In these larvae, genesin large chromosomal puffs called Balbiani rings are abundantly transcribedinto nascentpremRNAs that associate with hnRNP proteins and are processedinto coiled mRNPs with an mRNA of =75 kb (Figure 8-23a, b). These giant mRNAs encode large glue proteins that adhere the developinglarvae to a leaf. After processingof the pre-mRNA in Balbiani ring hnRNPs, the resulting mRNPs move through nuclear pores to the cytop l a s m . E l e c t r o n m i c r o g r a p h s o f s e c t i o n so f t h e s e c e l l s show mRNPs that appear to uncoil during their passage through nuclear pores and then bind to ribosomesas they enter the cytoplasm. This uncoiling is probably a consequenceof the remodeling of mRNPs as the result of phosphorylation of mRNP proteins by cytoplasmickinasesand the action of an RNA helicaseassociatedwith NPC cytoplasmic filaments,as discussedin the previous section.The o b s e r v a t i o n t h a t m R N P s b e c o m e a s s o c i a t e dw i t h r i b o somesduring transport indicatesthat the 5' end leads the way through the nuclear pore complex. Detailed electron microscopic studies of the transport of Balbiani ring

mRNPs through nuclear pore complexesled to the model depictedin Figure 8-23c.

Are Not Exported in Spliceosomes Pre-mRNAs from the Nucleus It is critical that only fully processedmature mRNAs be exported from the nucleusbecausetranslation of incompletely processedpre-mRNAs containing introns would produce defectiveproteins that might interfere with the functioning of the cell. To prevent this, pre-mRNAs associatedwith snRNPs in spliceosomesusually are prevented from being transportedto the cytoplasm. In one type of experiment demonstratingthis restriction' gene encoding a pre-mRNA with a single intron that nora mally is splicedout was mutated to introduce deviationsfrom Mutation of either the 5' the consensussplice-sitesequences. or the 3'invariant splice-sitebasesat the ends of the intron resultedin pre-mRNAs that were bound by snRNPs to form spliceosomes;however, RNA splicing was blocked, and the ore-mRNA was retainedin the nucleus.In contrast, mutation

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of both the 5'and 3'splice sitesin the samepre-mRNA resulted in export of the unspliced pre-mRNA, although less efficiently than for the splicedmRNA.'fi/hen both splicesites were mutated, the pre-mRNAs were not efficiently bound by snRNPs,and, consequently,their export was not blocked. Recentstudiesin yeasthave shown that a nuclear protein that associates with a nucleoporinin the NPC nucleaibasket is requiredto retain pre-mRNAs associatedwith snRNPsin the nucleus.If either this protein or the nucleoporin to which it binds is deleted,unsplicedpre-mRNAs are exported. Many casesof thalassemia,an inherited diseasethat resultsin abnormallylow levelsof globin proterns,are due to mutationsin globin-genesplicesitesthat decreasethe efficiency of splicing but do not prevent association of the pre-mRNA with snRNPs. The resulting unspliced globin pre-mRNAs are retained in reticulocyte nuclei and are rapidly degraded.I

HIV Rev ProteinRegulatesthe Transport o f U n s p l i c e dV i r a l m R N A s As discussedearlier,transport of mRNPs containing marure, functional mRNAs from the nucleusto the cytoplasm entails a complex mechanismthat is crucial to gene expression(see Figures8-21, 8-22, and 8-23). Regulationof this transport theoreticallycould provide another meansof genecontrol, although it appearsto be relativelyrare. Indeed,the only known examplesof regulatedmRNA export occur during the cellular

responseto conditions (e.g.,heat shock)that causeprotein denaturation or during viral infection when virus-inducedalterations in nuclear transport maximize viral replication. Here we describethe regulation of mRNP export mediated by a protein encodedby human immunodeficiencyvirus (HIV). A retrovirus, HIV integratesa DNA copy of its RNA genomeinto the host-cellDNA (seeFigure4-49).The integrated viral DNA, or provirus, contains a single transcription unit, which is transcribedinto a singleprimary transcript by cellular RNA polymeraseII. The HIV transcript can be spliced in alternative ways to yield three classesof mRNAs: a 9-kb unsplicedmRNA; :4-kb mRNAs formed by removal of one intron; and:2-kb mRNAs formed by removal of two or more introns (Figure 8-24). After their synthesisin the host-cell nucleus, all three classesof HIV mRNAs are transported to the cytoplasm and translated into viral proteins; some of the 9-kb unspliced RNA is used as the viral genomein progeny virions that bud from the cell surface. Sincethe 9-kb and 4-kb HIV mRNAs contain splicesites, they can be viewed as incompletely spliced mRNAs. However, as discussedearlier,associationof such incompletely spliced mRNAs with snRNPs in spliceosomesnormally blocks their export from the nucleus.Thus HIV, as well as other retroviruses, must have some mechanism for overcoming this block, permitting export of the longer viral mRNAs. Some retroviruseshave evolved a sequencecalled the constitutiue trdnsport element (CTE), which binds to the TAP-Nxt1 mRNP exporter with high affintty,thereby permitting export

H I Vp r o v i r u s

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9-kb Unspliced

9kb

4-kb S i n g l ys p l i c e d

2-kb M u l t i p l ys p l i c e d

-+ 2 kb Nucleoplasm

A FIGURE8-24 Transport of HIV mRNAs from the nucleus to the cytoplasm. The HIVgenome,which containsseveralcoding regions,is transcribedinto a single9-kb primarytranscriptSeveral =4-kb mRNAsresultfrom alternativesplicingout of any one of severalintrons(dashedlines)and several=2-kb mRNAsfrom splicing out of two or more alternativeintrons After transportto the

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cytoplasm,the variousRNAspeciesare translatedinto differentviral proteins Revprotein,encodedby a 2-kb mRNA,interactswith the Rev-response element(RRE) in the unspliced and singlysplicedmRNAs, stimulatingtheir transportto the cytoplasm[Adapred fromB R Cullen andM H Malim,1991,Trends BiochemScl 16:346I

of unsplicedretroviral RNA into the cytoplasm. HIV solved the problem differently. Studies with HIV mutants showed that transport of unspliced 9-kb and singly spliced4-kb viral mRNAs from the nucleus to the cytoplasm requires the virus-encoded Rev protein. Subsequentbiochemicalexperimentsdemonstrated that Rev binds to a specificRev-responseelement (RRE) present in HIV RNA. In cells infected with HIV mutants lacking the RRE, unspliced and singly spliced viral mRNAs remain in the nucleus, demonstrating that the RRE is required for Rev-mediated stimulation of nuclear export. Rev contains a leucine-richnuclear export signal that interacts with transporter exportinl. As discussed in Chapter 13, this results in export of the unspliced and singly spliced HIV mRNAs through the nuclear pore complex. I

Transport of mRNA Across the Nuclear Envelope r Most mRNPs are exported from the nucleus by a heterodimeric mRNP exporter that interacts with FG-repeats of FG-nucleoporins(see Figure 8-20). The direction of transport (nucleus-+ cytoplasm) may result from dissociation of the exporter-mRNP complex in the cytoplasm by phosphorylation of mRNP proteins by cytoplasmic kinases and the action of an RNA helicase associatedwith the cytoplasmic filaments of the nuclear pore complexes (see Figure8-20). r The mRNP exporter binds to most mRNAs cooperatively with SR proteins bound to exons and REF associated with exon-junction complexesthat bind to mRNAs following RNA splicing, and to additional mRNP proteins. r Pre-mRNAs bound by a spliceosomenormally are not exported from the nucleus, ensuring that only fully processed,functional mRNAs reach the cytoplasm for translation.

Cytoplasmic Mechanisms f[ of Post-transcriptional Control Before proceeding,let's quickly review the stepsin gene ex'We pression at which control is exerted. saw in the previous chapter that regulation of transcription initiation is the principal mechanism for controlling the expressionof genes.In preceding sections of this chapter, we also learned that the expression of protein isoforms is controlled by regulating alternative RNA splicing. Although nuclear export of fully and correctly processedmRNPs to the cytoplasm is rarely regulated, the export of improperly processedor aberrantly remodeled pre-mRNPs is prevented, and such abnormal transcripts are degraded by the exosome. However, retroviruses, including HIV, have evolved mechanismsthat permit pre-mRNAs that retain splice sites to be exported and translated(seeFigure 8-24).

In this section we consider other mechanismsof posttranscriptional control that contribute to regulating the expression of some genes.Most of these mechanismsoperate localization in the cytoplasm, controlling the stability or.!7e begin by of mRNA or its translation into protein. discussingtwo recently discoveredand related mechanisms of gene control that provide powerful new techniques for manipulating the expressionof specificgenesfor experimental and therapeutic purposes. These mechanisms are controlled by short, -21 nucleotide, single-strandedRNAs called micro RNAs (miRNAs) and short interfering (siRNAs). Both base-pairwith specifictarget mRNAs, either inhibiting their translation (miRNAs) or causingtheir degradation (siRNAs). Humans express=1,000 miRNAs. Most of theseare expressedin specificcell types at particular times during embryogenesisand after birth. Many miRNAs can target more than one mRNA. Consequently,thesenewly discovered mechanismscontribute significantly to the regulation of gene expression. siRNAs, involved in the process called RNA interference, are also an important cellular defenseagainstviral infection and excessivetransposition by transposons.

Micro RNAsRepressTranslation of SpecificmRNAs Micro RNAs (miRNAs) were first discoveredduring analysis of mutations in the lin-4 and let-7 genesof the nematode C. elegans,which influence development of the organism. Cloning and analysis of wild-type lin-4 and let-7 revealed that they encode no protein products but rather RNAs only 2t and 22 nucleotides long, respectively.The RNAs hybridize to the 3' untranslated regions of specific target mRNAs. For example, the lin-4 miRNA, which is expressed early in embryogenesis,hybridizes to the 3' untranslatedregions of both the lin-14 and /lz-28 mRNAs in the cytoplasm, thereby repressingtheir translation by a mechanism discussedbelow. Expressionof lin-4 miRNA ceaseslater in development, allowing translation of newly synthesizedlin-14 and lin-28 mRNAs at that time. Expressionof /er-7 miRNA occurs at comparable times during embryogenesisof all bilaterally symmetric animals. The role of lin-4 and let-7 miRNAs in coordinating the timing of early developmental events in C. elegans is discussedin Chapter 22. Hete we focus on what is currently understood about how miRNAs represstranslation. miRNA regulation of translation appears to be widespreadin all multicellular plants and animals. In the past few years, small RNAs of 20-26 nucleotideshave been isolated, cloned, and sequenced from various tissues of multiple model organisms.Recentestimatessuggestthe expressionof one-third of all human genesis regulated by -1,000 human miRNAs isolated from various tissues.The potential for regulation of multiple mRNAs by one miRNA is great because base pairing between the miRNA and the sequencein the 3'-ends of mRNAs that they regulate need not be perfect (Figure 8-25). In fact, considerableexperimentationwith

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( a ) m i R N A- + t r a n s l a t i o ni n h i b i t i o n p

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CXCR4 miRNAandtargetmRNA(H. sapiensl

miR-166andPHAVOLUIA mRNA(A.thalianal

FIGURE 8-25 Basepairingwith target RNAsdistinguishes miRNAand siRNA.(a)miRNAs hybridize imperfectly withtheir targetmRNAs, repressing translation of the mRNANucleotides 2 to 7 of an miRNA(highlighted blue)arethe mostcritical for targeting

perfectly it to a specific mRNA(b)siRNAhybridizes with itstarget mRNA, causing cleavage of the mRNAat the position indicated by the redarrow,triggering itsrapiddegradation fromp D fndapted Zamore andB Haley, 2005,Sclence 309:1 519 l

syntheticmiRNAs has shown that complementarity between the six or seven5' nucleotides of an miRNA and its target mRNA 3' untranslated region are most critical for target mRNA selection. Most miRNAs are processedfrom RNA polymerase II transcriptsof severalhundred to thousandsof nucleotides in length called pri (for primary transcript)-miRNAs (Figwe 8-26). Pri-miRNAs can contain the sequenceof one or more miRNAs. miRNAs are also processedout of some excisedintrons and from 3' untranslatedregions of some pre-mRNAs. Within these long transcripts are sequences that fold into hairpin structures of -70 nucleotides in length with imperfect base pairing in rhe stem. A nuclear RNase specific for double-strandedRNA called Drosha acts with a nuclear double-stranded RNA-binding protein called DGCRS in humans (Pasha in Drosophila) and cleavesthe hairpin region out of the long precursor RNA, generating a pre-miRNA. Pre-miRNAs are recognized and bound by a specific nuclear transporter, ExportinS, which interacts with the FG-domains of nucleoporins, allowing the complex to diffuse through the inner channel of the nuclearpore complex, as discussedabove (seeFigure 8-20), and in Chapter 13. Once in the cytoplasm, a cytoplasmic double-strandedRNA-specific RNase calledDicer acs with a cytoplasmic double-strandedRNA-binding protein called TRBP in humans (for Tar binding protein; called Loquocious in Drosopbila) to further processthe pre-miRNA into a double-strandedmiRNA. The double-strandedmiRNA is approximately two turns of an A-form RNA helix in length, with strands 21--23 nucleotides long and two unpaired 3'-nucleotides at each end. Finally, one of the two strands is selectedfor assemblyinto a mature RNA-lnduced silencing complex (RISC) containing a single-stranded

mature miRNA bound by a multidomain Argonaute protein, a member of a protein family with a recognizableconserved sequence.SeveralArgonaute proteins are expressed in some organisms,especiallyplants, and are found in distinct RISC complexeswith different functions. The miRNA-RISC complexesassociatewith target mRNPs by basepairing betweenthe Argonaute-bound mature miRNA and complementary regions in the 3'-untranslated regions (3'-UTRs) of target mRNAs (seeFigure 8-25). Inhibition of target mRNA translation requires the binding of two or more RISC complexesto distinct complementary regions in the target mRNA 3'-UTR. It has been suggestedthat this may allow combinatorial regulation of mRNA translation by separately regulating the transcription of two or more different pri-miRNAs, which are processedto miRNAs that are required in combination to suppressthe translation of a specifictarget mRNA. The binding of several RISC complexes to an mRNA inhibit translation initiation by a mechanismcurrently being analyzed.Recent discoveriesshowed that binding of RISC complexescausesthe bound mRNPs to associatewith dense cytoplasmicdomains many times the sizeof a ribosomecalled cytoplasmicRNA-processingbodies,or simply P bodies.P bodies, which will be describedin greater detail below, are sites of RNA degradation that contain no ribosomes or translation factors, potentially explaining the inhibition of translation. The associationwith P bodies may also explain why expressionof an miRNA often decreasesthe stability of a targeted mRNA. As mentioned earlier, approximately 1000 different human miRNAs have beenobserved,many of them expressed only in specific cell types. Determining the function of these miRNAs is currently a highly active area of research.

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Drosha Pasha pre-miR-1-1

Figure 8-16). It binds to the 3' splicesite region in the premRNAs of many genes,leading to exon skipping or use of alternative 3' splice sites.I7hen miR-133 is expressedin differentiating myoblasts, the PTB concentration falls without a significant decreasein the concentrationof PTB mRNA. As a result, alternativeisoforms of multiple proteins important for muscle-cellfunction are expressedin the differentiatedcells. Other examplesof miRNA regulation in various organisms are being discoveredat a rapid pace' Knocking out the dicer geneeliminatesthe generationof miRNA in mammals. This causesembryonic death early in mouse development. However, when dicer is knocked out only in limb primordia, the influence of miRNA on the development of the nonessentiallimbs can be observed(Figure8-27). Although all major cell types differentiated and fundamental aspects of limb patterning were maintained, development was abnormal-demonstrating the importance of miRNAs in regulatingthe proper level of translation of multiple mRNAs. Of the :1,000 human miRNAs, 53 appearto be unique to primates. It seems likely that new miRNAs arose readily during evolution by the duplication of a pri-miRNA gene followed by mutation of basesencodingthe mature miRNA. miRNAs are particularly abundant in plants-more than 1.5 million distinct miRNAs have been characterizedin Arabidopsis tbaliana!

R N AI n t e r f e r e n c eI n d u c e sD e g r a d a t i o n Y RNAs y o m p l e m e n t a rm o f P r e c i s e lC mrH-l-l

a' s'-pQAUAQUUQVuuaunuGccc4un|iltIt|||t Il

RNA interference(RNAi) was discoveredunexpectedlyduring attempts to experimentally manipulate the expression of specificgenes.Researcherstried to inhibit the expression of a gene in C. elegansby microiniecting a single-stranded, complementary RNA that would hybridize to the encoded mRNA and prevent its translation, a method called antisense inhibition. But in control experiments,perfectly base-paired double-strandedRNA a few hundred base pairs long was

5' 3'-oUGUAUGAAGAAAUGUA O GGUp-

I M a t u r em i R - 1 - 1 b o u n dt o a n Argonaute protein

RISC

Thisdiagram shows FIGURE 8-26 miRNAprocessing. miRNATheprtmary andprocessing of themiR-1-1 transcription (pri-miRNA) ll The istranscribed by RNApolymerase miRNA transcript withits Drosha nuclear double-stranded specific endoribonuclease (Pasha proteinDGCRB in partner double-stranded RNA-binding generating maketheinitialcleavages in thepri-miRNA, Drosophila) pre-miRNA by to thecytoplasm a =70nucleotide thatisexported in isfurtherprocessed nuclear transporter Exportin 5 Thepre-miRNA witha two-base singleto a double-stranded miRNA thecytoplasm protein withtheDSRNA-binding 3' endby Dicerinconlunction stranded (Loquatiousin is Finally, Drosophila) oneof the two strands TRBP intoan RISC complex, whereit isboundbyanArgonaute incorporated protein[Adapted 2005, Sclence309:1519 fromP D Zamoreand B Haley, ]

In one example, a specific miRNA called miR-133 is induced when myoblastsdifferentiateinto musclecells.miRthe translationof PTB, a regulatory splicing 133 suppresses factor that functions similarly to Sxl in Drosophila (see

Wild type

Dicer mutant

8-27 miRNAfunctionin limb FIGURE A EXPERIMENTAL normal(/eft)andDicer comparing development.Micrographs day-13mouse (nghf)limbsof embryonic development knockout protein, of joint a marker for the Gd5 immunostained embryos by embryos mouse in developing out is knocked Dicer formation of the Dicergene of Creto inducedeletron expression conditional etal,2005,Proc B D Harfe 5-42)[From onlyin thesecells(seeFigure Natl Acad Sci IJSA102:10898l

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much more effectiveat inhibiting expressionof the genethan the antisensestrand alone. Similar inhibition of geneexpression by an introduced double-strandedRNA soon was observed in plants. In each case, the double-stranded RNA induced degradation of all cellular RNAs containing a sequencethat was exactly the same as one strand of the double-stranded RNA. Becauseof the specificity of RNA interference in targeting mRNAs for destruction, it has become a powerful experimental tool for studying gene function (seeFigure 5-45). Subsequent biochemical studies with extracts of Drosophila embryos showed that a long double-stranded RNA that mediatesinterferenceis initially processedinto a double-stranded short interfering RNA (siRNA). The strands in siRNA contain 21-23 nucleotides hybridized to each other so that the two basesat the 3' end of each strand are single-stranded.Further studies revealed that the cytoplasmic double-strandedRNA-specific ribonucleasethat cleaveslong double-strandedRNA into siRNAs is the same Dicer enzymeinvolved in processingpre-miRNAs after their nuclear export to the cytoplasm (seeFigure 8-27). This discovery led to the realization that RNA interference and miRNA-mediated translational repression are related processes.In both cases,the mature short single-stranded RNA, either mature siRNA or mature miRNA, is assembled into RISC complexesin which the short RNAs are bound by an Argonaute protein. I7hat distinguishesa RISC complex containing an siRNA from one containing an miRNA is that the siRNA base-pairsextensivelywith its target RNA and inducesits cleavage,whereas a RISC complex associatedwith an miRNA recognizes its target through imperfect basepairing and results in inhibition of translation. The Argonaute protein appears to be responsiblefor cleavageof target RNA; one domain of the Argonaure protein is homologous to RNase H enzymesthat degrade the RNA of an RNA-DNA hybrid (seeFigure 5-14). When the 5' end of the short RNA of a RISC complex base-pairsprecisely with a target mRNA over a distanceof one turn of an RNA helix (10-12 base pairs), this domain of Argonaute cleavesthe phosphodiester bond of the target RNA across from nucleotides10 and 1.1of the siRNA (seeFigure 8-25). The cleaved RNAs are releasedand subsequentlydegraded by cytoplasmic exosomesand 5' exoribonucleases.If base pairing is not perfect, the Argonaute domain doesnot cleave or releasethe target mRNA. Instead,if severalmiRNA-RISC complexes associatewith a target mRNA, its translation is inhibited and the mRNA becomesassociatedwith p bodies, where, as mentioned earlier, it is probably degraded by a different and slower mechanismthan the degradation pathway initiated by RISC cleavageof a perfectly complementary target RNA. When double-strandedRNA is introduced into the cytoplasm of eukaryotic cells, it entersthe pathway for assembly of siRNAs into a RISC complex becauseit is recognizedby the cytoplasmic Dicer enzyme and TRBp double-stranded RNA binding protein that processpre-miRNAs (seeFigure 8-25). This processof RNA interferenceis believedto be an ancient cellular defenseagainst certain viruses and mobile 350

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genetic elements in both plants and animals. Plants with mutations in the genes encoding Dicer and RISC proteins exhibit increasedsensitivityto infection by RNA virusesand increasedmovement of transposons within their genomes. The double-stranded RNA intermediates generated during replication of RNA viruses are thought to be recognizedby the Dicer ribonuclease,inducing a RNAi responsethat ultimately degradesviral mRNAs. During transposition, transposonsare inserted into cellular genesin a random orientation, and their transcription from different promoters produces complementary RNAs that can hybridize with each other, initiating the RNAi system that then interferes with the expressionof transposon proteins required for additional transpositions. In plants and C. elegans the RNAi response can be induced in all cells of the organism by introduction of double-strandedRNA into just a few cells. Such organismwide induction requires production of a protein that is homologous to the RNA replicasesof RNA viruses.This finding suggeststhat double-strandedsiRNAs are replicated and then transferred to other cells in theseorganisms.In plants, transfer of siRNAs might occur through plasmodesmata,the cytoplasmicconnectionsbetweenplant cellsthat traversethe cell walls between them (seeFigure 1,9-38).Organism-wide induction of RNA interferencedoesnot occur in Drosophila or mammals, presumably becausetheir genomes do not encodeRNA replicasehomologs. In mammalian cells,the introduction of long RNA-RNA duplex moleculesinto the cytoplasm results in the generalized inhibition of protein synthesisvia the PKR pathway, discussedfurther below. This greatly limits the use of long double-strandedRNAs to experimentally induce an RNAi response against a specific targeted mRNA. FortunatelS researchersdiscovered that one strand of double-stranded siRNAs of 21-23 nucleotidesin length with two-base 3' singlestranded regions leads to the generation of mature siRNA RISC complexeswithout inducing the generalizedinhibition of protein synthesis. This has allowed researchersto use synthetic double-strandedsiRNAs to "knock down" the expressionof specificgenesin human cellsas well as in other mammals. This method of siRNA knockdown is now widely used in studies of diverse processes,including the RNAi pathway itself. RNA| Inhibition of Transcription In plants and the fission yeast Schizosaccharomyces pombe, double-stranded RNA also induces the formation of heterochromatin on geneswith the same sequenceas the double-strandedRNA, inhibiting their transcription. Nuclear proteins homologous to cytoplasmic Dicer and Argonaute proteins generatenuclear siRNA complexescomposedof different proteins from the cytoplasmicRISC complexes.Thesenuclear siRNA complexes are thought to be targeted to specific genesby base pairing with nascentpre-mRNAs during their transcription. This interaction induces the methylation of histone H3 at lysine 9, generating a binding site for HP1 proteins and the subsequentassemblyof heterochromatin,as discussedin Chapter 6 (seeFigure 6-34).In plants, the DNA in these

heterochromatic regions also is methylated, contributing to the formation of heterochromatin. Components of the RNAi system are also required for the formation of heterochromatin at centromeresand the proper function of centromeres in S. pombe, plants, and cultured mammalian cells. Centromeres from most organisms contain highly repetitive DNA sequences.Consequently,the RNAi system that leads to heterochromatizationof repeated genes in S. pombe and plants may be exploited generallyby most eukaryotes for the proper formation of the DNA-protein kinetochore complex formed at centromeresand critical for cell division (Chapter20).

CytoplasmicPolyadenylationPromotes Translationof SomemRNAs In addition to repressionof translation by miRNAs, other protein-mediatedtranslational controls help regulateexpression of some genes.Regulatory sequences,or elements,in mRNAs that interact with specificproteinsto control translation generallyare presentin the untranslatedregion (UTR) at the 3' or 5' end of an mRNA. Here we discussa type of protein-mediatedtranslational control involving 3' regulatory elements. A different mechanisminvolving RNA-binding proteins that interact with 5' regulatory elementsis discussedlater. Translation of many eukaryotic mRNAs is regulated by sequence-specific RNA-binding proteins that bind cooperatively to neighboring sites in 3' UTRs. This allows them to function in a combinatorial manner,similar to the cooperative binding of transcription factors to regulatory sites in an enhancer or promoter region. In most casesstudied,translation is repressedby protein binding to 3' regulatory elementsand regulationresultsfrom derepressionat the appropriatetime or place in a cell or developingembryo. The mechanismof such repressionis best understoodfor mRNAs that must undergo cytoplasmic polyadenylation before they can be translated.

Cytoplasmic polyadenylation is a critical aspectof gene expression in the early embryo of animals. The egg cells (oocytes) of multicellular animals contain many mRNAs, encoding numerous different proteins, that are not translated until after the egg is fertilized by a sperm cell. Some of these "stored" mRNAs have a short poly(A) tail, consisting of only -20-40 A residues,to which just a few moleculesof cytoplasmic poly(A)-binding protein (PABPI) can bind. As discussedin Chapter 4, multiple PABPI moleculesbound to the long poly(A) tail of an mRNA interact with the eIF4G initiation factor, thereby stabilizing the interaction of the mRNA 5' cap with eIF4E, which is required for translation initiation (seeFigure 4-28b). Becausethis stabilization cannot occur with mRNAs that have short poly(A) tails, such mRNAs stored in oocytes are not translated efficiently. At the appropriate time during oocyte maturation or after fertilization of an egg cell, usually in responseto an external signal, approximately 150 A residuesare added to the short poly(A) tails on thesemRNAs in the cytoplasm, stimulating their translation. Recent studies with mRNAs stored in Xenopus oocytes have helped elucidatethe mechanismof this type of translational control. Experiments in which short-tailed mRNAs are injected into oocytes have shown that two sequencesin their 3' UTR are required for their polyadenylation in the cytoplasm: the AAUAAA poly(A) signal that is also required for the nuclear polyadenylation of pre-mRNAs and one or more copiesof an upstreamU-rich cytoplasmicpolyadenylation element (CPE). This regulatory element is bound by a highly conservedCPE-binding protein (CPEB) that contains an RRM domain and a zinc-finger domain. According to the current model, in the absenceof a stimulatory signal, CPEB bound to the U-rich CPE interactswith the protein Maskin, which in turn binds to eIF4E associated with the mRNA 5' cap (Figure 8-28,left). As a result' eIF4E cannot interact with other initiation factors and the 40S

ly active Translational

T r a n s l a t i o n a l dl yo r m a n t

TAAUAAA-A UUUUAU

t-

A FIGURE 8-28 Model for control of cytoplasmic pofyadenylation and translationinitiation.(Left)ln immature polyadenylation mRNAs containing the U-richcytoplasmic oocytes, (CPE) protein(CPEB) haveshortpoly(A) element tails CPE-binding interactions depicted, mediates repression of translation throughthe at the 5' endof the whichprevent assembly of an initiation complex Hormone stimulation activates a protein mRNA(R/EIht) of oocytes it to release CPEB, causrng MaskinThe kinase thatphosphorylates

thenbinds factor(CPSF) specificity andpolyadenylation cleavage a n dt h e t o t h e p o l y ( As)i t e ,i n t e r a c t i nwgi t h b o t hb o u n dC P E B (PAP). Afterthe poly(A)tail form of poly(A)polymerase cytoplasmic poly(A)-binding of the cytoplasmic multiple copies islengthened, which with elF4G, protein| (PABPI) canbindto it andinteract factorsto bindthe40Sribosome with otherinitiation functions andJ D fromR Mendez translation subunitandinitiate lAdapted Biol 2:521 Rev. Mol Cell 2001.Nature I Richter.

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ribosomal subunit, so translation initiation is blocked. During oocyte maturation, a specific CPEB serineis phosphorylated, causing Maskin to dissociatefrom the complex. This allows cytoplasmic forms of the cleavageand polyadenylation specificity factor (CPSF)and poly(A) polymeraseto bind to the mRNA cooperatively with CPEB. Once the poly(A) polymerase catalyzesthe addition of A residues, PABPI can bind to the lengthenedpoly(A) tail, leading to the stabilizedinteraction of all the participants neededto initiate translation (Figure 8-28, right; seealso Figure 4-28). In the case of Xenopus oocyte maturation, the protein kinase that phosphorylatesCPEBis activatedin responseto the hormone progesterone.Thus timing of the translationof storedmRNAs encoding proteins neededfor oocyte maturation is regulated by this external signal. Considerableevidenceindicatesthat a similar mechanism of translational control plays a role in learning and memory. In the central nervous system, the axons from a thousand or so neurons can make connections(synapses) with the dendrites of a single postsynaptic neuron (Figure 23-23). When one of these axons is stimulated, the postsynaptic neuron "remembers" which one of thesethousandsof synapseswas stimulated. The next time that synapseis stimulated, the strengthof the responsetriggeredin ihe postsynaptic cell differs from the first time. This changein response has been shown to result largely from the translational activation of mRNAs stored in the region of the synapse,leading to the local synthesisof new proteins that increasethe size and alter the neurophysiological characteristicsof the synapse.The finding that CPEB is present in neuronal dendrites has led to the proposal that cytoplasmic polyadenylation stimulates translation of specific mRNAs in dendrites, much as it does in oocytes.In this case,presumably,synaptic activity (rather than a hormone) is the signal that induces phosphorylation of CPEB and subsequentactivation of translation.

Degradationof mRNAsin the Cytoplasm Occursby SeveralMechanisms The concentration of an mRNA is a function of both its rate of synthesisand its rate of degradation. For this reason, if two genesare transcribed at the same rate, the steady-state concentration of the corresponding mRNA that is more stablewill be higher than the concentration of the other. The stability of an mRNA also determineshow rapidly synthesis of the encoded protein can be shut down. For a stable mRNA, synthesisof the encoded protein persistslong after transcription of the geneis repressed.Most bacterialmRNAs are unstable, decaying exponentially with a typical half-life of a few minutes. For this reason,a bacterial cell can rapidly adjust the synthesisof proteins to accommodatechangesin the cellular environment. Most cells in multicellular organisms, on the other hand, exist in a fairly constant environment and carry out a specificset of functions over periods of days to months or even the lifetime of the organism (nerve cells, for example). Accordingly, most mRNAs of higher eukaryoteshave half-lives of many hours. However, some proteins in eukaryotic cells are required only for short periods and must be expressedin bursts. For example, as discussedin the chapter introduction, certain signaling moleculescalled cytokines, which are involved in the immune responseof mammals, are synthesizedand secreted in short bursts. Similarly, many of the transcriprion factors that regulatethe onset of the S phaseof the cell cycle, such as c-Fos and c-Jun, are synthesizedfor brief periods only (Chapter 20). Expression of such proteins occurs in short bursts becausetranscription of their genescan be rapidly turned on and off, and their mRNAs have unusually short half-lives, on the order of 30 minutes or less. Cytoplasmic mRNAs are degraded by one of the three pathways shown in Figure 8-29. For most mRNAs, the deadenylation-dependentpatbway is followed: the length

Decapping pathway (deadenylation-independent)

Deadenylation-dependent pathways

AAAAAA

AAAAAA

Endonucleolytic pathway

AAAAAA I

e.oonu"leotytic creavase

I

A FIGURE 8-29 Pathwaysfor degradationof eukaryotic mRNAs.ln the deadenylation-dependenl(niddle) pathways, the poly(A) tailis progressively (orange) shortened by a deadenylase until it reaches a lengthof 20 or fewerA residues, at whichpointthe interaction with PABPI isdestabilized, leading to weakened interactions betweenthe 5' capandtranslation-initiation factors. Thedeadenylated mRNAthenmayeither(1)be decapped anddegraded by a 5, -+ 3,

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exonuclease or (2) be degraded by a 3' -+ 5' exonuclease in (right)arecleaved cytoplasmic exosomes. SomemRNAs internally by an endonuclease andthefragments degraded by an exosome. Other (/eft)aredecapped mRNAs beforetheyaredeadenylated andthen degraded by a 5' + 3' exonuclease. fromM Tucker and lAdapted R parker, 2ooo,Ann Rev. Biochem 69:571 I

of the poly(A) tail gradually decreaseswith time through the action of a deadenylatingnuclease.Sfhen it is shortened sufficiently, PABPI molecules can no longer bind and stabilizethe interaction of the 5' cap and translation initiation factors (see Figure 4-28b). The exposed cap then is removed by a decapping enzyme (Dcp1/Dcp2 in S. cereuisiae),and the unprotectedmRNA is degradedby a 5' -+ 3' exonuclease(Xrn1 in S. cereuisiae).Removal of the poly(A) tail also makes mRNAs susceptibleto degradation by cytoplasmicexosomescontaining 3' -+ 5'exonucleases. The 5' J 3' exonucleasespredominate in yeast, and the 3' -+ 5' exosome predominatesin mammalian cells. The decappingenzymesand 5' -+ 3' exonucleaseare concentrated in the P bodies,regions of the cytoplasm of unusually high density (seeFigure 8-31). Some mRNAs are degraded primarily by a deadenylation-independentdecappingpathway (seeFigure 8-29). This is becausecertain sequencesat the 5' end of an mRNA seem to make the cap sensitiveto the decappingenzyme.For these mRNAs, the rate at which they are decapped controls the rate at which they are degradedbecauseonce the 5' cap is removed, the RNA is rapidly hydrolyzed by the 5' -+ 3' exonuclease. The rate of mRNA deadenylation varies inversely with the frequency of translation initiation for an mRNA: the higher the frequency of initiation, the slower the rate of deadenylation.This relation probably is due to the reciprocal interactions between translation initiation factors bound at the 5' cap and PABPI bound to the poly(A) tail. For an mRNA that is translated at a high rate, initiation factors are bound to the cap much of the time, stabilizing the binding of PABPI and thereby protecting the poly(A) tail from the deadenylationexonuclease. Many short-lived mRNAs in mammalian cellscontain multiple, sometimesoverlapping copiesof the sequenceAUUUA in their 3'-untranslated region. Specific RNA-binding proteins have been found that both bind to these 3' AU-rich sequencesand also interact with a deadenylating enzyme and with the exosome.This causesrapid deadenylationand subsequent3' --> 5' degradationof these mRNAs. In this mechanism, the rate of mRNA degradation is uncoupled from the frequency of translation. Thus mRNAs containing the AUUUA sequencecan be translatedat high frequencyyet also be degraded rapidly, allowing the encoded proteins to be expressedin short bursts. As shown in Figure 8-29, some mRNAs are degraded by an endonwcleolyticpathuay that does not involve decapping or significant deadenylation.One example of this type of pathway is the RNAi pathway discussedabove (see Figure 8-25). Each siRNA-RISC complex can degrade thousands of targeted RNA molecules. The fragments generated by internal cleavage then are degraded by exonucreases. P Bodies As mentioned above, P bodies are sitesof translational repressionof mRNAs bound by miRNA-RISC complexes. They are also the major sites of mRNA degradation in the cytoplasm. These denseregions of cytoplasm contain

the decapping enzyme (Dcp1/Dcp2 in yeast), activators of decapping(Dhh, Pat1, Lsml-7 in yeast),the major 5' -+ 3' exonuclease(Xrn1), as well as denselyassociatedmRNAs. P bodies are dynamic structuresthat grow and shrink in size dependingon the rate at which mRNPs associatewith them, the rate at which mRNAs are degraded, and the rate at which mRNPs exit P bodies and reenter the pool of translated mRNPs.

ProteinSynthesisCan Be GloballyRegulated Like proteins involved in other processes,translation initiation factors and ribosomal proteins can be regulatedby posttranslational modifications such as phosphorylation. Such mechanismsaffect the translation rate of most mRNAs and hencethe overall rate of cellular protein synthesis. TOR Pathway The TOR pathway was discoveredthrough researchinto the mechanismof action of rapamycin, an antibiotic produced by a strain of Streptomycesbacteria,useful for suppressingthe immune response in organ transplant patients. The target of rupamycin (TOR)was identified by isolating yeast mutants resistant to rapamycin inhibition of cell growth. TOR is a large (-2400 amino acid residue) protein kinase that regulatesseveralcellular processesin yeast cells in responseto nutritional status. In multicellular eukaryotes,metazoan TOR (wTOR) also respondsto multiple signals from cell-surface-signalingproteins to coordinate cell growth with developmentalprograms as well as nutritional status. Current understandingof the mTOR pathway is summarized in Figure 8-30. Active mTOR stimulates the overall rate of protein synthesisby phosphorylating two critical proteins that regulate translation directly. mTOR also activates transcription factors that control expression of ribosomal components,tRNAs, and translation factors, further activating protein synthesisand cell growth. Recall that the first step in translation of a eukaryotic mRNA is binding of the eIF4 initiation complex to the 5' cap via its eIF4E cap-binding subunit (see Figure 4-24). The concentrationof active eIF4E is regulated by a small family of homologous elF4E-binding proteins (4E-BPs) that inhibit the interaction of eIF4E with mRNA 5' caps. 4E-BPs are direct targets of mTOR. !(hen phosphorylated by mTOR, 4E-BPs releaseeIF4E, stimulating translation initiation. mTOR also phosphorylates and acttvatesanother protein kinase that phosphorylatesthe small ribosomal subunit protein S6 (S6K) and probably additional substrates, leading to a further increase in the rate of protein synthesis. Translation of a specific subset of mRNAs that have a string of pyrimidines in their 5' untranslated regions (called TOP mRNAs for tract of oligopyrimidine) is stimulated particularly strongly by mTOR. The 5'TOP mRNAs encoderibosomal proteins and translation elongation factors. mTOR also activates the RNA polymerase I transcription factor TIF1A, stimulating transcription of the large rRNA precursor (seeSection 8.5 below). mTOR also activatestranscription

CAOLN T R O L OF POST.TRANSCRIPTION C Y T O P L A S M IM C ECHANISMS

o oo Nutrients o o o o

Growth factor receptor

Stress hypoxia

Exterior

Low o o o

Cytoplasm

o

o Low nutrients Rapamycin

Ribosome biogenesis

Transcription

Macroautophagy

FIGURE 8-30 mTORpathway.mTORisan activeproteinkinase mTORproteinkinase activates activity. Lownutrientconcentratton whenboundby a complex alsoregulates RhebGTPase activity by a mechanism of Rhebandan associated thatdoesnot GTP(/ower /eff) ln contrast, mTORisinactive require TSCl/TSC2 ActivemTORphosphorylates 4E-BP, whenboundby a complex causing it to of Rheb associated with GDP(lowerright).Whenactive, release elF4E, stimulating translation initiationlt alsophosphorylates theTSCl/TSC2 (S6K), Rheb-GTPase protein(Rheb-GAP) activating andactivates 56 kinase whichin turnphosphorylates ribosomal causes hydrolysis of proteins, Rheb-bound GTPto GDBtherebyinactivating stimulating translation. Activated mTORalsoactivates mTORTheTSCl/TSC2 (arrows) Rheb-GAP isactivated t r a n s c r i p t ifoanc t o r fso r R N Ap o l y m e r a sl e, lsl ,a n dl l l ,l e a d i ntgo by phosphorylation by AMPkinase (AMPK) whencellular energychargeis lowandby othercellular synthesis andassembly of ribosomes, tRNAs, andtranslation factors stress responses. Signal-transduction pathways Intheabsence of mTORactivity, allof theseprocesses areinhibited activated by cellgroMhfactorreceptors surface leadto phosphorylation d T O Ri n h i b i tm s a c r o a u t o p h awg hy i, c hi s of inactivating I n c o n t r a sat ,c t i v a t em siteson TSCl/TSC2, inhibiting itsGAPactivityConsequently, stimulated in cellswith inactive mTOR[Adapted fromS Wullschleger they leavea higherfractionof cellular et al, 2006, Cell 124:471 l Rhebin the GTPconformation that

by RNA polymerase III, although the mechanism is not clear. In addition, mTOR activatestwo RNA polymeraseII activators that stimulate transcription of ribosomal protein and translation factor genes. Finally, mTOR stimulates processingof the rRNA precursor(Section8.5). As a consequence of phosphorylation of these several mTOR substrates,the synthesisand assemblyof ribosomes as well as the synthesisof translation factors and tRNAs are greatly increased.Alternatively, when mTOR kinase activity is inhibited, these substratesbecome dephosphorylated,greatly decreasingthe rate of protein synthesisand the production of ribosomes, translation factors, and tRNAs, thus halting cell growth. mTOR activity is regulated by a monomeric small G protein in the Ras protein family called Rheb. Like other small G proteins, Rheb is in its active conformation when it is bound to GTP. Rheb .GTP binds the mTOR complex.

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activating mTOR kinase activity, probably by inducing a conformation changein its kinase domain. Rheb is in turn regulatedby a heterodimercomposedof subunitsTSCl and TSC2, named for their involvement in the medical syndrome /uberous sclerosis complex, as discussedbelow. In the active conformation, the TSC1/TSC2 heterodimer functions as a GTPaseactivatingprotein for Rheb (Rheb-GAP), causing hydrolysis of the Rheb-bound GTP to GDP. This converts Rheb to its GDP-bound conformation, which binds to the mTOR complex and inhibits its kinase activiry. Finally, the activity of the TSCI 1TSC2Rheb-GAP is regulated by severalinputs, allowing the cell to integrate different cellular signaling pathways to control the overall rate of protein synthesis.Signaling from cell-surfacegrowth factor receptors leads to phosphorylation of TSCIiTSC2 at inhibitory sites,causingan increasein Rheb'GTP and activation of mTOR kinase activity. This type of regulation

through cell-surface receptors links the control of cell growth to developmental processescontrolled by cell-cell rnteractrons. mTOR activity also is regulated in response to nutritional status. When energy from nutrients is not sufficient for cell growth, the resulting fall in the ratio of ATP to AMP concentrationsis detectedby the AMP kinase. The activated AMP kinase phosphorylatesTSCIiTSC2 at activating sites, stimulating its Rheb-GAP activity and consequentlyinhibiting mTOR kinase activity and the global rate of translation. Hypoxia and other cellular stressesalso activate the TSCI/ TSC2 Rheb-GAP. Finally, the concentration of nutrients in the extracellular space also regulatesRheb by an unknown mechanismthat does not require the TSC1/TSC2 complex. In addition to regulating the global rate of cellular protein synthesisand the production of ribosomes,tRNAs, and translation factors, mTOR regulates at least one other processinvolved in the responseto low levels of nutrients: macroautophagy. Starved cells degrade cytoplasmic constituents, including whole organelles,to supply energy and precursors for essential cellular processes.During this process alarge, double-membranestructure engulfs a region of cytoplasm to form an autophagosome,which then fuses with a lysosome where the entrapped proteins, lipids, and other macromoleculesare degraded,completing the process of macroautophagy.Activated mTOR inhibits macroautophagy in growing cells when nutrients are plentiful. Macroautophagy is stimulated when mTOR activity falls in nutrient-deprived cells.

Severalviruses encode proteins that activate mTOR early after viral infection. The resulting stimulation of translation has an obvious selective advantagefor these cellular parasites.I

elF2 Kinases eIF2 kinasesalso regulatethe global rate of cellular protein synthesis. Figure 4-24 summarizes the steps in translation initiation. Translation initiation factoi eIF2 brings the charged initiator tRNA to the small ribosome subunit P site. eIF2 is a trimeric G protein and consequently exists in either a GTP-bound or a GDPboundconformation. Only the GTP-bound form of eIF2 is able to bind the charged initiator IRNA and associate with the small ribosomal subunit. The small subunit with bound initiation factors and charged initiator IRNA then interactswith the eIF4 complex bound to the 5' cap of an mRNA via its eIF4E subunit. The small ribosomal subunit then scansdown the mRNA in the 3'direction until it reaches an AUG initiation codon that can base-pair with the initiator IRNA in its P site. When this occurs,the GTP bound by eIFZis hydrolyzed to GDP and the resulting eIF2'GDP complex is released.GTP hydrolysis results in an irreversible "proofreading" step that prepares the small ribosomal subunit to associatewith the large subunit only when an initiator IRNA is properly bound in the P site and is properly base-pairedwith the AUG start codon. Before eIF2 can participate in another round of initiation, its bound GDP must be replaced with a GTP. This process is catalyzed by the translation initiation factor eIF2B, a guanine nucleotide exchange factor (GEF) specific for eIF2. A mechanism for inhibiting general protein synthesisin Genesencoding componentsof the mTOR pathway ffi stressedcells involves phosphorylation of the eIF2 ct subunit a.e mutated in many human cancers,resulting in cell 3l at a specificserine.Phosphorylation at this site does not ingrowth in the absenceof normal growth signals.TSCl and with eIF2 function in protein synthesis directly. terfere TSC2 (Figure 8-30) were initially identified becauseone or phosphorylated eIF2 has very high affinity for the Rather, genetic the other of the proteins is mutant in a rare human nucleotide exchange factor, eIF2B' which canguanine eIF2 with this syndrome: tuberous sclerosiscomplex. Patients phosphorylated eIF2 and consequently is the rilease not The disorder develop benign tumors in multiple tissues. GTP exchange of additional eIF2 catalyzing from blocked TSC2 TSCl or diseaseresultsbecauseinactivation of either is an excessof eIF2 over eIF2B, phosthere Since factors. hetTSC1/TSC2 eliminates the Rheb-GAP activity of the of elF2 results in inhibition of all fraction a of phorylation unregulated high and erodimer,resulting in an abnormally eIF2 accumulatesin its remaining ' The eIF2B. cellular ihe achigh, unregulated resulting level of Rheb GTP and the in protein synparticipate cannot which form, GDP-bound of cell-surface in components Mutations tivity of mTOR. protein synthesis' cellular all nearly inhibiting thereby thesis, inhibilead to pathways that receptor signal-transduction translaallow that regions have 5' mRNAs some Howeve! common also are Rheb-GAP activity TSC1/TSCZ tion of tion initiation at the low eIF2-GTP concentration that rein human tumors and contribute to cell growth and replication in the absenceof normal signals for growth and proliferation. High mTOR protein kinase activity in tumors correlates with a poor clinical prognosis.Consequently,mTOR inhibitors are currently in clinical trials to test their effecthesestress-induced Proteins. tiveness for treating cancers in coniunction with other Human cells contain four eIF2 kinasesthat phosphorymodes of therapy. Rapamycin and other structurally related late the same inhibitory eIF2a serine' Each of these is regumTOR inhibitors are potent suppressorsof the immune related by a different type of cellular stress,inhibiting protein sponsebecausethey inhibit activation and replication of synthesisand allowing cells to divert the large fraction of T lymphocytesin responseto foreign antigens(Chapter24).

CA OLN T R O L OF POST-TRANSCRIPTION C Y T O P L A S M IM C ECHANISMS

cellular resourcesdevoted to protein synthesisin growing cells for use in responding to the stress. The GCN2 (general control non-derepressible2) eIF2k i n a s ei s a c t i v a t e db y b i n d i n g u n c h a r g e d iRNRr. The concentration of uncharged tRNAs increaseswhen cells are starved for amino acids,activating GCN2 elF2-kinaseactivity and greatly inhibiting protein synthesis. PEK (pancreaticeIF2a kinase) is activatedwhen proteins translocatedinto the endoplasmicreticulum (ER) do not fold properly becauseof abnormalities in the ER lumen environment. Inducersinclude abnormal carbohydrateconcentration becausethis inhibits the glycosylation of many ER proteins and inactivating mutations in an ER chaperonerequired for proper folding of many ER proteins (Chapters 13 and 14). Heme-regulatedinhibitor (HRI) is activatedin develooing red blood cells when the supply of heme prostheric group is too low to accommodatethe rate of globin protein synthesis. This negative feedback loop lowers the rate of globin protein synthesis until ir matches the rate of heme synthesis.HR1 is also activated in other types of cells in responseto oxidative stressor heat shock. Finally, protein kinase RNA activated (pKR) is acivated by double-strandedRNAs longer than -30 basepairs. Under normal circumstancesin mammalian cells, such doublestranded RNAs are produced only during a viral infection. Long regions of double-strandedRNA are generated in replication intermediates of RNA viruses or from hybridization of complementary regions of RNA transcribed from both strands of DNA virus genomes.Inhibition of protein synthesisprevents the production of progeny virions, protecting neighboring cells from infection. InrerestinglS adenovirusesevolved a defenseagainst pKR: they express prodigious amounts of an -1,60-nucleotide virusassociated(VA) RNA with long double-strandedhairpin regions. VA RNA is transcribed by RNA polymeraseIII and exported from the nucleus by Exportin5, the exportin for pre-miRNAs (seeFigure 8-27).VA RNA binds to pKR with high affinity, inhibiting its protein kinase activity and preventing the inhibition of protein synthesisobservedin iells infected with a mutant adenovirus from which the VA gene wasdeleted.

Sequence-Specific RNA-BindingproteinsControl SpecificmRNATranslation In contrast to global mRNA regulation, mechanismshave also evolved for conrolling the translation of certain specific mRNAs. This is usually done by sequence-specificRNAbinding proteins that bind to a particular sequenceor RNA

Control of intracellular iron concentration by the iron response element-binding protein (IRE-BP) is an elegant example of a singleprotein that regulatesthe translation of 356

.

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one mRNA and the degradation of another. precise regulation of cellular iron ion concentrationis critical to the cell. Multiple enzymesand proteins contain Fe2* as a cofactor, such as enzymesof the Krebs cycle (seeFigure 12-10) and electron-carryingproteins involved in the generation of ATP by mitochondria and chloroplasts (Chapter 12). On the other hand, excessFe2* generatesfree radicalsthat react with and damagecellular macromolecules.Ifhen intracellular iron sroresare low, a dual-control systemoperates to increasethe level of cellular iron; when iron is in excess, the system operatesto prevent accumulation of toxic levels of free ions. One component in this system is the regulation of the production of ferritin, an intracellular iron-binding protein that binds and storesexcesscellular iron. The 5' untranslated region of ferritin mRNA contains iron-response elements (IREs)that have a stemJoop structure.The IRE-binding protein (IRE-BP) recognizesfive specific basesin the IRE loop and the duplex nature of the stem. At low iron concentrations, IRE-BP is in an active conformation that binds to the IREs (Figure 8-31a). The bound IRE-BP blocks the small ribosomal subunit from scanning for the AUG start codon (see Figure 4-24), thereby inhibiting translation initiation. The resulting decreasein ferritin means less iron is complexed with the ferritin and is therefore available to ironrequiring enzymes.At high iron concentrations,IRE-BP is in an inactive conformation that does not bind to the 5, IREs, so translation initiation can proceed.The newly synthesized ferritin then binds free iron ions, preventing their accumulation to harmful levels. The other part of this regulatory system controls the import of iron into cells. In vertebrates,ingested iron is carried through the circulatory systembound to a protein called transferrin. After binding to the transferrin receptor (TfR) in the plasma membrane, the transferrin-iron complex is brought into cells by receptor-mediatedendocytosis (Chapter 14). The 3' untranslated region of TfR mRNA contains IREs whose stemshave AU-rich destabilizing sequences(Figure 8-31b). At high iron concentrations, when the IRE-BP is in the inactive, nonbinding conformation, these AU-rich sequencespromote degradation of TfR mRNA by the same mechanismthat leads to rapid degradation of other short-lived mRNAs, as described previously. The resulting decreasein production of the transferrin receptor quickly reducesiron import, thus protecting the cell from excessiron. At low iron concenrrations, however, IRE-BP can bind to the 3, IREs in TfR mRNA. The bound IRE-BP blocks recognition of the destabilizing AU-rich sequences by the proteins that would otherwise rapidly degradethe mRNAs. As a result, production of the transferrin receptor increasesand more iron is brought into the cell. Other regulated RNA-binding proteins may also function to control mRNA translation or degradation,much like the dual-actingIRE-BP.For example,a heme-sensitiveRNAbinding protein controls translation of the mRNA encoding aminolevulinate (ALA) synthase,a key enzyme in the synthesisof heme. In vitro studieshave shown that the mRNA

( a ) F e r r i t i nm R N A

lREs

cooH

C o d i n gr e g i o n

An

+ Translated ferritin

5'

( b ) T f Rm R N A

A^ JF

No translation initiation

Another mechanism called nonsense-mediateddecay causesdegradation of mRNAs in which one or more exons have been incorrectly skipped during splicing. Such exon skipping often will alter the open reading frame of the mRNA 3' to exon junction, resulting in introduction of out-ofih.i-ptop"t frame miJsensemutations and an incorrect stop codon' For nearly all properly splicedmRNAs' the stop codon is in the last ."otr. Th. p.ocessof nonsense-mediateddecay (NMD) results in the rapid degradation of mRNAs with stop codons that occur before the last splice junction in the mRNA since in most cases,such mRNAs arise from errors in RNA splicing A search for possible molecular signals that might indicate the positions of splice junctions in a processedmRNA led to thi discovery of exon-junction complexes. As noted

lREs AU-rich region

\/ t t/-

t

5'

A" aF

/--' - - ar ) Ir r t

tants indicate that one of the proteins in exon-junction complexes (Upf3) functions in nonsense-mediateddecay' In the Degraded iytoplasm, this component of exon-junction complexes inmononucleotides teractswith a protein (Upf 1) that causesthe mRNA to associate with P bodies,repressingtranslation of the mRNA' An

'r't)l?

Little degradation

8-31 lron-dependentregulationof mRNAtranslation A FIGURE protein element-binding and degradation.Theironresponse (lRE-BP) of ferritinmRNA(a)anddegradatton translation controls iron (TfR)mRNA(b) At low intracellular of transferrin-receptor (lREs) in elements bindsto iron-response IRE-BP concentrations At highiron regionof thesemRNAs. the 5' or 3' untranslated and change a conformational IRE-BP undergoes concentrations, precisely cannotbrndeithermRNAThedualcontrolby IRE-BP the levelof f reeironionswithincells.Seethetextfor regulates discussion.

encoding the milk protein casein is stabilized by the hormone prolactin and rapidly degradedin its absence.

with the mRNA, resulting in nonsense-mediateddecay'

Localizationof mRNAsPermitsProduction of Proteinsat SpecificRegionsWithin the Cytoplasm

SurveillanceMechanismsPreventTranslation mRNAs of lmproperlyProcessed Translation of an improperly processedmRNA could lead to the 3' untranslatedregion of the mRNA' As mentioned earlier, in neurons, localization of specific the translation of improperly processedmRNA molecules.We have previously mentioned two such surveillancemechanisms: the recognition of improperly processedpre-mRNAs in the nucleus and their degradation by the exosome and the general restriction against nuclear export of incompletely spliced pre-mRNAs that remain associatedwith a spliceosome. CA OLN T R O L OF POST-TRANSCRIPTION C Y T O P L A S M IM C ECHANISMS

357

EXPERIMENTAL FTGURE 8-32 A specificneuronalmRNA focafizesto synapses.Sensory neuronsfromthe seaslugAplysia californica-among the largest neurons in theanimalkingdom_ werecultured with targetmotorneurons sothat processes fromthe sensory neuronformedsynapses with processes fromthe motor neuron. Themicrograph at the leftshowsmotorneuronprocesses visualized with a bluefluorescent (green) dye GFp-VAMp was expressed In sensory neurons andmarksthe location of synapses formedbetween sensory andmotorneuronprocesses (arrows). The micrograph on the rightshowsredfluorescence from in situ h y b r i d i z a t i o fna n a n t i s e n s o rmi nR N Ap r o b eS e n s o r r n sa neurotransmrtter expressed by thesensory neurononly;sensory neuronprocesses arenot otherwise visualized in thispreparation, but theylieadjacent to the motorneuronprocesses. Thein situ hybridization results indicate thatsensorin mRNAislocalized to y Zhao, synapses V Lyles, [From andK C Martin. 2006,Neuron 49:3231 properties of this one synapseout of hundreds to thousands of synapsesmade by a neuron.

r Both miRNAs and siRNAs contain 21-23 nucleotides, are generated from longer precursor molecules, and are assembled into a multiprotein RNA-induced silencing complex (RISC) that either repressestranslation of target mRNAs or cleavesthem (seeFigures 8-26 and 8-27). r Cytoplasmic polyadenylation is required for translation of mRNAs with a short poly(A) tail. Binding of a specificprotein to regulatoryelementsin their 3' UTRs represses translation of thesemRNAs. Phosphorylation of rh[ RNA-binding protein, induced by an external signal, leadsto lengtheningof the 3' poly(A) tail and translation(seeFigure 8-29). Most mRNAs are degradedas the result of the gradual ortening of their poly(A) tail (deadenylation)followed by exosome-mediated3' -+ 5' digestion or removal of the 5' cap and digestionby a 5' -+ 3' exonuclease(seeFigure 8-30). Eukaryotic mRNAs encoding proteins that are expressed short burstsgenerallyhave repeatedcopiesof an AU-rich sequencein their 3' UTR. Specificproteinsthat bind to these elementsalso interactwith the deadenylatingenzymeand cytoplasmicexosomes,promoting rapid RNA degradation. r Binding of various proteins to regulatory elementsin the 3' or 5' UTRs of mRNAs regulatesthe translation or degradation of many mRNAs in the cytoplasm. r Translation of ferritin mRNA and degradation of transferrin receptor (TfR) mRNA are both regulatedby the same iron-sensitiveRNA-binding protein. At low iron concentrations, this protein is in a conformation that binds to specific elementsin the mRNAs, inhibiting ferritin mRNA translation or degradation of TfR mRNA (seeFigure 8-32). This dual control preciselyregulatesthe iron level within cells. r Nonsense-mediateddecay and other mRNA surveillance mechanismsprevent the translation of improperly processed mRNAs encoding abnormal proteins that might interfere with functioning of the correspondingnormal proteins. r Some mRNAs are directed to specific subcellular locations by sequencesusually found in the 3, UTR, leading to localization of the encodedproteins.

ff,l example of this mechanism of mRNA localization occurs dur_ ing cell division in S. cereuisiap-as we describein Chapter 21.

Cytoplasmic Mechanismsof post-transcriptional Control

Processing of rRNAandIRNA

Approximately 80 percent of the total RNA in rapidly growing mammalian cells (e.g.,cultured HeLa cells)is rRNA, and 15 percent is IRNA; protein-coding mRNA rhus consrrtutes only a small portion of the total RNA. The primary transcripts produced from most rRNA genes and from IRNA genes, like pre-mRNAs, are extensively processedto yield the mature, functional forms of theseRNAs.

r Translation can be repressedby micro-RNAs (miRNAs), which form imperfect hybrids with sequencesin the 3, un_ translated region (UTR) of specifictarget mRNAs. r The related phenomenon of RNA interference, which probably evolved as an early defensesystemagainstviruses and transposons,Ieads to degradation of mRNAs that form perfecthybrids with short interferins RNAs (siRNAs). 358

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dination of all three nuclear RNA polymerases.The 2gS and 5.8S rRNAs associatedwith the large ribosomal subunit and the single 18S rRNA of the small subunit are transcribedby

RNA polymeraseI. The 55 rRNA of the large subunit is transcribedby RNA polymeraseIII, and the mRNAs encodingthe ribosomal proteins are transcribedby RNA polymeraseII. In addition to the four rRNAs and :70 ribosomal proteins' at least 150 other RNAs and proteins interact transiently with the two ribosomal subunits during their assemblythrough a series of coordinated steps. Furthermore, multiple specific basesand ribosesof the mature rRNAs are modified to optimize their function in protein synthesis.Although most of the stepsin ribosomal subunit synthesisand assemblyoccur in the nucleolus(a subcompartmentof the nucleusnot boundedby a membrane), some occur in the nucleoplasmduring passage from the nucleolusto nuclearpore complexes.A quality-control step occursbefore nuclearexport so that only fully functional subunits are exported to the cytoplasm,where the final steps of ribosome subunit maturation occur. tRNAs also are processedfrom precursor primary transcripts in the nucleus and modified extensivelybeforethey are exportedto the cytoplasm and usedin protein synthesis.First we'll discussthe processing and modification of rRNA and the assembly and nuclearexport of ribosomes.Then we'll considerthe processing and modification of tRNAs.

+ .I E

Nucleolar chromatin

a (!

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G e n e sF u n c t i o na s N u c l e o l a r Pre-rRNA O r g a n i z e r sa n d A r e S i m i l a ri n A l l E u k a r y o t e s The 28S and 5.8S rRNAs associatedwith the large (605) ribosomal subunit and the 18S rRNA associatedwith the small (40S)ribosomal subunit in higher eukaryotes(and the functionally equivalent rRNAs in all other eukaryotes) are encoded by a single type of pre-rRNA transcription unit. In human cells,transcriptionby RNA polymeraseI yieldsa 45S (:13.7 kb) primary transcript (pre-rRNA), which is processedinto the mature 285, 18S,and 5.8S rRNAs found in cytoplasmic ribosomes.Sequencingof the DNA encoding pre-rRNA from many speciesshowed that this DNA shares several properties in all eukaryotes.First, the pre-rRNA genesare arranged in long tandem arrays separatedby nontranscribed spacer regions ranging in length from :2 kb in frogs to :30 kb in humans (Figure 8-33). Second, the genomic regions corresponding to the three mature rRNAs are alwaysarrangedin the same5' -> 3' order: 18S,5'8S' and 28S. Third, in all eukaryoticcells(and evenin bacteria),the pre-rRNA gene codes for regions that are removed during processingand rapidly degraded.These regions probably contribute to proper folding of the rRNAs but are not required once the folding has occurred. The general structure of pre-rRNAs is diagrammedin Figure 8-34. The synthesisand most of the processingof pre-rRNA occurs in the nucleolus. Sfhen pre-rRNA genes initially were identified in the nucleolusby in situ hybridization, it was not known whether any other DNA was required to form the nucleolus.Subsequentexperimentswith transgenic Drosophila strains demonstrated that a single complete pre-rRNA transcription unit induces formation of a small nucleolus.Thus a singlepre-rRNA geneis sufficienttobe a nucleoldr organizer, and all the other components of the ribosome diffuse to the newly formed pre-rRNA. The structure

8-33 Electronmicrographof pre' FIGURE a EXPERIMENTAL rRNAtranscriptionunits from the nucleolusof a frog oocyte. associated pre-rRNA molecules multiple Each"feather"represents (pre-RNP) emerging complex pre-ribonucleoprotein with proteinin a "knob" of each the 5'-end at dense Note the unit. a transcrrption from transcription Pre-rRNA pre-RNP thoughtto be a processome nascent spacer by nontranscribed in tandem,separated unitsarearranged Jr] andO J Miller, of Y osheim chromatin. of nucleolar [Courtesy regions

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8-34 Generalstructureof eukaryoticpre-rRNA A FIGURE (red)encode the 185, units.Thethreecodingregions transcription or higher eukaryotes of ribosomes in found 5 8S,and28SrRNAs regions coding these of order The species in other theirequivalents of the in the lengths in the genomeisalways5' -+ 3' Variations in (blue)account for the majordifference regions spacer transcribed organisms' in different units transcription of pre-rRNA the lengths P R O C E S S I NOGF r R N A A N D I R N A

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of the nucleolus observed by light and electron microscopy results from the processingof pre-RNA and the assemblyof ribosomal subunits.

S m a l lN u c l e o l a R r N A sA s s i s ti n p r o c e s s i n g Pre-rRNAs Ribosomal subunit assemblg maturation, and export to the cytoplasm are best understood in the yeast S. cereuisiae. However, nearly all the proteins and RNAs involved are highly conservedin multicellulareukaryotes,where the fundamental aspectsof ribosome biosynthesis are likely to be the same. As for pre-mRNAs, nascent pre-rRNA uanscnprs are immediatelybound by proteins,forming preribosomalribonucleoproteinparticles(pre-rRNPs).For reasonsnot yet known, cleavageof the pre-rRNA doesnot beginuntil transcription of

the pre-rRNA is nearly complete. In yeast,it takes approximately six minutes for a pre-rRNA to be transcribed. Once transcription is complete, the rRNA is cleaved, and bases and riboses are modified in about 10 seconds.In a rapidly growing yeastcell, -40 pairs of ribosomal subunits are synthesized,processed,and transported to the cytoplasm every second.This extremely high rate of ribosome synthesisin the face of the seemingly long period required to transcribe a pre-rRNA is possible becausepre-rRNA genes are packed with RNA polymerase I molecules transcribing the same gene simultaneously(seeFigure 8-33) and becausethere are 100-200 such geneson chromosomeXII, the yeastnucleolar otganrzeL The primary transcript of -7 kb is cut in a seriesof cleavage and exonucleolytic steps that ultimately yield the mature rRNAs found in ribosomes(Figure 8-35). During processing,

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185 5.8Ss 25S FIGURE 8-35 rRNAprocessing. Endoribonucleases that make internal cleavages arerepresented asscissors. Exoribonucleases that digestfromoneend,either5, or 3,, areshownaspac-Men. Most2,_ O-ribose (CH:)andgeneration methylation of pseudouridines in the 350

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modificationof pre-rRNA. 8-36 SnoRNP-directed A FIGURE (a)A snoRNA in ribose2'-Oisinvolved calledboxC+D snoRNA to two different hybridize in thissnoRNA Sequences methylation. sites at the indicated methylation directing in the pre-rRNA, regions (b)BoxH+ACAsnoRNAs fold intotwo stemloopswith internal

to the hybridizes bulgesin thestems.Pre-rRNA single-stranded pseudouridylation' site of a demarcating bulges, single-stranded by the box directed (c)Conversion fromuridineto pseudouridine J 2001 , EMBO of part(b).lPart(a)f romr' Kiss, H+ACA snoRNAs 114:1 (b) 20:3617Part fromU I Meier,2005,Chromosoma |

pre-rRNA also is extensivelymodified, mostly by methylation of the 2'-hydroxyl group of specificribosesand conversionof specific uridine residuesto pseudouridine.These post-transcriptionalmodificationsof rRNA are probably important for protein synthesisbecausethey are highly conserved.Mrtually all of these modifications occur in the most conserved core structure of the ribosome, which is directly involved in protein synthesis.The positions of the specificsitesof 2'-O-methylation and pseudouridineformation are determinedby approxRNA species, imately 150 different small nucleolus-restricted (snoRNAs), which hybridize called small nucleolar RNAs transiently to pre-rRNA molecules. Like the snRNAs that function in pre-mRNA processing,snoRNAs associatewith proteins,forming ribonucleoproteinparticlescalledsnoRNPs. One class of more than 40 snoRNPs (box C+D snoRNAs) positions a methyltransferaseenzymenear methylation sitesin the pre-mRNA. The multiple different box C+D snoRNAs direct methylation at multiple sitesthrough a similar mechanism. They share common sequenceand structural features and are bound by a common set of proteins. One or two regions of each of thesesnoRNAs are preciselycomplementary to sites on the pre-rRNA and direct the methyltransferaseto specificribosesin the hybrid region (Figure 8-36a). A second maior class of snoRNPs (box H+ACA snoRNAs) positions the enzyme that converts uridine to pseudouridine' This conversion involves rotation of the pyrimidine ring (Figure 8-36c). Baseson either sideof the modified uridine in the prerRNA base-pair with basesin the bulge of a stem in the H+ACA snoRNAs, leaving the modified uridine bulged out

of the helical double-strandedregion, like the branch point A bulges out in pre-mRNA spliceosomalsplicing (seeFigure 8-10). Other modifications of pre-rRNA nucleotides'such as adenine dimethylation, are carried out by specific proteins without the assistanceof guiding snoRNAs. The U3snoRNA is assembledinto a large snoRNP con-

from pre-mRNAs. Nuclear 5' -+ 3' exoribonucleases(Rat1; Xrnl) also remove someregionsof 5' spacer' SomesnoRNAs are expressedfrom their own promoters

exist onlv to expresssnoRNAs from excisedintrons' P R O C E S S I NOGF T R N AA N D I R N A

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malns associatedwith the region that is cleavedinto the pre_ cursor of the largeribosomal subunit. Most of the ribosomal proteinsof the small 40S riboso_

subunit occurs in the cytoplasm: exonucleolyric processing of the 20S rRNA into mature small subunit 1gS rRNA by the cytoplasmic 5' -+ 3, exoribonucleaseXrnl and the .

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transiently with the maturing subunits aredepicted in different colors, asshownin the key fFrom H Tschochner andE Hurt,2OO3. Trends CellBiol 13:2551

dimethylationof two adjacentadeninesnear the 3, end of 18SrRNA by the cytoplasmicenzymeDim1. In contrast to the pre-40S particle, the precursor of the large subunit requiresconsiderablymore remodelingthrough many more transient interactions with nonribosomal proteins before it is sufficiently mature for export to the cytoplasm. Consequently,it takes a considerably longer period for the maturing 605 subunit to exit the nucleus than for the 40S subunit (30 minutescomparedto 5 minutesfor export of the small subunit in cultured human cells). Multiole presumptive RNA helicasesand small G proteins urro.i"r. ated with the maturing pre-60Ssubunits.Some RNA helicasesare necessaryto dislodgethe snoRNps that base-pair perfectly with pre-rRNA over up ro 30 base pairs. Oiher RNA helicasesmay function in the disruption of proteinRNA interactions. The requirement for so many GTpases suggeststhat there are many quality-control checkpoints in the assembly and remodeling of the large subunit RNp, where one step must be completed before a GTpase is

activated to allow the next step to proceed.Members of the AAA ATPasefamily are also bound transiently.This classof proteins is often involved in large molecular movementsand may be required to fold the complex, large rRNA into the proper conformation. Somestepsin 605 subunit maturation occur in the nucleoplasm,during passagefrom the nucleolus to nuclear pore complexes (seeFigure 8-37). Much remains to be learned about the complex, fascinating, and essential remodeling processesthat occur during formation of the ribosomalsubunits. The large ribosomal subunit is one of the largest structures to passthrough nuclear pore complexes.Maturation of the large subunit in the nucleoplasmleads to the generation of binding sites for a nuclear export adapter called Nmd3. Nmd3 is bound by the nuclear tranporter Exportinl (also called Crml). This is another quality-control step since only correctly assembled subunits can bind Nmd3 and be exported. The small subunit of the mRNP exporter (Nxt1) also b..o-.t associatedwith the nearly mature large ribosomal subunit. Thesenucleartransportersinteractwith FG-domains of FG-nucleoporins.This mechanism allows penetration of the molecular meshwork that makes up the central channel of the NPC (seeFigure 8-20). Severalspecific nucleoporins without FG-domains are also required for ribosomal subunit export and may have additional functions specific for this task. The dimensionsof ribosomal subunits(-25-30 nm in diameter) and the central channel of the NPC are comparable, so passagemay not require distortion of either the ribosomal subunit or the channel. Final maturation of the large subunit in the cytoplasm includes removal of these export factors. As for the export of most macromoleculesfrom the nucleus, including tRNAs and pre-miRNAs (but not most mRNPs), ribosome subunit export requiresthe function of a small G protein called Ran, as discussedin Chapter 13.

The splicing mechanismsused by group I introns, group II introns, and spliceosomesare generally similar, involving two transesterificationreactions, which require no input of

catalysis.The group I intron functions like a metalloenzyme to piecisely orient the atoms that participate in the two transesterificationreactionsadjacentto catalytic Mg2* ions' Considerableevidencenow indicates that splicing by group II introns and by snRNAs in the spliceosomealso involves bound catalytic Mg2* ions. In both the group I and II selfsplicing introns and probably in the spliceosome,RNA funca ribozyme, an RNA sequencewith catalytic ability' tions "s

Pre-tRNAsUndergoExtensiveModification in the Nucleus Mature cytosolictRNAs, which average75-80 nucleotidesin length, aie producedfrom larger precursors-(pre-tRNAs)synthesizedfy nNe polymeraseIII in the nucleoplasm'Mature tRNAs also contain numerous modified basesthat are not

an endonucleolytic cleavage specified by the IRNA threedimensional structure rather than the start site of transcrip-

S e l f - S p l i c i nG g r o u pI I n t r o n sW e r et h e F i r s t Examplesof CatalyticRNA During the 1970s, the pre-rRNA genes of the protozoan Tetrahymena thermophila were discovered to contain an intron. Careful searchesfailed to uncover even one prerRNA genewithout the extra sequence'indicating that splicing is required to produce mature rRNA in theseorganisms' In 1,982, in vitro studies showing that the pre-rRNA was spliced at the correct sitesin the absenceof any protein provided the first indication that RNA can function as a catalyst. like enzymes. A whole raft of self-splicingsequencessubsequentlywere found in pre-rRNAs from other single-celledorganisms, in mitochondrial and chloroplast pre-rRNAs, in several premRNAs from certain E. coli bacteriophages'and in some bacterial tRNA primary transcripts. The self-splicingse-

self-splicingintron, designatedgroup II' P R O C E S S I NOGF r R N A A N D I R N A

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J FIGURE8-38 Splicing mechanismsin group I and group ll self-splicingintrons and spliceosome-catalyzedsplicing of pre_ mRNA. The intron is shown in gray,the exonsto be joined in red. In group I Introns,a guanosinecofactor(G)that is not part of the RNA chajnassociates with the activesite.The 3,_hydroxyl group of this guanosineparticrpates in a transesterificatlon reactionwith the phosphateat the 5' end of the intron;this reactionrsanatogous to

thatinvolving the 2'-hydroxyl groupsof branch-site As in groupll introns andpre-mRNA introns g_g). spliced (seeFigure in spliceosomes Thesubsequent transesterification that linksthe 5, and3, exonsis s i m i l ai rn a l lt h r e es p l i c i nm g echanism Nso t et h a ts p l i c e d _ ogur ot u pI intronsarelinearstructures, unlikethe branched intronproducts in theothertwo cases. fromp A. Sharp, [Adapted 1987,Science235:769.1

As shown in Figure 8-39, the pre-tRNA expressed from . the yeast tyrosine IRNA (tRNATt.; gene contains a 14_base intron that is not present in mature tRNATy.. Some other eukaryotic IRNA genes and some archaeal IRNA genes also contain introns. The introns in nuclear pre_tRNAs are shorter than those in pre-mRNAs and lack the consensus

tRNAs generally are associatedwith proteins and spend little time free in the cell, as is also the case for mRNAs and rRNAs.

N u c l e a rB o d i e sA r e F u n c t i o n a l t S y pecialized N u c l e a rD o m a i n s High-resolution visualization of plant- and animal_cellnu_ clei by electron microscopy and subsequentstaining with fluorescentlylabeled antibodies has revealed domains in nuclei in addition to chromosome territories and nucleoli. Thesespecializednuclear domains, called nuclear bodies.are not surrounded by membranes but are nonethelessregions of high concentrarions of specific proteins and RNAs that form distinct, roughly spherical structures within the nu_ cleus. The most prominent nuclear bodies are nucleoli, the sites of ribosomal subunit synthesisand assemblydiscussed earlier. Severalorher types of nuclear bodies also have been describedin structuralstudies. Experiments with fluorescently labeled nuclear proteins have shown that the nucleus is a highly dynamic environ_ ment, with rapid diffusion of proteins through the nucleo_ plasm. Proteins associatedwith nuclear bodies are often also observedat lower concentration in the nucleoplasm outside

364

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Pre-tRNATYT of 8-39 Changesthat occurduringthe processing FIGURE intron(blue)in theanticodon A 14-nucleotide tyrosinepre-tRNA. (green) at the sequence by splicingA 16-nucleotide loopisremoved by at the 3' endarereplaced P U residues by RNase 5' endiscleaved bases (red)foundin all maturetRNAsNumerous the CCAsequence

the nuclear bodies, and fluorescencestudies indicate that they diffusein and out of the nuclearbodies.Basedon these measurementsof molecularmobility in living cells,nuclear bodies can be mathematically modeled as the expected steady state for diffusing proteins that interact with sufficient affinity to form self-organizedregions of high concentrations of specificproteins but with low enough affinity for each other to be able to diffuse in and out of the structure. Electron micrographsshow thesestructuresappear to be a heterogeneous,spongelikenetwork of interacting compo'We discussa few of these nuclear bodies here as exnents. amplesof thesenucleardomains. Cajaf Bodies Calal bodies are :0'2-L pm sphericalstructures that have been observedin large nuclei for more than a century (Figure 8-40). Current research indicates that like nucleoli,Cajal bodiesare centersof RNP-complexassembly for spliceosomalsnRNPs and other RNPs. Like rRNAs' snRNAs undergo specificmodifications, such as the conversion of specificuridine residuesto pseudouridineand addition of methyl groups to the 2'-hydroxyl groups of specific riboses.Thesepost-transcriptionalmodificationsare important for the proper assemblyand function of snRNPs in premRNA splicing. These modifications occur in Cajal bodies, where they are directed by a class of snoRNA-like guide RNA molecules called scaRNAs (small Caial body-associated RNAs). There is also evidencethat the Caial body is the site of reassemblyof the U4N6NS tri-snRNP complexes required for pre-mRNA splicing from the free U4, U5, and

bases modified to characteristic in the stemloopsareconverted out during spliced are that introns pre-tRNAs contain (yellow). Notall shown theothertypesof changes but theyallundergo processing, h e r eD : d i h y d r o u r i d iinl ,e=; p s e u d o u r i d i n e

U6 snRNPs releasedduring the removal of each intron (see Figure 8-11). SinceCajal bodiesalso contain a high concen,ri io., ol the uTsnRNP involved in the specialized3'-end processingof the major histone mRNAs, it is likely that this o..nr. in Calal bodies, as may the assemblyof pro..r, "l'ro RNP. the telomerase

3 kDa ,l::l:ll:

8-40 Nuclearbodiesare differentiallypermeableto A FIGURE showsa Eachpairof panels moleculesin the bulk nucleoplasm' which was nucleus, oocyte singleareathrougha livingXenopus molecular indicated of the dextran withfluorescent injected previously of the upperpanelisa confocal mass(3-2000kDa),Eachsection of dextran isa measure of fluorescence rmagein whichthe intensity hasbeen wheredextran (i.e.,darkerareas showregions concentration interference panel is a differential lower of the Eachsection excluded). nucleoli, indicate imageof thesamefield.openarrowheads contrast speckles nuclear (CBs) attached with bodies cajal arrowheads closed cells' thanin mostsomatic oocytes whicharemuchlargerin Xenopus (e completely g., almost kDa) 3 mass of low molecular Dextrans and speckles morefromnuclear CBsbutwereexcluded penetrated Bar' mass molecular with increased of dextran Exclusion nucleoli. Mol BiolCell15:2021 K E Handwerger,2OO5, 1Opm, [From P R O C E S S I NOGF r R N A A N D I R N A

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Nuclear Speckles Nuclear speckleswere observed with fluorescently labeled antibodies to snRNp proteins and other proteins involved in pre-mRNA splicing as -25-50 irregular,amorphous structures0.5-2 pm in diameter that are distributed through the nucleoplasm of vertebrate cells.

Promyelocytic Leukemia (pML) Nuclear Bodies The PML gene was originally discovered when chromosomal translocations within the gene were observed in the leukemic cells of patients with the rare diseasepromyelol_ cytic leukemia (PML). When antibodies speci?icfor the PML protein were used in immunofluorescence ml_ croscopy studies, the protein was found to localize to -10-30 roughly sphericalregions0.3-1 pm in diameterin the nuclei of mammalian cells. Multiple functions have been proposed for PML nuclear bodies,but a consensusis

r Synthesisand processingof pre-rRNA occur in the nucleolus. The 55 rRNA component of the large ribosomal subunit is synthesizedin the nucleoplasm by RNA polymeraseIIL r :150 snoRNAs associatedwith proteins in snoRNps base-pair with specific sites in pre-RNA, directing ribose methylation, modification of uridine to pseudouridine,and cleavage at specific sites during rRNA processing in the nucleolus.

r Pre-tRNAs synthesizedby RNA polymerase III in the nucleoplasm are processedby removal of the S,-end sequence,addition of CCA to the 3' end, and modification of multiple internal bases(seeFigure 8-39). r Some pre-tRNAs contain a short intron that is removed by a protein-catalyzedmechanism distinct from the solicing of pre-mRNA and self-splicingintrons. pecies of RNA molecules are associatedwith provarious types of ribonucleoprotein particles both in leus and after export to the cytoplasm.

responsegenes. Recent results indicate that pML nuclear bodies are also sites of protein post-translational modification through the addition of a small, ubiquitin-like protein called SUMOl (small zbiquitin-like miiety-1), whlch can control the activity and subcellular localization of the modified protein. Many transcriptional rcpressors are sumoylated,and mutation of their site of sumoylation re_ duces their repressionactivity. These results suggestthat PML nuclear bodies may be involved in a mechanism of transcriptional repressionthat remains to be studied and understood. In addition to PML nuclear bodies,the first nuclear bod_ ies to be observed,the nucleoli may have specializedregions of substructurethat are dedicatedto functironsother thin ri_ bosome biogenesis.There is evidencethat immature SRp ri_ bonucleoprotein complexes involved in protein secretion and ER membraneinsertion (Chapter 13) are assembledin nucleoli and then exported to the cytoplasm, where their fi_ nal maturation takes place.

Processing of rRNAand IRNA r. A.l.arg1precursorpre-rRNA (45Sin humans)synthesizedby RNA polymerase I undergoes cleavage, exonucle_ olytic digestion,and basemodificationsto yi.ld -"t.rr. 28S,185,and5.8SrRNAs,whichassociate with ribosomal proteinsinto ribosomalsubunits. 365

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r Nuclear bodies are functionally specializedregionsin the nucleus where interacting proteins form self-or ganized, structures.Many of these,like the nucleolus,are regions of assemblyof RNP complexes.

In this and the previous chapter, we have seen that in eu_

RNA sequencesand provides various avenuesfor regulating synthesis of a polypeptide chain. Much remains to be learned about the structure, operation, and regulation of such complex machines as spliceosomesand the cleavage/ polyadenylation apparatus. Recent examples of the regulation of pre-mRNA splic_ . ing raise the question of how extracellular signals might control such events, especiallyin the nervous system of vertebrates.A case in point is the remarkable situation in

of Slo pre-mRNA. The challengingtask facing researchersis to discoverhow such cell-cellinteractions regulatethe activity of RNA-processingfactors. The mechanism of mRNP transport through nuclear pore complexesposesmany intriguing questions.Future researchwill likely reveal additional activities of hnRNP and nuclear mRNP proteins and clarify their mechanisms of action. For instance, there is a small gene family encoding proteins homologous to the large subunit of the mRNA exporter. What are the functions of these related proteins? Do they participate in the transport of overlapping sets of mRNPs? Some hnRNP proteins contain nuclear-retention signals that prevent nuclear export when fused to hnRNP proteins with nuclear-export signals (NESs).How are these hnRNP proteins selectivelyremoved from processedmRNAs in the nucleus,allowing the mRNAs to be transported to the cytoplasm? The localization of certain mRNAs to specificsubcellular locations is fundamental to the developmentof multicellular organisms.As discussedin Chapter 22, during development an individual cell frequently divides into daughter cells that function differently from each other. In the languageof developmental biology, the two daughter cells are said to have different developmentalfates. In many cases'this difference in developmental fate results from the localization of an mRNA to one region of the cell before mitosis so that after cell division, it is present in one daughter cell and not the other. Much exciting work remains to be done to fully understand the molecular mechanisms controlling mRNA localization that are critical for the normal development of multicellular organisms. Someof the most exciting and unanticipated discoveries in molecular cell biology in recent years concern the existence and function of miRNAs and the processof RNA interference.RNA interference(RNAi) provides molecular cell biologists with a powerful method for studying gene function. The discoveryof -1000 miRNAs in humans and other organisms suggeststhat multiple significant examples of translational control by this mechanismawait to be characterized. Recent studies in S. pombe and plants link similar DNA methylation and short nuclear RNAs to the control of 'Will similar processes the formation of heterochromatin. control geneexpressionthrough the assemblyof heterochromatin in humans and other animals?'What other regulatory processesmight be directed by other kinds of small RNAs? Sincecontrol by these mechanismsdependson base pairing between miRNAs and target mRNAs or genes'genomic and suggestgenesthat may bioinformatic methods will probably'What other processesin be controlled by thesemechanisms. addition to translation control' mRNA degradation, and heterochromatin assemblymight be controlled by miRNAs? These are iust a few of the fascinating questions concerning RNA processing,post-transcriptional control, and nuclear transport that will challenge molecular cell biologistsin the coming decades.The astoundingdiscoveriesof entirely unanticipated mechanisms of gene control by miRNAs remind us that many more surprisesare likely in the future.

KeyTerms 5' cap325 alternativesplicing337 cleavage/polyadenylation complex335 recognrtlon cross-exon 333 complex Dicer348 Drosha348 335 exosome expoftin 347 342 FG-nucleoporins groupI introns353 groupII introns334 importin 344 elementiron-response bindingprotein (rRE-BP)356 micro RNAs (miRNAs) 347 mRNP expofiet343 357 mRNA surveillance

nuclearpore complex (NPC)342 poly(A)tail335 pre-mRNA326 pre-rRNA359 rrbozyme363 RNA editing341 RNA-inducedsilencing complex(RISC)348 RNA interference (RNAi)34e RNA splicing329 short interferingRNAs (siRNA)347 siRNA knockdown351 smallnuclearRNAs (snRNAs)330 smallnucleolarRNAs (snoRNAs)361 332 spliceosome SRproteins333

Reviewthe ConcePts l. Describe three types of post-transcriptional regulation of protein-coding genes. 2. You are investigatingthe transcriptional regulation of

not? 'S(hat is the evidencethat transcription termination by 3. RNA polymeraseII is coupled to polyadenylation? 4. It has been suggestedthat manipulation of HIV antitermination might provide for effectivetherapiesin combating 'S(hat effect would a mutation in the TAR sequence AIDS. that abolishesTat binding have on HIV transcription after HIV infection and why? A mutation in Cdkg that abolishes activity? 5. Describe how the discovery that introns are removed during splicing was made. How are the locations of exonintron junctions Predicted? 6. \7hat is the difference between hnRNAs' snRNAs, miRNAs. siRNAs, and snoRNAs? 7. \7hat are the mechanisticsimilarities between group II intron self-splicing and spliceosomal splicing? \fhat is the evidencethat there may be an evolutionary relationship between the two? T H EC O N C E P T S . REVIEW

367

8. rWheredo researchersbelieve most transcription and RNA-processing eventsoccur? \fhat is the evidenceto suDport this? 9. You obtain the sequenceof a gene containing 10 exons, 9 introns, and a 3' UTR containing a polyadenylation consensussequence.The fifth intron also contains a polyadenylation site. To test whether both polyadenylation sites are used, you isolate mRNA and find a longer transcript from muscle tissueand a shorter mRNA transcript from all other tissues.Speculateabout the mechanisminvolrred in the oroduction of thesetwo transcrlDrs.

that then undergo drug-induced cell death was compared to that of control cells. The experiment was repeatedin cells in which Dicer expression was knocked down using Dicer siRNA. The data obtained are shown in the graph below. \fhy did the scientistswho conductedthis study examine the effects of silencing Dicer? Under what conditions does the LAT geneprotect the cells from apoptosis? ! o o6

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@ celts transfectedwith LAr expresstonvector

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11. A protein complex in the nucleus is responsiblefor transporting mRNA moleculesinto the cytoplaim. Describe the proteins that form this exporter and whar two protern groups are likely behind the mechanism involved in the directional movement of the mRNp and complex into the cytosol. 12. RNA knockdown has become a powerful tool in the arsenal of methods to deregulat. g.n. expression. Briefly describehow gene expressioncan be knocked down. What effect would introducing siRNAs to TSC1 have on human cells? L3. Speculateabout why plants deficient in Dicer activity show increasedsensitivity to infection by RNA viruses. 14. lVhat is the evidencethat some mRNAs are directed to accumulatein specificsubcellular locations?

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celts transfectedwith LAr e x p r e s s i o nv e c t o ra n d e x p r e s s i n gD i c e rs i R N A

b. Cells were transfected with an expression vector expressing the Pst-Msu fragment of the LAT gene from which the region between the two Sty restriction sites was deleted (ASty; diagram below). Ifhen these cells were induced to undergo apoptosis, they died at the same rate as did non-transfected cells. In additional studies, cells were transfectedwith an expressionvector expressingthe Sty-Sty region of the LAT gene. These cells exhibited the same resistance to apoptosis as did cells transfected with the pstMsu fragment. What can be deduced from these findings about the region of the LAT gene required to protect cels from apoptosis? P s t / M l uf r a g m e n t

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Most humans are infected with herpes simplex virus_1 (HSV-1), the causative agenr of cold sores. The HSV_1 genomeencodesabout 100 genes,most of which are expressed in infected host cells at the site of oral sores. The infectious

LAT gene Exon 1

Exon2

5'-crcGccGccccGcccccGccccccccGGAcccAAGGGGcccccGcccccGeccctli_ g, s t . r n( b ' a r m ) I LOOP S t e m( 3 ' a r m ) |

These latently infected neurons are the source of active

type HSV-1. To determineif LAT functions to block apopto_ sis by encoding a miRNA, the following studies *.r. dtn. (seeGupta er al., 2006, Nature C+2:52-g5). a. A cell line was rransfectedwith an expressionvector that expressesa Pst-Msu (part b, diagram belowl fragment of the LAT gene. The percentageof these transfecteJ cells 368

.

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c. RNA encodedwithin the Sty-Styregion is predicted to form a stem loop (part b, diagram above). Northern blot analysiswas performed on total-cell RNA isolatedfrom control cells (mock), cells infected with wild-type HSV-1, cells infected with an HSV-1 deletion mutant from which the sequence between the two Sty sites in the LAT gene was deleted (ASty), and cells infected with a rescuedASty virus into which the deleted region was re-insertedinto the viral genome (StyR). The probe used for the Northern blot was the labeled 3' stem region of the LAT RNA in the Sty-Sty region, as diagrammed in part (b). The RNAs recognizedby this probe were either -55 nucleotidesor 20 nucleotides,as shown in the Northern blot below. -il/hy were two differentsize RNAs detected?\7hen a second probe was used that was the labeled 5' stem region of the RNA sequenceshown

in part (b), only the -S5-nucleotide RNA was detected. What can you deduce about the processingof RNA ex'What enzyme likely produced pressedfrom the LAT gene? the -55 nucleotide RNA? In what part of the cell? What enzyme likely produced the 20-nucleotide RNA? In what part of the cell? Control wt HSV-1 A Sty

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d. TGF-B mRNA encodes a protein, transforming growth factor B, that inhibits cell growth and inducesapoptosis.The 3' untranslatedregion (3'-UTR) of TGF-B mRNA can form an imperfect duplex with RNA encoded by the 5' stem region of the LAT Sty-Sty domain (miR-LAT), as shown below. In what way might the expression levels of TGF-p differ in cells infected with wild-type HSV-1 com'What can you infer about latent pared to uninfected cells? HSV-1 infections from thesestudies? miR-LAr A

| | CAG

rGF-B-3,urR

References of EukaryoticPre-mRNA Processing Bentley,D. L. 2005. Rules of engagement:co-transcriptional recruitmentof pre-mRNA processingfactors. Curr. Opin. Cell Biol. 17251.-256. Buratowski, S. 2005. ConnectionsbetweenmRNA 3' end processingand transcriptiontermination. Curr. Opin. Cell Biol. 17:257-261.. structural the message: Gu, M., and C. D. Lima. 2005. Processing insights into capping and decappingmRNA. Curr' Opin. Struct. Biol15:99-106. Houseley,J., J. LaCava,and D. Tollervey'2006. RNA-quality control by the exosome.Nat. Reu.Mol. Cell Biol.7:29-539. Hsieh, J., A. J. Andrews, and C. A. Fierke.2004. Roles of protein subunitsin RNA-protein complexes:lessonsfrom ribonuclease P. Biopolymers73:79-89. Lambowitz, A. M., and S. Zimmerly. 2004' Mobile group II introns. Annu. Reu.Genet.3821.-35. Lehmann,K., and U. Schmidt.2003. Group II introns: structure and catalytic versatilityof large natural ribozymes.Crit. Reu. Biochem.Mol. Biol. 38:249-303. Moore, M. J. 2005. From birth to death:the complex lives of eukaryotic mRNAs. Science309:15 14-1 5 18' Sharp,P.A. 2005. The discoveryof split genesand RNA splicing. TrendsB io chem. Sci. 3O279 -28 t.

Shatkin,A. J., and J. L. Manley. 2000. The endsof the affair: capping and polyadenylation. Nature Struct. Biol. 7 2838-842' RNA processingand its regulaSoller,M. 2006. Pre-messenger tion: a genomicperspective.Cell Mol. Life Sci.63:796-81'9' Valadkhan,S. 2005. snRNAs as the catalystsof pre-mRNA splicing.Curr. Opin. Chem.Biol.9:603-608. snRNAs: Villa, T., J. A. Pleiss,and C. Guthrie.2002. Spliceosomal lO9:149-1'52' Cell core? the catalytic at chemistry Mg(2*)-dependent Regulation of Pre-mRNA Processing Black, D. L.2003' Mechanismsof alternativepre-mRNA splicing. Ann. Reu.Biocbem. 72:291-336. Blencowe,B. J. 2005. Alternative splicing:new insightsfrom giobal analyses.Cell 126:37-47. Buratti, E., M. Baralle,and F. E. Baralle.2006. De{ectivesplicand therapy:searchingfor mastercheckpointsin exon ing, disease d"Jinition.NucleicAcidsRes.34:3494-3510Lee, C., and Q. \7ang. 2005. Bioinformaticsanalysisof alternative splicing.Brief Bioinform. 6:23-33. Licatalosi,D. D., and R. B. Darnell' 2006. Splicingregulationin neurologicdisease.Neuron 52293-101'. Maniatis, T., and B. Tasic' 2002. Alternative pre-mRNA splicing and proteomeexpansionin metazoans.Nature 418:236-243' Sanford,J. R., J. Ellis, and J. F. Caceres.2005. Multiple roles of splicingfactors in RNA processing'Biochem' arginine/serine-rich Soc.Trans.33:443446. Xing, Y., and C. Lee. 2006. Alternative-splicingand RNA selector eukaryottcgenomes' tion pressure-evolutionary consequences N at.-Reu.G enet.7 :499-5 09 . Transport of mRNA Across the Nuclear Envelope Cole, C. N., and J. J. Scarcelli.2006. Transport of messenger RNA from the nucleusio the cytoplasm.Curr. Opin' Cell Biol' 78:299-306. Darzacg,X., R. H. Singer,and Y. Shav-Tal.2.005'Dynamicsoftranscriptionand mRNA eiport. Curr. Opin- Cell Biol' 17:332-339' trans-. Dimaano,C., and K. S.Ullman' 2004. Nucleocytoplasmic oort, intes.atine mRNA production and turnover with export through ih. .ru.l.i, po... Moi. cail B iol. 24:3069-3076. Fried, H., and U. Kutay. 2003. Nucleocytoplasmictransport: taking an inventory.Cell Mol. Life Sci. 60t1'659-1688' Huang, Y., and J' A. Steitz.2005. SRprisesalong a messenger's journey.Mol. Cell. 77:613-615. Izaurralde,8.2004. Directing mRNA export. Nat' Struct' MoL Biol. ll:21.0-272. Kuersten.S.. and E. B' Goodwin. 2005. Linking nuclearmRNP assemblyand cytoplasmicdestiny.Biol. Cell. 97:469-478' Lim, R. Y., and B. Fahrenkrog.2006.The nuclearpore complex up close.Curr. Opin. Cell Biol. 18:342-347. Maco, B., B. Fahrenkrog,N. P. Huang, and U' Aebi' 2005' Nuclear pore-complexstructuri and plasti:ity r9ve115{by electronand atomfo force -^i.to..opy. Methods Mol. Biol.322:273-288' Reed,R., and H. Cheng.2005. TREX, SR proteins and export of mRNA. Curr. Opin. Cell Biol. 17:269-273. Ribbeck, K., and D. Gorlich. 2001. Kinetic analysisof tra-nslocation through nuclearpore complexes.EMBO J ' 2O:L320-1'330' Rodriguez,M. S., C. Dargemont,and F. Stutz' 2004' Nuclear export of RNA. Blol. Cell 96:639-655. Saguez,C., J. R. Olesen,and T. H. Jensen'2005' Formation of mRNP: escapingnucleardestruction' Cun' Opin' .*pori.o-p.t.nt 17:287-293. Biol. Cell Sommer.P..and U. Nehrbass.2005. Quality control of messenger ribonucleoproteinparticlesin the nucleusand at the pore' Curr' Opin. Cell Biol. l7:294-30t. Tran, E. J., and S' R. Wente.2006. Dynamic nuclearpore complexes: life on the edge' Cell 125:t041'-1'053 ' REFERENCES

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P o S T - T R A N S C R | P T | O NGAELN EC O N T R O L

Ruvkun, G. B. 2004. The tiny RNA world. HarueyLect.99:1.-21.. Schmauss,C. 2003. Serotonin2C receptors:suicide,serotonin, and runaway RNA editing. Neuroscientist9:237J42. Shikanai,T.2006. RNA editing in plant organelles:machinery, physiologicalfunction and evolurion.Cell Mol. Life Sci.63$98-708. Simpson,L., S. Sbicego,and R. Aphasizhev.2003.Uridine insertion/deletionRNA editing in trypanosomemitochondria: a complex business.RNA 92265-276. Sontheimer,E. J., and R. W. Carthew.2005. Silencefrom within: endogenoussiRNAs and miRNAs. Cell 122:9-12. St. Johnston,D. 2005. Moving messages: the intracellularlocalization of mRNAs. Nat. Reu.MoL Cell Biol.6:363-375. Tang, G. 2005. siRNA and miRNA: an insight into RISCs. TrendsBiochem.Sci.30:106-114. Ule, J., and R. B. Darnell. 2006. RNA binding proreinsand the regulationof neuronal synapticplasticity.Curr. Opln. Neurobiol. L6:102-11.0. Valencia-Sanchez, M. A., J. Liu, G. J. Hannon, and R. parker. 2006. Control of translationand mRNA degradationby miRNAs and siRNAs. GenesDeu. 20:51.5-524. Verdel,A., and D. Moazed. 2005. RNAi-directed assemblyof heterochromatinin fission yeast.FEBS Lett. 579:5872-5878. $Tullschleger, S., R. Loewith, and M. N. Hall. 2006. TOR sig. naling in growth and metabolism.Cell 124:471484. . Zamore, P.D., and B. Haley. 2005. Ribo-gnome:the big world of small RNAs. Sclezce309:1.519-1,524. Processing of rRNA and IRNA _. Anderson,J. T. 2005. RNA turnover: unexpectedconsequences of being tailed. Curr. Biol. l5:R635-R638. Decatur,W. A., and M. J. Fournier.2003. RNA-euided nucleotidemodification of ribosomal and other RNAs. /. Biol. Chem. 278:695-698. Dez, C., and D. Tollervey.2004.Ribosomesynthesismeetsthe cell cycle. Curr. Opin. Miuobiol. T:631-637. Evans,D., S. M. Marquez,and N. R. Pace.2006. RNasep: interfaceof the RNA and protein worlds. TrendsBiochem. Sci. 3l:333-347. Fatica,A., and D. Tollervey.2002.Making ribosomes.Curr. Opin. Cell Biol. 14:313-318. Handwerger,K. E., and J. G. Gall. 2006. Subnuclearorqanelles: new insights into form and function. Trends Cell Biot. 16:15-26. Hopper, A. K., and E. M. Phizicky.2003. IRNA rransfersto rhe limelight. GenesDeu. 17i.62-780. Mayer, C., and I. Grummt. 2006. Ribosomebiogenesisand cell growth: mTOR coordinatestranscription by all threi classesof nuclear RNA polymerases.Oncogene2006 25:6384-6391.. Stahley,M. R., and S. A. Strobel.2006. RNA splicing:group I intron crystal structuresrevealthe basisof splicesite selecti,cnand metal ion catalysis.Curr. Opin. Struct. Biol. 16:319-326. Tschochner,H., and E. Hurt. 2003. Pre-ribosomeson the road from the nucleolusro rhe cytoplasm.TrendsCettBiot. L3:255-263.

Microtubules

Golgi

Actinfibers

CHAPTER

Mitochondria

VISUALIZING, FRACTIONATING, AND CULTURING CELLS thelocation of DNAandmultiple microscopy shows Fluorescence proteins and fluorescent tagging withinthesamecellHere, molecules reveal fluorescent usingdifferent techniques staining (green) proteins andactin(red),DNA ct-tubulin thecytoskeletal (purple) (yellow), The (blue), andmitochondria theGolgicomplex images of eachstructure alongthetoparefalse-colored images imagemerges theseseparate Thelarger individually stained BNG thefullcell.Scale bars,20 pm [From images to depict etal, 2006,Science312:217 Giepmans, | lmost 200 years ago Matthias Schleiden and / \ theodore Schwannused a primitive light microscope /1ro show that individual cells constitute the fundamental unit of life, and light microscopy has been an increasingly important researchtool for biologists ever since. Sophisticated light microscopes developed in the last two decades now enable cell biologists to reveal the myriad movements of cells ranging from the translocation of chromosomesand vesiclesto cell crawling and swimming. Electron microscopy provides a much higher resolution of cell ultrastructure than light microscopy,but the technology requires that the cell be fixed and sectionedand thus all cell movements are frozen in time. Electron microscopy revealed that all eukaryotic cells-be they of fungal, plant' or animal origin-are divided into similar multiple membranelimited compartments termed organelles.In the first section of this chapter we describethe basic structuresand functions of the major organellesin animal and plant cells. In the second and third sectionsof this chapter we discuss many modern techniques in light and electron microscopy that are suitable for detecting and imaging particular structural features of the cell. Developmentsin both light and electron microscopS togetherwith those for generating monoclonal antibodies,have enabledmodern cell biologiststo detect specificproteins in fixed cells' thus providing a static image of their location within cells, as illustrated in the chapter opening figure. Such studiesled to the important concept that the membranesand interior spacesof each type A

of organelle contain a unique group of proteins that are essential for the organelleto carry out its unique functions. In addition, we see how certain chimeric proteins-consisting of a protein of interest covalently linked to a naturally fluoprotein-enable biologists to image movements of ,.r.*t individual proteins in live cells. Introduction of digital systems also has resulted in the improved quality of microscopic images,as well as digital storageand retrieval. Digital algorithms also permit three-dimensionalreconstructionsof ..il .o-pottents from two-dimensional images, and allow visualization and quantification of specific proteins and other moleculesin cells.

OUTLIN E 9.1

Organellesof the EukarYoticCell

9.2

Light Microscopy:VisualizingCellStructure a n d L o c a l i z i n gP r o t e i n sW i t h i n C e l l s

380

9.3

Methods ElectronMicroscoPY: andApplications

388

9.4

Purificationof Cell Organelles

391

9.5

lsolation,Culture,and Differentiation of MetazoanCells

371

Parallel developmentsin subcellular fractionation have enabled cell biologists to isolate individual organellesto a high degree of purity. These techniques,detailed in the fourth section of this chapter,continue to provide important information about the protein composition and biochemical function of organelles.For example, the use of proteomic approaches including mass spectrometry to determine the identity of all of the major proteins present in preparatrons of purified mitochondria has revealedmany novel functions for this organelle. Many technicalconstraints hamper studieson cells in intact animals and plants. One alternativeis the use of intact organs that are removed from animals and treated to maintain their physiologic integrity and function. However, the organization of organs, even isolated ones, is sufficiently complex to posenumerousproblemsfor research.Thus molecular cell biologists often conduct experimental studieson cells isolated from an organism. tn the fifth secion of this chapter we learn how to isolate certain types of cells to high purity from a complex mixture of cells. In many cases,isolated cells can be maintained in the laboratory under conditions that permit their survival and growth, a procedure known as culturing. Cultured cells have severaladvantagesover intact organismsfor cell biology research.Cells of a singlespecifictype can be grown in culture, experimental conditions can be better controlled, and in many casesa single cell can be readily grown into a colony of many identicalcells.The resultingsrrain of cells,which is genetically homogeneous,is called a clone. In culture, certain lines of undifferentiated mammalian cells can be induced to differentiate over a period of days to a specifictype of cell such as muscle or nerve when switched to a different culture medium. As we learn in the last section of this chaoter, such lines of cellsprovide powerful tools for understanding how specific types of differentiated cells are formed in the body.

Organelles of the EukaryoticCell

fl|

The major organellesin animal and plant cells are depicted in Figure9-1. Unique proteinsin the interior and membranes of each type of organelle largely determine its specific functional characteristics,which are examined in more detail in later chapters. Those organellesbounded by a single membrane are coveredfirst, followed by the three types that have a double membrane-the nucleus, mitochondrion, and chloroplast. The internal organizationof organelles,and the structure of the plasma membrane, is organized by the fibrous cytoskeleton; Chapters 1,7 and 18 discussthesefibers in detail.

T h e P l a s m aM e m b r a n eH a sM a n y C o m m o n F u n c t i o n si n A l l C e l l s Although the lipid composirion of a membrane largely determines its physical characteristics,its complement of pro_ teins is primarily responsiblefor a membrane.sfunctional 372

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c H A p r E R9

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properties. In all cells, the plasma membrane acts as a permeability barrier that prevents the entry of unwanted materials from the extracellular milieu and the exit of needed metabolites. Specific membrane transport proteins in the plasma membrane permit the passageof nutrients into the cell and metabolic wastes out of it; others function to maintain the proper ionic composition and pH (=7.2) of the cytosol, the aqueous portion of the cytoplasm excluding organelles,membranes, and insoluble cytoskeletal components. The structure and function of proteins that make the plasma membrane selectivelypermeable to different moleculesare discussedin Chapters10 and 11. Unlike animal cells, most bacterial, and all fungal and plant cells are surrounded by a rigid cell wall and lack the extracellular matrix found in animal tissues. The plasma membrane is intimately engagedin the assembly of cell walls, which in plants are built primarily of cellulose.The cell wall prevents the swelling or shrinking of a cell that would otherwise occur when it is placed in a medium that is less concentrated (hypotonic) or more concentrated (hypertonic), respectively,than the cell interior. For this reason, cells surrounded by a wall can grow in media having an osmotic strength much less than that of the cytosol. The properties,function, and formation of the plant cell wall are coveredin Chapter 19. In addition to these universal functions, the plasma membranehas other crucial roles in multicellular organisms. Few of the cells in multicellular plants and animals exist as isolated entities; rather, groups of cells with related specializations combine to form tissues.In animal cells, specialized areasof the plasma membrane, called cell junctions, contain proteins and glycolipids that form specific structures between cells that strengthentissuesand allow the exchangeof metabolites between cells. Certain plasma membrane proteins anchor cells to componentsof the extracellular matrix, the mixture of fibrous proteins and polysaccharidesthat provides a bedding on which sheets of epithelial cells or small glands lie. \Weexamine both of thesemembrane functions in Chapter 19. Still other proteins in the plasma membrane act as anchoring points for many of the cytoskeletal fibers that permeate the cytosol, imparting shape and strength to cells (Chapters 17 and 18). The plasma membranesof many types of eukaryotic cells also contain proteins that function as receptors by binding specific signaling molecules, such as hormones, growth factors, and neurotransmitters, leading to various cellular responses.These proteins, which are critical for cell development and functioning, are describedin severallater chapters. Finally, peripheral cytosolic proteins that are recruited to the membrane surface function as enzymes,intracellular signal transducers,and structural proteins for stabilizing the membrane.

EndosomesTakeUp SolubleMacromolecules from the Cell Exterior Although transport proteins in the plasma membrane mediate the movement of ions and small moleculesinto and out

v t s u A l t z t N c ,F R A C ' ' . N A T ' N GA,N D c u L T U R ' N G cELLs

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L v s o s o m e s ,w h i c h h a v e a n a c i d i c l u m e n , d e g r a d e m a t e r i a l i n t e r n a l i z e db v t h e c e l l a n d w o r n - o u t c e l l u l a rm e m b r a n e sa n d o r g a n elle s .

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N u c l e a re n v e l o p e ,a d o u b l e m e m b r a n e ,e n c l o s e st h e c o n t e n t s o f t h e n u c l e u s ;t h e o u t e r n u c l e a rm e m b r a n e i s c o n t i n u o u s with the rough ER.

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N u c l e o l u si s a n u c l e a rs u b c o m p a r t m e n tw h e r e m o s t o f t h e c e l l ' sr R N A i s s y n t h e s i z e d .

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N u c l e u si s f i l l e d w i t h c h r o m a t i n c o m p o s e d o f D N A a n d p r o t e i n s ;s i t e o f m R N A a n d t R N A s y n t h e s i s .

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S m o o t h e n d o p l a s m i cr e t i c u l u m ( E R )s y n t h e s i z e sl i p i d s a n d d e t o x i f i e sc e r t a i nh y d r o p h o b i cc o m p o u n d s .

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R o u g h e n d o p l a s m i cr e t i c u l u m( E R )f u n c t i o n s i n t h e s y n t h e s i s , processinga , n d s o r t i n g o f s e c r e t e dp r o t e i n s ,l y s o s o m a l p r o t e i n s ,a n d c e r t a i nm e m b r a n e p r o t e i n s .

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C o t g i c o m p l e x p r o c e s s e sa n d s o r t s s e c r e t e dp r o t e i n s , l v s o s o m a lp r o t e i n s ,a n d m e m b r a n e p r o t e i n ss y n t h e s i z e do n t h e r o u g hE R .

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S e c r e t o r yv e s i c l e ss t o r e s e c r e t e dp r o t e i n sa n d f u s e w i t h t h e o l a s m a m e m b r a n et o r e l e a s et h e i r c o n t e n t s .

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P e r o x i s o m e sd e t o x i f yv a r i o u s m o l e c u l e sa n d a l s o b r e a kd o w n f a t t v a c i d st o p r o d u c ea c e t y lg r o u p s f o r b i o s y n t h e s i s .

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C y t o s k e l e t afli b e r s f o r m n e t w o r k sa n d b u n d l e st h a t s u p p o r t c e l l u l a rm e m b r a n e s ,h e l p o r g a n i z eo r g a n e l l e s ,a n d p a r t i c i p a t e in cell movement. M i c r o v i l l ii n c r e ; s e s u r f a c ea r e a f o r a b s o r p t i o no f n u t r i e n t s f r o m s u r r o u n d i n gm e d i u m . s a i n t a i nt h e C g l l w a l l , c o m p o s e d l a r g e l yo f c e l l u l o s e , . h e l p m c e l l ' s s h a p e a n d p r o v i d e sp r o t e c t i o na g a i n s t m e c h a n i c a l stress. V a c u o l es t o r e sw a t e r , i o n s , a n d n u t r i e n t s ,d e g r a d e s m a c r o m o l e c u l e sa, n d f u n c t i o n s i n c e l l e l o n g a t i o nd u r i n g growth. C h l o r o p l a s t sw , h i c h c a r r y o u t p h o t o s y n t h e s i sa, r e s u r r o u n d e d b v a d o u b l e m e m b r a n e a n d c o n t a i na n e t w o r k o f i n t e r n a l m e m b r a n e - b o u n d e ds a c s .

overviewof a "typical"animalcell FIGURE 9-1 Schematic (top) and plant cell(bottom)and their majorsubstructures. , df i b r o u s l i l lc o n t a i a n l lt h eo r g a n e l l egsr,a n u l eas n N o te v e r yc e l w

in canbe present shownhere,andothersubstructures structures p r o m i n ence i n t h e a n d i n s h a p e c o n s i d e r a b l y s o m eC e l l sa l s od i f f e r substructures and organelles various of

of the cell across the lipid bilayer, proteins and some other soluble macromoleculesin the extracellular milieu are internalized by endocytosis.In this process,a segmentof the plasma membraneinvaginatesinto a "coated pit," whose cytosolic face is lined by a specificset of proteins; severaltypes of endocytosis,each involving different sets of proteins, are known. In receptor-mediatedendocytosis, for example, certain receptor proteins in the plasma membrane bind macromoleculesin the cell exterior and then becomeincorporated into the invaginating coated pit. The pit pinches from the membrane into a small membrane-boundedvesicle that contains extracellular material-both soluble and bound to receptors-and is deliveredto an early endosome, a sorting station of membrane-limited tubules and vesicles (Figure 9-2a,b). From this compartment' some membrane proteins are recycled back to the plasma membrane; other membrane proteins are transported to a late endosome

where further sorting takes place' The endocytic pathway ends when a late endosomedeliversits membrane and internal contents-including material from the extracellular solution-to lysosomes for degradation. The entire endocytic pathway is describedin somedetail in Chapter 14.

Are Acidic OrganellesThat Contain Lysosomes a Battery of DegradativeEnzymes Lysosomesprovide an excellentexample of the ability of intracellular membranes to form closed compartments in which the composition of the lumen (the aqueousinterior of the compartment) differs substantially from that of the surrounding cytosol. Found exclusively in animal cells, lysosomesare responsiblefor degradingcertain componentsthat have becomeobsoletefor the cell or organism' (The vacuoles in plant and fungal cells have many of the same functions as O R G A N E L L EO SF T H E E U K A R Y O T I C E L L

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hydrolytic enzymes proteins, degrade nucleic acids,andotherlarge m o l e c u l e(sb )A n e l e c t r om n i c r o g r a pohf a s e c t i o o nf a c u l t u r e d m a m m a l i acne l lt h a th a dt a k e nu p s m a lgl o l dp a r t i c l ecso a t e dw i t h the eggproteinovalbumin. (black Gold-labeled ovalbumin spots) is (EE) foundin earlyendosomes (LE), andlateendosomes butverylittle (AV) (c)Electron is presentin autophagosomes micrograph of a section of a ratlivercellshowing a secondary lysosome containing fragments (M)anda peroxisome of a mrtochondrion (p).lpart(b)from T.E Tjelle et al, 1996,J CellSci109:2905 Part(c)courtesy of D Friend l

animal lysosomes.)The processby which an agedorganelle is degradedin a lysosomeis called autophagy (,,eating oneself"). Materials are taken into a cell not only by endocytosis but also by phagocytosis,a processin which large, insoluble particles (e.g., bacteria) are envelopedby the plasma membrane and internalized(seeFigure 9-2a).ln borh cases,the internalizedmaterial may be degradedin lysosomes. Lysosomescontain a group of enzymesthat degradepolymers, releasingtheir monomeric subunits. For example, nucleases degrade RNA and DNA to mononucleotides; proteasesdegradea variety of proteins and peptidesto amino acids;phosphatasesremovephosphategroups from mononucleotides, phospholipids, and other compounds; still other enzymes degrade complex polysaccharidesand glycolipids into smaller units. All the lysosomalenzymeswork most effi-

Lysosomesvary in size and shape, and severalhundred may be presentin a typical animal cell (Figure 9-3). In effect, they function as siteswhere various materialsto be degraded collect. Primary lysosomesare roughly sphericaland do not contain obvious particulate or membrane debris. Secondary lysosomes,which are larger and irregularly shaped, result from the fusion of primary lysosomeswith late endosomes and other membrane-boundedorganellesand vesicles.They contain particles or membranes in the process of being digested(Figure9-2c). Severalhuman diseasesare causedby defectsin specific lysosomalenzymesbecausetheir substratesaccumulate insidethe organelle.For example, Tay-Sachsdiseaseis caused by a defect in one enzyme catalyzing a step in the lysosomal breakdown of gangliosides.The resulting accumulation of these glycolipids, especially in nerve cells, has devastating consequences.The symptoms of this inherited diseaseare usually evident before the age of 1. Affected children commonly becomedementedand blind by age 2 and die before their third birthday. Nerve cells from such children are greatly enlargedwith swollen lipid-filled lysosomes.I

PeroxisomesDegradeFatty Acids a n d T o x i cC o m p o u n d s protein complexesin the cytosol (seeFigure 3-29). 374

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All animal cells (except erythrocytes) and many plant cells contain peroxisomes,a classof roughly sphericalorganelles, 0.2-1.0 pr.min diameter (Figure 9-4). Peroxisomescontain

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9-4 Electronmicrographshowing variousorganelles FIGURE (P)lie in closeproximity to in a rat liver cell.Twoperoxisomes reticulum (M)andthe roughandsmoothendoplasmic mitochondria that is a polysaccharide (ER)Alsovisible of glycogen, areaccumulations P Lazarow of in animals ] molecule glucose-storage primary [courtesy the and mitochondriain a A FIGURE 9-3 Locationof lysosomes many of the sametypes of enzymesas well as additional ones culturedliving bovinepulmonaryarteryendothelialcell.The usedto convert fatty acids into glucoseprecursors.I ically luorescing dyethat isspecif with a green-f cellwasstained dyethat isspecifically anda red-fluorescing boundto mitochondria, c e t i c u l u ml s a N e t w o r k T h e E n d o p l a s m iR usinga The image was sharpened into lysosomes. incorporated laterin the chapter computerprogramdiscussed deconvolution of InterconnectedInternal Membranes Inc] Probes Invitrogen/ Molecular N : nucleus[Courtesy Generally,the largestmembrane in a eukaryotic cell encloses the endoplasmic reticulum (ER)-an extensive network of closed, flattened membrane-bounded sacs called cisternae severalox.idases-enzymesthat use molecular oxygen to ox(seeFigure9-1). The endoplasmicreticulumhas a number of idize organic substances,in the process forming hydrogen functions in the cell but is particularly important in the synperoxide (HzOz), a corrosive substance.Peroxisomesalso thesis of lipids, membrane proteins, and secretedproteins' contain copious amounts of the enzyme catdlase,which deThe smooth endoplasmicreticulwm is smooth becauseit gradeshydrogen peroxide to yield water and oxygen: lacks ribosomes.In contrast' the cytosolic face of the rowgh ca ta lase endoplasmicreticulum is studded with ribosomes' ZH2O2 ----+ 2H2O + 02 The Smooth Endoplasmic Reticulum The synthesisof mitofatty acids and phospholipidstakes place in the smooth ER' In contrast with the oxidation of fatty acids in genAlthough many cellshavevery little smooth ER, this organelle chondria, which producesCO2 and is coupled to the yields is abundant in hepatocytes'Enzymesin the smooth ER of the eration of ATP, peroxisomal oxidation of fatty acids (see Figliver also modify or detoxify hydrophobic chemicalssuch as acetyl groups and is not linked to ATP formation oxipesticidesand carcinogensby chemicallyconvertingthem into peroxisomal ure 1.2-L2).The energy releasedduring groups are more water-soluble,conjugatedproducts that can be excreted acetyl dation is converted into heat, and the in the from the body. High dosesof such compounds result in alarge are used they transported into the cytosol, where In most proliferation of the smooth ER in liver cells. metabolites. and other synthesis of cholesterol principal organelle peroxisome is the the eukaryotic cells, The Rough Endoplasmic Reticulum Cytoplasmic riboin which fatty acids are oxidized, thereby generating presomes bound to the rough ER synthesizecertain membrane cursors for important biosyntheticpathways. Particularly and organelle proteins and virtually all proteins to be secreted in liver and kidney cells,various toxic moleculesthat enter in the nascentpolypeptide from the cell (Chapter 13). Sequences the bloodstreamalso are degradedin peroxisomes,producing harmlessproducts. Plant seedscontain glyoxisomes,small organellesthat oxidize stored lipids as a source of carbon and energy for growth. They are similar to peroxisomes and contain O R G A N E L L EO SF T H E E U K A R Y O T I C E L L

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FIGURE 9-5 Characteristic featuresof cellsspecialized to secretelargeamountsof particularproteins(e.g.,hormones, (a)Electron antibodies). micrograph of a thinsection of a hormonesecreting cellfromthe rat pituitaryThebasalendof thecell(botfom) isfilledwithabundant roughERandGolgisacs, wherepolypeptide hormones aresynthesized andpackaged At theopposite apical end of thecell(top)arenumerous secretory vesicles, whichcontain recently madehormones (b)Diagram eventually to besecreted of a typicalsecretory celltracingthe pathwayfollowedbya protein(small reddots)to besecreted, lmmediatelv aftertheirsvnthesis on

interior space,or lumen. There the protein folds, assistedby folding catalysts termed chaperones.Secretedproteins are modified in severalways by enzymeslocalizedin the ER lumen, including covalentaddition of sugars(glycosylation)and formation of disulfide bonds. Newly made membrane proteins remain associatedwith the rough ER membrane, and proteins to be secretedaccumulate in the lumen of the organelle. AII eukaryotic cells contain a discernible amounr of rough ER becauseit is neededfor the synthesisof plasma membraneproteins and secretedproteins that compiise the extracellularmatrix. Phospholipidsare also synthesizedin associationwith the rough ER. Rough ER is particularly abundant in specializedcellsthat produce an abundanceof specificproteins to be secrered.For example, plasma cells produce antibodies, pancreatic acinar cells synthesizedigestiveenzymes,and cellsin the pancreaticisletsof Langerhans produce the polypeptide hormones insulin and glucagon.In thesesecretorycellsand others, alarge part of the cytosol is filled with rough ER and secretoryvesicles ( F i g u r e9 - 5 ) . 376

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(smallblackdots)of the roughER,secreted ribosomes proteins arefound inthelumenof theroughERTransport vesicles budoff andcarrythese proteins (lf ), wherethe proteins to the Golgicomplex areconcentrated andpackaged intoimmature ([) These secretory vesicles vesicles then coalesce to form largermaturesecretory vesicles that losewaterto the cytosol, leaving an almostcrystalline mixtureof secreted proteins i n t h e l u m e n( B ) A f t e rt h e s ev e s i c l easc c u m u l aut en d e tr h ea p i c a l surface, theyfusewiththeplasma membrane andrelease theircontents (exocytosis) in response to appropriate hormonal or nervestimulation (4) IPaft(a)courtesy of Biophoto Associates l

The Golgi ComplexProcesses and SortsSecreted a n d M e m b r a n eP r o t e i n s Severalminutes after proteins are synthesizedin the rough ER, most of them leave the organelle within small membrane-boundedtransportvesicles.Thesevesicles,which bud from regions of the rough ER not coated with ribosomes, carry the proteins to another membrane-limited organelle, the Golgi complex (seeFigure 9-5), named after the Italian microscopistCamillio Golgi. Three-dimensional reconstructions from serial sections of a Golgi complex revealthis organelleto be a seriesof flattenedmembranevesiclesor sacs(cisternae), surroundedby a number of more or lesssphericalmembrane-limitedvesicles (Figure 9-6). The stack of Golgi cisrernaehas three defined regions-the cis, the medial, and the trans. Translort vesicles from the rough ER fuse with the cls region of ihe Golgi complex, where they deposit their protein contents. As detailed in Chapter 14, these proreins then progress from the cis to the medial to the trons region. Within each region are different luminal enzymesthat modify proteins to be secreted

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Model of a Golgi Complex &) vlA"o: Three-Dimensional > FIGURE 9-6 Modelof the Golgicomplexbasedon threedimensionalreconstruction of electronmicroscopyimages. (whitespheres) Transport vesicles that havebuddedoff the rough (lightblue)of the Golgicomplex ERfusewith the crsmembranes in Chapter14,proteins movefromthe Bymechanisms described crsregionto the medralregionandfinallyto the transregionof vesicles budoff the trans-Golgi the GolgicomplexEventually, (orange and membranes andred);somemoveto the cellsurface likethe rough TheGolgicomplex, othersmoveto lysosomes. prominent reticulum, isespecially in secretory cells endoplasmic B J Marsh et al , 2001,ProcNat'lAcadSciUSA98:2399 I [From

and membrane proteins differentln depending on their structuresand their final destinations. After proteins to be secretedand membrane proteins are modified in the Golgi complex, they are transported out of the complex by a secondset of vesicles,which bud from the trans stde of the Golgi complex. Some vesiclescarry membrane proteins destinedfor the plasma membrane or soluble proteins to be releasedinto the extracellular medium; others carry soluble or membrane proteins to lysosomesor other organelles.Continuous budding of vesiclesin this manner would resultin accumulationof phospholipidsat the plasma membrane, but endocytic vesicles(seeFigure 9-2) return plasma membrane phospholipids to lysosomesor to the Golgi. Yet other vesiclesbud from Golgi vesiclesand fuse with earlier Golgi vesiclesor with the rough ER. How intracellular transport vesicles"know" with which membranesto fuse and where to deliver their contents is also discussedin Chapter 14.

small moleculesstored within it. Becausethe solute concentration is much higher in the vacuole lumen than in the cytosol or extracellular fluids, water tends to move by osmotic flow into vacuoles. This influx of water, which causesthe vacuole to expand, createshydrostatic pressure' or turgor, inside the cell. This pressureis balanced by the mechanical

P l a n tV a c u o l e sS t o r eS m a l lM o l e c u l e s a n d E n a b l ea C e l lt o E l o n g a t eR a p i d l y Most plant cells contain at least one membrane-limited internal vacuole.The number and sizeof vacuoles dependon both the type of cell and its stageof development; a single vacuole may occupy as much as 80 percent of a mature plant cell (Figure9-7). A varietyof transportproteinsin the vacuolar membrane allow plant cells to accumulateand store water, ions, and nutrients (e.g., sucrose,amino acids) within vacuoles(Chapter11). Like a lysosome,the lumen of a vacuole contains a battery of degradativeenzymesand has an acidic pH, which is maintained by similar transport proteins in the vacuolar membrane. Thus plant vacuoles may also have a degradativefunction similar to that of lysosomes in animal cells. Similar storage vacuolesare found in green algae and many microorganismssuch as fung^. Like most cellular membranes,the vacuolar membrane is permeableto water but is poorly permeableto the

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9-7 Electronmicrographof a thin sectionof a A FIGURE leaf cellshowinga prominentvacuole.In thiscell,the single muchof the cellvolumePartsof f ive occupies largevacuole c e lw l a l la l s oa r ev i s i b l eN o t et h e i n t e r n a l t h e a n d chloroplasts of Biophoto in the chloroplasts. [Courtesy subcompartments Laboratory National I C Ledbetter/Brookhaven Associates/Myron CELL O R G A N E L L EO SF T H E E U K A R Y O T I C

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A FIGURE 9-8 Electronmicrographof a mitochondrion. M o s tA T Pp r o d u c t i oin n o n p h o t o s y n t h ecterlcl st a k e sp r a c er n m i t o c h o n d rT i ah ei n n e rm e m b r a n w e ,h i c hs u r r o u n dt hs e m a t r i x space, hasmanyinfoldings, calledcristaeSmallcalcium-containing matrixgranules alsoareevident[From D W Fawcett, 1981 , TheCell, 2ded, Saunders, p a21l

resistanceof the cellulose-containingcell walls thar surround plant cells. Most plant cells have a rurgor of 5-20 atmospheres (atm); their cell walls must be strong enough to react to this pressurein a controlled way. Unlike animal cells, plant cells can elongate extremely rapidly, at rates of 20-75 prmlh. This elongation, which usually accompaniesplant growth, occurs when a segmentof the somewhat elasticcell wall stretchesunder the pressurecreated by water taken into the vacuole.I

T h e N u c l e u sC o n t a i n st h e D N A G e n o m e ,R N A SyntheticApparatus,and a FibrousMatrix The nucleus, the largest organelle in animal cells, is surrounded by two membranes,each containing many different types of proteins. The inner nuclear membrane defines the nucleus itself. In most cells, the outer nuclear membrane is continuous with the rough endoplasmic reticulum, and the space between the inner and outer nuclear membranes is

ture of nuclear pores and the regulatedtransport of material through them are detailed in Chapter 8. 378

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Most of the cell'sribosomal RNA is synthesizedin the nucleolus,a subcompartmentof the nucleusthat is not bounded by a phospholipid membrane. Ribosomal proteins, like all nuclear-encoded proteins,are made in the cytosol.Most enter the nucleus via nuclear pores and are added to ribosomal RNAs within the nucleolus. The finished or partly finished ribosomalsubunits,exit through a nuclearpore into the cytosol for use in protein synthesis(Chapter 4). In mature erythrocytes from nonmammalian vertebratesand in other types of "resting" cells,the nucleusis inactiveor dormant and minimal synthesisof DNA and RNA takesplace.Similarly,mRNA and IRNA is synthesizedin the nucleus;following extensiveprocessingwithin the nucleus, particles containing these RNAs also exit the nucleusinto the cytosol through nuclearpores. The packagingof nuclear DNA into chromosomesis describedin Chapter 6. Only during cell division are individual chromosomesvisible by light microscopy.In the electronmicroscope,the nonnucleolarregions of the nucleus,called the nucleoplasm, can be seen to have dark- and light-staining areas.The dark areas,which are often closelyassociatedwith the nuclearmembrane,contain condensedconcentratedDNA, called heterochromatin (seeFigure 6-33a). Fibrous proteins called lamins form a two-dimensional network along the inner surface of the inner membrane, giving it shapeand apparently binding DNA to it. The breakdown of this network occurs early in cell division, as we detail in Chapter 20.

MitochondriaAre the PrincipalSitesof ATP Productionin Aerobic NonphotosyntheticCells Most eukaryotic cells contain many mitochondria (sing., mitochondrion), which occupyup to 25 percentof the volume of the cytoplasm (seeFigure 9-3). Thesecomplex organelles, the main sitesof ATP production during aerobic metabolism, are generally exceededin size only by the nucleus,vacuoles, and chloroplasts (which also produce ATP). The two membranesthat bound a mitochondrion differ in compositionand function. The outer membrane,composedof about half lipid and half protein, containsporin proteins (see Figure 10-18) that render the membranepermeableto molecules having molecular weights as high as 10,000. The inner membraneis much lesspermeable;its surfacearea is greatly increasedby alarge number of infoldings, or cristae, that protrude into the matrix, or central space(Figure9-8). In nonphotosynthetic cells, the principal fuels for ATp synthesisare fatty acids and glucose.The complete aerobic degradation of glucose to CO2 and H2O is coupled to the synthesisof about 30 moleculesof AIP (the exact number is in question). In eukaryotic cells, the initial stagesof glucose degradation take place in the cytosol, where 2 ATP molecules per glucose molecule are generated. The terminal stagesof oxidation and the coupled synthesisof ATP are carried out by enzymesin the mitochondrial matrix and inner membrane (Chapter 12). As many as 28 ATP moleculesper glucose molecule are generated in mitochondria. Similarly virtually all the AIP formed in the oxidation of fatty acidsto CO2 is generatedin mitochondria. Thus mitochondria can be regardedas the "power plants" of the cell.

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then incorporated into the organellesby processesdescribed in Chaoter 13.

ChloroplastsContainInternal Compartments in Which Photosynthesis TakesPlace Except for vacuoles, chloroplasts are the largest and the most characteristic organelles in the cells of plants and green algae.They can be as long as 10 pcmand are typically 0.5-2 p"n thick, but they vary in size and shape in different cells, especially among the algae. In addition to the double membranethat bounds a chloroplast, this organellealso contains an extensiveinternal systemof interconnectedmembrane-limited sacs called thylakoids, which are flattened to form disks (Figure 9-9). Thylakoids often form stacks called grana and are embedded in a matrix space, the stroma. The thylakoid membranes contain green pigments (chlorophylls) and other pigments that absorb light, as well as enzymes that generate ATP during photosynthesis.Some of the ATP is used to convert CO2 into three-carbon intermediates by enzymeslocated in the stroma; the intermediates are then exported to the cytosol and convertedinto sugars.I The molecular mechanismsby which ATP is formed in mitochondria and chloroplastsare very similar, as explained in Chapter 12. Chloroplasts and mitochondria have other featuresin common: both often migrate from place to place within cells, and they contain their own DNA, which encodes some of the key organellar proteins (Chapter 6). The proteins encoded by mitochondrial or chloroplast DNA are synthesizedon ribosomes within the organelles.However, most of the proteins in each organelleare encodedin nuclear DNA and are synthesizedin the cytosol; these proteins are P l a s m am e m b r a n e

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Organelles of the Eukaryotic Cell r AII eukaryotic cells contain a nucleus as well as numerous other organellesin their cytoplasm (seeFigure 9-1). r The nucleus, mitochondrion, and chloroplast are bounded by two bilayer membranesseparatedby an intermembrane space.All other organellesare surrounded by single membranes. I The plasma membrane acts as a permeability barrier. It contains a multitude of proteins that transport nutrients and waste molecules or that bind components of the extracellular matrix or, in plants, the cell wall. The plasma membranesof many eukaryotic cells also contain receptor proteins that bind specificsignaling molecules. r Endosomesinternalize plasma membrane proteins and soluble materials from the extracellular medium, and sort the internalizedmaterials back to the membrane or to lysosomesfor degradation. r Lysosomeshave an acidic interior and contain various hydrolasesthat degrade worn-out or unneeded cellular components and some ingestedmaterials (seeFigure 9-2). r Peroxisomes are small organelles containing enzymes that oxidize various organic compounds without the production of ATP. By-products of oxidation are used in biosynthetic reactions. r Secretedproteins and membraneproteins are synthesized on the rough endoplasmicreticulum' a network of flattened membrane-boundedsacsstuddedwith ribosomes.

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r Proteins synthesizedon the rough ER first move to the Golgi complex, where they are processedand sorted for transport to the cell surfaceor other destinations(seeFigure 9-5). r Plant cellscontain one or more large vacuoles,which are storagesitesfor ions and nutrients. Osmotic flow of water into vacuoles generates turgor pressure that pushes the plasma membrane againstthe cell wall. r The nucleus housesthe genome of a cell. The inner and outer nuclear membranes are fused at numerous nuclear pores, through which materials pass between the nucleus and the cytosol. The outer nuclear membrane is continuous with that of the rough endoplasmicreticulum.

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The FIGURE 9-9 Electronmicrographof a plant chloroplast. (grana), (thylakoids) vesicles arefusedintostacks internalmembrane in the cell whichresidein a matrix(thestroma)All the chlorophyll wherethe light-induced in the thylakoidmembranes, is contained of production of ATPtakesplaceduringphotosynthesis [Courtesy National Laboratory Associates/M C Ledbetter/Brookhaven Biophoto l

r Mitochondria have a highly permeableouter membrane and an inner membranethat is extensivelyfolded. Enzymes in the inner mitochondrial membrane and central matrix spacecarry out the terminal stagesof sugar and lipid oxidation coupled to AIP synthesis' r Chloroplasts contain a complex system of thylakoid membranesin their interiors. Thesemembranescontain the pigments and enzymesthat absorb light and produce ATP during photosynthesis.

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< EXPERIMENTAL FIGURE 9-10 Opticalmicroscopes are commonlyconfiguredfor both bright-field(transmitted),phase-contrast, (a)In a typical and epifluorescence microscopy. lightmicroscope, the specimen is usually mountedon a transparent glassslideandpositioned on the movable specimen stageThethreeimagingmethods require different illumination systems and condensers; brightfieldandepifluorescence microscopy usethe samelight-gathering anddetection (b)In bright-field systems, lightmicroscopy, lightfroma tungsten lamptsfocused on thespecimen by a condenser lensbelowthestage;the lighttravels the pathway shownin yellow(c)ln phasecontrast microscopy, incident lightpasses throughan annular diaphragm, whichfocuses a circular (ring)of lighton thesampleLightthatpasses annulus unobstructed throughthespecimen is focused by the oblective lens onto gray the thick ring phase plate, of the which absorbs some of the W | il directlightandaltersitsphaseby one-quarter of a wavelength. (bends) lf a specimen refracts or diffracts the light,the phaseof somelightwavesisaltered(greenlines) andthe lightwavesare redirected throughthethin,clearregionof the phaseplateTherefracted andunrefracted lightare recombined at the imageplaneto formthe image(d)In epifluorescence microscopy, ultraviolet light (greenline)froma mercury lamppositioned abovethestageisfocused on thespecimen bythe objective lensFilters in the lightpathselect a particular wavelength of ultraviolet lightfor illumination andarematched to capture onlylightemittedbythe specimen of a predetermined wavelength that islongerthanthatof the incident light(redline)

LightMicroscopy: Visualizing @ CellStructureand Localizingproteins Within Cells For many years, light microscopy has been an essentialpart of virtually all researchon eukaryotic cells. Basic light microscopesare used to enumeratethe number of cells under study, and simple staining procedures enable the study of live cells. More specializedtechniques in lighr mrcroscopy enable the investigator to visualize movements such as the crawling of cells along a substrate,extensionof nerve axons, and movementsof chromosomesand organelleswithin cells.

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Using techniquesof recombinant DNA, researcherscan express,in a cell or in an organism, a chimera of any desired protein of interest linked to a naturally fluorescentprotein. Usually a chimera exhibits the normal function of the desired protein, and its location in living cells can be observed over time. Using immunofluorescence,researcherscan determine the location of specificproteins in fixed cells, as well as any localization changesin responseto changesin the cell's environment. Finally, many microscopic images of the same cell can be stored in a computer data base;digital reconstructions permits three-dimensionalreconstructionsof cell components from two-dimensional images,and a determination

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of whether two or more oroteins are found in the same subcellular compartment.

The Resolutionof the Light Microscope fs About O.2p,m All microscopesproduce a magnified image of a small object, but the nature of the imagedependson the type of microscope employedand on the way in which the specimenis prepared. The compound microscope,used in conventionalbright-field light microscopy,contains severallensesthat magnify the image of a specimenunder study (Figure 9-10a, b). The total magnificationis a product of the magnificationof the individual lenses:if the objectiuelens,the lensclosestto the specimen, magnifies 100-fold (a 100x lens, the maximum usually employed) and the proiection lens, sometimescalled the ocular or eyepiece,magnifies 10-fold, the final magnification recorded by the human eye or on film will be 1000-fold. However, the most important property of any microscope is not its magnification but its resolving power, or resolution-the ability to distinguish between two very closely positioned objects. Merely enlarging the image of a specimenaccomplishesnothing if the image is blurred. The resolution of a microscope lens is numerically equivalent to D, the minimum distance between two distinguishable objects.The smallerthe value of D, the betterthe resolution. The value of D is given by the equation

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Owing to limitations on the values of a, )", and N based on the physical properties of light, the limit of resolution of a light microscope using visible light is about 0.2 trr.m (200 nm). No matter how many times the image is magnified, a conventional light microscope can never resolve objects that are less than =0.2 p'm apart or reveal details smaller than =0.2 pr,min size. But exotic new light microscopesdescribedbelow can resolve two fluorescent obiects even if they are as close together as =20 nm. A conventional light microscopecan be usedto track the location of a small bead of known sizeto a precision of only a few nanometers.If we know the precise size and shapeof an obiect-say, a 5-nm sphereof gold that is attached to an antibody in turn bound to a cell-surfaceprotein-and if we use a video camera to record the microscopic image as a digital image, then a computer can calculate the position of the center of the object to within a few nanometers.In this way, computer algorithms can be used to make observations at a more preciselevel-in this casethe movement of a cell-surface protein labeled with the gold-tagged antibody-than would be possible based on the light microscope'sresolution alone. This technique has also been used to measure nanometer-sizesteps as molecules and vesicles move along cytoskeletalfilaments(seeFigures1'7-21,17-26, and 17-27).

and DifferentialInterference Phase-Contrast ContrastMicroscopyVisualizeUnstained (e-1) L i v i n gC e l l s

where a is the angular aperture,or half-angle,of the cone of light entering the objective lens from the specimen;N is the refractive index of the medium between the specimen and the objective lens (i.e., the relative velocity of light in the medium compared with the velocity in air); and \ is the wavelength of the incident light. Resolution is improved by using shorter wavelengthsof light (decreasingthe value of \) or gathering more light (increasingeither N or a). Note that the magnification is not part of this equation.

Two common methods for imaging live cells and unstained tissuesgeneratecontrast by taking advantageof differences in the refractive index and thickness of cellular materials' These methods, called phase-contrastmicroscopy and dif' (or Noferential interference contrast (DIC) microscopy difthat produce images microscopy), interference marski of cell features different reveal and in appe arance fer live, cultured of images compares Figure 9-11 architecture. cells obtained with thesetwo methods and standard brightfield microscopy.

by FIGURE 9-11 Livecellscanbe visualized EXPERIMENTAL techniquesthat generatecontrastby interference. microscopy cells viewedby macrophage These micrographs showlive,cultured (DlC) (/eft),differential contrast interference bright-fieldmicroscopy (righ\ Ina phase(middle), microscopy microscopy andphase-contrast

darkandlight byalternating image,cellsaresurrounded contrast imagedin a aresimultaneously details andout-of-focus bands;in-focus Ina DICimage,cellsappearin pseudorelief microscope. phase-contrast a DICimageisan regionisimaged, onlya narrowin-focus Because andJ Evans N Watson of ] the object. [Courtesy opticalslicethrough

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Phase-contrastmicroscopy (Figure 9-10c) generatesan image in which the degreeof darknessor brightnessof a region of the sample depends on the refractiue index of that region. Light moves more slowly in a medium of higher refractive index. Thus, a beam of light is refracted (bent) once as it passesfrom air into a transparentobject and again when it departs. Consequently, part of a light wave that passesthrough a specimenwill be refracted and will be out of phase (out of synchrony) with the part of the wave that does not passthrough the specimen.How much their phases will differ dependson the differencein refractive index along the two paths and on the thickness of the specimen.If the two parts of the light wave are recombined, the resultant light will be brighter if they are in phase and less bright if they are out of phase.The refractedand unrefractedlight are recombined at the image plane to form the image. Phasecontrast microscopy is suitable for observing single cells or thin cell layers, but not thick tissues.It is particularly useful for examining the location and movement of larger organellesin live cells. DIC microscopy is based on interferencebetween polarized light and is the method of choice for visualizing extremely small details and thick objects.Contrast is generated by differences in the index of refraction of the object and its surrounding medium. In DIC images, objects appear to cast a shadow to one side. The "shadow" primarily representsa differencein the refractive index of a specimenrather than its topography.DIC microscopy easily definesthe outlines of large organelles,such as the nucleus and vacuole.In addition to having a "relief"-like appearance,a DIC image is a thin optical section, or slice, through the object. Thus details of the nucleus in thick specimens (e.9., an intact Caenorhabditis elegans roundworm; seeFigure 21.-4)can be observedin a seriesof such optical sections, and the three-dimensionalstructure of the object can be reconstructedby combining the individual DIC images. Both phase-contrastand DIC microscopy can be used in time-lapse microscopy, in which the same cell is photographed at regular intervals over periods of severalhours. This procedure allows the observerto study cell movemenr, provided the microscope'sstagecan control the temperature of the specimenand the gas environmenr.

Fluorescence MicroscopyCan Localize a n d Q u a n t i f yS p e c i f i cM o l e c u l e si n L i v eC e l l s Perhapsthe most versatileand powerful technique for localizing proteins within a cell by light microscopy is fluorescent staining of cellsand observationby fluorescencemtcroscopy. A chemical is said to be fluorescentif it absorbslight at one wavelength (the excitation wavelength) and emits light (fluoresces)at a specific and longer wavelength. In modern fluorescencemicroscopes,only fluorescentlight emitted by the sample is used to form an image; light of the exciting wavelength induces the fluorescencebut is then not allowed to passthe filters placed betweenthe objective lens and the eye or camera (Figure 9-10d). 382

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Expression of Fluorescent Proteins in Live Cells and Organisms A naturally fluorescentprotein found in the jellyfish Aequorea uictoria can be exploited to visualize live cells and specific proteins within them. This 238-residue protein, called green fluorescent protein (GFP), contains a serine, tyrosine, and glycine sequencewhose side chains spontaneously cyclize to form a green-fluorescing chromophore. rWith the use of recombinant DNA techniques discussedin Chapter 5, the GFP gene can be introduced into living cultured cells or into specific cells of an entire animal. Cells containing the introduced gene will produce GFP and thus emit a green fluorescencewhen irradiated; this GFP fluorescence can be used to localize the cells within a tissue or even a whole organism, as illustrated in Figure 9-12 for a primitive metazoan organism, the Cnidairian Hydra uulgans. In a particularly useful application of GFP, a cellular protein of interest is "tagged" with GFP to localize it. In this technique, the gene for GFP is fused to the gene for a particular cellular protein, producing a recombinant DNA encoding one long chimeric protein that contains the entirety of both proteins. GFP fusion proteins frequently retain normal function, even with the large GFP polypeptide appended. Thus GFP fusions are exrremely useful

A EXPERf MENTAT FIGURE 9-12 Transgenichydra H. vulgaris expressinggreenfluorescentprotein (GFP).A recombinant plasmidin whichthe promoterof the H vulgaris B-actingene drivesexpression of the GFPgenewasmicroinjected intoembryos at the two- to eight-cell stage.In certainhydralines,the plasmid integrated intothe genomeof oneor a few adjacent cellsthat t h e nu n d e r w e ndti v i s i o nT.h eG F P - p r o d u cci negl l s( g r e e nw) e r e visualized by fluorescence microscopy In the specimen shownhere, GFPwasconfinedto certaininternalendodermal epithelial cells (/nset)A patchof GFP*endodermal epithelial cellsvisualized by confocalmicroscopy Scalebar,20 pm With both imaging techniques, the GFP-producing cellscanbe followedduringgrowth anddifferentiation of theorganism[From J Wittlieb eral,2006,proc Nat'l Acad Sci USA 103:6208 l

vtsuAltztNG, FRACT|ONAT|NG A,N D C U L T U R T NC GE L L S

tools for looking at the normal function and trafficking of native proteins. Cells in which this recombinant DNA has b e e n i n t r o d u c e d w i l l s y n t h e s i z et h e c h i m e r i c p r o t e i n whose green fluorescencerevealsthe subcellular location of the protein of interest.This GFP-taggingtechnique,for example, has been used to visualize the expression and distribution of lamin A, a protein that lines the inner, or intranucleaq surface of the inner nuclear membrane; it also forms tubule-like structuresthat protrude into the nuc l e u s( F i g u r e9 - 1 3 ) . In a variation of this technique,a cDNA encoding a protein of interest is modified at its C-terminus by the addition of a segmentthat encodesfour cysteineresiduesin a defined Following expressequence(Cys-Cys-Xaa-Xaa-Cys-Cys). sion of the recombinant protein in cultured cells, a chemically modified red-fluorescingdye (ReAsH) is added to the culture; this dye forms stable covalent bonds with the four cysteinesin the tetracysteinesequence.Becausethis sequence is not found in natural proteins, the dye binds to no other cellular proteins. After the cells are washed in buffers to remove unattached dye, the protein of interest can be visualized by fluorescencemicroscopy on either live or fixed cells. The subcellularlocalizationof the taggedprotein in the same cellscan also be visualizedat a higherresolutionby electron microscopy. Figure 9-14 shows the use of this fluorescenttagging technique to localize connexin, a plasma membrane protein that is found in gap junctions. These cell-surface structurespermit the rapid diffusion of small, water-soluble molecules between the cytoplasms of two adjacent cells (Chapter 19). Another exampleof this taggingtechniqueis shown in the chapter opening figure. In this case,HeLa cells were transfectedwith a cDNAs encoding a B actin with a tetracysteinetag and then stained with ReAsH dye to label the B actin.

Podcast;Light and ElectronMicroscopy

9-14 Detectionof tetracysteine' FIGURE ^| EXPERIMENTAL a gap-junctionprotein,by both light and taggedConnexin43, (Cx43)was Connexin43 ThecDNAencoding electronmicroscopy. a short encodes that a sequence with C-terminus at the extended The sequence. peptidecontaining the Cys-Cys-Xaa-Xaa-Cys-Cys in HeLacells,whichwerethen cDNAwasexpressed recombinant bindsonly dyethatcovalently a red-fluorescing with ReAsH, stained (a) (TC) Confocal sequence. tetracysteine with thisspecific to proteins of adjacent plasma membranes the of segments two reveals image the gapjunctions thatcontain by multiple cells thatareconnected bywhitearrows)Thecells Cx43-TCprotein(brightlinesindicated treatedwith the fixedwith2% glutaraldehyde, weresubsequently Under illumination intense to and subjected dyediaminobenzidine, that a photoconversion dyeundergoes the ReAsH theseconditions, whichcanbe seen,after in formationof a denseprecipitate, results (b)Inthiselectron microscope in theelectron thecellsaresectioned, (a) photoconversion, after in shown of thesamearea micrograph the gapiunctions(c)Highrepresent thedarklines(blackarrows) many in panel(b)reveals depicted viewof thesection magnification of membranes proteins in theplasma gapjunction Cx43-containing the high cells(darkstain)Thisimagehighlights thetwo adjacent methodBar labeling with the tetracysteine obtainable resolutron i n ( a )1, O U mi n; ( b )1, p m ;a n d i n ( c 1) ,0 0 n ml F r o m G G a i e t t ,a e t a l 296:503; see also B N G Giepmanset al , 2006, Sclence 2OO2,Science V o l 3 1 2 , p a g e2 0 7 l

FIGURE 9-13An opticalsectionof a living EXPERIMENTAL CHO-K1cellexpressinga recombinantlaminA-GFPchimeric with laminA (white) protein.Shownhereistheoval-shaped nucleus A i sa l s of o u n di n i n t r aa- n d r u c l e amr e m b r a nLea m i n l i n i n gt h ei n n e n Therestof thecelldoesnotcontarn tubule-like structures transnuclear et al, fusionprotein andthusisblack[rromJ L V Broers anylamin-GFP 1999,J CellSci112:3463 l

Determination of Intracellular Ca2* and H+ Levels with lon-sensitive Fluorescent Dyes The concentration of Ca2* or H+ within live cellscan be measuredwith the aid of fluorescent dyes, or fluorochromes, whose fluorescencedeoends on the concentration of these ions. As discussedin i"te, .hapters, intracellular Ca2* and H* concentrations have pronounced effectson many cellular processes.For instance,many hormones and other stimuli causea rise in cytosolic Ca2* from the restinglevelof about 10-' M to 10-" M, which induces various cellular responsesincluding the c o n t r a c t i o no f m u s c l e . The fluorescentdye fwra-2, which is sensitiveto Ca"-, contains five carboxylate groups that form ester linkages with ethanol.The resultingfura-Zesteris lipophilic and can

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l m a g i n gS u b c e l l u l aD r e t a i l sO f t e n R e q u i r e st h a t the SamplesBe Fixed,Sectioned,and Stained

EXPERIMENTAL FIGURE 9-15 Fura-2, a Ca2*-sensitive fluorochrome,can be usedto monitorthe relativeconcentrations of cytosolicCa2*in differentregionsof live cells.(left)Ina moving leukocyte, a Ca2*gradientisestablished. (green) Thehighestlevels areat the rearof thecell,wherecortical contractions takeplace, and (blue)areat the cellfront,whereactinundergoes the lowestlevels polymerization. (Right)Whena pipettefilledwith chemotactic placed molecules to thesideof thecellinduces thecellto turn,the Ca2*concentration momentarily increases throughout thecytoplasm anda newgradient isestablished Thegradient isoriented suchthat the regionof lowestCa2*(blue)liesin thedirection thatthecellwill turn,whereas a regionof highCa2*(yellow) always formsat thesite thatwillbecome the rearof thecell [From R A Brundage etal, 1991, Science 254:703; courtesy of F.Fayl

Live cells and tissuesgenerally lack compounds that absorb light and are thus nearly invisible in a light microscope.Although such specimenscan be visualized by the specialtechniqueswe just discussed,thesemethods do not reveal the fine details of structure.Microscopy of live cells also requires thar cells be housed in special glass-faced chambers,called culture chambers,that can be mounted on a microscope stage. For these reasons, cells are often fixed, sectioned,and stained to reveal subcellular structures. Specimensfor light and electron microscopy are commonly fixed with a solution containing chemicals that cross-linkmost proteinsand nucleicacids.Formaldehyde,a common fixative, cross-links amino groups on adjacent molecules; these covalent bonds stabilize protein-protein and protein-nucleic acid interactions and render the molecules insoluble and stable for subsequentprocedures.After fixation, a sample used for light microscopy is usually embeddedin paraffin and cut into sections0.5-50 ir,m thick (Figure9-16).For electronmicroscopysamplesare imbedded in liquid plastic and, after hardening, sections 50-100 nm thick are cut. Alternativeln the samplecan be frozenwithout

Block Specimen

diffuse from the medium across the plasma membrane into cells. \Tithin the cytosol, esteraseshydrolyze fura-2 ester, yielding fura-Z whose free carboxylate groups render the molecule nonlipophilic and thus unable to cross cellular membranes, so it remains in the cytosol. Inside cells, each fura-2 molecule can bind a single Ca2* ion but no other celIular cation. This binding, which is proportional to the cytosolic Ca2* concentration over a certain range, rncreases the fluorescenceof fura-2 at one particular wavelength.At a second wavelength, the fluorescenceof fura-2 js the same whether or not Ca2+ is bound and provides a measureof the total amount of fura-Z in a region of the cell. By examining cells continuously in the fluorescencemicroscope and measuring rapid changes in the ratio of fura-2 fluorcscence at these two wavelengths, one can quantify rapid changes in the fraction of fura-2 that has a bound Ca2* ion and thus in the concentration of cytosolic Ca2+ (Figure 9-15). Fluorescentdyes (e.g., SNARF-1)-th^t urc sensitiveto the H* concentrationcan similarly be used to monitor the

recrossthe organelle membrane, they accumulate in the Iumen in concentrations many fold greater than in the cytosol. Thus this type of fluorescent dye can be used to specifically stain lysosomesin living cells, as Figure 9-3 snows, 384

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Microscopeslide

Copper mesh grid

A EXPERIMENTAL FIGURE 9-16 Tissuesfor microscopyare commonlyfixed,embeddedin a solidmedium,and cut into thin sections. A fixedtissueisdehydrated by soaking in a series of alcohol-water solutions, endingwith an organic solvent compatible with the embedding mediumToembedthetissuefor sectioning, thetissueis placedin liquidparaffin for lightmicroscopy or in liquid plastic for electron microscopy. Afterthe blockcontaining thespecimen hashardened, it ismounted on thearmof a microtome andslices are cutwith a knife.Typical sections cutfor lightmicroscopy are0 5-50 pm thick.Thosecutfor electron microscopy aregenerally 50-100nm thick Thesections arecollected eitheron microscope (light slides microscopy) or coppermeshgrids(electron microscopy) andstalned with an appropriate agent

v t s u A l t z t N GF, R A C T | O N A T | N AG N ,D C U L T U R T N CG ELLS

prior fixation and then sectioned;such treatment preserves the activity of enzymesfor later detection by cytochemical reagents. Cultured cells growing on glass coverslips, as describedlater, are thin enough so they can be fixed in situ and visualized by light microscopy without the need for sectioning. A final step in preparing a specimen for light microscopy ls to starn rt so as to visualize the main structural features of the cell or tissue. Many chemical stains bind to molecules that have specific features.For example, hematoxylin binds to basic amino acids (lysine and arginine) on many different kinds of proteins, whereas eosin binds to acidic molecules (such as DNA and side chains of aspartate and glutamate). Becauseof their different binding properties, these dyes stain various cell types sufficiently differently that they are distinguishable visually. lf an enzymecatalyzesa reaction that producesa colored or otherwise visible precipitate from a colorless precursor,the enzyme may be detectedin cell sectionsby their colored reaction products. Such staining techniques, although once quite common, have been largely replaced by other techniquesfor visualizing particular proteins, as discussednext.

MicroscopyCan Detect lmmunofluorescence Proteins in Fixed Cells Specific The common chemical dyes just mentioned stain nucleic acids or broad classesof proteins, but investigators often want to detect the presenceand location of specificproteins. A widely used method for this purpose employs specific antibodies that are detectedby fluorescence.In one version of this technology, the antibody-generally a monoclonal antibody-is covalently linked to a fluorochrome. Commonly used fluorochromes include rhodamine and Texas red, which emit red light; Cy3, which emits orange light; and fluorescein, which emits green light. These fluorochromes can be chemically coupled to purified antibodies specificfor almost any desiredmacromolecule.'Whena fluorochrome-antibody complex is added to a permeabilizedcell or tissue section,the complex will bind to the correspondingantigens, which then light up when illuminated by the exciting waveIength, a technique called immunofluorescencemicroscopy (Figure 9-17). Staining a specimenwith different dyes that fluoresce at different wavelengths allows multiple proteins as well as DNA to be localized within the same cell (see chapter opening figure). In a variation of the immunofluorescence technique, a monoclonal or polyclonal antibody is applied to the fixed tissue section, followed by a second fluorochrome-tagged antibody that binds to the common (Fc) segment of the first antibody. For example, a "second" antibody (called "goat anti-rabbit") is preparedby immunizing a goat with the Fc segmentthat is common to all rabbit IgG antibodies; when coupled to a fluorochrome, this second antibody preparation will detect any rabbit antibody used to stain a tissue or cell. Becauseseveral goat anti-rabbit antibody moleculescan bind to a singlerabbit antibody moleculein

, 2oP'm , 9-17 Aspecificprotein can be FIGURE A EXPERIMENTAL localizedin fixed tissuesectionsby immunofluorescence wallwasstainedwith A sectionof the rat intestinal microscopy. andwith redfluorescence, a nonspecific blue,whichgenerates Evans glucose a for GLUT2, specific antibody green-fluorescing yellow a micrograph, fromthisfluorescence protein. As evident transport cells sidesof the intestinal in the basalandlateral ispresent GLUT2 packed of closely composed but isabsentfromthe brushborder, lumen.Capillaries facingthe intestinal on theapicalsurface microvilli tissuebeneath a looseconnective runthroughthe laminapropria, 259:C279' Am.J Physio al, 1990, layer. theepithelial lseeB Thorenset of B,Thorens courtesy l

a section, the fluorescenceis often brighter than if just a protein-specific antibody directly coupled to a fluorochrome is used. In another widely used version of this technolog5 cells are transfected with a cDNA encoding a recombinant protein that has appended,generally to one end' a short sequenceof amino acids called an epitope tag. Two commonly used epitope tags are called FLAG' encoding the amino acid sequenceDYKDDDDK (single-letter code), and myc, encoding the sequenceEQKLISEEDL. Commercial fluorochrome-coupled monoclonal anti-epitope antibodies can then be used to detect the recombinant protein in the cell. New types of fluorescencemicroscopy, such as one with the exotic name of STED (stimulatedemissiondepletion). enabletwo fluorescentobiectsto be resolvedeven if they are as closetogether as =20 nm, well below the =200nm resolution limit for standard light microscopy. For example, nerve cells contain a class of membrane-lined vesicles, termed synaptic vesicles, that ate too small (=40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. However, STED can resolve individual vesiclesin nerve cells. This technique should also enable investigators to detect single fluorescent protein molecules in the membranes of purified orqanelles.

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C o n f o c aal n d D e c o n v o l u t i o nM i c r o s c o p yE n a b l e Visualization o f T h r e e - D i m e n s i o nO a lb j e c t s

scanning processcan often take a few minutes and so is not well-suited to imaging fast biological processesin liv, C o n v e n t i o n a l f l u o r e s c e n c em i c r o s c o p y h a s r w o m a j o r ing samples.A revolutionary newly developedvariation of limitations. First, the fluorescent light emitted by a samlaser-scanningconfocal microscopy is Nipkow confocal ple comes from molecules above and below the plane of microscopy.This method usesa spinning disk with multifocus; thus the observer seesa blurred image caused by ple pinholes that split the laser into hundreds of beamlets, the superpositionof fluorescentimagesfrom moleculesat increasingthe rate at which the image is collectedby a facmany depths in the cell. The blurring effect makes it diffitor of 10 or more. cult to determine the actual molecular arrangements(FigImagesof the highest possibleresolution currently availu r e 9 - 1 8 a ) .S e c o n d t, o v i s u a l i z et h i c k s p e c i m e n sc, o n s e c u - able can be obtained by deconvolution,a computationally tive (serial) secrions must be prepared, imaged, and intensive mathematical processwhereby blurred objects are aligned to reconstruct structuresin thick tissues.Two apsharpened.Similar algorithms are used by astronomersto p r o a c h e sc a n b e u s e d t o a v o i d t h e p r e p a r a t i o n o f s e r i a l sharpen images of distant stars. In deconuolution mis e c t i o n sa n d t o o b t a i n h i g h - r e s o l u t i o nt h r e e - d i m e n s i o n a l croscopy,a seriesof images of an object are taken at differinformation. ent focal planes with a conventional fluorescencemicroOne approach, called confocal microcopy differs from scope or confocal microscope. A separateseriesof images conventional fluorescencemicroscopy by the use of a pinare made from a test slide containing tiny fluorescentbeads hole located in front of the detector that blocks light not smaller (e.g.,0.2 trrmin diameter)than the resolutionof the originating from that focal plane. The resulting imagesdo microscope.Each bead representsa pinpoint of light that benot contain blurs from structures above and below the comesa blurred object becauseof the imperfect optics of the c u r r e n tp o s i t i o no f t h e f o c a l p l a n e ( F i g u r e9 - 1 8 b ) .T h e m a microscope;from theseimagesa point spreadfunction is dejority of confocal microscopesuse a laser as the source of termined that enablesthe investigator to calculatethe distriillumination; lasers provide a defined excitation wavebution of fluorescentpoint sourcesthat generatedthe "blur" length and becauseof their focused energy are often well in the object image. In other words, the light generaringthe suited to penetrating thick specimens.Since a laser is foblurred sample image from adjacent focal planes is reasc u s e d o n a s i n g l e p o i n t o n t h e s p e c i m e n ,i t m u s t b e signed to the correct focal plane via iterative comparlson scannedacrossand down to build an image. The intensity with the point spreadfunction. Imagesrestored by deconvo, of light from these in-focus areas is recor"dedby a photolution display impressivedetail without any blurring as illusmultiplier tube, and the image is stored in a comouter.The trated in Figure9-19.

( a )C o n v e n t i o n af l u o r e s c e n cm e icroscopy

(b) Confocalfluorescencemicroscopy

' lmaged volume

A EXPERIMENTAL FIGURE 9-18 Confocalmicroscopyproducesan in-focus optical section through thick cells.A mitoticfertilizedegg from a seaurchin(Psammechinus) was lysedwith a detergenr,exposeo to an anti-tubulin antibody,and then exposedto a fluorescein-tagged antibodythat bindsto the anti-tubulinantibody.(a)When viewedby conventional f luorescence microscopy, the mitoticspindleis blurred

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Thisblurringoccursbecausebackground fluorescence is detectedfrom tubulinaboveand belowthe focal planeas depictedin the sketch (b) Theconfocalmicroscopic imageis sharp,particularly in the center of the mitoticspindleln thiscase,fluorescence is detectedonlyfrom molecules in the focalplane,generatinga verythin opticalsection fromJ G Whiteet al , 1987,J CellBiol 104:41] [Micrographs

V t S U A L t Z t N GF, R A C T | O N A T I N G A ,N D C U L T U R T NC GE L L S

Graphicsand InformaticsHaveTransformed Modern Microscopy In the past decade,digital cameras have largely replaced optical camerasto record microscopy images.Digital imagescan be stored in a computer and manipulated by conventional photographic software as well as by specialized algorithms. As mentioned above, deconvolution algorithms can sharpen an image by restoring out-of-focus photons to their origin. Other computer algorithms, which previously required supercomputing facilities' enable visualizationof intricate three-dimensionalstructures reconstructed on desktop computers (see Figure 9-1'9). Informatics including image analysis algorithms and statistical approachesallow quantitation of shapes,movements and signal intensitieswithin objects such as cells or organelles.

Light Microscopy:Visualizing Cell Structure and LocalizingProteins Within Cells r The limit of resolution of a light microscope is about 200 nm. r Becausecells and tissuesare almost transparent' various types of stains and optical techniquesare used to generate sufficientcontrastfor imaging' r Phase-contrastand differential interference contrast (DIC) microscopy are used to view the details of live' unstained cells and to monitor cell movement (seeFigure 9-11). 'Sfhen proteins tagged with naturally occurring green r fluorescent protein (GFP) or its variants are expressedin live cells, they can be visualized in a fluorescencemicroscope(seeFigure9-12). r \fith the use of dyes whose fluorescenceis proportional to the concentration of Ca2* or H* ions' fluorescencemicroscopycan measurethe local concentration of Ca2* ions and intracellular pH in living cells.

FIGURE 9-19 Deconvolutionfluorescence EXPERIMENTAL yieldshigh-resolution opticalsectionsthat can be microscopy image.A macrophage into one three-dimensional reconstructed for DNA specific reagents with fluorochrome-labeled cellwasstained ( b l u e )m , i c r o t u b u l(egsr e e n a) ,n da c t i nm i c r o f i l a m e n( rtes d )T. h e focalplanes imagesobtainedat consecutive series of f luorescent (optical in threedimensions through thecellwererecombined sections) (a)In thisthree-dimensional the reconstruction of the rawimages, D N A .m i c r o t u b u l easn.da c t i na o p e aar sd i f f u s ez o n e si n t h ec e l l . (b)Afterapplication algorithm to the images, of thedeconvolution f t h ef i b r i l l aor r g a n i z a t i oonf m i c r o t u b u laens dt h e l o c a l i z a t i o n r e a d i lvyi s i b l ien t h e r e c o n s t r u c t i o n a c t i nt o a d h e s i o nbse c o m e Whitehead Institute of J Evans, ] [Courtesy

r In immunofluorescencemicroscopy, specific proteins and organelles in fixed cells are stained with fluorochrome-labeledmonoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes (see chapter opening figure). r Confocal microscopy and deconvolution microscopy use different methods to optically section a specimen, thereby reducing the blurring due to out-of-focus fluorescence light (see Figures 9-18 and 9-19). Both methods provide much sharper images, particularly of thick specimens, than does standard fluorescence or light microscopy. r Advances in computation and graphics enable measurements to be extracted from microscopeimages.

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Microscopy: Methods !f,l Electron andApplications Electron microscopy of cells and tissues provides a much higher resolution of cell ultrastrucrure than can be obtained by light microscopy. However, the technology requires that the cell be fixed and sectioned,and thus living cells cannot be imaged. Researchersweigh these kinds of trade-offs and selectfrom a variety of methods to produce imagesthat answer their questions. For example, immunoelectron microscopycan be usedto localize specificproreins at high resolution within cells. Two proteins, but generally not more, can be detected simultaneously,though the procedure is technically challenging. By comparison, fluorescencemicroscopy can be used to detect four or more proteins at the same time (seechapter opening figure) albeit at a lower resolution. Preparations of certain purified particles, such as viruses and ribosomes,can be visualized by electron microscopy without prior fixation or staining if the sample is frozen in liquid nitrogen and maintained in the frozen state while being observed.

Figure 9-16). The generationof the image dependson differentialscatteringof the incident electronsby moleculesin 'Without the preparation. staining, the beam of electrons passesthrough a specimen uniformlS and so the entire sample appears uniformly bright with little differentiation of components.To obtain useful imagesby TEM, samples are commonly stainedwith heavy metals such as lead and uranium, during or just after the fixation step. Metalstained areas appear dark on a micrograph becausethe metalsscatter(diffract) most of the incident electronslscattered electrons are not focused by the electromasnetic

TEM Tungstenfilament (cathode)

-

Resolutionof Transmission ElectronMicroscopy ls VastlyGreaterThan That of Light Microscopy The fundamental principles of electron microscopy are slmilar to those of light microscopy; the major differenceis that electromagneticlensesfocus a high-velocityelectron beam instead of the visible light used by optical lenses.In the transmission electron microscope (TEM), electrons are emitted from a filament and acceleratedin an electric field. A condenser lens focusesthe electron beam onto the sample; objectiveand projector lensesfocus the electronsthat passthrough the specimenand project them onto a viewing screenor other detector (Figure 9-20, left). Becauseatoms in air absorb electrons,the entire tube between the electron source and the detector is maintained under an ultrahigh vacuum. The short wavelength of electronsmeansthat the limit of resolution for a transmissionelectron microscopeis theoretically 0.005 nm (lessthan the diameterof a singleatom), or 40,000 times better than the resolution of a light microscope, and 2 million times better than that of the unaided human eye. However, the effective resolution of the transmission electron microscope in the study of biological systems is considerablylessthan this ideal.Under optimal conditions, a resolution of 0.10 nm can be obtained with transmissionelectron microscopes,about 2000 times better than the best resolution of light microscopes.Severalexamples of cells and subcellularstructuresimaeed bv TEM were includedin Section9.1. BecauseTEM requiresvery thin, fixed sections(about 50 nm), only a small part of a cell can be observedin any one section.Sectionedspecimensare preparedin a manner similar to that used for light microscopy,by using a knife capableof producing sections50-100 nm in thickness(see

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Specimen EXPERIMENTAL FIGURE 9-20 In electronmicroscopy, images are formed from electronsthat passthrough a specimenor are scatteredfrom a metal-coatedspecimen,In a transmlssron (TEM), electron microscope electrons areextracted froma heated filament, accelerated by an electric field,andfocused on the specimen by a magnetic condenser lens Electrons that passthrough the specrmen arefocused by a series of magnetic objective and projector lenses to forma magnified imageof the specimen on a detector, whichmaybe a fluorescent viewingscreen, photographic film,or a charged-couple-device (CCD)cameraIn a scanning (5EM), electron microscope electrons arefocused by condensor and objective lenses on a metal-coated specimen Scanning coilsmove the beamacross the specimen, andelectrons scattered fromthe metalarecollected by a photomultiplier tubedetectorIn bothtypes of microscopes, because electrons areeasily scattered by air m o l e c u l et h s ,ee n t i r e c o l u m ni sm a i n t a i n eadt a v e r yh i g hv a c u u m ,

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9-21 Gold particlescoatedwith FIGURE < EXPERIMENTAL proteinA are usedto detectan antibody-boundprotein by (a)Firstantibodies are transmissionelectronmicroscopy. in a antigen(e g , catalase) with theirspecific allowedto interact sectionof fixedtissueThenthe sectionistreatedwith electroncoatedwith proteinA fromthe bacterium densegoldparticles of the S aureusBindingof the boundproteinA to the Fcdomains protein, the target of location the makes antibodymolecules n i c r o s c o p( eb .)A s l i c e n t h i sc a s ev, i s i b l ien t h ee l e c t r om c a t a l a si e andthen sectioned, of livertissuewasfixedwith glutaraldehyde, catalaseThegold in part(a)to localize treatedasdescribed arelocated of catalase (blackdots)indicating the presence particles J CellBiol etal, 1981, H J Geuze in peroxisomes [From exclusively

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lensesand do not contribute to the image. Areas that take up less stain appear lighter. Osmium tetroxide preferentially stains certain cellular components, such as memb r a n e s( s e eF i g u r e 1 0 - 6 ) . Specific proteins can be detected in thin sections using protein-specificantibodies.In this technique the cells are lightly fixed (to avoid denaturing epitopes on the desired protein) and then hozen at the temperature of liquid nitrogen and sectioned.After thawing, an antibody is applied to the section, which then is treated with electron-densegold particles coated with protein A, a bacterial protein that binds the Fc segmentof all antibodymolecules(Figure9-21). Becausethe gold particles diffract incident electrons, they appearas dark dots.

CryoelectronMicroscopyAllows Visualization of ParticlesWithout Fixationor Staining Standard transmissionelectron microscopy cannot be used to study live cells becausethey are generallytoo vulnerable to the required conditions and preparatory techniques. [n particular, the absenceof water causesmacromolecules to become denatured and nonfunctional. However, hydrated, unfixed, and unstained biological specimenscan be viewed directly in a transmission electron microscopeif the sample is frozen. In this technique

of cryoelectron microscopy, an aqueous suspenslonol a sample is applied to a grid in an extremely thin film, frozen in liquid nitrogen, and maintained in this state by means of a special mount. The frozen sample then is placed in the electron microscope.The very low tempera' C ) k e e p sw a t e r f r o m e v a p o r a t i n g ,e v e n i n a ture (- 196 vacuum. Thus, the sample can be observedin detail in its native, hydrated statewithout fixing or heavy metal staining, although cells and some viruses subjected to this will be killed. By computer-basedaveraging of tr*t-.ttt hundreds of images, a three-dimensional model can be generatedalmost to atomic resolution. For example, this method has been used to generatemodels of ribosomes (seeFigure 4-26), the muscle calcium pump discussedin Chapter 11, and other large proteins that are difficult to crystallize. Many virusescontain coats' or capsids,that contain multiple copiesof one or a few proteins arrangedin a symmetric ^ir^y.lia cryoelectronmicroscope,imagesof theseparticles can be viewed from a number of angles.A computer analysis of multiple images can make use of the symmetry of the particle to ihe calculate three-dimensionalstructure of the iapsid to about 5-nm resolution. Examples of such images are shown in Figure 4-44. An extension of this technique,cryoelectrontomography, allows researchersto determine the three-dimeniional architecture of organellesor even whole cells embedded in ice, that is, in a state close to life' In this technique, the specimen holder is tilted in small increthe axis perpendicularto the electron beam; -.nt, "rotr.td of thus images the object viewed from different directions are obtained (Figure 9-22a, b). The images are then merged computationally into a three-dimensionalreconstruition termed a tomogram (Figure 9-22c)' A disadvantage of cryoelectrontomography is that the samplesmust be relativelythin, about 200 nm; this is much thinner than the samples(200 p'm thick) that can be studied by confocal light microscopy.

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< EXPERIMENTAL FIGURE 9-22 Structure of the nuclearporecomplex(NPC)by cryoelectrontomography.(a)In electron tomography, a semicircular series of twoprojection dimensional imagesis recorded fromthe three-dimensional specimen that is located at the center;the specimen istilted whilethe electron opticsanddetector remainstationary Thethree-dimensional structure iscomputed fromthe individual two-dimensional images that areobtained whenthe objectis imagedby electrons comingfromdifferent (arrows directions in leftpanel) Theseindividual images areused to generate a 3-dimensional imageof the object(arrows, rightpanel)(b)lsolated n u c l ef ir o mt h ec e l l u l asrl i m em o l d Dictyostelium drscoideum werequick-frozen i n l i q u i dn i t r o g eann dm a i n t a i n ei ndt h i s stateasthe sample wasobserved in the electron microscope. Thepanelshowsthree s e q u e n t i tai l t e di m a g e sD. i f f e r e n t orientations of NPCs(arrows) areshownin top-view(leftand center) andside-view (nghf).Ribosomes connected to theouter nuclear membrane arevisible, asisa patch (c)Computerof roughER(arrowheads) generated surface-rendered representation of a segment of the nuclear envelope (yellow) membrane studded with NPCs (blue)lpart(a)after5 Nickell etal. 2006. Nature Rev.Mol Cell Biol 7.225 Parts(b) and (c) f r o m M B e c ke I a l , 2 0 0 4 , S c i e n c e 3 0 6 : 1 3 8 7l

ElectronMicroscopyof Metal-Coated SpecimensCan RevealSurfaceFeatures o f C e l l sa n d T h e i rC o m p o n e n t s Information about the shapesof purified viruses,fibers, enzymes, and other subcellular particles can be obtained with TEM by using metal shadowing. In this preparative technique,,a thin layer of metal, such as platinum, is evaporated on a fixed or rapidly frozen biological sample (Figure9-23a). teatment with acid and bleachdissolvesaway thecell, leaving a metal replica that is viewed in a transmissionelectron mi_ croscope(Figure9-23b). AlternativelS the scanning electron microscope (SEM) _. allows investigators to view the surfacesof unsectioned metal-coatedspecimens.An intense electron beam inside the microscope scansrapidly over rhe sample. Molecules in the coating are excited and releasesecondaryelectrons that are focusedonto a scintillation detector;the resulting signal is displayedon a cathode-raytube much like , .orl ventional television (seeFigure 9-20, right). The resulting scanning electron micrograph has a three-dimensional a p p e a r a n c eb e c a u s et h e n u m b e r o f s e c o n d a r ye l e c t r o n s produced by any one point on the sample dependson the angle of the electron beam in relation to the surface

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(Figure 9-24). The resolving power of scanning electron microscopes,which is limited by the thicknessof the metal coating, is only about 10 nm, much lessthan that of transmission lnstruments.

Electron Microscopy:Methods and Applications r Specimens for transmission electron mrcroscopy (TEM) generally must be fixed, dehydrated, embedded, sectioned, and then stained with electron-denseheavy metals. r Cryoelectron microscopy allows examination of hydrated, unfixed, and unstained biological specimensdirectly in a transmission electron microscope; samples are frozen in liquid nitrogen and maintained in this state. r Surfacedetails of objectscan be revealedby transmission electron microscopy of metal-coatedspecimens. r Scanning elecrron microscopy (SEM) of metal-coated unsectionedcells or rissuesproducesimagesthat appear to be three-dimensional.

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9-23 Metalshadowingmakessurface FIGURE A EXPERIMENTAL detailson very smallobjectsvisible by transmissionelectron (a)Thesampleis spreadon a micasurtace andthen microscopy. ( trr ) T h es a m p l g e r i di sc o a t e dw i t h evaporato d r i e di n a v a c u u m o r g o l d ,e v a p o r a t e d a t h i nf i l mo f a h e a v ym e t a ls, u c ha sp l a t i n u m he l le y a t e dm e t afl i l a m e n(tE ) T os t a b i l i zt e f r o ma n e l e c t r i c a h f i l m e v a porated w i t h a c a r b o n i s t h e n c o a t e d r e p l i c at h, e s p e c i m e n a l a t e r i ai sl t h e n f r o ma n o v e r h e aedl e c t r o d(eB ) T h eb i o l o g i c m ( E l ) ,w h i c hi sv i e w e di n a T E M I n d i s s o l v ebdy a c i da n db l e a c h the carbon-coated of suchpreparations, electronmrcrographs h fss i m p l em e t a l areaa s p p e alri g h t - t h er e v e r soef m i c r o g r a p o p r e p a r a t i o n s h e a v i e smt e t a sl t a i n i n g o f i n w h i c h t h e a r e a s stained a p p e atrh e d a r k e s (t b )A p l a t i n u m - s h a d o wr eepdl i coaf t h e s u b s t r u c t u fr iabl e r so f c a l f s k icno l l a g e nt h, e m a j o rs t r u c t u r a l p r o t e i no f t e n d o n sb, o n e a , n ds i m i l atri s s u e sT h ef i b e r sa r ea b o u t pattern(white 64-nmrepeated a characteristic 200 nm thick; p a r a l l el iln e si)sv i s i b l a e l o n gt h e l e n g t ho f e a c hf i b e r[.c o u r t e os fy

Purificationof CellOrganelles

Many studies on cell structure and function require samples of a particular type of subcellular organelle' As one i*ample, a recent proteomic study on mitochondria purified fiom mouse brain, heart, kidney' and liver revealed 591 mitochondrial proteins, including 163 proteins not previously associatedwith this organelle.Severalproteins were found in mitochondria only in specificcell types' Determining the functions associatedwith these newly identified mitochondrial proteins is a maior objective of current researchon this organelle.In this section'we describe severalcommonly used techniquesfor separatingdifferent organelles.

D i s r u p t i o no f C e l l sR e l e a s eTs h e i r O r g a n e l l e s and Other Contents The initial step in purifying subcellular structures is to releasethe cell's contents by rupturing the plasma membrane and the cell wall, if present.First, the cells are suspendedin a solution of appropriate pH and salt content, usually isotonic sucrose(0.25 M) or a combination of salts similar in

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through a very narrow spacebetween a plunger and the vessel wall; the pressureof being forced betweenthe wall of the vesseland the plunger ruptures the cell. Recall that water flows into cellswhen they are placed in a hypotonic solution, that is, one with a lower concenrration of ions and small moleculesthan found inside the cell. This

Disrupting the cell produces a mix of suspendedcellular components, the homogenate, from which the desired organellescan be retrieved.Becauserat liver containsan abundnnce of a single cell type, this tissuehas been used in many classicstudiesof cell organelles.However,the sameisolation principles apply to virtually all cells and tissues,and modifications of these cell-fractionation techniquescan be used to separateand purify any desiredcomponents.

CentrifugationCan SeparateMany Types of Organelles In Chapter 3, we consideredthe principles of centrifugation and the usesof centrifugation techniquesfor separatingproteins and nucleic acids. Similar approachesare used forieparating and purifying the various organelles,which differ in both sizeand density and thus undergo sedimentationat differentrates. Most cell-fractionation procedures begin with differential centrifugation of a filtered cell homogenate ar in-

creasingly higher speeds(Figure 9-25). After centrifugation at each speedfor an appropriare time, the liquid that remains at the top of the vessel,called the supernaranr,ls poured off and centrifuged at higher speed.The pelleted fractions obtained by differential centrifugation generally contain a mixture of organelles,although nuclei and viral particles can sometimes be purified completely by this procedure. An impure organelle fraction obtained by differential centrifugation can be further purified by equilibrium density-gradient centrifugation, which separates cellular components according to their density.After the fraction is resuspended,it is layered on top of a solution that contains a gradient of a dense nonionic substance(e.g., sucrose or glycerol). The tube is centrifuged at a high speed (about 40,000 rpm) for severalhours, allowing each particle to migrate to an equilibrium position where the density of the surrounding liquid is equal to the density of the particle (Figure 9-26). The different layers of liquid are then recovered by pumping out the contents of the centrifuge tube through a narrow piece of tubing. Becauseeach organelle has unique morphological features, the purity of organellepreparationscan be assessed by examination in an electron microscope. Alternatively, organelle-specificmarker moleculescan be quantified. For example, the protein cytochrome c is present only in mitochondria; so the presenceof this protein in a fraction of lysosomeswould indicate irs conramination by mitochondria. Similarly, catalaseis present only in peroxisomes;acid phosphatase,only in lysosomes;and ribosomes, only in the rough endoplasmicreticulum or the cyrosol.

> EXPERIMENTAL FTGURE 9-25 Differentiat centrifugationis a commonfirst step in fractionatinga cell homogenate.The h o m o g e n a rt e s u l t i nfgr o md i s r u p t i ncge l l si s usually filteredto removeunbroken cellsandthen centrifuged at a fairlylowspeedto selectively pelletthe nucleus-thelargest organelle The undeposited (thesupernatant) materiai isnext centrifuged at a higherspeedto sediment the m i t o c h o n d r icah, l o r o p l a sltyss, o s o m easn, d peroxisomes. Subsequent centrifugation in the u l t r a c e n t r i f uagte1 0 0 , 0 0 0f9o r 6 0 m i n u t e s resultsin deposition of theplasma membrane, fragments of theendoplasmic reticulum, and largepolyribosomes Therecovery of ribosomal subunits, smallpolyribosomes, andparticles such a sc o m p l e x eosf e n z y m erse q u i r easd d i t i o n a l c e n t r i f u g a t ia o tns t i l lh i g h e sr p e e d sO. n l yt h e c y t o s o l - t h es o l u b l a e q u e o upsa r to f t h e c y t o p l a s m - r e m a i n st h es u p e r n a t aanftt e r centrifugation at 300,0009 for 2 hours

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9-26 A mixed organellefraction FIGURE < EXPERIMENTAL by equilibriumdensity-gradient separated can be further utilizingrat liver,materialin centrifugation.In thisexample, ( s e eF i g u r 9 e - 2 5 )i s o tn1 5 , 0 0 0 9 t h e p e l l eftr o mc e n t r i f u g a t i a more gradient increasingly of on a and layered resuspended centrifugation During tube. centrifuge in a solutions densesucrose to itsappropriate migrates hours,eachorganelle for several e q u i l i b r i udme n s i tay n dr e m a i ntsh e r eT oo b t a i na g o o ds e p a r a t t o n with a the liveris perfused from mitochondria, of lysosomes the tissue before of detergent a smallamount solutioncontaining intothe is taken period, detergent perfusion this During is disrupted. maktngthem to the lysosomes, andtransferred cellsbyendocytosis a "clean" be andpermitting lessdensethantheywouldnormally frommitochondria. of lysosomes separatron

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AntibodiesAre Useful Organelle-Specific i n P r e p a r i n gH i g h l yP u r i f i e dO r g a n e l l e s Cell fractions remaining after differential and equilibrium density-gradientcentrifugation usually contain more than one type of organelle.Monoclonal antibodiesfor various organelle-specificmembrane proteins are a powerful tool for further purifying such fractions. One example is the pu-

isolated by low-speed centrifugation (Figure 9-27)' A telated technique uses tiny metallic beads coated with specific antibodies. Organellesthat bind to the antibodies, and are thus linked to the metallic beads, are recovered from

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, 9-27 Smallcoatedvesiclescan be FIGURE EXPERIMENTAL purified by binding of antibody specificfor a vesiclesurface a suspension proteinand linkageto bacterialcells.Inthisexample, specific an antibody with is incubated rat liver from of membranes of certaincytosolic a proteinthatcoatsthe outersurface for clathrin. of Staphylococcus Tothismrxtureis addeda suspension vesicles. proteinA, which contains membrane whosesurface aureusbacteria

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(a)Interaction of bindsto the Fcconstantregionof antibodies. links vesicles boundto clathrin-coated proteinA with antibodies complexes cells.Thevesicle-bacteria to the bacterial thevesicles (b) thin-section A ugation centrif low-speed by canthenbe recovered to an bound vesicles clathrin-coated reveals micrograph electron I CellBiol93:846Micrograph etal, 1982, E Merisko cell [See S aureus c o u r t e s yo f G P a l a d el P U R I F I C A T I OO N F C E L LO R G A N E L L E S

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the preparation by adhesion to a small magnet on the side of the test tube.

tain a particular glucosetransporter(GLLT4) that is localized to the membraneof one of theserypesof vesicle.\Wheninsulin is added to the cells, these vesiclesfuse with the cell-surface membrane and increasethe number of glucoserransporters able to take up glucose from the blood. As we will see in Chapter 15, this processis critical to maintaining the appropriate concentration of sugar in the blood. The GLUIa-

animal in the laboratory under conditions that permit their survival and growth for at least severaldivisions. Certain types of primary cells, especially those from embryos, can undergo differentiation in culture. As an example, we will describehow muscle cell precursors grown in culture can differentiate and form apparently normal musclecells,providing a good systemfor studying this developmentalprocess. Although many types of primary cells undergo only a limited number of divisions in culture, some accumulate cancercausing (oncogenic)mutations that allow them to be cultured indefinitely. In many casesa single cell can be readily grown into a colony of identical cells, a process called cell cloning. Becausethesecells are genetically homogeneous,they are pa:. ticularly suitable for many types of biochemical and genetic studies. Certain cloned cells can undergo differentiation into specificcellstypes such as adipocytes(fat-storingcells),nerve, or muscle, allowing studieson the mechanism of cell differentiation to be conducted on homogenous cell populations. Many times in this chapter we have shown how monoclonal antibodies facilitate cell biological experiments; at the end of this section we describehow special cultured cells are used to generatetheseantibodres.

Flow CytometrySeparatesDifferent CellTypes Purification of Cell Organelles r Disruption of cells by vigorous homogenization, sonication,.or other techniquesreleasestheir organelles.Swelling of cells in a hypotonic solution weakens the plasma membrane,making it easierro ruprure. r Sequentialdifferential centrifugation of a cell homogenate yields fractions of partly purified organellesthat diifer in mass and density (seeFigure 9-25). r Equilibrium density-gradientcentrifugation, which sepa_ rates cellular components according to their densities,ian further purify cell fractions obtained by differential centrifugation (seeFigure9-26). r Immunological techniques using antibodies against organelle-specificmembrane proteins are particulaily useful in purifying organellesand vesiclesof similar sizesand den_ sities (seeFigure 9-27).

lsolation,Culture, ![ and Differentiationof MetazoanCells Most animal and plant rissuescontain multiple types of cells, but biochemical and molecular investigationi b.rt u..o-_ plished on homogenouscell populationi. In the"r. first part of this section we describe a powerful instrument, the flulrescence_ 394

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Some cell types differ sufficiently in density that they can be separatedon the basisofthis physical property. Nfhite blood cells (leukocytes)and red blood cells (erythrocytes),for instance, have very different densities becauseerythrocvtes have no nucleus;thus thesecellscan be separatedfy .quitiUrium density centrifugation (describedabove). Becausemost cell types cannot be differentiated so easilS other techniques such as flow cytometry must be used to separatethem. A flow cytometer identifies different celli by measuringthe light that they scatterand the fluorescencethat they emit as-they flow through a laser beam; thus it can quantify the numbers of cellsof a particular type from a mixture. Indeed,a fluorescenceactivated cell sorter (FACS),which is basedon flow cytometry can selectone or a few cells from thousands of other cells and sort them into a separateculture dish (Figure 9-28). For example, if an antibody specificto a certain cell-surfacemolecule is linked to a fluorescent dye, any cell bearing this molecule will bind the antibody and will then be separaredfrom other cells when it fluorescesin the FACS. Having been sorted from other cells,the selectedcellscan be grown in culture. The FACS procedure is commonly usedto purify the different types of white blood cells, each of which bears on its surface one or more distinctive proteins and will thus bind monoclonal antibodies specific for that protein. Only the T cells of the immune system,for instance,have both CD3 and Thyl .2 proteins on their surfaces.The presenceof thesesurface proteins allows T cells to be separatedeasily from other

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typesof blood cellsor spleencells(Figure9-29).Smallmagnetic beadsmay be usedin a variation of this processthat doesnot involve flow cytometry. The beadsare coated with a monoclonal antibody specificfor a surfaceprotein such as CD3 or Thyl.2. Only cells with theseproteins will stick to the beads and can be recoveredfrom the preparation by adhesionto a small magneton the side of the test tube. Other uses of flow cytometry include the measurement of a cell's DNA and RNA content and the determination of its generalshapeand size.The FACS can make simultaneous of the sizeof a cell (from the amount of scatmeasurements

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9-28 FluorescenceFIGURE < EXPERIMENTAL activatedcell sorter (FACS)separatescells that are labeleddifferentiallywith a fluorescentreagent.StepE: A concentrated cellsis mixedwith a buffer of labeled suspension (thesheathf luid)sothatthe cellspasssingle-file . t e pA : B o t ht h e t h r o u g ha l a s e lri g h tb e a m S lightemittedandthe lightscattered fluorescent frommeasurements by eachcellaremeasured; light,the sizeandshapeof the of the scattered StepS: Thesuspension cellcanbe determined whichformstiny isthenforcedthrougha nozzle, cell At the a single at most containing droplets given a negative is droplet each timeof formation, e l e c t r icch a r g ep r o p o r t i o ntaol t h e a m o u not f wrthno of itscell.Step4: Droplets fluorescence charges electric chargeandthosewith different f ieldandcollected by an electric areseparated m i l l i s e c o ntdoss o r te a c h o n l y t a k e s i t Because c e l l sp e rh o u rc a n d r o p l e ta,sm a n ya s 1 0 m i l l i o n fromD R Parks passthroughthe machinelAdapted Biol26:2831 Cell Meth 1982, andL A Herzenberg,

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tered light) and the amount of DNA that it contains (from the amlunt of fluorescenceemitted from a DNA-binding dye). Measurementsof the DNA content of individual cells .rs.d to follow replication of DNA as the cells progress "ie through the cell cycle (Chapter 20).

C u l t u r eo f A n i m a l C e l l sR e q u i r e sN u t r i e n t - R i c h l o l i dS u r f a c e s M e d i a a n d S p e c i aS In contrast with most bacterial cells, which can be cultured quite easily,animal cells require many specializednutrients and often speciallycoated dishesfor successfulculturing' To permit the iurvival and normal function of cultured tissues Lr cells, the temperature, pH, ionic strength, and accessto 9-29 T cellsboundto fluorescenceFIGURE < EXPERIMENTAL tagged antibodiesto two cell-surfaceproteinsare separated trom ottrerwhite blood cellsby FACS.Spleencellsfroma mouse for antibodyspecific monoclonal weretreatedwith a redfluorescent monoclonal green fluorescent a with protein and cell-surface theCD3 protein, Ihyl '2. Asthece||s cel|-surface for a second specific antibody of thegreenand intensity the machine, througha FACS *ere passed Eachdot was recorded cell each emittedby redfluorescence (vertical green fluorescence plot the of This cell. single a represents of spleen (horizontal axis)for thousands redfluorescence versus axis) bothCD3 cellsshowsthatabouthalfof them-the T cells-express The quadrant) (upper-right on theirsurfaces andThyl.2proteins (lower-left quadrant)' fluorescence low exhibit which cells, remaining andareothertypes of theseproteins levels onlybackground "*pr"r, scaleon bothaxes. lcourtesy of whitebloodcells.Notethe logarithmic o { C h e n g c h e nZ g h a n g ,W h i t e h e a dI n s t i t u t eI

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essentialnutrients must simulate as closely as possible the conditions within an intact organism. Isolated animal cells are typically placed in a nurrient-rich liquid, called the cul_ ture medium, within speciallytreated plastic dishesor flasks. The cultures are kept in incubators in which the tempera_ ture, atmosphere,and humidity can be controlled. To reduce

ganismsand other airborne contaminants.

fa91o1sinclude the polypeptide hormone insulin; rransferrin, which supplies iron in a bioaccessibleform; and numerous growth factors. In addition, certain cell types requlre spe_ cializedprotein growth factors not present in serum. For in_

when calcium is removed; other cell-adhesionmoleculesthat are not calcium dependent need to be proteolyzed for the cells to separate.The releasedcells are then placed in dishes in a nutrient-rich, serum-supplementedmedium, where they can adhere to the surface and one another. The same protease/chelatorsolution is usedto remove adherentcells from a culture dish for biochemicalstudiesor subculturing (transfer to another dish). Fibroblasts are the predominant cells in connectivetissue a_ndnormally produce ECM components such as collagen that bind to cell-adhesionmolecules,thereby anchoring cells to a surface. In culture, fibroblasts usually divide more rapidly than other cells in a tissue,eventually becoming the predominant cell type in a primary culture unless special precautions are taken to remove them when isolatinq other typesof cells.

sion, the cells induce synthesisof dozens of muscle,specific proteins necessaryfor further muscle development furr.tion. Similar mononucleated cells, termed ,itrllit, "nJ cells, are found in adult muscle and can fuse to form multinucleated myotubes and simultaneously induce synthesis of muscle-

ling muscle development. Certain cells from blood, spleen,or bone marrow adhere poorly, if at all, to a culture dish but nonethelessgrow well in culture. In the bodg such nonadherent cells are held in

can be maintained or grown in suspensionas singlecells.

PrimaryCell CulturesCan Be Used to Study Cell Differentiation Normal animal tissues(e.g.,skin, kidneg liver) or whole em_ bryos are commonly used to establishprimary cell cwbures. To prepare tissuecellsfor a primary .ulrr.., rhe cell_celland cell-matrix interactions must be broken. To do so, tissue

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from an embryo or an adult animal are cultured, most of the adherentones will divide a finite number of times and then ceasegrowing (cell senescence). For instance,human fetal fibroblastsdivide about 50 times

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FIGURE 9-30 Differentiationin cultureof A EXPERIMENTAL (myoblast) cellsinto musclecells. primarymousesatellite undernondifferentiating cellswerecultured mousesatellite Primary p r o l i f e r a t i oc n o n d i t i o n( sa , c o) r d i f f e r e n t i a t icoonn d i t i o n( sb , d ) ( a , b )P h a s e - c o n t riamsat g es h o wf o r m a t i o on f m u l t i n u c l e a t e d

before they ceasegrowth (Figure 9-3lal. Starting with 106 cells, 50 doublings can produce 106 x 2)u, or more than 1020cells, which is equivalent to the weight of about 1000 people.Normally, only a very small fraction of thesecellsare used in any one experiment.Thus, eventhough its lifetime is limited, a singleculture, if carefully maintained, can be studied through many generations.Such a lineage of cells originating from one initial primary culture is called a cell strain. Researchwith cell strains is simplified by the ability to freeze and successfullythaw them at a latet time for experimental analysis.Cell strains can be frozen in a state of sus-

(c,d)Staining with a redduringmuscledifferentiation syncytia reveals thatthis chain heavy myosin ic for specif antibody f iuoiescing Staining differentiation during protein is induced muscle-specific nucleiBar: 100pm [Courtesy dye(blue)detects with Hoechst CharlesEmersonandJenniferChen,BostonBiomedicaIResearc

pended animation and stored for extended periods at liquid nltrogen temperature' provided that a preservativethat prevents the foimation of damagittg ice crystals is used' Although some cells do not survive thawing, many do survive and resumegrowth.

TransformedCellsCan Grow Indefinitely in Culture To be able to clone individual cells, modify cell behavior,or select mutants, biologists often want to maintain cell

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"rft3?" FIGURE 9-31 Stagesin the establishment of a cellculture. (a)Whencellsisolated fromhumantissue areinitiallv cultured. some cellsdieandothers(mainly fibroblasts) startto grow;overall, the growthrateincreases (phasel) lf the remaining cellsareharvested, diluted, andreplated intodishes againandagain,thecellstrain continues to divideat a constant ratefor about50 cellgenerations (phase ll),afterwhichthegrowthratefallsrapidly. Intheensuing period(phase lll),allthecellsin theculturestopgrowing(senescence) (b)Ina culture prepared frommouseor otherrodentcelrs, initiar cerl death(notshown)jscoupled withtheemergence growing of healthy cells.Asthesedividing cellsaredilutedandallowedto conttnue growth,theysoonbeginto losegrowthpotential, andmoststop growing(i e , theculturegoesintosenescence) Veryrarecels undergooncogenic mutations thatallowthemto survlve ano continue dividing untiltheirprogeny overgrow theculture, These cells constitute a cellline,whichwillgrowindefinitely if it isappropriately dilutedandfedwithnutrients. Suchcellsaresaidtobe immortal cultures for many more rhan 100 doublings. Suchprolonged growth is exhibited by cells derived from some tumors. In addition, rare cells in a population of primary cells may un_ dergo spontaneous oncogenic mutations, leading to onco_ genic transformation (Chapter 25). Such cells,iaid to be oncogenically transformed or simply transformed, are able to grow indefinitely. A culture of cells with an indefinite life span is consideredimmortal and is called a cell line. The HeLa cell line, the first human cell line, was origi_ nally obtained in 1952 from a malignant tumor (carcinoma ) of the uterine cervix. Although primary cell cultures of nor_ mal human cells rarely undergo transformation into a cell line, rodent cells commonly do. After rodent cells are grown in culture for severalgenerations,the culture goesinto senes_ cence (Figure 9-31b). During rhis period, mlst of the cells stop growing, but often a rapidly dividing transformed cell 398

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arisesspontaneouslyand takes over, or overgrows, the culture. A cell line derived from such a transformed variant will grow indefinitely if provided with the necessarynutrienrs. Regardlessof the source, cells in immortalized lines often have chromosomes with abnormal DNA sequences.In addition, the number of chromosomesin suchcellsis usually greater than that in the normal cell from which they arose. and the chromosomenumber expandsand contractsas the cells continue to divide in culture. A noteworrhy exception is the Chinesehamster ovary (CHO) line and its derivatives,which have fewer chromosomesthan their hamster progenitors. Cells with an abnormal number of chromosomesare said tobe aneuploid.

SomeCell LinesUndergoDifferentiation in Culture Most cell lines have lost some or many of the functions characteristicof the differentiatedcells from which they were derived. Such relatively undifferentiated cells are poor models for investigating the normal functions of specific cell types. Better in this regard are severalmore-differentiatedcell'lines that exhibit many properties of normal nontransformed cells. Theselines include the human liver tumor (hepatoma) HepG2 line, which synthesizesmost of the serum proteins made by normal liver cells (hepatocytes)and has been em_ ployed in studiesidentifying transcription factors that regulate synthesisof liver proteins. More useful for cell and developmentalbiologistsare cell lines that grow without acquiring characteristicsof a differentiated cell, yet can undergo differentiation into a particular cell type when placed in a different culrure medium. Becauselarge numbers of such cells can be induced to undergo synchronized differentiation, they are often used in biochemicaland molecularsrudies. One example is a line of transformed mouse myoblasts termed C2C1,2 cells. Derived from adult mouse muscle, these_cellsdivide rapidly and induce none of the principai muscle-specificproteins when grown in media rich in growth factors. When the culture is placedin a medium with

tion, these cells have been particularly valuable in uncovering the roles of many rranscription factors in muscle development (Chapter 22). Similarl5 the murine 3T3-L1 preadipocytecell line grows with fibroblastJike morphology in culture. Vhen switchedto a medium containing insulin and dexamethasone(a glucocor_ ticoid steroid hormone), 3T3-L1 cells undergo synchronized differentiation into adipocytes, as shown both by accumulation of intracellular lipids (Figure 9-33a-d) and induction of adipocyte-specificmRNAs (Figure 9-33e). Becausepnmary adipocytesdo nor divide in culture, thesecell lines ari widely used in biochemical,molecular,and cell bioloeical studieson the developmenrand function of adiposecells.

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Days FIGURE 9-32 Differentiationin cultureof A EXPERIMENTAL C2C12cells,a line of transformedmousemyoblasts'(a)C2C12 fetal in a mediumcontaining but do not differentiate cellsproliferate growth of mitogenic highconcentrations calfserum,whichcontains C2C12cellsin a mediumwith horseserum, factors(b)Afterplacing manyof of suchgrowthfactors, lower concentrations whichcontains the thatexpress syncytia thecellsfusedto formmultinucleate with a greenmyosinheavychain,detected muscle-specific of DNAwith for thisproteinStaining specific antibody fluorescing (c) pm. During 20 Bar denotes the nuclei blueDAPIdyereveals of certainmRNAs 2 cellsthe levels C2C'1 of cultured differentiation the asshownin thesegraphsForinstance, markedly, rncrease o-actin (a muscle-specific factor), transcription for myogenin mRNAs

glucose andthe GLUT4 filaments), (majorcomponent of contractile 5 to increased fat cells) (found and in muscle only transporter for growing proteins specific encodrng mRNAs 50 fold.In contrast, were and B-actin, glucose transporter cells,suchasthe GLUT1 glycolytic enzyme the as such OthermRNAs, downregulated (G3PDH), were dehydrogenase 3 phosphate glyceraldehyde reverse by using measured were mRNAs Individual unaffected. mRNAinto DNA,followedby to copytotalcellular transcriptase (PCR) of specific amplification chainreaction polymerase quantitative present in amount the to normalized were Theresults cDNAs. Rao' andPrakash Evans (a)and(b)courtesyJames growingcellslParts et al, 1998' fromT.Shimokawa Part(c)adapted Institute Whitehead ResComm246:287 I BioPhYs Biochem

As detailed in Chapter 19, many cell types function only when closelylinked to other cells.Key examplesare the sheetlike layers of epithelial tissue, called epithelia (sing., epithelium),which cover the external and internal surfacesof organs.Typically,the distinct surfacesof a polarizedepithelial cell are called the apical (top), basal (base or bottom), and lateral (side) surfaces(seeFigure 19-8). The basal surface

usually contactsan underlying extracellularmatrix called the basal iamina, whose composition and function are discussed in Section 19.3. Severaltypes of cell junctions interconnect adjacentepithelialcells and anchor them to the basal lamina' Cultured iells called Madin-Darby canine kidney (MDCK) cells arc often used to study the formation and function of epithelial cells. \flhen grown in specializedcontainers,these

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< E X P E R I M E N TFA L U R9E- 3 3 A IG line of 3T311preadipocytes can differentiateinto adipocytesand expressadipocyte-specific mRNAsin culture.Fibroblast-like 3T3-11 preadtpocytes growingin standard serum-containing proliferation medium (a,c)wereswitched to a differentiation m e d i u mc,o n t a i n i ni n g s u l i nt h, es t e r o i d h o r m o ndee x a m e t h a s oanned, i s o b u t y l m e t h y l x a n ta hn i ni n eh , ibitor o f c A M Pp h o s p h o d i e s t e r faosre , B d a y s( b , d )(.a , b )D i f f e r e n t r a r ) icroscopy i n t e r f e r e nc o e n t r a s( tD l C m r e v e a lt sh e c o n s i d e r a b l e m o r p h o l o g i ccahla n g eisn t h ec e l l s d u r i n gd i f f e r e n t i a t i o( cn, d )S t a i n i n g with Oil RedO reveals droplets of (red)in differentiated triglycerides but not undifferentiated cells,(e)Northern blotanalysis demonstrates expression of two keyadipocyte genes, encoding the transcription factorPPARI andthe insulin-responsive glucose transporter GLUT4 in thedifferentiated 3T3-11 cells(ngrht /ane)but not in the undifferentiated 3T3-11preadipocytes (leftlane)B-Actinwasuseoasa conrrol to showloading of equalamounts of RNAin thetwo gellanes[part a-d c o u r t e s yJ a m e sE v a n sa n d H u a n g m i n gX i e , WhiteheadInstitute Part(e)from N L Harvey e t a l , 2 0 0 5 , N a t u r eG e n 3 7 : 1 0 7 2 1

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ties of cells in complex rissuesand organismsonly on the basisof experimentswith isolated, cultured cells.

H y b r i dC e l l sC a l l e dH y b r i d o m a sp r o d u c e A b u n d a n tM o n o c l o n aA l ntibodies A major disadvantageof cultured cells is that they are not in their normal environment and hence their activities are not regulatedby the other cellsand rissuesas rhey would be in an intact organism.For example,insulin producedby the pancreashas an enormous effect on liver glucosemetab_ olism; however, this normal regulatory mechanismdoes not operatein a purified population of liver cells(calledhepato_ cytes)grown in culture.In addition, as alreadydescribed,the three-dimensionaldistribution of cells and extracellular matrix around a cell influences its shape and behavior. Becausethe immediateenvironmentof coitured cellsdiffers radically from this "normal', environment,their properties may be affected in various ways. Thus care must always be exercisedin drawing conclusionsabout the normal proper_

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In addition to serving as researchmodels for studies on cell function, cultured cells can be convertedinto ,,factories" for producing specificproreins. In Chapter 5, we describedhow this is done by introducing genesencoding insulin, growth factors, and other therapeutically useful proteins into bacterial or eukaryoticcells(seeFigures5-31 and 5-32). Here we consider the use of special cultured cells to generatemonoclonal antibodies,which we have seenare experimentaltools widely used in many aspectsof cell biological research.In-

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9-34 Madin-Darbycaninekidney FIGURE A EXPERIMENTAL (MDCK)cellsgrown in specialized containersprovidea useful experimentalsystemfor studyingepithelialcells.MDCKcellsform filtercoated membrane whengrownon a porous epithelium a polarized of thebasallamina andothercomponents on onesidewithcollagen on culture dishshownhere,themedium Withtheuseof thespecial canbe sides of themonolayer) andbasal eachsideof thefilter(apical across the of molecules manipulated andthemovement experimentally thecellsform thatinterconnect celljunctions layermonitoredSeveral Ca2' contains sufficient onlyif thegrowthmedium normal antibody-producingB lymphocyte in a mammal is capableof producinga singletype of antibody that can bind to a specific chemical structure (called a determinant or epitope) on an antigenmolecule.If an animal is injectedwith an antigen,many B lymphocytesthat make antibodiesrecognizing that antigen are stimulated to grow and secretethe antibodies.Each antigen-activatedB lymphocyte forms a clone of cells in the spleenor lymph nodes,with each cell of the clone producing the identical antibody-that is, a monoclonal antibody.Becausemost natural antigenscontain multiple epitopes,exposureof an animal to an antigen usually stimulates the formation of multiple different B-lymphocyte clones, each producing a different antibody. The resulting mixture of antibodiesthat recognizedifferent epitopeson the same antigen is said to be polyclonal. Stch polyclonal antibodiescirculatein the blood and can be isolatedas a group. Although polyclonal antibodiesare useful for a variety of experiments,monoclonal antibodiesare required for many types of experimentsand medical applications. Unfortunately,the biochemicalpurification of any one type of monoclonal antibody from blood is not feasiblefor two main reasons:the concentrationof any given antibody is quite low, and all antibodieshave the samebasicmoleculararchitecture(seeFigure3-19). Becauseof their limited life span, primary culturesof normal B lymphocytesare of limited usefulnessfor the production of monoclonal antibodies.Thus the first step in producing a monoclonal antibody is to generate immortal, antibody-producingcells.This immortality is achievedby fusing normal B lymphocytesfrom an immunized animal with transformed, immortal lymphocytes calledmyeloma cells that themselvessynthesizeneither the heavy (H) nor the light (L) polypeptidesthat constituteall antibodies(seeFigure 3-19). During cell fusion, the plasmamembranesof two cellsfusetogether,allowing their cytosols and organellesto intermingle. Treatment with certain viral glycoproteins or the chemical

polyethyleneglycol promotes cell fusion. Some of the fused cells undergo division, and their nuclei eventually coalesce, producing viable hybrid cells wtth a single nucleusthat coniains chromosomesfrom both "parents." The fusion of two cells that are geneticallydifferent can yield a hybrid cell with novel characteristics.For instance,the fusion of a myeloma cell with a normal antibody-producing cell from a rat or mouse spleen yields a hybrid that proliferates into a clone calleda hybridoma. Like myeloma cells,hybridoma cellsgrow rapidly and are immortal. Each hybridoma producesthe monoclonal antibody encodedby its BJymphocyteparent. The second step in this procedure for producing monoclonal antibody is to separate'or select,the hybridoma cells from the unfused parental cells and the self-fusedcells generated by the fusion reaction. This selection is usually performed by incubating the mixture of cells in a specialculture medium, calledselectionmedium, that permits the growth of only the hybridoma cells becauseof their novel characteristics. If the myeloma cellsusedfor the fusion cany a mutation that blocks a metabolic pathway, a selectionmedium can be used that is lethal to them and not their lymphocyte fusion Dartnersthat do not have the mutation. In the immortal hytrid cells,the functional genefrom the lymphocyte can supply the missing gene product, and thus the hybridoma cells will be able to grow in the selection medium. Becausethe lymphocytesused in the fusion are not immortalized and do not divide rapidly, only the hybridoma cells will proliferate rapidly in the selectionmedium and can thus be readily isolated from the initial mixture of cells. Figure 9-35 depicts the general procedure for generating and selectinghybridomas.In this case' normal B lymphocytesin a sampleof spleencells are fused with myeloma cells that cannot grow in HAT mediwm' a common selection medium. Only the myeloma-lymphocyte hybrids can survive and grow for an extended period in HAT medium for reasons describedshortly' Thus, this selectionmedium permits

chromatographyto isolate and purify proteins from complex mixtures (seeFigure 3-37c).They can also be usedto label and thus iocatea particular protein in specificcellsof an organ and miwithin cultured cellswith the use of immunofluorescence of with the use fractions cell in specific or croscopytechniques also antibodies Monoclonal (see 3-38). Figure immunoblotting have become important diagnostic and therapeutic tools in medicine. For example, monoclonal antibodiesthat bind to and inactivatetoxins secretedby bacterialpathogensare used Other monoclonal antibodiesare specificfor to treat diseases. cell-surfaceproteins expressedby certain types of tumor cells' Severalof theseanti-tumor antibodiesare widely usedin cancer therapy,including monoclonal antibody againsta mutant in somebreast form of the Her2 receptorthat is overexpressed cancers(seeChapter16, Figure16-18).

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Technique Animation:preparingMonoclonalAntibodi", {lltl Injectmouse w i t h a n t i g e nX

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< EXPERIMENTAL FIGURE 9-35 Useof cellfusionand selectionto obtain hybridomasproducingmonoclonal antibodyto a specificprotein.Step[: lmmortalmyeloma cells thatlackHGPRT, an enzyme required for growthon HATmedium, arefusedwith normalantibody-producing spleen cellsfroman animalthatwasimmunized with antigenX. Thespleencellscan makeHGPRT. Step[: Whenplatedon HATmedium, unfused and self-fused cellsdo not grow:the mutantmyeloma cellsbecause theycannotmakepurines throughan HGPRT-dependent metabolic "salvage" pathway(seeFigure 9-36),andthespleen cellsbecause theyhavea limitedlifespanin cultureThusonlyfusedcellsformed froma myeloma cellanda spleen cellsurvive on HATmedium, proliferating intoclones calledhybridomas Eachhybridoma produces a singleantibodyStepB: Testing of individual clones identifies thosethat recognize antigenX Aftera hybridoma that produces a desired antibody hasbeenidentified, theclonecanbe cultured to yieldlargeamounts of thatantibody.

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HATMedium ls CommonlyUsedto lsolate H y b r i dC e l l s The principles underlying HAT selectionare important not only for understandinghow hybridoma cells are isolated but also for understanding several other frequently used selection methods, including selectionof the embryonic stem (ES) cells used in generatingknockout mice (seeFigure 5-40). HAT medium containsDypoxanthine(a purine), 4minopterin, and rhymidine. Most animal cells can synthesizethe purine and pyrimidine nucleotidesfrom simpler carbon and nitrogen compounds (Figure 9-36, top). The folic acid antago_ nists amethopterin and aminopterin block thesebiochemiial pathways; they interfere with the donation of methyl and formyl groups by tetrahydrofolic acid in the early stagesof the_synthesis of glycine, purine nucleosidemonophosphates, and thymidine monophosphate. These drugs are calledan_ 402

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tifolates becausethey block reactionsof tetrahydrofolate, an active form of folic acid. Many cells, however, are resistant to antifolates becausethey contain enzymesthat can synthesizethe necessary nucleotides by a different route from purine bases and thymidine (Figure 9-36, bottom). Two key enzymesrn these nucleotide saluage pathways are thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Cells that produce these enzymes can grow on HAT medium, which supplies a salvageable purine and thymidine, whereas those lacking either of tneseenzymescannot. Cells with a TK mutation that prevents the production of the functional TK enzymecan be isolated becausesuch cells are resistant to the otherwise toxic thymidine analog S-bromodeoxyuridine. Cells containing TK convert this compound into 5-bromodeoxyuridine monophosphate, which is then converted into a nucleoside triphosphate by other enzymes.The triphosphate analog is incorporated by DNA polymeraseinto DNA, where it exerts its toxic effects. This pathway is blocked in TK- mutanrs, and thus they are resistant to the toxic effects of S-bromodeoxyuridine. Similarly, cells lacking the HGPRT enzyme,such as the HGpRT myeloma cell lines used in producing hybridomas, can be isolated becausethey are resistant to the otherwise toxic guanine analog 6-thioguanrne. Normal cells can grow in HAI medium becauseeven though the aminopterin in the medium blocks de novo synthesis of purines and TMP, the thymidine in the medium is transported into the cell and converted into TMp by TK and the hypoxanthine is transported and converted into usableDurines by HGPRT. On the other hand, neither TK- nor HGPRTceLlscan grow in HAI medium becauseeach lacks an enzyme of the salvagepathway. However, hybrids formed by the fusion of these two mutants will carry a normal TI( gene from the HGPRT- parent and a normal HGPRT gene from the TKparent. The hybrids will thus produce both functional salvagepathway enzymesand will grow on HAT medium.

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9-36 De novo and salvagepathwaysfor nucleotide A FIGURE (AMP, purinenucleotides synthesis.Animalcellscansynthesize e M Pf)r o ms i m p l ecro m p o u n dbsy d e c M P ,I M P a ) n dt h y m i d y l a (t T (blue)Several requirethe transfer of thesereactions novopathways form of of a methylor formyl("CHO")groupfroman activated (e g., tvs,N10-methylenetetrahydrofolate), asshown tetrahydrofolate suchasaminopterin Antifolates, in the upperpartof the diagram.

lsolation, Culture, and Differentiation of Metazoan Cells r Flow cytometry can identify different cells on the basis of the light that they scatter and the fluorescencethat they cell sorter (FACS)is useful emit. The fluorescence-activated in separatingdifferent types of cells (seeFigures 9-28 and 9-29).

of tetrahydrofolate, blockthe reactivation andamethopterin, Manyanimalcellscanalso synthesis. purineandthymidylate preventing purinebasesor nucleosides (red)to incorporate pathways usesalvage normal in the medium, arepresent lf theseprecursors andthymidine. cultured However, presence of antifolates grow in the even cellswill APRLoTTK--rcfthe salvage cellslackingoneof the enzymes-HGPRL antifolates. in mediacontaining pathways will not survive r The fusion of an immortal myeloma cell and a single B lymphocyte yields a hybrid cell that can proliferate indefinitely, forming a clone called a hybridoma (seeFigure 9-35). Becauseeach individual B lymphocyte produces antibodies specificfor one antigenic determinant (epitope),a hybridoma produces only the monoclonal antibody synthesizedby its original B-lymphocyte parental cell. r HAT medium is commonly used to isolate hybridoma cells and other types of hybrid cells.

r Growth of vertebratecells in culture requires rich media containing essentialamino acids, vitamins, fatty acids, and peptide or protein growth factors; the last are frequently provided by serum. r Most cultured vertebrate cells will grow only when attached to a negatively charged substratum that is coated with components of the extracellular matrix. r Primary cells, which are derived directly from animal tissue, have limited growth potential in culture and may give rise to a cell strain (seeFigure 9-31). Some primary cells can undergo differentiation into a specific type of cell. r Transformed cells, which are derived from animal tumors or arise spontaneouslyby transformation of primary cells,grow indefinitely in culture, forming cell lines. r Certain cell lines can be induced to undergo differentiation in culture to generatemuscle, adipose, epithelial, and other types of cells and are widely used in cell biological studies(seeFigures9-30,9-32, and 9-33).

Light microscopy will continue to be a major tool in cell biologS providing images that relate to both the interactions proteins and the movements' or mechanics,involved "-o.g cell processes.The use of more fluorescentlabels various in will allow visualization of five or six different types and tags Vith more labeledproteins, the simultaneously. molecules of complex interactions among proteins and organellesinside cells will becomebetter understood. Very recent advancesin light microscopy are opening entire new areasfor investigation.As an example, the two-photon fluorescencemicroscope enablesvisualization of fluorescent proteins (e.g., GFP expressedfrom a reporter gene) in thick iissuesamples.With this technologythe propertiesof individin tissuesinside living animals. ual cellscan assessed Although the limit of resolution of a light microscope using visible light is about 0.2 pm (200 nm), new types of

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fluorescencemicroscopy,such as STED (stimulated emission depletion),can resolvetwo fluorescentobiects as close together as =20 nm. For example, we noted that synaptic vesicles are too small (=40 nm in diameter) and too densely packed to be resolvedby availablefluorescencemicroscopes. Howeveq STED can resolve these individual vesicles.This technique will also enable investigatorsto detect single fluorescent protein molecules in the membranes of purified organelles.Another type of fluorescencemicroscopy under development,termed total internal reflection (TIR) fluorescencemicroscopy, enablesdetection of single fluorochrometagged proteins on the surfaceof living cells. Improvements in cell culture technology will allow both primary cells and cultured lines to be studied in more natural contexts, not on glass cover slips but in three-dimensional gelsof extracellularmatrix molecules.This technique will permit cultured cells such as liver and severalhormoneproducing cells to achieveand maintain their differentiated state for days, enabling many types of experimentsto be performed. Bioengineersalso are fabricating artificial tissues basedon a syntheticthree-dimensionalarchitecrurelncorDorating layers of different cells. Eventually such artificial iissueswill provide replacementsfor defective tissuesin sick, injured, or aging individuals. In parallel, investigatorswill use advancedmicrofabrication techniques to culture minute numbers of cells in microliter volumes on a glassslide consistingof microfabricated wells and channels.With this technique, reagenrs in nanoliter volumes can be introduced and exposedto selected parts of individual cells; rhe responsesof the cells can then be detected by light microscopy and,analyzed by powerful image-processingsoftware. In these types of studies cells can be screenedrapidly with millions of different chemicalcompounds, thus facilitarine the discovery o f n e w d r u g s , d e r e c t i o no f s u b t l e p h e n o r y l e s o f m u r a n r cells (e.g., tumor cells), and development of comprehensive models of cellularprocesses. Thus advancesin bioengineering will make major contributions not only to our understandingof cell and tissuefunction but also to the qualitv o f h u m a nh e a l t h . Finally the electron microscope will become the dominant insrrument for studying the structure of multiprotein machinesin vitro and in situ. Tomographic methods applied to single cells and molecules combined with automated reconstruction methods will generatemodels of protein-based structures that cannot be determined by x-ray crystallography. High resolution three-dimensionalmodels of molecules in cells will help explain the intricate biochemical interactrons among proteins.

KeyTerms autophagy 374 bright-field light mrcroscopy381 cell line 398 cell strain 397 404

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chimericproteins3Zl chloroplast 379 cryoelectron microscopy 389 culturing 372 cytosol372

differential centrifugation 392 differential interference conrrast(DIC) microscopy381 endoplasmicreticulum (ER)325

immunofluorescence microscopy 385 lumen 373 Iysosome373 membrane transport protern 372 metal shadowing 390

endosome373 mitochondrion 378 equilibrium density-gradient monoclonal antrbody 394 centiftgation 392 nucleolus378 extracellular matrix organelles371 (ECM)372 peroxisome324 fluorescence-activated cell phase-contrast sorter (FACS)394 microscopy381 fluorescentstaining 3 82 resolution 381 gap junctions383 scanningelectron Golgi complex 376 microscope(SEM)390 HAT medium 401 transmissionelectron hybridoma 401 m i c r o s c o p e( T E M ) 3 8 8

Review the Concepts l. One of the defining features of eukaryotic cells is the presenceof organelles.I7hat are the major organellesof eukaryotic cells, and what is the function of each?What is the cytosol? Sfhat cellular processesoccur within the cytosol? 2. The internalization of proteins, soluble macromolecules and large particles is a fundamental processrequired by all cells.The sizeof the moleculeoften dictateshow ir getsinternalized. Describehow phagocytosisdiffers from endocytosis and what generallyhappensto the internalizedmaterial. 3. Cell organellessuch as mitochondria, chloroplasts, and the Golgi apparatuseach have unique structures.How is the structure of each organellerelated to its function? 4. Much of what we know about cellular function deoends on experimentsutilizing specificcells and specificprrrc 1..g., '$fhat organelles)of cells. techniquesdo scientistscommonly use to isolate cells and organellesfrom complex mixtures, and how do thesetechniqueswork? 5. Both light and electron microscopy are commonly used to visualize cells, cell structures,and the location of specific molecules. Explain why a scientist may choose one or the other microscopy technique for use in research. 6. The magnification possiblewith any type of microscope is an important property, but its resolution, the ability to distinguish between two very closely apposed objects, is even more critical. Describewhy the resolving power of a microscopeis more important for seeingfiner details than its magnification. \fhat is the formula to describethe resolution of a microscopelens and what are the limitations placed on the values in the formula? 7. Sfhy are chemical srains required for visualizing cells and tissueswith the basic light microscope?What advantage do fluorescent dyes and fluorescencemicroscopy provide in

V I S U A L I Z I N GF, R A C T I O N A T I N G A ,N D C U L T U R I N G CELLS

comparison to the chemical dyes used to stain specimensfor light microscopy? What advantagesdo confocal scanning microscopy and deconvolution microscopy provide in comparison to conventional fluorescencemicroscopy? 8. In certain electron microscopy methods, the specimenis not directly imaged. How do these methods provide information about cellular structure, and what types of structures do they visualize? 9. Vhat is the difference between a cell strain, a cell line, and a clone? 10. Explain why certain cell lines are used to study the differentiation and function of muscle or fat cells. 1L. Explain why the process of cell fusion is necessaryto produce monoclonal antibodies used for research.

a. Name the marker molecule and give the number of the fraction that is most emiched for each of the following cell components: lysosomes;peroxisomes;mitochondria; plasma membrane; rough endoplasmic reticulum; smooth endoplasmicreticulum. b. Is the rough endoplasmicreticulum more or less densethan the smooth endoplasmicreticulum? lfhy? c. Describean alternativeapproachby which you could identify which fraction was enrichedfor which organelle. d. How would addition of a detergent to the homogenate, which disrupts membranes by solubilizing their lipid and protein components, affectthe equilibrium densitygradientresults?

Analyze the Data

References

Mouse liver cells were homogenizedand the homogenate subjected to equilibrium density-gradient centrifugation with sucrosegradients.Fractions obtained from thesegradients were assayedfor marker molecules(i.e., moleculesthat are limited to specificorganelles).The results of theseassays are shown in the figure. The marker moleculeshave the following functions: Cytochrome oxidase is an enzyme involved in the processby which ATP is formed in the complete aerobic degradation of glucose or fatty acids; ribosomal RNA forms part of the protein-synthesizing ribosomes;catalase catalyzesdecomposition of hydrogen peroxide; acid phosphatasehydrolyzes monophosphoric estersat acid pH; cytidylyl transferaseis involved in phospholipid biosynthesis;and amino acid permeaseaids in transDort of amino acids acrossmembranes.

Organelles of the EukarYotic Cell

E

tr

=ou

E x o

E

x9 + o

10 Fractionnumber 50% CurveA = cytochromeoxidase C u r v e B = r i b o s o m aRl N A Curve C = catalase

15

20 Sucrose0%

Curve D = acid phosphatase Curve E = cytidylyltransferase C u r v eF = a m i n o a c i d p e r m e a s e

Bainton, D. 1981. The discoveryof lysosomes.J. Cell Biol' 9lz66s-76s. Cuervo, A. M., and J. F. Dice. 1998. Lysosomes:a meetingpoint of proteins,chaperones,and proteases.J. Mol. Med. 76:6-1'2. de Duve, C. 1'996.The peroxisomein retrospect.Ann. NY Acad. Sci.804:1-10. Foster,L. J., et al. 2006. A mammalian organellemap by protein correlationprofiling. Cell t25 :187-1,99. Holtzman, E. 1989. Lysosomes.Plenum Press. Lamond, A., and \0. Earnshaw.1998. Structureand function in the nucleus.Science2802547-553' Mootha, V.K., et al. 2003. Integratedanalysisof protein composition, tissuediversity,and generegulationin mousemitochondria. Cell tl5z629-640. Palade,G. 197S.Intracellular aspectsof the processof protein synthesis.Science1.89:347-358.The Nobel Prizelectureof a pioneer in the study of cellular organelles' 'Wanders. R.. and H. R. Waterham.2006. Biochemistryof mammalian peroxisomesrevisited.Ann' Reu.Biochem. 7 52295-332' Light Microscopy: Visualizing Cell Structure and Localizing Proteins Within Cells Chen, X., M. Velliste,and R. F Murphy. 2006' Automated interpretation of subcellular patterns in fluorescencemicroscope imagesfor location proteomics. Cytometry (Part A) 69,4.: 631.-640. microscopywith Egner,A., and S. Hell. 2005. Fluorescence super-iesolvedoptical sections.Trends Cell Biol- 15:207-215. Gaietta.G.. et al. 2002. Multicolor and electronmicroscopic imaging of connexin trafficking' Science296:503-507. Giepmans,B. N. G., et aI.2006- The fluorescenttoolbox for protein location and function. Science3t2z2l7-224' assessing microscopyof living plant cells' Gilroy, S. 1997. Fluorescence Ann. Reu.Plant Physiol. Plant Mol. Biol- 48:165-190. 'l'997. Video Microscopy,2d ed' Inou6, S., and K. Spring. Plenum Press. Matsumoto. B.. ed. 2002. Methods in Cell Biology.Yol. T0: Cell Biological Applications of Confocal Microscopy.AcademicPress' Misteli, T., and D. L. Spector. 1997' Applications of the green fluorescentprotein in cell biology and biotechnology.Nature Biotech.15:961-964. Pepperkok,R., and J. Ellenberg.2006. High-throughput fluo..r...ri. microicopy for systemsbiology. Nature Reu.Mol' Cell Biol.7:690-696. R E F E R E N C E 5.

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Sako,Y., S. Minoguchi, and T. Yanagida.2000.Single-molecule imaging of EGFR signalling on the surface of living celli. Nature Cell Btol.2:168-172. Simon,S.,and J. Jaiswal.2004. Potentialsand pitfallsof fluorescent quantum dots for biologicalimaging.TrendsCell Biol. 14:497-504. Sluder,G., and D. Wolf, eds. 1998. Methods in Cell Biology. Vol. 55: Video Microscopy.AcademicPress. So, P.T.C.,et al. 2000. Two-photon excitation fluorescencemicroscopy.Ann. Reu.Biomed.tng. Z:3VO-42T. \fillig, K. I., et al. 2005. STED microscopyrevealsthat synaprotagmin remainsclusteredafter synapticvesicleexocytosis.Nature 440:935-939. Electron Microscopy: Methods and Applications Beck,M., et al. 2004. Nuclear pore complex structure and dynamics revealedby cryoelectrontomography. Science306:j.387-1390. FreS T. G., G. A. Perkins,and M. H. Ellisman.2006. Electron tomography of membrane-boundcellular organelles.Ann. Reu. Biophy. Biomol. Struc. 352199-224. . . Hlatq M. A. Principlesand Techniquesof Electron Microscopy, 4th ed. 2000. CambridgeUniversity Press. .. Koster,A., and J. Klumperman. 2003. Electron microscopyin cell biology: integratingsrructureand function. Nature Reu.Mol. Cell Biol.4:SS6-5510. Medalia, O., et al. 2002. Macromoleculararchitecturein eukaryotic cellsvisualizedby cryoelectrontomography.Science 298:1209-1213. Nickell, S., et al. 2006. A visual approachto proteomics.Nature Reu.Mol. Cell Biol.7:225-230. Purification of Cell Organelles Battye,F. L., and K. Shortman. 1991. Flow cyrometryand cellseparationprocedures.Curr. Opin. lmmunol. 3:238-241. de Duve, C. 1,975. Exploring cellswith a centrifuge.Science 189:.1,86-1.94. The Nobel Prize lectureof a pioneerin the study of cellular organelles.

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de Duve, C., and H. Beaufay.1981. A short history of tissue fractionation.J. Cell Biol. 9tz293s-299s. Howell, K. E., E. Devaney,and J. Gruenberg.1,989.Subcellular fractionation of tissueculture cells.TrendsBiochem. Sci.t4:4448. Ormerod, M. G., ed. 1990. FIow Cytometry:A Practical Approach. IRL Press. Rickwood, D. 1992. Preparatiue Centrifwgation: A Practical Approach. IRL Press. lsolation, Culture, and Differentiation of Metazoan Cells Bissell,M. J., A. Rizki, and I. S. Mian. 2003. Tissuearchitecture: the ultimate regulator of breastepithelialfunction. Curr. Opin. Cell Biol. l5:753-762. Davis, J. M., ed. 1.994.Basic Cell Cultwre:A Practical Approach. IRL Press. Edwards,B., et al. 2004. Flow cyrometryfor high-throughput, high-contentscreening.Curr. Opin. Chem. Biol. 8:392-398. Goding, I.W. 1996. Monoclonal Antibodies:Principlesand Practice.Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry, and Immunology, 3d ed. Academic Press. Griffith, L. G., and M. A. Swartz.2006. Capturing complex 3D tissuephysiologyin vitro. Nature Reu.Mol. Cell. Biol.7z211J24. Krutzik, P.,et al. 2004. Analysisof protein phosphorylationand cellular signalingeventsby flow cytometry: techniquesand clinical applications.Clin. Immwnol. 1L0z206- 221. Paszek,M. J., and V. M. Weaver.2004.The tensronmounrs: mechanicsmeetsmorphogenesisand malignancy.!. Mammary Gland Biol. Neoplasia9:325-342. Shaq A. J., ed. 1996. Epithelial Cell Cubure.IRL Press. Tyson, C. A., and J. A. Frazier, eds. L993. Methods in Toxicology. . Vol. I (Part A): In Vitro Biological Systems.Academic Press.Descri6es methods for growing many types of primary cells in culture.

V t S U A L t Z t N GF, R A C T | O N A T | N G A ,N D C U L T U R T NC GE L L S

cLASSIC

EXPERIMENT

ORGANELLES SEPARATING Beaufayet al., 1964,BiochemJ.92=191

In the 1950s and 1960sscientistsused two techniquesto study cell organelles: microscopy and fractionation. Christian de Duve was at the forefront of cell fractionation. In the early 1950s he used centrifugation to distinguish a new organelle, the lysosome, from previously characterizedfractions: the nucleus, the mitochondrial-rich fraction, and the microsomes.Soon thereafter he used equilibrium-density centrifugation to uncover yet another organelle.

Background Eukaryotic cells are highly organized and composed of cell structures known as organelles that perform specific functions. Although microscopyhas allowed biologiststo describethe location and appearanceof various organelles,it is of limited use in uncoveringan organelle'sfunction. To do this, cell biologists have relied on a technique known as cell fractionation. Here, cells are broken open, and the cellular componentsare separated on the basis of size, mass, and density using a variety of centrifugation techniques.Scientistscould then isolate and analyze cell components of different densities, called fractions. Using this method, biologists had divided the cell into four fractions: nuclei, mitochondrial-rich fraction, microsomes,and cell sap. De Duve was a biochemist interested in the subcellular locations of metabolic enzymes. He had already completed a large body of work on the fractionation of liver cells, in which he had determined the subcellular location of numerous enzymes.By locating theseenzymesin specificcell fractions, he could begin to elucidate the function of the organelle.He noted that his

work was guided by two hypotheses: the "postulate of biochemical homogeneity" and "the postulate of single location." In short, these hypotheses proposethat the entire compositionof a subcellular population will contain the same enzymes and that each enzyme is located at a discretesite within the cell. Armed with these hypotheses and the powerful tool of centrifugation. de Duve further subdivided the mitochondrial-rich fraction. First, he identified the light mitochondrial fraction, which is made up of hydrolytic enzymesthat are now known to composethe lysosome.Then, in a seriesof experiments describedhere, he identified another discrete subcellular fraction, which he called the peroxisome, within the mitochondrial-rich fraction.

The Experiment De Duve studied the distribution of enzymesin rat liver cells. Highly active in energy metabolism, the liver contains a number of useful enzymes to study. To look for the presenceof various enzymesduring the fractionation, de Duve relied on known tests, called enzyme assays,for enzYme activity. To retain maximum enzyme activity, he had to take precautions, which included performing all fractionation steps at OoC becauseheat denaturesprotein, compromising enzyme actlvrty. De Duve used rate-zonal centrifugation to separatecellular components by successivecentrifugation steps. He removed the rat's liver and broke it apart by homogenization. The crude preparation of homogenizedcells was then subjectedto relatively low-speed centrifugation. This initial step separated the cell nucleus,which collectsas sediment at the bottom of the tube'

from the cytoplasmic extract' which remains in the supernatant. Next, de Duve further subdivided the cytoplasmic extract into heavy mitochondrial fraction, Iight mitochondrial fraction, and microsomal fraction. He accomplished separating the cytoplasm by employing successive centrifugation steps of increasingforce. At each step he collected and stored the fractions for subsequentenzyme analysis' Once the fractionation was comPlete, de Duve performed enzyme assaysto determine the subcellular distribution of eachenzyme.He then graphically plotted the distribution of the enzyme throughout the cell. As had been shown previously, the activity of cytochrome oxidase, an important enzyme in the electron transfer system' was found primarily in the heavy mitochondrial fractions. The microsomal fraction was shown to contain another characterized enzYme, previously The light mitoglucose-5-phosphatase. chondrial fraction, which is made up of the lysosome,showed the characteristic acid phosphataseactivity. Unexpectedly, de Duve observed a fourth pattern when he assayeduricase activity. Rather than following the pattern of the referenceenzymes,uricaseactivity was sharply concentrated within the light mitochondrial fraction. This sharp concentration, in contrast to the broad distribution, suggested to de Duve that the uricase might be secluded in another subcellular population separate from the lYsosomal enzymes. To test this theorS de Duve employed a technique known as equilibrium density-gradient centrifugation, which separates macromolecules on the basis of density. Equilibrium density-gradientcentrifugation can be performed using a number of different

ORGANELLES SEPARATING

407

) 1.09 A^

1.11

ot

Organelle fraction Lysosomes ( 1 . 1 2g / c m 3 ) M i t o c h o n d r i -a | ( 1 . 1 8g / c m s )

oxidase, segregatedinto the samefractions as uricase.Becauseeach of these enzymes either produced or used hydrogen peroxide, de Duve proposed that this fraction represented an organelle responsiblefor the peroxide metabolism and dubbed it the peroxisome.

Discussion

De Duve's work on cellular fractionation provided an insight into the function of cell structures as he sought to Before After map the location of known enzymes. centrifugation centrifugation Examining the inventory of enzymesin A FIGURE 1 Schematic a given cell fraction gave him clues to depictionof the separationof the lysosomes, mitochondria, and peroxisomes by equilibriumdensitycentrifugation. The mitochondrial-rich fraction fromrate-zonal centrifugation wasseparated in a sucrose gradient, andthe organelles wereseparated on the basisof density[From Lodish etal, Molecular CellBiology,3d ed, W H Freeman and Company, p 166] Peroxisomes.f ( 1 . 2 3g / c m s ) |

gradients,includingsucroseand glycogen. In addition, the gradient can be made up in eitherwater or "heavy water," which contains the hydrogen isotope deuterium in place of hydrogen. In his experiment de Duve separated the mitochondrial-rich fraction prepared by rare-zonalcentrifugation in each of these different gradients (Figure 1). If uricasewere part of a separate subcellularcompartment,it would separate from the lysosomal enzymes in each gradient tested.De Duve performed the fractionations in this series of gradients, then performed enzyme assaysas before.In eachcase,he found uricasein a separatepopulation than the lysosomal enzyme acid phosphataseand the mitochondrial enzyme cytochromeoxidase (Figure2).By rep e a t e d l yo b s e r v i n gu r i c a s ea c r i v i t yi n a distinct fraction from the acivity of the lysosomal and mitochondrial enzymes,de Duve concluded that uricase was part of a separateorganelle. The experiment also showed that two other enzymes,catalaseand o-amino acid

408

C H A P T E R9

|

its function. His careful work resulted in the uncovering of two organelles: the Iysosomeand the peroxisome. His work also provided important clues to the organelles'function. The lysosome, where de Duve found so many potentially destructiveenzymes.is now known to be an important site for degradation of biomolecules.The peroxisome has been shown to be the site of fatty acid and amino acid oxidation, reactions that produce a large amount of hydrogen peroxide. In 1974, de Duve received the Nobel Prize for Physiology and Medicine in recognition of his pioneering work.

Cytochromeoxidase

c" o4

6r Uricase

o2 o) ol

0)

Acid phosphatase

20 40 60 80 Percent heightin tube a FIGURE 2 Graphicalrepresentation of the enzymeanalysisof productsfrom a sucrosegradient.Themitochondrial-rich fraction wasseparated asdepicted in Figure10.,1 , andthenenzyme assays wereperformed Therelative concentration of activeenzyme is plottedon they axis;the heightin thetubeisplottedon the x axisThepeakactivities of (rop)andacidphosphatase cytochrome oxidase (bottom) areobserved nearthetop of the tube Thepeakactivityof uricase (middle)migrates to the bottomof the tube [Adaptedfrom Beaufay et al , 1964,Biochem J 92:191l

v t S u A L t z t N G ,F R A C T | O N A T | N G A ,N D C U L T U R T N C GE L L S

CHAPTER

BIOMEMBRANE STRUCTURE bilayersunoundedby water,as Molecularmodelof a phospholipid @ NationalInstitutes dynamrcs calculations determined by molecular Inc Researchers, of Health/Photo

embranesparticipate in many aspectsof cell structure and function. The plasma membranedefinesthe cell and separatesthe inside from the outside. In eukaryotes, membranesalso define the intracellular organelles such as the nucleus and lysosome.These biomembranesall have the same basic architecture-a phospholipid bilayerbut they are not static; their function is not to prevent all exchange across a border. Each cellular membrane has its own set of proteins that allow it to carry out its multitude of specificfunctions (Figure 10-1). Prokaryotes, the simplest and smallest cells, are about t-2 pm in length and are surrounded by a single plasma membrane;in most casesthey contain no internal membranelimited subcompartments(seeFigure 1-2a). However, this single plasma membrane contains hundreds of different types of proteins that are integral to the function of the cell. Some of these proteins catalyze ATP synthesisand initiation of DNA replication, for instance. Others represent the many types of membrane transport proteins that enable specific ions, sugars, amino acids, and vitamins to cross the otherwise impermeable phospholipid bilayer to enter the cell and that allow specific metabolic products to exrt. In eukaryotic cellsthe plasma membrane (Figure 10-2) is not a site for ATP generation or DNA synthesis.Eukaryotic plasma membranesare studded with a multitude of membrane transpoft proteins that allow selectiveimport and export of small molecules and ions. Receptorsin the plasma membrane are proteins that allow the cell to recognizechemical signals-

many sent by neighboring cells-present in its environment and adlust its metabolism or, especiallyduring development, its pattern of gene expressionin response.Other specialized plaima membrane proteins allow the cell to adhere to other cells and to components in the surrounding fibrous extracellular matrix. Many plasma membrane proteins bind components of the cytoskeleton, the densenetutork of protein filaments that permeatesthe cytosol and mechanically supports cellular membranes,interactions that are essential for the cell to assumeits specific shape and for many types of cell movements. The plasma membrane bends, folds, and flexes in three dimensions. Some segments bleb inward, incorporating components from the extracellular medium into intracellular vesicles(seeFigure 9-2). Virusessuch as HIV bud outward from the cell-surface membrane, enveloping themselves with a bit of the plasma membrane that contains virus-specificproteins (Figure 10-3).

OUTLIN E 10.1

B i o m e m b r a n e sL:i p i d C o m p o s i t i o n and StructuralOrganization

4't1

10.2

Biomembranes:ProteinComponents and BasicFunctions

10.3

P h o s p h o l i p i dS s ,p h i n g o l i p i d sa,n d C h o l e s t e r o l : 429 Synthesisand IntracellularMovement

409

Lipid-anchor protein I

Peripheral memDrane protein Hydrophilic phospholipid h e a dg r o u p Phospholipid bilayer

Cytosol Lipid-anchored protein I n t e g r am l embrane protein

Hydrophilic fatty acyl P e r i p h e r a l s i d ec h a i n s memorane protein

FIGURE 10-1 Fluidmosaicmodelof biomembranes. A bilayer anchored proteins aretethered to oneleafletby a longcovalently -3 nm thickprovides of phospholipids the basicarchitecture of all attached hydrocarbon proteins chainPeripheral associate withthe cellular membranes; membrane proteins giveeachcellular membrane membrane primarily by specific noncovalent interactions with itsuniquesetof f unctionsIntegral (transmembrane) proteins proteins span integral or membrane lipids.lAfterD Enqelman. 2005, /Vature the bilayer andoftenformdimersandhigher-order oligomersLipid438:578-80 l Each organellein a eukaryotic cell (Figures1-2, 10-2 and 10-4)containsa unique complementof protelns-some embeddedin its membrane(s),others containedin its aqueous interior space,the lumen-that enable it to carry our lrs characteristic cellular functions. For instance, the internal pH of the lysosome, an organelle that contains many P l a s m am e m b r a n e

degradativeenzymes,is about 5, compared to pH 7.2 of the cytosol, the aqueous part of the cytoplasm. The lysosome membrane contains ATP-powered H* pumps that use the energy of hydrolysis of ATP phosphoanhydride bonds to pump protons from the cytosol into the lumen of the lysosome, lowering its pH. We begin our examination of biomembranesby considering their lipid components. These not only affect membrane shapeand function but also play important roles in anchoring proteins to the membrane, modifying membrane protein activities,and transducingsignalsto the cytoplasm. 'We then consider the structure of membrane proteins. Many, such as transport proteins and receptors, have large segmentsthat are imbedded in the hydrocarbon core of the

HIV core

Lysosome Multivesicular body A FIGURE 10-2 Cellularmembranes plasma The memorane defines theexterior of thecellandcontrols themovement of molecules betweenthe cytosolandthe extracellular mediumDifferent typesof organelles andsmaller vesicles enclosed withintheirown distinctive membranes carryout special functrons suchasgeneexpression, energy production, membrane synthesis, andintracellular transport.

410

CHAPTER 1O I

B I O M E M B R A NSET R U C T U R E

Plasma memDrane

FIGURE 10-3 Eukaryotic cellmembranes are dynamic structures. An electron micrograph of the plasma membrane of an HIV-infected cell,showingHIVparticles buddingintotheculture medium. As theviruscorebudsfromthecell,it becomes enveloped by a membrane derived fromthe cell'splasma membrane that contains specific viralproteins[From W Sundquist andU vonSchwedler. University of Utahl

A FIGURE 10-4 Stackedmembranes of the Golgicomplex. Notethe irregular shapeandcurvature of thesemembranes lFrom C Hopkins andJ Burden, lmperial London College l

phospholipid bilayer, and we will focus on the principal classesof such membrane proteins. Finally, we consider how phospholipids and cholesterol are synthesizedin cells and distributedto the many membranesand organelles.Cholesterol is an essentialcomponent of the plasma membrane of all animal cells but is toxic to the organism if present in CXCCSS.

Biomembranes: LipidComposition and StructuralOrganization In Chapter 2 we learnedthat phosphoglyceridesare the principal building blocks of most biomembranes.Like the two other principal classesof membrane lipids, sphingolipids and cholesterol(Figure10-5),phosphoglycerides are amphipathic molecules-they consist of two segmentswith very different chemical properties. In phosphoglyceridesand sphingolipids the hydrocarbon "tails" of the fatty acid side chains are hydrophobic and partition away from water, whereasthe "head groups" are strongly hydrophilic, or water loving, and tend to interact with water molecules. Steroids such as cholesterol, in contrast, are mostly hydrophobic except for one hydrophilic hydroxyl group. AII three types of phospholipids have the necessaryqualities to form membranesand play different roles in the function of the cell.

persed in aqueous solution, the phospholipids aggregate into one of three forms: sphericalmicellesand liposomes and sheetlikephospholipid bilayers, which are two moleculesthick (Figure 10-6). The type of structureformed by a pure phospholipid or a mixture of phospholipidsdepends on several factors, including the length of the fatty acyl chains, their degreeof saturation, and temperature.In all three structures, the hydrophobic effect causesthe fatty acyl chainsto aggregateand excludewater moleculesfrom the "core." Micelles are rarely formed from natural phosphoglycerides, whose fatty acyl chains generally are too bulky to fit into the interior of a micelle. However, micelles are formed if one of the two fatty acyl chains is removed from the phosphoglyceride by hydrolysis, forming a lysophospholipid.In aqueoussolution, common detergents and soapsform micellesthat behaveas tiny ball bearings, thus giving soap solutions their slippery feel and lubricating properties. Under suitable conditions, phospholipids of the composition present in cells spontaneously form symmetric phospholipid bilayers.Each phospholipid layer in this lameilar structureis called a leaflet.The hydrophobic fatty acyl chains in each leaflet minimize contact with water by aligning themselvestightly together in the center of the bilayer, forming a hydrophobic core that is about 3-4 nm thick (Figure 10-6b). The close packing of thesenonpolar 'Sfaals interactions between tails is stabilized by van der thesehydrocarbon chains. Ionic and hydrogen bonds stabilize the interaction of the phospholipid polar head groups with one another and with water. Electron microscopy of thin membrane sectionsof cells stained with osmium tetroxide, which binds strongly to the polar head groups of phospholipids,revealsthe bilayer structure (Figure 10-6a). A cross section of a single membrane stained with osmium tetroxide looks like a railroad track: two thin dark lines (the stained head group complexes)with a uniform light space of about 2 nm between them (the h y d r o p h o b i ct a i l s ) . A phospholipid bilayer can be of almost unlimited sizefrom micrometers (pm) to millimeters (mm) in length or width-and can contain tens of millions of phospholipid molecules. Becauseof their hydrophobic core, bilayers are virtually impermeable to salts, sugars,and most other small hydrophilic molecules. The phospholipid bilayer is the basic structural unit of nearly all biological membranes; although they contain other molecules (e.g., cholesterol, glycolipids, proteins), biomembraneshave a hydrophobic core that separatestwo aqueoussolutions and acts as a permeability barrier.

P h o s p h o l i p i dS s p o n t a n e o u s lFy o r mB i l a y e r s

P h o s p h o l i p i dB i l a y e r sF o r ma S e a l e d C o m p a r t m e nS t u r r o u n d i n ga n l n t e r n a l A q u e o u sS p a c e

The amphipathic nature of phospholipids,which governs their interactions,is critical to the structureof biomembranes. When a suspensionof phospholipids is mechanicallydis-

Phospholipid bilayers can be generatedin the laboratory by simpleexperimentalmanipulations;theseutilize either chemically pure phospholipidsor lipid mixtures of the composition

B I O M E M B R A N E LSI:P I DC O M P O S I T I OANN D S T R U C T U R AOLR G A N I Z A T I O N

41'l

(a) Phosphoglycerides

H e a dg r o u p H

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A FIGURE 10-5 Threeclasses of membranelipids.(a)Most phosphoglycerides (red), arederivatives of glycerol 3-phosphate whichcontains two esterified fattyacylchains thatconstitute the hydrophobic "tail" anda polar"headgroup"esterified to the phosphate. Thefattyacidscanvaryin lengthandbe saturated (no (one,two, or threedoublebonds). doublebonds)or unsaturated (PC), In phosphatidylcholine the headgroupischolineAlsoshown arethe molecules attachedto the phosphate groupin threeother commonphosphoglycerides: phosphatidylethanolamine (PE), phosphatidylserine (PS), (Pl)Plasmalogens andphosphatidylinositol containonefattyacylchainattached to glycerol by an esterlinkage andoneattached by an etherlinkage; thesecontainsimilar head g r o u p s a s p h o s p h o g l y c e(rbi )dSepsh i n g o l i p i d s a r e d e r i v a t i v e s o f (red),an aminoalcohol sphingosine with a lonqhvdrocarbon chain 412

CHAPTER 1O I

B I O M E M B R A NSET R U C T U R E

stisqrasterol

Various fattyacylchainsareconnected to sphingosine by an amide (SM),whichcontaina phosphocholine bond Thesphingomyelins headgroup,arephospholipids Othersphingolipids areglycolipids in whicha singlesugarresidue or branched oligosaccharide isattached to thesphingosine backbone. Forinstance, thesimpleglycolipid (GlcCer) glucosylcerebroside hasa glucose headgroup.(c)The (cholesterol), majorsterols in animals fungi(ergosterol), andplants (stigmasterol) differslightlyin structure, butallserveaskeycomponents of cellular membranes Thebasicstructure of steroids isa four-ring (yellow). hydrocarbon Likeothermembrane lipids, sterols are amphipathic groupisequivalent Thesinglehydroxyl to the polar headgroupin otherlipids; theconjugated ringandshorthydrocarbon c h a i n f o r m t h e h y d r o p h o b i c[ S t aei el Hs p r o n g e t,a2l0 0 1N , atureRev. Mot Celt Biot2:5041

(at

that mediate the transport of specific molecules across this otherwise impermeable bilayer. The secondproperty of the bilayer is its stability. The bilayer structure is maintained by hydrophobic and van der Waals interactions between the lipid chains. Even though the exterior aqueousenvironment can vary widely in ionic strength and pH, the bilayer has the strength to retain its characteristic architecture. Third, all phospholipid bilayers can spontaneouslyform sealedclosed compartments where the aqueous space on the inside is

M e m b r a n eb i l a y e r

Phospholipid bilayer

P o l a rh e a d g roups

'l

g o l un, u u a / groups (c)

P h o s p h o l i p i disn s o l u t i o n

"1r.., Disperse phospholipids in water

FIGURE 10-6 The bilayerstructureof biomembranes. (a)Electron micrograph of a thin sectionthroughan erythrocyte "railroad membrane stained withosmiumtetroxide Thecharacteristic presence track"appearance of the membrane indicates the of two polarlayers, consistent withthe bilayer for phospholipid structure (b)Schematic membranes interpretation of the phospholipid bilayer in whichpolargroupsfaceoutwardto shield the hydrophobic fatty acyltailsfromwaterThehydrophobic effectandvanderWaals interactions betweenthe fattyacyltailsdrivethe assembly of the bilayer(Chapter 2).(c)Cross-sectional viewsof two otherstructures has formedbydispersal of phospholipids in waterA spherical micelle interior composed entirely of fattyacylchains; a a hydrophobic liposome consists of a phospholipid bilayer surrounding an spherical aqueous centerlPart(a)courtesy of J D Robertson ]

found in cell membranes(Figure 10-7). Studieson these bilayers have shown that they possessthree important properties. First, the hydrophobic core is an impermeable barrier that prevents the diffusion of water-soluble (hydrophilic) solutesacrossthe membrane.Importantly, this simple barrier function is modulated by the presenceof membrane proteins

D i s s o l v ep h o s p h o l i p i d s i n s o l v e n ta n d a p p l y t o s m a l lh o l e in oartition

Liposome

10-7 Formationand studyof FIGURE a EXPERIMENTAL of biological pure phospholipidbilayers.(Iop)A preparation of suchasa mixture solvent, istreatedwith an organic membranes (3:1),whichselectively the solubilizes andmethanol chloroform remain andcarbohydrates Proteins phospholipids andcholesterol. by evaporation is removed Thesolvent residue. in an insoluble in water,they (Bottom/eft)lf the lipidsaremechanically dispersed with an shownin crosssection, forma liposome, spontaneously (Bottom also right)A planarbilayer, compartment. internal aqueous canformovera smallholein a partition shownin crosssection, canbe usedto study phases; suchbilayers two aqueous separating to the otherthroughthe fromonesolution of solutes the movement membrane

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Mitochondrion

Matrix Intermembranespace Exoplasmic face

Cytosolic face

< FIGURE 10-8 Thefacesof cellular membranes. Theplasmamembrane, a s i n g l eb i l a y em r embranen , c l o s et h s ec e l l I n t h i sh i g h l ys c h e m a tri ce p r e s e n t a t i o n , (greenstipple) internal cytosol andexternal (purple) (red) environment definethe cytosolic (black) andexoplasmic facesof the bilayer Vesicles andsomeorganelles havea single membrane andtheirinternal aoueous soace (purple) istopologically equivalent to the outside of thecell.Threeorganelles-the nucleus, mitochondrion, andchloroplast (whichis not shown)-areenclosed by two membranes separated bya smallintermembrane Theexoplasmic space. facesof the innerand outermembranes aroundtheseorganelles borderthe intermembrane spacebetween them Forsimplicity, the hydrophobic membrane interior isnotindicated in thisdiaqram

P l a s m am e m b r a n e

Exterior

IntermembranesDace

-

separatedfrom that on the outside. An "edge" of a phospholipid bilayer, as depicted in Figure 10-6b with the hydrocarbon core of the bilayer exposedto an aqueoussolution, is unstable; the exposed fatty acyl side chains would be in an energeticallymuch more stable state if they were not adjacent to water molecules but surrounded by other fatty acyl chains (hydrophobic effect; Chapter 2). Thus in aqueous solution, sheetsof phospholipid bilayers sponraneouslyseal their edges,forming a spherical bilayer that enclosesan aqueous central compartment. The liposome depicted in Figure 10-6 is an example of such a structure viewed in cross sectton. This physical chemical property of a phospholipid bilayer has important implications for cellular membranes: no membrane in a cell can have an "edge" with exposed hydrocarbon fatty acyl chains. All membranes form closed compartments, similar in basic architecture to liposomes. Becauseall cellular membranesenclosean entire cell or an internal compartment, they have an internal face (the surface oriented toward the interior of the compartment) and an externalface (the surfacepresentedto the environment). More commonl5 we designatethe two surfacesof a celluIar membrane as the cytosolic face and the exoplasmic face. This nomenclature is useful in highlighting the topological equivalence of the faces in different membranes, as diagrammed in Figures 10-8 and 10-9. For example. the

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Exoplasmic

Cytosolic face

A FIGURE 10-9 Facesof cellularmembranesare conserved during membranebuddingand fusion.Redmembrane surfaces arecytosolic faces;blackareexoplasmic faces.Duringendocytosis a segment of the plasma membrane budsinwardtowardthecytosol pinches andeventually off a separate vesicle. Duringthisprocess the cytosolic faceof the plasma membrane remains facingthe cytosol andtheexoplasmic faceof the newvesicle membrane facesthe lumenDuringexocytosis an intracellular vesicle fuseswith the plasma (exoplasmic membrane, andthe lumenof thevesicle face) connects with theextracellular mediumProteins thatspanthe membrane retaintheirasymmetric orientation duringvesicle budding andfusionevents; in particular thesamesegment always facesthe cyrosol

exoplasmic face of the plasma membrane is directed away from the cytosol, toward the extracellular spaceor external environment, and definesthe outer limit of the cell. The cytosolic face of the plasma membrane faces the cytosol. Similarly for organellesand vesiclessurroundedby a singlemembrane,the cytosolic face facesthe c1'tosol.The exoplasmic face is always directed away from the cytosol and in this caseis on the inside of the organelle in contact with the internal aqueous space,or lumen. That the inside, or lumen, of thesevesiclesis topologically equivalentto the extracellularspaceis most easilyunderof the stood for vesiclesthat ariseby invagination(endocytosis) plasma membrane. This processresults in the external face of the plasma membrane becoming the internal face of the vesicle membrane, and in the vesiclethe cytosolic face of the plasma membraneremainsfacing the cytosol (seeFigure 10-9). Two distinct membranessurround three organelles-the nucleus, mitochondrion, and chloroplast; the exoplasmic surface of each membrane faces the spacebetween the two membranes.This can perhaps best be understood by reference to the endosymbiont hypothesis,discussedin Chapter 1, which posits that mitochondria and chloroplasts arose early in the evolution of eukaryotic cells by the engulfment of bacteria capable of oxidative phosphorylation or photosynthesis,respectively(seeFigure6-20). Thus the exoplasmic face of the mitochondrial inner membrane, derived from the exoplasmicface of the ancestralbacterial plasma membrane, as well as the exoplasmic face of the outer mitochondrial membrane,derived from the exoplasmicface of the ancestral plasma membrane, both face the intermembranespace. Natural membranes from different cell types exhibit a variety of shapes,which complementa cell'sfunction (Figure 10-10; seeFigures10-3 and 10-4).The smooth,flexiblesurface of the erythrocyte plasma membrane allows the cell to squeezethrough narrow blood capillaries.Somecells have a long, slenderextensionof the plasma membrane,called a cilium or flagellum, which beatsin a whiplike manner.This motion causesfluid to flow acrossthe surfaceof a sheetof cells or a sperm cell to swim toward an egg. Membranes are dynamic structures;Figure 10-3 shows an HIV virus budding from the surfaceof a human cell. In infectedcells,specificviral proteins becomeinsertedinto the plasma membrane,and segmentsof the plasma membrane envelop the viral core, or nucleocapsid, that contains the viral RNA genome as the virus buds from the cell. The membrane-coatedvirus then pinches off from the plasma membrane and is releasedinto the surrounding medium. Internal cellular membranessuch as the Golgi complex (seeFigure 10-4) are constantly budding off membrane vesiclesinto the cytosol. Thesethen fuse with other membranesto transport the luminal contentsfrom one organelleto another (Chapter 14).

B i o m e m b r a n eC s o n t a i nT h r e eP r i n c i p a l Lipids of Classes As noted above, a typical biomembrane is assembledfrom three classes of amphipathic lipids: phosphoglycerides,

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in differentcell 10-10Variationin biomembranes FIGURE of the covers the surface membrane types.(a)A smooth,flexible micrograph. electron in this scanning as seen cell erythrocyte discoid cellsthat linethe (b)Tuftsof cilia(Ci)projectfromthe ependymal Inc. Researchers, @omiKron/Photo brainventricles lPart(a)Copyright A andOrgans: 1979,Tissues andR H Kardon, Part(b)fromR G Kessel Company] Freeman and W H Microscopy, Electron of Scanning Text-Atlas sphingolipids, and steroids, which differ in their chemical structures,abundance,and functions in the membrane. the most abundant class of lipids in Phosphoglycerides, of glycerol 3-phosphate(see derivatives are membranes, most moleculeconsistsof Figure 10-5a).A typical phosphoglyceride a hydrophobic tail composedof two fatty acyl chains esterified to the fvvo hydroxyl groups in glycerol phosphate and a polar head group attached to the phosphate group' The two fatty acyl chains may differ in the number of carbons that they contain (commonly 1.6or 18) and their degreeof saturation(0, 1, or 2 double bonds).A phosphoglycerideis classifiedaccording to the nature of its head group. In phosphatidylcholines,

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the most abundant phospholipids in the plasma membrane, the head group consistsof choline, a positively chargedalcohol, esterifiedto the negarivelycharged phosphate.In other phosphoglycerides,an OH-containing molecule such as ethanolamine, serine, and the sugar derivative inositol is linked to the phosphate group. The negatively charged phosphate group and the positively charged groups or the hydroxyl groups on the head group interacr strongly with water. At neutral pH, some phosphoglycerides (e.g., phosphatidylcholineand phosphatidylethanolamine) carry no net electriccharge,whereasothers (e.g.,phosphatidylinositol and phosphatidylserine)carry a single net negative charge. Nonetheless,the polar head groups in all phospholipidscan pack togetherinto the characteristicbilayer structure. The plasmalogensare a group of phosphoglyceridesthat contain one fatty acyl chain attached to carbon 2 of glycerol by an esterlinkage and one long hydrocarbon chain attached to carbon 1 of glycerol by an ether (C-O-C) rather than an ester linkage. The abundance of plasmalogensvaries among tissuesand speciesbut is especiallyhigh in human brain and heart tissue. The additional chemical stability of the ether linkage in plasmalogens, compared to the ester linkage, or the subtle differencesin their three-dimensional structure compared with that of other phosphoglycerides may have as yet unrecognizedphysiologic significance. A secondclassof membranelipid is the sphingolipids.All of thesecompounds are derived from sphingosine,an amino alcohol with a long hydrocarbon chain, and contain a longchain faty acid attached in an amide linkage to the sphingosine amino group. Like phosphoglycerides,sphingolipids have a phosphate-based polar head.In sphingomyelin,the most abundant sphingolipid, phosphocholine is attached to the terminal hydroxyl group of sphingosine(seeFigure 10-5b).Thus sphingomyelin is a phospholipid, and its overall structure is quite similar to that of phosphatidylcholine. Sphingomyelinsare similar in shape to phosphoglyceridesand can form mixed bilayers with them. Other sphingolipids are amphipathic glycolipids whose polar head groups are sugars that are not linked via a phosphategroup. Glucosylcerebroside, the simplest glycosphingolipid, contains a singleglucoseunit attached to sphingosine. In the complex glycosphingolipids called gangliosides,one or two branched sugar chains (oligosaccharides) containing sialic acid groups are attached to sphingosine. Glycolipids constitute 2-10 percent of the total lipid in plasma membraneslthey are most abundant in nervoustissue. Cholesterol and its analogs constiture the third important class of membrane lipids, the steroids.The basic structure of steroidsis a four-ring hydrocarbon. The structuresof the principal yeast sterol (ergosterol)and plant phytosterols (e.g., stigmasterol) differ slightly from that of cholesterol, the major animal sterol (seeFigure 10-5c).The small differences in the biosynthetic pathways of fungal and animal sterols and in their structures are the basis of most antifungal drugs currently in use. Cholesterol, Iike the rwo orher sterols, has a hydroxyl substituent on one ring. Although cholesterolis almost entirely hydrocarbon in composition, it is amphipathic becauseits hydroxyl group can interact with water. Cholesterol is especially abundant in the plasma 4'16

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membranesof mammalian cells but is absent from most prokaryotic and all plant cells.As much as 30-50 percent of the lipids in plant plasma membranes consist of certain steroids unique to plants. Between50 and 90 percent of the cholesterolin most mammalian cells is presentin the plasma membrane and associatedvesicles.Cholesterol and other sterols are too hydrophobic to form a bilayer structure on their own. Instead, at concentrationsfound in natural membranes,these sterols must intercalate between phospholipid moleculesto be incorporated into biomembranes. In addition to its structural role in membranes,cholesterol is the precursor for severalimportant bioactive molecules.They include bile acids,which are made in the liver and help emulsify dietary fats for digestionand absorption in the intestinesl steroid hormones produced by endocrine cells (e.g.,adrenalgland, ovar5 testes);and vitamin D produced in the skin and kidneys. Another critical function of cholesterol is its covalent addition to Hedgehogprotein, a key signaling moleculein embryonic development(Chapter 16).

Most Lipidsand Many ProteinsAre Laterally M o b i l ei n B i o m e m b r a n e s In the two-dimensional plane of a bilayer, thermal motion permits lipid molecules to rotate freely around their long axes and to diffuse Iaterally within each leaflet. Because such movements are lateral or rotational, the fatty acyl chains remain in the hydrophobic interior of the bilayer. In both natural and artificial membranes,a typical lipid molecule

Heat

Gel-likeconsistency

Fluidlikeconsistency

si

FIGURE 10-11Geland fluid forms of the phospholipid bilayer.(Iop)Depiction of gel-to-fluid transitionPhospholipids with longsaturated fattyacylchains tendto assemble intoa highly gel-like ordered, bilayer in whichthereislittleoverlap of the nonpolar tailsin thetwo leaflets. Heatdisorders the nonpolar tails andinduces a transition froma gelto a fluidwithina temperature rangeof onlya few degrees. As thechainsbecome disordered, the (Bottom) bilayer alsodecreases in thickness. Molecular models of phospholipid monolayers in gelandfluidstates, asdetermined by molecular dynamrcs calculations. based onH Heller etal, 1993. lBottom J PhysChem97:8343 l

exchangesplaces with its neighbors in a leaflet about 107 times per second and diffuses several micrometers per second at 37 "C. Thesediffusion ratesindicatethat the viscosity of the bilayer is 100 times as great as that of waterabout the same as the viscosity of olive oil. Even though lipids diffuse more slowly in the bilayer than in an aqueous solvent, a membrane lipid could diffuse the length of a typical bacterialcell (1 pm) in only 1 secondand the length of an animal cell in about 20 seconds.Vhen fluid artificial pure phospholipid membranesare cooled below 37'C, the lipids can undergo aphase transition from a liquidlike (fluid) state to a gel-like (semisolid)state, analogousto the liquidsolid transition when liquid water freezes(Figure 10-11). Below the phase transition temperature, the rate of diffusion of the lipids drops precipitously. At usual physiologic temperatures,the hydrophobic interior of natural membranes generally has a low viscosity and a fluidlike consistency, in contrast to the gel-like consistency observed at lower temperatures,

In pure membrane bilayers, phospholipids and sphingolipids rotate and move laterallg but they do not spontaneously migrate, or flip-flop, from one leaflet to the other' The energetic barrier is too high; migration would require moving the polar head group from its aqueous environment through the hydrocarbon core of the bilayer to the aqueoussolution on the other side. Specialmembraneproteins discussedin Chapter 1.1 are required for membrane lipids and other polar molecules to flip from one leaflet to the other. The lateral movements of specific plasma-membrane proteins and lipids can be quantified by a technique called fluorescencerecouery after photobleaching /FRAP/. Phospholipids containing a fluorescent substituent are used to monitor lipid movement. For proteins, a fragment of a monoclonal antibody that is specificfor the exoplasmic domain of the desired protein and that has only a single antigenbinding site is tagged with a fluorescent dye. \fith this method, described in Figure 10-1.2, the rate at which

(a)

Fluorescentreagent

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ri 3 3000 6

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FIGURE A EXPERIMENTAL 10-12Fluorescence recoveryafter photobleaching(FRAP)experimentscan quantify the lateral movementof proteinsand lipidswithin the plasma protocolStep(E): Cellsarefirst membrane.(a)Experimental to a specific with a fluorescent reagent thatbindsuniformly labeled membrane lipidor proteinStep(E): A laserlightisthenfocused the bound irreversibly bleaching on a smallareaof thesurface, in the illuminated area thefluorescence reagent andthusreducing patchrncreases of the bleached Step(B): Intime,thefluorescence fluorescent surface molecules diffuseintoit and asunbleached of bleached onesdiffuseoutwardTheextentof recovery patchisproportronal to thefractionof in the bleached fluorescence

150

(b)Results thataremobilein the membrane. molecules labeled with a treated cells hepatoma with human experiment of FRAP receptor icfor the asialoglycoprotein specif antibody fluorescent to returned of thefluorescence proteinThefindingthat 50 percent molecules of the receptor that 50 percent areaindicates the bleached patchweremobileand50 percent membrane in the illuminated is recovery rate of fluorescence the Because wereimmobile moveintothe molecules to the rateat whichlabeled oroportional of a proteinor lipidin the coefficient region, the diffusion bleached etal, Y I Henis fromsuchdata.[See canbe calculated membrane 111:14091 J CellBiol. 1990,

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o/o) (MOt C0MP0$Tl0N SOURCUI.()CATION

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PC : phosphatidylcholine; PE : phosphatidylethanolamine; PS: phosphatidylserine; SM - sphingomyelin. souRcE:S7.DowhanandM.Bogdanov,2002,inD.E.VanceandJ.E Vance,eds.,Biochemistryof Lipids,Lipoproteins,andMembranes,Elsevier.

membrane molecules move-the diffusion coefficient-can be determined, as well as rhe proportion of the molecules that are laterallymobile. The results of FRAP studieswith fluorescence-labeled phospholipidshave shown that in fibroblast plasma membranes, all the phospholipids are freely mobile over distancesof about 0.5 pm, but most cannot diffuse over much longer distances.These findings suggestthat protein-rich regions of the plasma membrane about 1 pm in diameter separatelipid-rich regionscontaining the bulk of the membrane phospholipid. Phospholipids are free to diffuse within such regions but not from one lipid-rich region to an adjacent one. Furthermore,the rate of lateral diffusion of lipids in the plasma membrane is nearly an order of magnitude slower than in pure phospholipid bilayers: diffusion consrantsof 10-8 cm2/sand I0-7 cm2lsare characteristic of the plasma membrane and a lipid bilayer, respectively.This difference suggeststhat lipids may be tightly but not irreversibly bound to certain integral proteins in some membranes, as indeed has recently been d e m o n s t r a t e d( s e eF i g u r e 1 0 - 1 7 b e l o w ) .

L i p i dC o m p o s i t i o nI n f l u e n c e st h e p h y s i c a l Propertiesof Membranes A typical cell contains myriad types of membranes,each with unique properties bestowed by its particular mix of Iipids and proteins. The data in Table 10-1 illustrate the variation in lipid composition in different biomembranes.Several

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phenomena contribute to these differences.For instance, there are differencesin the relative abundancesof phosphoglyceridesand sphingolipids between membranesin the endoplasmic reticulum (ER), where phospholipids are synthesized, and the Golgi, where sphingolipids are synthesized. The proportion of sphingomyelin as a percentageof total membrane lipid phosphorus is about six times as high in Golgi membranesas it is in ER membranes.In other cases, the movement of membranesfrom one cellular compartment to another can selectivelyenrich certain membranesin lipids such as cholesterol.In responding to differing environments throughout an organism, different types of cells generate membranes with differing lipid compositions. In the cells that line the intestinal tract, for example, the membranes that face the harsh environment in which dietary nutrients are digested have a sphingolipid-to-phosphoglyceride-tocholesterol ratio of 1:1,;1 rather than the 0.5:1.5:1 ratio found in cells subject to less stress.The relatively high concentration of sphingolipid in this intestinal membrane may increaseits stability becauseof extensivehydrogen bonding by the free -OH group in the sphingosine moiety (see F i g u r e1 0 - 5 ) . The degreeof bilayer fluidity dependson the lipid composition, structure of the phospholipid hydrophobic tails, 'Waals and temperature. As aheady noted, van der interactions and the hydrophobic effect causethe nonpolar tails of phospholipids to aggregate.Long, saturated fatty acyl chains have the greatest tendency to aggregate,packing tightly together into a gel-like state. Phospholipids with

10-13Effectof lipid compositionon bilayer < FIGURE (SM)bilayer thicknessand curvature.(a)A puresphingomyelin as phosphoglyceride such from a formed isthickerthanone (PC).Cholesterol effecton hasa lipid-ordering phosphatidylcholine but doesnot theirthickness thatincreases phosphoglyceride bilayers (b)Phospholipids SMbilayer. of the moreordered affectthethickness shapeandformmoreor lessflat suchasPChavea cylindrical headgroupssuchas with smaller those whereas monolayers, (PE)havea conical shape(c)A bilayer phosphatidylethanolamine leafletandwith PEin the with PCin theexoplasmic enriched wouldhavea natural membranes, face,asin manyplasma cytosolic Mol CellBiol Sprongetal curvature ,2001,NatureRev lAdaptedfromH 22504l SM and cholesterol

short fatty acyl chains, which have less surface area and therefore fewer van der'Waals interactions, form more fluid bilayers. Likewise, the kinks in cis- unsaturated fatty acyl chains (Chapter2) result in their forming lessstablevan der Waals interactions with other lipids, and hence more fluid bilayers,than do straight saturatedchains,which can pack more tightly together. Cholesterolis important in maintaining the appropriate fluidity of natural membranes,a property that appearsto be essentialfor normal cell growth and reproduction. Cholesterol restricts the random movement of phospholipid head groups at the outer surfacesof the leaflets, but its effect on the movement of long phospholipid tails depends on concentration.At cholesterolconcentrationspresentin the plasma membrane, the interaction of the steroid ring with the long hydrophobic tails of phospholipidstends to immobilize these lipids and thus decreasebiomembrane fluidity. At lower cholesterolconcentrations,however,the steroid ring separatesand dispersesphospholipid tails, causing the inner regions of the membrane to become slightly more fluid. The lipid composition of a bilayer also influencesits thickness, which in turn may influence the distribution of other membrane components,such as proteins, in a particular membrane.The resultsof biophysical studieson artificial membranesdemonstratethat sphingomyelin associatesinto

a more gel-like and thicker bilayer than phospholipids do (Figure 10-13a). Cholesteroland other moleculesthat decreasemembrane fluidity also increasemembrane thickness. Becausesphingomyelintails are akeady optimally stabilized, the addition of cholesterolhas no effect on the thicknessof a sphingomyelin bilayer. Another property dependenton the lipid composition of a bilayer is its curvature, which dependson the relative sizes of the polar head groups and nonpolar tails of its constituentphospholipids.Lipids with long tails and largehead groups are cylindrical in shape; those with small head groupsare cone shaped(Figure10-13b).As a result,bilayers composed of cylindrical lipids are relatively flat, whereas those containing large numbers of cone-shapedlipids form curved bilayers(Figure 10-13c).This effect of lipid composition on bilayer curvature may play a role in the formation of highly curved membranes,such as sites of viral budding (seeFigure 10-3) and formation of internal vesiclesfrom the plasma membrane (seeFigure 10-9) and in specialized stable membrane structures such as microvilli. Severalproteins bind to the surfaceof phospholipid bilayers and cause the membrane to curve; such proteins are important in formation of transport vesiclesthat bud from a donor membrane (Chapter 14).

Lipid Compositionls Different in the Exoplasmic and CytosolicLeaflets A characteristicof all membranesis an asymmetryin lipid composition across the bilayer. Although most phospholipids are present in both membrane leaflets' they are commonly more abundant in one or the other leaflet. For instance,in plasma membranesfrom human erythrocytes and certain canine kidney cells grown in culture' almost all the sphingomyelin and phosphatidylcholine, both of which form less fluid bilayers, are found in the exoplasmic leaflet. In contrast, phosphatidylethanolamine,phosphatidylserine, and phosphatidylinositol, which form more fluid bilayers, are preferentially located in the cytosolic leaflet. This segregationof lipids acrossthe bilayer

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phosphatidylcholinein the exoplasmic leaflet. Movement of this phosphoglyceride,and perhaps others, from one leaflet to the other in some natural membranesis catalyzedby ATPpowered transport proteins called flippases,which are discussedin Chapter 11. The preferential location of lipids on one face of the bilayer is necessaryfor a variety of membrane-basedfunctions. For example,the head groups of all phosphorylated forms of phosphatidylinositol (see Figure 10-5; PI) face the cytosol. Stimulation of many cell-surfacereceptorsby o _______>| their corresponding hormone results in activation of the c:o cytosolic enzyme phospholipase C, which can then hyI drolyze the bond connecting the phosphoinositolsto the (cH,) -n | diacylglycerol.As we will see in Chapter 15, both water cHg soluble phosphoinositols and membrane-embeddeddiaA FIGURE cylglycerol participate in intracellular signaling pathways 10-14 Specificityof phospholipases. Eachtypeof p h o s p h o l i p acsl e a v eosn eo f t h e s u s c e p t i bbl e that affect many aspects of cellular metabolism. Phoso n d ss h o w ni n r e d T h eg l y c e r ocla r b o na t o m sa r ei n d i c a t ebdy s m a lnl u m b e r sI n phatidylserinealso is normally most abundant in the cyi n t a c ct e l l so, n l yp h o s p h o l i p iidnst h ee x o p l a s ml ieca f l eot f t h e tosolic leaflet of the plasma membrane.In the initial stages plasmm a e m b r a naer ec l e a v ebdy p h o s p h o l i p a si netsh e of platelet stimulation by serum, phosphatidylserine is s u r r o u n d i nmge d i u mP h o s p h o l i p aCs,e a c y t o s o le i cn z y m ec,l e a v e s briefly translocatedto the exoplasmicface, presumably by c e r t a i np h o s p h o l i p iidnst h e c y t o s o l li e c a f l eot f t h e p l a s m a a flippase enzyme,where it activatesenzymesparticiparmembrane ing in blood clotting.

C h o l e s t e r oal n d S p h i n g o l i p i d C s l u s t e rw i t h S p e c i f i cP r o t e i n si n M e m b r a n eM i c r o d o m a i n s may influence membrane curvature (see Figure 10-13c). Unlike particular phospholipids, cholesterol is relatively evenly distributed in both leaflets of cellular membranes. The relative abundanceof a particular phospholipid in the t w o l e a f l e t so f a p l a s m a m e m b r a n e c a n b e d e t e r m i n e d experimentally on the basis of the susceptibilityof phospholipids to hydrolysis by phospholipases,enzymesthat cleavevarious bonds in the hydrophilic ends of phospholipids (Figure 10-14). \7hen added to the external medium, phospholipasescannot cross the membrane, and thus they cleave off the head groups of only those lipids present in the exoplasmic face; phospholipids in the cytosolic leaflet are resistant to hydrolysis becausethe enzymescannot penetrateto the cytosolic face of the plasma memDrane. How the asymmetric distribution of phospholipids in membrane leafletsarisesis still unclear.As noted, in pure bilayers phospholipids do not sponraneouslymigrate, or flipflop, from one leaflet to the other. To a first approximation, the asymmetry in phospholipid distribution results from synthesisof these lipids in the endoplasmic reticulum and Golgi. Sphingomyelinis synthesizedon the luminal (exoplasmic) face of the Golgi, which becomesthe exoplasmic face of the plasma membrane. In contrast, phosphoglycerides are synthesizedon the cytosolic faceof the ER membrane, which is topologically identical with the cytosolic face of the plasma membrane(seeFigure 10-8). Clearly,this explanation does not account for the preferential location of

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Membrane lipids are not randomly distributed (evenly mixed) in eachleaflet of a bilayer.One hint that lipids may be organized within the leaflets was the discovery that the lipids remaining after the extraction of plasma membranes with nonionic detergents predominantly conrain two species:cholesteroland sphingomyelin.Becausethesetwo lipids are found in more ordered, less fluid bilayers, researchers hypothesized that they form microdomains, termed lipid rafts, surrounded by other, more fluid phospholipids that are more readily extracted by detergents. Some biochemical and microscopicevidencesupports the existenceof lipid rafts, which in natural membranesare typically 50 nm in diameter. Rafts can be disrupted by methyl-B-cyclodextrin,which specificallyextracts cholesterol out of membranes,or by antibiotics, such as filipin, that sequestercholesterolinto aggregateswithin the membrane. Such findings indicatethe importance of cholesterol in maintaining the integrity of these rafts. As judged by their presencein the complex of cholesterol and sphingomyelin remaining after detergent extraction, lipid rafts in plasma membranesare thought to be enrichedfor a subset of plasma membraneproteins,including those that participate in sensing extracellular signals and transmitting them into the cytosol. Thus by bringing many key proteins into close proximity these lipid-protein complexes may facilitate signalingby cell-surfacereceptorsand the subsequent activation of cytosolic events. However, much remains to be learned about the structure and bioloeical function of lipid rafts.

Biomembranes:Lipid Composition and Structural Organization r The eukaryotic cell is demarcatedfrom the external environment by the plasma membrane and organized into membrane-limited internal compartments (organellesand vesicles). r The phospholipid bilayer, the basic structural unit of all biomembranes,is a two-dimensional lipid sheet with hydrophilic facesand a hydrophobic core, which is impermeable to water-solublemoleculesand ions (seeFigure 10-6). r The primary lipid components of biomembranesare phosphoglycerides,sphingolipids, and sterols such as cholesterol(seeFigure 10-5). r Most lipids and many proteins are laterally mobile biomembranes. r Membranes can undergo phasetransitions from the fluid to gel-like states depending on the temperature and composition of the membrane. r Different cellular membranesvary in lipid composition (see Table 10-1). Phospholipids and sphingolipids are asymmetricallydistributed in the two leafletsof the bilayer, whereas cholesterol is fairlv evenlv distributed in both leaflets. r Natural biomembranesgenerally have a viscous consistencywith fluidlike properties.In general,membrane fluidity is decreasedby sphingolipidsand cholesteroland increasedby phosphoglycerides.The lipid composition of a membrane also influencesits thickness and curvature (see F i g u r e1 0 - 1 3 ) . r Lipid rafts are microdomains containing cholesterol, sphingolipids,and certain membrane proteins that form in the plane of the bilayer. These aggregatesmight facilitate signaling by certain plasma-membranereceptors.

Protein Biomembranes: and BasicFunctions Components Membrane proteins are defined by their location within or at the surfaceof a phospholipid bilayer.Although every biological membrane has the same basic bilayer structure, the proteins associatedwith a particular membrane are responsible for its distinctive activities. The kinds and amounts of proteins associatedwith biomembranesvary depending on cell type and subcellular location. For example, the inner mitochondrial membrane is 75 percent protein; the myelin membrane that surrounds nerve axons, only 18 percent.The high phospholipid content of myelin allows it to electricallyinsulate the nerve from its environment, as we discussin Chapter 23. The importance of membrane proteins is suggested from the finding that approximately a third of all yeastgenes

encode a membrane protein. The relative abundance of genesfor membrane proteins is greater in multicellular organisms in which membrane proteins have additional functions in cell adhesion. The lipid bilayer presentsa distinctive two-dimensional hydrophobic environment for membrane proteins. Someproteins contain segmentsthat arc imbedded within the hydrophobic core of the phospholipid bilayer; other proteins are associated with the exoplasmic or cytosolic leaflet of the bilayer. Protein domains on the extracellular surface of the plasma membrane generally bind to extracellular molecules, including external signalingproteins, ions, and small metabolites(e.g.,glucose, fatty acids), as well as proteins on other cells or in the external environment. Segmentsof the protein within the plasma membrane have a variety of functions, including those that form channelsand pores in transport proteins that move molecules and ions into and out of cells.Domains lying along the cytosolic face of the plasma membrane have a wide range of functions, from anchoring cytoskeletalproteins to the membraneto triggering intracellular signaling pathways. In many cases,the function of a membrane protein and the topology of its polypeptidechain in the membranecan be predicted on the basis of its similarity with other wellcharacterizedproteins. In this section' we examine the characteristicstructural featuresof membraneproteins and some 'We will describe the structures of of their basic functions. severalproteins to help you get a feel for the way membrane proteins interact with membranes.More completecharacterization of the properties of various types of membrane proteins is presentedin later chapters that focus on their structures and activitiesin the context of their cellular functions.

ProteinsInteractwith Membranes in Three DifferentWaYs Membrane proteins can be classifiedinto three categoriesintegral, lipid-anchored, and peripheral-on the basisof the nature of the membrane-proteininteractions(seeFigure 10-1)' Integral membrane proteins, also called transrnembrane proteins, span a phospholipid bilayer and comprise three segments.The cytosolic and exoplasmic domains have hydrophilic exterior surfaces that interact with the aqueous solutions on the cytosolic and exoplasmic faces of the membrane. These domains resemble segments of other water-solubleproteins in their amino acid composition and structure. In contrast, the membrane-spanning segments usually contain many hydrophobic amino acidswhose side chains protrude outward and interact with the hydrophobic hydrocarbon core of the phospholipid bilayer. In all transmembrane proteins examined to date, the membranespanning domains consist of one or more a helicesor of multiple B strands. Becausethey must be inserted into membranes, the ribosomal synthesisand posttranslational processingof integral membrane proteins differs from that of soluble cytosolic proteins and is discussedseparatelyin Chapters 13 and 1'4.

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Lipid-anchored membraneproteins are bound covalently to one or more lipid molecules.The hydrophobic segmentof the attachedlipid is embeddedin one leaflet of the membrane and anchors the protein to the membrane. The polypeptide chain itself does not enter rhe phospholipid bilayer. Peripheral membrane proteins do not directly contact the hydrophobic core of the phospholipid bilayer. Instead they are bound to the membraneeither indirectly by interactionswith integral or lipid-anchored membraneproteins or directly by interactionswith lipid headgroups.Peripheralproteins can be bound to either the cytosolic or the exoplasmicfaceof the plasmamembrane. In addition to theseproteins, which are closelyassociated with the bilayer,cytoskeletalfilamentscan be more looselyassociated with the cytosolic face, usually through one or more peripheral (adapter) proteins. Such associationswith the cytoskeleton provide support for various cellular membranes, helping to determine cell shapeand mechanicalproperties, and play a role in the two-way communication betweenthe cell interior and the exterior, as we learn in Chapter 17. Finally, peripheral proteins on the outer surfaceof the plasma membrane and the exoplasmicdomainsof integralrn.rntr"n. proreinsare often anached to components of the extracellular matrix or to the cell wall surrounding bacterial and plant cells, providing a crucial interface betlveenthe cell and its environmenr.

Most Transmembrane ProteinsHave M e m b r a n e - S p a n n i nagH e l i c e s Solubleproteins exhibit hundreds of distinct localizedfolded structures, or motifs (see Figure 3-9). In comparison, the repertoireof folded structuresin the transmembranedomains of integral membrane proteins is quite limited, with the hydrophobic ct helix predominating. Proteinscontaining membrane-spanning a-helical domains are stably embedded in membranes becauseof energeticallyfavorable hydrophobic and van der rWaalsinteractionsof the hydrophobic sidechains in the domain with specificlipids and probably also by ionic interactionswith the polar head groups of the phospholipids. A single cr-helical domain is sufficient to incorporate an integral membrane protein into a membrane. However, many proteins have more than one transmembranecr helix. Typically, a membrane-embeddedcr helix is composed of a continuous segment of 20-25 hydrophobic (uncharged) amino acids (seeFigure 2-14).The predictedlength of such an ct helix (3.75 nm) is just sufficient to span the hydrocarbon core of a phospholipid bilayer. In many membrane proteins, these helices are perpendicular to the plane of the membrane, whereas in others, the helicesrraversethe membrane at an oblique angle.The hydrophobic side chains protrude outward from the helix (Figure 10,15) and form van (bI

(a)

Extracellu lar domain

M e mb r an e - s p a nning helices

Cytosolic domain

A FIGURE10-15 Structureof glycophorin A, a typical singlepasstransmembrane protein. (a)Diagramof dimericglycophorin showingmajorsequencefeaturesand its relationto the membrane The single23-residue membrane-spanning o helixin eachmonomeris composedof amino acidswith hydrophobic (uncharged) sidechains (redand greenspheres). By bindingnegatively chargedphospholipid headgroups,the positively chargedarginlneand lysineresidues (blue spheres) nearthe cytosolicsideof the helixhelpanchorglycophorin in the membraneBoththe extracellular and the cytosolicdomains a r e r i c h i n c h a r g e dr e s i d u eas n d p o l a ru n c h a r q e dr e s i d u e st h :e

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extracellular domainisheavily glycosylated, withthecarbohydrate (green sidechains diamonds) attached to specific serine, threonine, (b)Molecular andasparagine residues modelof thetransmemprane domainof dimeric glycophorin corresponding to resrdues 73-96.The hydrophobic sidechains of theo helixin onemonomer aresnownIn pink;thosein theothermonomer, in greenResidues depicted as space-filling participate structures in intermonomer vanderWaals interactions thatstabilize (b)adapted thecoiled-coil dimer[part from K R M a c K e n z i e e t a l ,1 9 9 7 ,S c i e n c e 2 T 6 : 1 3I1

H a l fh e l i c e s

(b) Back

Exterior

Membrane

Cytosol

10-16Structuralmodelsof two multipassmembrane FIGURE in certainbacteria proteins.(a)Bacteriorhodopsin, a photoreceptor the traverse in bacteriorhodopsin hydrophobic crhelices Theseven A perpendicular to the planeof the membrane lipidbilayer roughly (black) light to onehelixabsorbs attached covalently retinalmolecule in eukaryotic cellsalso receptors of G protein-coupled Thelargeclass theirthree-dimensional crhelices; hassevenmembrane-spanning (b)Two to thatof bacteriorhodopsin. isthoughtto besrmilar structure to each channel Glpf,rotated180"with respect viewsof theglycerol Note to the planeof the membrane. otheralongan axisperpendicular

the thatareat obliqueangles, o helices membrane-spanning several membrane the through onlyhalfway thatpenetrate two helices helix andonelongmembrane-spanning (purple withyellowarrows), withyellowline)' in the middle(purple witha "break"or distortion "core"iscolored red The in the hydrophilic molecule Theglycerol coreof the positioned in the hydrocarbon wasapproximately structure protein the of 3 mm slab most hydrophobic byfindingthe membrane (a)AfterH Luecke etal, plane.lPart to the membrane perpendicular

der.il/aals interactions with the fatty acyl chains in the bilayer.In conrrast,the hydrophilic amide peptide bonds are in the interior of the cr helix (seeFigure :-4;; e".h carbonyl (C:O) group forms a hydrogen bond with the amide hythe amino acid four residuestoward the Cdrogen "-to--of helix. These polar groups are shieldedfrom terminus of the the hydrophobic interior of tLe membrane. To help you get a better senseof the structuresof proteins with a-helical domains,we will briefly discussthree Jifferent kinds of such proteins: glycophorin A, G protein-coupled receptors,a^d aqrr"pori.i(*"i.riglycerol channels). blycophorin- A, the major p-rotein in the erythrocyte plasma membrane, is a ..pr.r.., itive single-passtiansmembraneprotein, which containsonly one membrane-spanning cr helix (Figure10-15).The transmembranehelix of one glycophorin A polypeptide associateswith the corresponding transmembrane helix in a second glycophorin A to form a coiled-coildimer (Figure10-15b).Suchinteractionsof membrane-spanningct helicesare a common mechanismfor creating dimeric membraneproteins, and many membraneproteins form oligomers (two or more polypeptides bound together noncovalently) by interactions between their memb r a n e - s p a ni n g h e l i c e s . A large and important group of integralproteinsis defined a helices;this inby the presenceof sevenmembrane-spanning cludes the large family of G protein-coupled cell-surfacereceptors discussedin Chapter 15. One such mwbipasstransmembrane protein whose structure is known in molecular

detail is bacteriorhodopsin' a protein found -in the membrane of certain photosynthetic bacteria; it illustrates the generalstructureof all theseproteins (Figure-10-16a)'Abiorption of light by the retinal group covalently attached to this protein causesa conformational change in the protein that results in the pumping of protons from-the cytosol across the bacterial membrane to the extracellular space' The proton concentration gradient thus generatedacrossthe membrane is used to synthesizeATP (Chapter 1'2)' In the high-resolution structure of bacteriorhodopsinthe positions of all the individual amino acids, retinal, and the surrounding lipids are clearly defined. As might be expected,virtually ali oi the amino acids on the exterior of the membrane-

1ggg,J Mol. Biol 291:899 Part(b) after J Bowie,20O5, Nature438:58'l-589, and D Fu et al , 2000, Sclence290:481-486.1

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the middle, and more strikingly, there are two c helixes that penetrate only halfway through rhe membrane. The N-termini of these helicesface each other (yellow N's in the figure), and together they span the membrane at an oblique angle. Thus some membrane-embeddedhelicesand other nonhelical structureswe will encounter laterdo not traversethe entire bilayer. As we will seein Chapter 1.1,these short helixes in aquaporins form part of the glycerol/water-selectivepore in the middle of each subunit. This highlights the considerablediversity in the ways membrane-spanningcr helices interact with the lipid bilayer and with other segmentsof the protein. The specificity of phospholipid-protein interactions is revealedin great detail in the structure of a different aquaporin, aquaporin 0 (Figure 10-17). Aquaporin 0 is the most abundant protein in the plasma membrane of the fiber cells that make up the bulk of the lens of the mammalian eye. Like orher aquaporins,it is a tetramer of identical subunits.The protein's surfaceis not coveredby a set of uniform binding sites for phospholipid molecules.Instead, fatty acyl side chains pack tightly against the

Podcast: AnnularPhospholipiO, $|

Membrane

FIGURE 10-17Annularphospholipids. Sideviewof the three-dimensional structure of onesubunitof the lens-specific aquaporin 0 homotetramer, crystallized in the presence of the phospholipid dimyristoylphosphatidylcholine, a phospholipid with 14 carbon-saturated fattyacylchainsNotethe lipidmorecures forminga bilayer shellaroundthe proteinTheproteinisshownas a surface plot(thelighterbackground molecule). Thelipid molecules areshownin space-fill format;the polarlipidhead groups(greyandred)andthe lipidfattyacylchains(blackand grey)forma bilayer with almostuniformthrckness aroundthe protein. Presumably, in the membrane, lipidfattyacylchains will coverthewholeof the hydrophobic surface of the protein; only the mostordered of the lipidmolecules will be resolved in the crystallographic structure[AfterA Lee,2005,Nature 438:569_570. and T. Gonen et al , 2005, Nature 439:633-688 l

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irregular hydrophobic outer surface of the proteinl some of the fatty acyl chains are straight, in the all-trans conformation (Chapter 2), whereas others are kinked in order to interact with bulky hydrophilic side chains on rhe surface of the protein. Some of the lipid head groups are parallel to the surface of the membrane, as is the casein purified phospholipid bilayers. Others, however, are oriented almost at right anglesto the plane of the membrane. Thus there can be specific interactions between phospholipids and membrane-spanning proteins, and the function of many membrane proteins can be affected by the specific types of phospholipid presentin the bilayer.

M u l t i p l e p S t r a n d si n P o r i n sF o r m M e m b r a n e - S p a n n i n" gB a r r e l s " The porins are a class of transmembrane proteins whose structurediffersradicallyfrom that of other integralproreins basedon a-helical transmembranedomains. Severaltypes of porins are found in the outer membrane of gram-negative bacteria such as E. coli and in the outer membranesof mitochondria and chloroplasts.The outer membrane prorecrsan intestinal bacterium from harmful agents (e.g., antibiotics, bile salts,and proteases)but permits the uptake and disposal of small hydrophilic molecules, including nutrients and waste products. Different types of porins in the outer membrane of an E. coli cell provide channelsfor the passageof specific types of disaccharidesor other small molecules as well as of ions such as phosphate.The amino acid sequences of porins contain none of the long hydrophobic segments typical of integral proteins with o-helical membrane-spanning domains. X-ray crystallography has revealed that porins are trimers of identical subunits. In each subunit, 16 B strands form a sheet that twists into a barrel-shaped structurewith a pore in rhe center (Figure 10-18). Unlike a typical water-solubleglobular protein, a porin has a hydrophilic interior and a hydrophobic exrerior; in this sense. porins are inside out. In a porin monomer, the outwardfacing side groups on each of the B strands are hydrophobic and form a nonpolar ribbonlike band that encirclesthe outside of the barrel. This hydrophobic band interacts with the fatty acyl groups of the membrane lipids or with other porin monomers. The side groups facing the inside of a iorin monomer are predominantly hydrophilic; they line the pore through which small water-solublemoleculescrossthe membrane. (Note that the aquaporins discussedabove, despite their name, are not porins and contain multiple trurrrr.ri-braneo. helices.)

CovalentlyAttached HydrocarbonChains A n c h o rS o m eP r o t e i n st o M e m b r a n e s In eukaryotic cells,severaltypes ofcovalently attachedlipids anchor some otherwise typically water soluble proteins to one or the other leaflet of the plasma membraneor other cellular membranes.In theselipid-anchored proteins, the lipid

group is bound through a thioether bond to the -SH group of a b-terminal cysteineresidue.In somecases'a secondgeranylgeranylgroup or a fatty acyl palmitate group is linked to a nearby cysteine residue. The additional hydrocarbon anchor is thought to reinforce the attachment of the protein to the membrane. For example, Ras' a GTPase superfamily protein that functions in intracellular signaling (Chapter 16), is recruited to the cytosolic face of the plasma membrane by such a double anchor.Rab proteins' which also belong to the GTPase superfamily, are similarly bound to the cytosolic surface of intracellular vesicles by prenyl anchors; these proteins are required for the fusion of vesicleswith their target membranes(ChaPtet 1"4). Some cell-surface proteins and specialized proteins with distinctivecovalently attachedpolysaccharidescalled

Periplasm

10-18Structuralmodelof one subunitof OmpX' A FIGURE a porin found in the outer membraneof E. coli' All porinsare t r i m e r itcr a n s m e m b r apnreo t e i n sE a c hs u b u n iitsb a r r esl h a p e d , w i t h B s t r a n dfso r m i n gt h ew a l la n da t r a n s m e m b r apnoer ei n t h e c e n t e rA b a n do f a l i p h a t i(ch y d r o p h o bainc dn o n c y c l isci)d ec h a r n s (ring-containing) (yellow) sidechains(red) anda borderof aromatic positionthe oroteinin the bilaver. Curr. Opin lAfterG E Schulz,20O0, StrucBiol 10:443|

Exterior

Cytosol

hydrocarbon chains are embedded in the bilayer' but the protein itself does not enter the bilayer. The anchors used to insert proteins at the cytosolic face are not used for the exoplasmicface and vice versa. One group of cytosolicproteins are anchoredto the cytosolic face of a membrane by a fatty acyl group (e.g., myristate or palmitate) covalently attachedto an N-terminal glycine residue(Figure 10-t9a). Retention of such proteins at the membrane by the N-terminal acyl anchor, called acylation, may play an important role in a membraneassociatedfunction. For example,v-Src, a mutant form of a cellular tyrosine kinase, induces abnormal cellular growth that can lead to cancer but only when it has a myristylatedN-terminus. A second group of cytosolic proteins are anchored to membranes by a hydrocarbon chain attached to a cysteine residueat or near the C-terminus(Figure 10-19b). Someof these chains arc prenyl anchors, built from S-carbon isoprene units, which, as detailed in the following section, are also used in the synthesisof cholesterol. In these proteins, a 1S-carbon farnesyl or 20-carbon geranylgeranyl

NHst

(a)Acylation

(b)Prenylation

proteins 10-19Anchoringof plasma-membrane A FIGURE groups' hydrocarbon linked covalently by to the bilayer with the plasma proteins (a)Cytosolic suchasv-Srcareassociated to the througha singlefattyacylchainattached membrane

(c)Thelipidanchoron the groups, bothof whichareunsaturated. isglycosylphosplasma membrane of the surface exoplasmic part(red)of this (GPl). Thephosphatidylinositol phatidylinositol The thatextendintothe bilayer. two fattyacylchains anchoicontains it to the links (purple) the anchor in unit phosphoethanolamine sugarunits,whichvaryin represent protein. Thetwo greenhexagons GPIanchorsThe in different nature,andarrangement number, of a yeastGPIanchorisshownin Figure13-14 structure complete M o l C e l lB i o l 2 : 5 0 4 1 l A d a p t e d f r o m H S p r o n g e t a l, 2 0 0 1 , N a t u r e R e v

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proteoglycans(Chapter 19) are bound to the exoplasmic face of the plasma membrane by a third type of anchor group, glycosylphosphatidylinositol (GpI). The exact structures of GPI anchors vary gready in different cell

L i p i do r protein

A antigen

L i p i do r p rotein

O antigen

L i p i do r protein

B antigen

(Figure 10-19c). Therefore GpI anchors are glycolipids. The GPI anchor is both necessaryand sufficient for bind-

proteinsand Glycolipids All Transmembrane Are AsymmetricallyOrientedin the Bilayer FIGURE 10-20 HumanABOblood group antigens.These antigens areoligosaccharide chains covalently attached to glycolipids or glycoproteins in the plasma membrane. Theterminaloligosaccharide sugars distinguish thethreeantigens. Thepresence or absence of the glycosyltransferases (Gal)or N-acetylgalactosamine thataddgalactose (GalNAc) to O antigendetermine a person's bloodtype faces.The orientation of different types of transmembrane proteinsis establishedduring their synthesis,as we describe in Chapter 13. Membrane proteins have never been observed to flip-flop across a membrane; such movemenr, requiring a transient movement of hydrophilic amino acid residues through the hydrophobic inteiior of rhe membrane, would be energeticallyunfavorable. Accordingly, the asymmetric topology of a transmembraneprotein, which is established during its biosynthetic insertion lnro a mem_ brane, is maintained throughout the protein's lifetime. As

drial membrane,chloroplastlamellae,and severalother in_ tracellular membranes.Becausethe carbohydratechains of glycoproteins and glycolipids in the plasma membrane 426

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extend into the extracellular space,they are available to interact with components of the extracellular matrix as well as lectins (proteins that bind specific sugars), growth factors. and antibodies. One important consequenceof such interactions is illustrated by the A, B, and O blood group antigens.These three structurally related oligosaccharidecomponents of certain glycoproteinsand glycolipids are expressedon the surfaces of human red blood cells and many other cell types (Figure 10-20). AII humans have the enzymes for synthesizingO antigen. Personswith type A blood also have a glycosyltransferaseenzyme that adds an extra modified monosaccharide called N-acetylgalactosamine to O antigen to form A antigen. Those with type B blood have a different transferasethat adds an extra galactoseto O antigen to form B antigen. People with both transferasesproduce both A and B antigen (AB blood type); those who lack thesetransferasesproduce O antigen only (O blood type). Peoplewhose erythrocyteslack the A antigen, the B antigen, or both on their surface normally have antibodies against the missing antigen(s)in their serum. Thus if a type A or O person receivesa transfusion of type B blood, antibodies against the B antigen will bind to the introduced red cells and trigger their destruction. To prevent such harmful reactions, blood group typing and appropriate matching of blood donors and recipients are required in all transfusi,ons ( T a b l e1 0 - 2 ) .

GRI)UP BI()OD

TYPES BTtlOl) RECEIVE CAN

ANTIBODIES SERUM

ONRBCSANTIGTNS

A

A

Anti-B

A and O

ts,

B

Anti-A

B and O

AB

A and B

None

All

O

O

Anti-A and anti-B

O

"See Figure 10-20 for antigen structures.

M o t i f s H e l pT a r g e tP e r i p h e r a l Lipid-Binding Proteinsto the Membrane Many water-soluble enzymesuse membrane phospholipids as their substratesand thus must bind to membranesurfaces' As exemplified by the phospholipases,many such enzymes initially bind to the polar head groups of membrane phospholipids to carry out their catalytic functions. As noted eariier, phospholipaseshydrolyze various bonds in the head groups of phospholipids (seeFigure 1.0-1'4).These enzymes have an important role in the degradation of damaged or aged cell membranes and also are active components in many snake venoms. The mechanismof action of phospholipase 42 illustrates how such water-solubleenzymescan reversibly interact with membranes and catalyzereactions at the interface of an aqueoussolution and lipid surface (interfacial chemistry).'Whenthis enzymeis in aqueoussolution, its Ca2*-containing active site is buried in a channel lined

Active site

(b)

FIGURE10-21 Interfacial binding surface and mechanism

the catalyticsite (Figure10-21b).

P r o t e i n sC a n B e R e m o v e df r o m M e m b r a n e s t olutions b y D e t e r g e n t so r H i g h - S a l S

hydrophobic part of a detergentmolecule is attracted to

acidsthat change,openinga channellinedwith hydrophobicamino moves phospholipid a As leadsfrom the bilayerto the catalyticsite the (green) to binds ion Ca2* into the channel,an enzyme-bound (red) to next cleaved be to bond ester the positioning headgroup, Curr'Opin the catalyticsite. lPart(a)adaptedfrom M H Gelbet al ' 1999' StrucBiol 9:428 Parl(b),seeD Blow,1991,Nature351:4441

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I O N I CD E T E R G E N T S H"C -t

o

H C - C H 2 - C H z- C O O - N a +

ll

H 3 C - ( C H 2 ) 1 1- O - S - O - N a +

o Sodium deoxycholate

Sodium dodecylsulfate (SDSI

N O N I O N I CD E T E R G E N T S

HsQ

1l H3c-c-cH2-?1 HsC

o-(cH2)7-CH3

9Hs

CHs

/o_rcnz_cH2_o)e5_H (Average)

(potyoxyet nyrrr lirlS i, Il lll-

pheno|) ""r'

A FIGURE 10-22 Structuresof four commondetergents.The hydrophobic partof eachmolecule isshownin yellow;the hydrophilic part,in blue Thebilesaltsodiumdeoxvcholate isa

water (Figure 10-22).Ionic detergents,such as sodium de_ oxycholate and sodium dodecylsulfate(SDS), contain a charged group; nonionic detergents,such as Triton X_100 and octylglucoside,lack a charged group. At very low con_ centrations, detergentsdissolve in pure water as isolated molecules.As the concentration incriases,the moleculesbe_ gin to form micelles-small. spherical aggregatesin which hydrophilic parts of the molecule, f".e Lut*ard and the hydrophobic parts clusrerin rhe center (seeFigure 10_6(c)). The critical micelle concentration (CMC) at which micelles form is characteristicof each detergentand is a function of the structuresof its hydrophobic and hydrophilic parts. Ionic detergents bind to the exposed hydrofhobi. ,._ gions of membrane proteins as well as to the hvdrophobic cores of water-solubleproteins. Becauseof rheir .iurg., these detergentsalso disrupt ionic and hydrogen borrds.Ai high concenrrations,for example, sodium Jodecylsulfate completely denatures proteins by binding to every side chain, a properry that is exploited in SDSgel electrophoresis (seeFigure 3-35). Nonionic detergenlsgenerally do not de_ nature proteins and are thus useful in extracting proteins from membranesbefore the proteins are purified.-Tirese de_ tergentsact in different ways at different concentrations. At high concentrations (above the CMC), they solubilize bio_ logical membranesby forming mixed micelies of detergent, phospholipid, and integral membrane proteins (Figure t0_ 23). At low concentrarions (below the CMC). these deter_ gents bind to the hydrophobic regions of most integral mem_ brane proteins, making them soluble in aqueoussolution. Treatmenr of cultured cells with a buffered salt solution containing a nonionic detergentsuch as Triton X_100 extracts water-solubleproteins as well as integral membraneprotelns. As noted earlier, the exoplasmic and cytosolic domains of integral membrane proteins g.rr.."ily hydrophilic and "r. 428 . cHAprE1 R0 | B T o M E M B R A s rNREu c r u R E

Octylglucoside (octyl-p-D-glucopyranoside) naturalproduct; theothersaresynthetic. Althoughionicdetergents commonly causedenaturation of proteins, nonionic detergents do not andarethususefulin solubilizing integral proteins membrane

soluble in water. The membrane-spanningdomains, how_ ever, are rich in hydrophobic and uncharged residues (see Figure 10-15). When separated from membranes, these exposed hydrophobic segmenrstend to interact with one

proteins can rhen be purified by affinity chromatography and other techniques used in purifying water-soluble pro_ teins (Chapter3). As discussedpreviously, most peripheral proteins are . bound to specific transmembraneproieins or membrane phospholipids by ionic or other weak interactions. Gener_ ally, peripheral proteins can be removed from the membrane by solutions of high ionic strength (high salt concentrations), which disrupt ionic bonds, or by chemicalsthat bind divaleni cations such as Mgt*. Unlike integral proteins, most periph_ eral proteins are soluble in aqueoussolution ,r..J rroi be "rrd solubilized by nonionic detergents.

Biomembranes:Protein Components and BasicFunctions r Biological membranes usually contain both integral (transmembrane)proteins and peripheral membrarr. p.o_ teins,which do not enter the hydrophobic core of the bilaver ( s e eF i g u r el 0 - I ) . r Most integral membrane proteins contain one or more membrane-spanninghydrophobic a helices bracketed by

of integral 10-23Solubilization < FIGURE membraneproteinsbY nonionic higherthan detergents.At a concentration (CMC),a concentration micelle itscritical lipidsandintegral solubilizes detergent proteins, formingmixedmicelles membrane protern, andlipid detergent, containing belowthe CMC, At concentrations molecules (eg., octylglucoside, detergents nonionic proteins membrane TritonX-100)candissolve the by coating withoutformingmicelles regions membrane-spanning

Concentration above CMC

Dissolved but not forming micelles

hydrophilic domains that extend into the aqueous solutions surrounding the cytosolic and exoplasmicfacesof the membrane(seeFigures10-15 and 1,0-16).

One focus will be on fatty acids, the precursors of the phos-

r Fatty acyl side chains as well as the polar headsof membrane lipids groups pack tightly and irregularly around the hydrophobic segmentsof integral membrane proteins. r All transmembraneproteins and glycolipids are asymmetrically oriented in the bilayer; invariably carbohydrate chains are attachedto the exoplasmicsurfaceof the proteln. r The porins, unlike other integral proteins, contain membrane-spanningB sheetsthat form a barrel-likechannel through the bilayer (seeFigure 10-18). r Long-chain lipids attachedto certain amino acids anchor some proteins to one or the other membrane leaflet (see F i g u r e1 0 - 1 9 ) . r The binding of a water-solubleenzyme (e.g.,a phospholipase, kinase, or phosphatase)to a membrane surface brings the enzyme close to its substrateand in some cases activatesit. Such interfacial binding is often due to the attraction between positive chargeson basic residuesin the protein and negativechargeson phospholipid head groups in the bilayer. r Transmembraneproteins are selectivelysolubilized and purified with the use of nonionic detergents.

SPhingoliPids, Phospholipids,

Synthesis and Cholesterol: Movement and Intracellular In this section,we considersome of the specialchallenges that a cell faces in synthesizingand transporting lipids, which are poorly soluble in the aqueousinterior of cells.

helps controls calcium metabolism; and other biologically activelipids.

o H 3 C- ( C H 2 ) nc- - o - c H 2

ol H3C-(CH2)"-c-o-cH

?l H3C-(CH2),-c-o-cH2 Triacylglycerol

Sfe focus our discussion of lipid biosynthesis and movement on the major lipids found in cellular membranes and their precursors.In lipid biosynthesis'watersoluble precursors are assembled into membraneintermediates that are then converted into "sroci"tei lipid products. The movement of lipids, espemembrane cially membtuni .o-ponents' between different org"n.il., is critical for maintaining the proper composition i.rd p.op..ties of membranes and overall cell structure, b.rt ou, understandingof such intracellular lipid transport is still rudimentarY' A fundamental principle of membrane biosynthesis is that cells synthesizenew membranes only by the expansion of existing membranes. Although some early steps in the

MOVEMENT SS P,H I N G O L I P I DASN,D C H O L E S T E R OSLY: N T H E S IASN D I N T R A C E L L U L A R PHOSPHOLIPID

429

synthesisof membrane lipids take place in the cytoplasm, the final steps are catalyzedby enzymesbound to preexisting cellular membranes,and the products are incoiporated into the membranesas they are generated.Evidencefor this phenomenon is seen when cells are briefly exposed to radioactiveprecursors(e.g.,phosphateor fatty all the "iidr;, phospholipids and sphingolipids incorporating these precursor substancesare associatedwith intracellular mem_

FIGURE 10-24 Bindingof a fatty acidto the hydrophobic pocketof a fatty-acid-binding protein(FABP). Thecrystal structure of adipocyte FABp(ribbondiagram) reveals that the hydrophobic bindingpocketisgenerated fromtwo B sheets that a r en e a r l a y t r i g h ta n g l e tso e a c ho t h e l f o r m i n ga c l a m - s h e l l - l i k e structureA fattyacid(carbons yellow;oxygens red)interacts noncovalently with hydrophobic aminoacidresidues withinthis

membranes.

Fatty Acids Synthesists Mediated by Severallmportant Enzymes

pocket. [SeeA Reese-Wagoner et al , j999, Biochim Biophys Acta 23:144 1(2-3). 106-1 16 l

urated and unsaturatedchains. Fatty acids are synthesizedfrom the two-carbon building .. block acetate,CH3COO-. In cells,both acetateand the in_ termediates in fatty acid biosynthesis are esterified to the large water-solublemoleculecoenzymeA (CoA), as exemplified by the srructure of acetyl CoA:

SmallCytosolicProteinsFacilitate Movement of Fatty Acids In order to be transported through the cell cytoplasm free, or unesterified,fatty acids (those unlinked to a CoA). com_ monly are bound by fatty-acid-binding proteins (FABps),

CoenzymeA (CoA)

Acetyl CoA is an important intermediatein the metabolism of glucose,fatty acids,and many amino acids,as detailed in Chapter 12. It also contributes acetyl groups in many biosynthetic pathways. Saturated fatty' aC-ids lno double bonds) containing 14 or 16 carbon atoms are made from acetyl CoA by two enzymes,acetyl-CoA carboxylase and fatty acid synthase.In animal cells,theseenzymesare found in the plants, they are found in chloroplasts.palmitoyl 7t9sof;.in CoA (16 carbon fatty acylgroup linked to C-oe)can be elon_ gated to 18-24 carbons by the sequential addition of two_ carbon units in the endoplasmicreticulum (ER) or sometimes in the mitochondrion. Desaturaseenzymes,also locatedin the ER, introduce double bonds at specificpositionsin somefatty acids, yielding unsaturated fatty acidi. Oleyl CoA (oleat! linked to CoA, see Table 2-4), for example,is formed by re_ moval of two H atoms from stearyl CoA. In contrast to free fatty.acids, fatty acyl CoA derivativesare soluble in aqueous solutions becauseof the hydrophilicity of the CoA sesment. 430

.

cHAprER to I

BToMEMBRA s rNREU c r u R E

which belong to a group of small cytosolic proteinsthat facilitate the intracellular movement of many Iipids.These proteins contain a hydrophobic pocket lined by B sheets (Figure 10-24). A long-chain fatty acid can fit into this pocket and interact noncovalently with the surrounding protetn.

I

con \,

?

- *6-5-CoA

F a t t Y a c YG l oA

CDP-choline

+

c-@@choline

H'"tCl --C H

,

OH OH

Glycerol phosphate Phosphatidic acid

z;'

2CoA<

Diacylglycerol

CMP

c--@ Choline

OH

.;

Cytosol

2.) >!Y

ER membrane

Lumen

E @ o-g x;:

GPAT LPAAT (acvltransferases) Choline

phospholipids Because synthesis. 10-25Phospholipid A FIGURE synthesis of theirmultistep molecules, the laststages areamphipathic and andthe cytosol a membrane between takeplaceat the interface enzymesStep([): Twofatty by membrane-associated arecatalyzed to the phosphorylated acidsfromfattyacylCoAareesterified leaflet. formedto theexoPlasmic acld,whosetwo long formingphosphatidic glycerol backbone, Step(Z): to the membrane. chains anchorthe molecule hydrocarbon

Incorporationof Fatty Acids into Membrane L i p i d sT a k e sP l a c eo n O r g a n e l l eM e m b r a n e s Fatty acids are not directly incorporated into phospholipids; rather, in eukaryotic cells they are first converted into CoA esters.The subsequentsynthesisof many diacyl glycetophospholipids from fatty acyl CoAs, glycerol 3-phosphate,and polar head-groupprecursorsis carried out by enzymesassociated with the cytosolic face of the ER membrane,usually the smooth ER, in animal cells(Figure10-25);the ER is described in detail in Chapters9 and L3. Mitochondria synthesizesome of their own membranelipids and import others. Sphingolipidsare derivativesof sphingosine,an amino alcohol that containsa long, unsaturatedhydrocarbonchain (Figure 10-5). Sphingosineis made in the ER, beginningwith the coupling of a palmitoyl group from palmitoyl CoA to serine; the subsequentaddition of a second fatty acyl group to form N-acyl sphingosine(ceramide)also takesplace in the ER. The later addition of a polar head group to ceramidein the Golgi yieldssphingomyelin, whose head group is phosphorylcholine, and various glycosphingolipids, in which the head group may be a monosaccharideor a more complex oligosaccharide(see Figure 10-5b). Somesphingolipidsynthesiscan also take place in mitochondria. In addition to serving as the backbone for sphingolipids, ceramide and its metabolic products are important signaling molecules that can influence cell growth' to stress,and apoptosis. resistance proliferation,endocytosis, After their synthesisis completed in the Golgi, sphingolipids are transported to other cellular compartments

through vesicle-mediatedmechanismssimilar to those dis.orr.J in Chapter 14. In contrast,phospholipids,as well as cholesterol,can move betweenorganellesby different mechanisms,describedbelow.

F l i p p a s eM s o v e P h o s p h o l i p i dfsr o m O n e MembraneLeafletto the OppositeLeaflet Even though phospholipids are initially incorporated into the cytosolic leaflei of the ER membrane' various phospholipids are asymmetrically distributed in the two leaflets of tlie ER membrane and of other cellular membranes' As noted above, phospholipids spontaneouslyflip-flop from one leaflet to the other only very slowly' For the ER membrane to expand by growth of both leaflets and have asymmetrically distributed phospholipids, its phospholipid components must be able to rapidly and selectivelyflip-flop from tne membraneleaflet to the other. Although the mechanisms

leafletto the other (seeFigure 1'1'-1'6). The usual asymmetric distribution of phospholipids in membrane leaflets is broken down as cells (e'g', red blood cells) become senescentor undergo apoptosis' For instance, phosphatidylserineand phosphatidylethanolamineare prefer..rtl"ity locatedin the cytosolic leaflet of cellular membranes'

SN D I N T R A C E L L U L AMRO V E M E N T : N T H E S IA , D C H O L E S T E R OSLY P H O S P H O L I P I DSSP, H I N G O L I P I DASN

O

431

are high, binding of cholesterolto this domain causesthe protein to bind to two other integral ER membrane proteins, Insig-1 and Insig-2. This in turn induces ubiquitinat i o n ( s e e F i g u r e 3 - 2 9 ) o f H M G - C o A r e d u c t a s ea n d i t s degradationby the proteosomepathway, reducingthe production of mevalonate,the key intermediatein cholesterol biosynthesis.

Cholesterolls Synthesizedby Enzymes i n t h e C y t o s o la n d E RM e m b r a n e Next we focus on cholesterol,the principal sterol in animal cells.The first stepsof cholesterolsynthesis(Figure 10-26)-

protein, even though both its subsrrateand its product are water soluble.The water-solublecatalyticdomain of HMG_

Atherosclerosis,frequently calledcholesterol-dependent clogging of the arteries, is characterized by the progressivedeposition of cholesteroland other lipids. cells. and e x t r a c e l l u l am r a r r i x m a t e r i a li n t h e i n n e rl a y e i o f r h e w a l l o f an artery. The resulting distortion of the artery's wall can Iead, either alone or in combination with a blood clot, ro major blockage of blood flow. Atherosclerosisaccounts for 75 percent of deaths due to cardiovascular diseasein the United States. Cholesterolis synthesizedmainly in the liver. perhaps . the most successfulanti-atherosclerosismedications aie the statins. Thesedrugs bind to HMG-CoA reductaseand directly inhibit its activity, thereby lowering cholesterol biosynthesis.As a consequence, the amount of low-density lipoproteins (see Figure 14-27)-the small, membraneenvelopedparticles containing cholesterol esterifiedto fatty

Acetyl CoA * acetoacetylCoA S-CoA

II

J

-o/'-AAs-coa

HMG-CoA ,HMG-CoA reductase J Mevalonate

J J OPP

lsoPentenyladenosine lsopentenylpyrophosphat"'z ' M a n y o t h e ri s o p r e n o i d s (lPP)

Squalene

V i t a m i nD * - B i l ea c i d s CholesterolI Steroid hormones ' Cholesterolesters M o d i f i e dp r o t e i n s( H e d g e h o g )

HO

FIGURE 10-26Cholesterol biosyntheticpathway.The regulated rate-controlling stepin cholesterol biosynthesis isthe conversion of B-hydroxy-B-methylglutaryl CoA(HMG_CoA) into mevalonic acidby HMG-CoA reductase, an ER_membrane protein. Mevalonate isthenconverted intoisopentenyl pyrophosphate (lpp), 432

CHAPTER 10

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B T o M E M B R A NSET R U C T U R E

which hasthe basicfive-carbonisoprenoidstructurelpp can be convertedinto cholesterol and into manyother lipids,often through the polyisoprenoid intermedlates shown here.Someof the numerous compoundsderivedfrom isoprenoidintermediates and cholesterol itselfare indicated.

acids that often and rightly are called "bad cholestslol"drops in the blood, reducing the formation of atherosclerotic plaques.I Mevalonate,the 6-carbon product formed by HMG-CoA reductase,is convertedin severalstepsinto the 5-carbon isoprenoid compound isopentenylpyrophosphate (IPP) and its stereoisomer,dimethylallyl pyrophosphate (DMPP) (see Figure 70-26). Thesereactionsare catalyzedby cytosolic enzymes, as are the subsequentreactionsin the cholesterol synthesispathway, in which six IPP units condenseto yield squalene,a branched-chain30-carbon intermediate.Enzymes bound to the ER membrane catalyzethe multiple reactions that convert squaleneinto cholesterol in mammals or into related sterols in other species.One of the intermediatesin this pathway, farnesylpyrophosphate,is the precursor of the prenyl lipid that anchors Ras and related proteins to the cytosolicsurfaceof the plasmamembrane(seeFigure10-19) as well as other important biomolecules(seeFigure 10-26\.

C h o l e s t e r oal n d P h o s p h o l i p i dAs r e T r a n s p o r t e d l echanisms B e t w e e nO r g a n e l l e sb y S e v e r a M As already noted, the final stepsin the synthesisof cholesterol and phospholipids take place primarily in the ER, are although some of thesemembranelipids (plasmalogens) produced in mitochondria and peroxisomes. Thus the plasma membraneand the membranesbounding other organellesmust obtain theselipids by meansof one or more inMembranelipids accompany tracellulartransportprocesses. both soluble and membrane proteins during the secretory pathway describedin Chapter 14; membranevesiclesbud from the ER and fuse with membranesin the Golgi complex, and other membrane vesiclesbud from the Golgi complex and fusewith the plasmamembrane(Figure10-27a).However,severallines of evidencesuggestthat there is substantial interorganelle movement of cholesterol and phospholipids through other mechanisms. For example, chemical inhibitors of the classicsecretorypathway and mutationsthat

impede vesiculartraffic in this pathway do not prevent cholesterolor phospholipidtransport betweenmembranes. A second mechanism entails direct protein-mediated contact of ER or ER-derivedmembraneswith membranesof other organelles(Figure 10-27b). In the third mechanism, small lipid-transfer proteins facilitate the exchangeof phospholipids or cholesterol between different membranes (Figr:.teIO-27c). Although such transfer proteins have beenidentified in assaysin vitro, their role in intracellular movements of most phospholipids is not well defined.For instance,mice with a knockout mutation in the gene encoding the phosphatidylcholine-transferprotein appear to be normal in most respects,indicating that this protein is not essentialfor cellularphospholipidmetabolism. As noted earlier, the lipid compositions of different organellemembranesvary considerably(seeTable 10-1).Some of th.r. differencesare due to different sitesof synthesis.For example,a phospholipid calledcardiolipin, which is localized to the mitochondrial membrane, is made only in mitochondria and little is transferredto other organelles.Differential transport of lipids also plays a role in determining the lipid compositions of different cellular membranes.For instance, even though cholesterol is made in the ER, the cholesterol molar ratio) is concentration (cholesterol-to-phospholipid -1.5-13-fold higher in the plasma membranethan in other organelles(ER, Golgi, mitochondrion, lysosome)' Although the mechanismsresponsiblefor establishingand maintaining thesedifferencesare not well understood' we have seenthat the distinctivelipid composition of eachmembranehas a maior influenceon its physical and biological properties'

and Cholesterol: Sphingolipids, Phospholipids, Movement Intracellular Synthesisand r Saturatedand unsaturatedfatty acidsof variouschain sphingolipids' of phospholipids, Iengthsare components and triglycerides. (c)

(b)

{a)

Binding proteln

m

,z\y\ \,/

Vesicle

Cytosol

of transportof 10-27Proposedmechanisms FIGURE In membranes. phospholipids between and cholesterol ln (a),vesicles lipidsbetweenmembranes transfer mechanism (b),lipidtransfer of directcontact isa consequence mechanism

Hypothetical proteins

Binding protein

Cytosol

Cytosol

by membrane-embedded thatis mediated betweenmemDranes by small,soluble (c),transfer is mediated proteinsIn mechanism 2002' andD Wustnel R Maxfield F from proteins lipid-transfer lAdapted I Clin lnvest 110:891 l

: N T H E S IASN D I N T R A C E L L U L AMRO V E M E N T P H O S P H O L I P I DSSP,H I N G O L I P I DASN,D C H O L E S T E R OSLY

e

433

Fatty acids are synthesizedfrom acetyl CoA by waterluble enzymesand modified by elongation and desaturation in the endoplasmicreticulum (ER). r The final stepsin the synthesisof glycerophospholipids, plasmalogens, and sphingolipids are catalyzed by membrane-associatedenzymesprimarily on the cytosolic face of the ER (seeFigure 10-25). r Each type of lipid is initially incorporated existing membraneson which it is made.

the pre-

r Most membranephospholipids are preferentially distributed in either the exoplasmic or the cytosolic leaflet. This a.symmetryresults in part from the action of phospholipid flippases. The initial stepsin cholesterolbiosynthesistake place in e cytosol, whereasthe last stepsare catalyzedby enzymes sociatedwith the ER membrane. r The rate-controlling step in cholesterol biosynthesisis catalyzedby HMG-CoA reductase,whose rransmembrane segmentsare embeddedin the ER membrane and contain a sterol-sensingdomain. r Considerableevidenceindicates that Golgi-independent vesicular transport, direct protein-mediated contacts between different membranes,soluble protein carriers, or all three may account for some inter-organelletransport of cholesteroland phospholipids(seeFigure 10-27).

for transporting cholesterol and phospholipids between organelle membranes remain poorly characterized.In particular, we lack a detailed understandingof how various transport proteins move lipids from one membrane leaflet to another (flippaseactivity) and into and out of cells.Such understanding will undoubtedly require a determination of many high-resolutionstructuresof thesemolecules,their capture in various stagesof the transport process,and careful kinetic and other biophysicalanalysesof their function, similar to the approachesdiscussedin Chapter 11 for elucidatingthe operation of ion channelsand ATP-poweredpumps. Recent advancesin solubilizing and crystallizing integral membrane proteins have led to the delineation of the molecular structuresof many important types of proteins, such as ion channels,ATP-powered ion pumps, and aquaporins, as we will seein Chapter 11. However, many important classes of membraneproteins have proven recalcitrant to even these new approaches.For example, we lack the structure of any protein that transports glucoseinto a eukaryotic cell. As we will learn in Chapters 15 and 16, many classesof receptors span the plasma membrane with one or more o helixes.perhaps surprisingl5 we lack the molecular structure of the transmembrane segment of any eukaryotic cell-surfacereceptor, and so many aspectsof the function of theseproteins are still mysterious. Elucidating the molecular structures of these and many other types of membrane proteins will clarify many aspectsof molecular cell biology.

KeyTerms One fundamental question in lipid biology concernsthe generation, maintenance,and function of the asymmetricdistribution of lipids within the leafletsof one membrane and the

acylation 425 brle acld 416 cholesterol 41.5 critical micelle concentration(CMC) 428 cytosolic face414 detergent 427 exoplasmic face414 external face414 flippase 420 fluorescencerecovery after photobleaching(FRAP)41 7

Major controversiessurround the existenceof lipid rafts in biological membranesand their function in cell signaling. Many biochemicalstudiesusing model membranesshow that

glycolipid 416 GPI anchor 426 HMG-CoA reductase432 hydrophobic41 1 hydrophilic 411 integral membrane protein 421

structure and are too small to be resolved by fluorescence microscopy.Proving their existencein cellswill require devel_ opment of new biophysicaland microscopictools. Despiteconsiderableprogressin our understandingof the cellular metabolism and movement of lipids, the mechanisms 434

o

cHAprE1 Ro I

BToMEMBRA s rNREU c r u R E

internalface414 Ieaflet411 lipid raft 420 liposome411 lumen4L0 micelle411 peripheralmembrane protein422 phosphoglyceide 415 phospholipidbllayer 4 11 plasmologen 415 porin 423 prenyl anchor425 receptorproteins409 sphingolipid 416 statrn432 sterol-sensin g domain432

Review the Concepts l. \flhen viewed by electron microscopy the lipid bilayer is often describedas looking like a railroad track. Explain how the structure of the bilayer createsthis image.

2. Biomembranes contain many different types of lipid molecules.What are the three main types of lipid molecules found in biomembranes?How are the three types similar, and how are they different? 3. Proteinsmay be bound to the exoplasmicor cytosolic face of the plasma membrane by way of covalently attached lipids. -What are the three types of lipid anchors responsiblefor tethering proteins to the plasma membrane bilayer, and which type is used by cell-surfaceproteins that face the external medium and by glycosylated proteoglycans? 4. Lipid bilayers are consideredto be two-dimensional fluids; what does this mean?'Whatdrivesthe movementof lipid molecules and proteins within the bilayer? How can such movement be measured?What factors affect the degree of membrane fluidity? 5. Phospholipid biosynthesisat the interface between the endoplasmicreticulum (ER) and the cytosol presentsa number of challengesthat must be solved by the cell. Explain how each of the following is handled. a. The substratesfor phospholipid biosynthesisare all water soluble, yet the end products are not. b. The immediate site of incorporation of all newly synthesized phospholipids is the cytosolic leaflet of the ER membrane, yet phospholipids must be incorporated into both leaflets. c. Many membrane systemsin the cell, for example' the plasma membrane, are unable to synthesizetheir own phospholipids, yet these membranesmust also expand if the cell is to grow and divide. 6. Fatty acidsmust associatewith lipid chaperonesin order to move within the cell. \7hy are these chaperonesneeded, and what is the name given to a group of proteins that are responsible for this intracellular trafficking of fatty acids? 'What is the key distinguishing feature of theseproteins that allows fatty acids to move within the cell? 7. What are the common fatty acid chains in glycerophospholipids, and why do these fatty acid chains differ in their number of carbon atoms by multiples of 2? 8. The biosynthesisof cholesterol is a highly regulated 'What is the key regulated enzyme in cholesterol process. biosynthesis?This enzyme is subject to feedback inhibition. What is feedback inhibition? How does this enzyme sense cholesterollevelsin a cell? 9. It is evident that one function of cholesterolis structural becauseit is the most common single lipid molecule in the plasma membrane of mammals. Yet cholesterol may also 'Strhat aspectsof cholesterol and its have other functions. metabolism lead to the conclusion that cholesterolis a multifunctional membrane lipid? 10. Phospholipidsand cholesterolmust be transportedfrom their site of synthesisto various membrane systemswithin cells. One way of doing this is through vesicular transport' as is the case for many proteins in the secretory pathway. However, most phospholipid and cholesterolmembrane-tomembrane transport in cells is not by Golgi-mediatedvesic-

'S7hat is the evidence for this statement? ular transport. \fhat appear to be the maior mechanismsfor phospholipid and cholesteroltransport? 11. Explain the following statement: The structure of all biomembranesdependson the chemical properties of phospholipids, whereas the function of each specific biomembrane dependson the specific proteins associatedwith that membrane. 12. Name the three groups into which membrane-associated proteins may be classified.Explain the mechanism by which each group associateswith a biomembrane. 13. Although both facesof a biomembraneare composed of the same general types of macromolecules,principally lipids and proteins, the two faces of the bilayer are not iJentical. What accounts for the asymmetry between the two faces?

Analyze the Data The behavior of receptor X (XR)' a transmembraneproteln present in the plasma membrane of mammalian cells, is being investigated.The protein has been engineeredas a fusion protein containing the green fluorescentprotein (GFP) at its N-terminus. GFP-XR is a functional protein and can replace XR in cells. a. Cells expressingGFP-XR or artificial lipid vesicles (liposomes)containing GFP-XR are subjectedto fluorescence recovery after photobleaching (FRAP). The intensity of the fluorescenceof a small spot on the surfaceof the cells (solid line) or on the surfaceof the liposomes(dashedline) is measured prior to and following laser bleaching (arrow)' The data are shown below

5000 o q)

c q)

o c) o 0) f L

1000 25

50 T i m e( s )

75

100

\What explanation could account for the differing behavior of GFP-XR in liposomesversusin the plasma membrane of a cell? b. Tiny gold particles can be attached to individual moleculesand their movement then followed in a light microscopeby single-particletracking. This method allows one to observe the behavior of individual proteins in a membrane. The tracks generatedduring a 5-secondobservational period by a gold particle attached to XR present in a cell ANALYZETHE DATA

435

(left) or in a liposome (middle) or to XR adheredto a microscopeslide (right) are shown below.

€ XR present in a cell

XR present in a liposome

XR presenton a m i c r o s c o p es l i d e

\fhat additional information do these data provide beyond what can be determinedfrom rhe FRAp data? c. Fluorescenceresonanceenergy transfer (FRET) is a technique by which a fluorescent molecule, following its excitation with the appropriatewavelengthof light, can transfer its emissionenergy ro and excite a nearby different fluorescent molecule (seeFigure 15-1,4).Cyan fluorescenr protein (CFP) and yellow fluorescentprotein (yFp) are relared to GFP but fluorescear cyan and yellow wavelengthsrather than at green. If CFP is excited with the appropnare wave, length of light and a YFP molecule is very near, rhen energy can be transferred from CFP emission and used to excite YFP, as indicated by a loss of emission of cyan fluorescence and an increasein emissionof yellow fluorescence.CFP-XR and YFP-XR are expressedrogetherin a cell line or are both incorporated into liposomes. The number of molecules of YFP-XR and CFP-XR per cm2 of membrane is equivalent in the cells and the liposomes.The cells and liposomesare then irradiated with a wavelengthof light that causesCFp but not YFP to fluoresce. The amount of cyan (CFp) and yellow (YFP) fluorescenceemitted by the cells (solid line) or liposomes(dashedline) is then monitored, as shown below.

10.2 Biomembranes: Protein Components and Basic Functions Bowie,J. Solvingrhe membraneprotein folding problem. 2005. Nature 438:581-589. Cullen,P.J.,G.E. Cozier,G. Banting,and H. Mellor. 2001. Modular phosphoinositide-bindingdomains:their role in signalling and membranetrafficking. Curr. Biol. 11:R882-R893. Engelman,D. Membranesare more mosaicthan fluid. 2005. Nature 438:578-580. Lanyi, J. K., and H. Luecke.2001. Bacteriorhod,oosin. Curr. Opin. Struc.Biol. 1l:415-519. Lee, A. G. A greasygrip. 2005. Nature 438:569-570. MacKenzie,K. R., J. H. Prestegard,and D. M. Engel,man.1997. A transmembranehelix dimer: structureand implications.Science 276:131-133. Mclntosh, T. J., and S. A. Simon. 2006. Rolesof bilayer material propertiesin function and distribution of membraneproreins Ann. Reu.Biophys. B iomolec.Struct. 3 5 :177-198. ^ Schulz,G- E. 2000. B-Barrelmembraneproteins. Curr. Opin. Struc. Biol. 10:443447. 10.3 Phospholipids, Sphingolipids, and Cholesterol: Synthesis and Intracellular Movement

20 o c o c

Yellow light

0) c o 1' "n o o q)

L

475 500 525 (nm)of emittedfluorescent Wavelength light

$7hat can be deducedabout XR from thesedata?

References 10.1 Biomembranes: Lipid Composition and Structural Organization . McMahon, H., and J. L. Gallop. 2005. Membrane curvature of dynamic cell mimbrane remodeling.Nature ii{ 1^e1hir^rigms 438:590-596. Mukherjee, S., and F. R. Maxfield. 2004. Membrane domains. Annu. Reu.Cell Deu.Biol.2O:839-866. _ Simons,K., and D. Toomre.2000. Lipid rafts and signaltrans_ duction. Nature Reu.Mol. Cetl Biol. t:Zi-4t.

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Simons,K., and \7. L. C. Vaz. Model sysrems,lipid rafts, and cell membranes.2004.Annw. Reu.Bio\hys. Biomolic. Strwct. 33:269-295. Thmm, L. K., V. K. Kiessling,and M. L. Wagner.2001.Membrane dynamics.Encyclopediaof Life Sciences.Nature Publishing Group. Vance,D. E., and J. E. Vance.2002. Biochemistryof Lipids, Lipoproteins, and Membranes, 4th ed. Elsevier. Van Meer, G.2006 Cellular lipidomics.EMBO J. 24:31.59-31.65. Yeager,P.L.2001,.Lipids. Encyclopediaof Life Sciences. Narure PublishingGroup. .. Zimmerberg,J., and M. M. Kozlov. 2006. How proteinsproduce cellufarmembranecurvature.Nature Reu.Mol. Cetl Biol.7:9-1,9.

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Bloch, K. 1965. The biological synthesisof cholesterol.Science 150:1.9-28. Daleke,D. L., and J. V. Lyles.2000. Identification and purification of aminophospholipidflippases.Biochim. Biophys. Acia t486:1.08-127. Futerman,A., and H. Riezman.2005. The inns and outs of sphingolipidsynrhesis.TrendsCell Biol. lS:312-318. Hajri, T., and N. A. Abumrad. 2002. Fatty acid rransport across membranes:relevanceto nutrition and metabolic pathology.Ann. Reu.Nutr. 22:383-41,5. Henneberry,A. L., M. M. \7right, and C. R. McMaster. 2002. The major sitesof cellular phospholipid synthesisand molecular determinanrs oI fatty acid and lipid head group specificity. Mol. Biol. Cell 13:3148-3161. . Holthuis, J. C. M., and T. P. Levine.2005. Lipid traffic: floppy drivesand a superhighwayNature Reu.Molec. Cblt Biot. 6JO9:r20. Ioannou, Y. A. 2001. Multidrug permeasesand subcellular cholesteroltransporr.Nature Reu.Mol. Cetl Biol. 2:657-66g. Kent, C. 1995. Eukaryotic phospholipid biosynthesis.Ann. Reu. Biochem.64:315-343. Maxfield, F. R., and I. Tabas.2005. Role of cholesteroland lipid organizationin disease.Nature 438:61,2-621,. Stahl,A., R. E. Gimeno, L. A. Tartaglia,and H. F. Lodish. 2001. F"y.".1d transport proteins:a current view of a growing family. TrendsEndocrinol. Metab. 12(6\:266-273. van Meer, G., and H. Sprong.2004. Membrane lipids and vesiculartraffic. Curr. Opin. Cell Biol. 16:373-378.

CHA PTER

TRANSMEMBRANE OF TRANSPORT IONSAND SMALL MOLECULES with palestripesledto the A studyof mutantzebrafish the that regulates transporter identificationof a sodium/calcium Micek darkness of humanskin @ Christina

I n all cells, the plasma membrane forms the permeability I barrier that separatesthe cytoplasm from the exterior enI vironment, thus topologically and biochemicallydefining a cell and distinguishingit from its surroundings.As a permeability barrier, the plasma membrane allows import of essential nutrients, ensuresthat metabolic intermediatesremain in the cell where they belong, and enableswaste products to leave the cell. By preventing the unimpeded movement of moleculesinto and out of cells, the plasma membrane maintains essentialdifferencesbetweenthe composition of the extracellular fluid and that of the cytoplasm; for example, the concentration of NaCl in the blood and extracellular fluids of animals is generallyabove 150 mM, similar to that of the seawaterin which all cells are thought to have evolved' whereas the Na+ concentration in the cytoplasm is tenfold lower. In contrast, the potassium ion concentration is higher in the cytoplasm than outside. The plasma membrane, like all cellular membranes, is basedon a bilayer of phospholipids into which proteins and other lipids are embedded.If the plasma membrane were a pure phospholipid bilayer, it would be an excellentchemical barrier, impermeable to virtually all ions, amino acids' sugars, and other water-solublemoleculesthat must selectively enter or leave a cell. In fact, only a few gasesand uncharged small moleculescan readily diffuse across a pure phospholipid membrane(Figure11-1).However,plasmamembranes must not only serve as barriers but also must play another' almost contradictory role. They must permit the selective transport of material and information between the cell's interior and exterior spaces,which often involves regulated entrv or exit of many diverse small biomolecules,ions, and

water. Plasma and other membranes serve as both barriers and conduits. Integral membrane proteins called transport proteins, embedded in the plasma membrane and other membranes within cells by multiple transmembranedomains, permit the controlled and selective transport of molecules and ions acrossthe membrane.In some cases,this involves molecules moving from a higher to a lower concentration, a thermodynamically spontaneousprocessthat does not require the input of energy.Examples include the movement of water or glucosefrom the blood into body cells. In other cases.moleculesmust be moved "uphill" across a membrane-against a concentration gradient-a thermodynamically unfavorable processthat can only occur when an external source of energy is available. Examples include

OUTLINE 11.1

Overviewof MembraneTransport

438

11.2

Uniport Transportof Glucoseand Water

441

1 1 . 3 A T P - P o w e r ePdu m p sa n d t h e I n t r a c e l l u l alro n i c Environment 1 1 . 4 N o n g a t e dl o n C h a n n e l sa n d t h e R e s t i n g M e m b r a n eP o t e n t i a l

458

andAntiporters by Symporters 11.5 Cotransport

465

Transport 11.6 Transepithelial

47O

437

Gases

K+

co2, N2,02 Exterior ,

Small uncharged polar molecules

il-

NH2-C-NH2

Water

Urea Large uncharged polar

1,"1""ut""

Cytosol

H,o

li

Slightly permeable

Glucose,fructose

K t , M g z * , C a 2 + ,C l - , HCO3-, HPO42-

Amino acids, ATP, glucose 6-phosphate, proteins, nucleic acids

A FIGURE 11-1 Relativepermeabilityof a purephospholipid bilayerto variousmolecules. A bilayer ispermeable to small hydrophobic molecules andsmalluncharged polarmolecules, slightly permeable to waterandurea,andessentrally impermeable to ions andto largepolarmolecules concentraring protons within lysosomesto generate a low pH in the lumen. Often this energy is provided by mechanistically coupling the energy-releasinghydrolysis of the terminal phosphoanhydride bond in ATp; such transporrers are called ATP-powered pumps. When such pumps rransport ions, suchas Na* and K+, they generateacrossthe membrane an electricalgradient, or potential, as well as a chemicalconcentration gradient. The energy stored in such gradientscan subsequently be used to do work or convey information. Other transport proteins couple the movement of one molecule or ion againstits concentration gradient with the movement of another down its gradient, using the energyreleased by the downhill movement of one moleculeor ion to thermodynamically drive the uphill movement of another. Frequently, several different types of transport proteins work in concert to achievea physiological function. An example is seenin Figure 11-2,where an ATp-powered rransporter pumps Na* our of the cell and K+ ions inward; this pump establishesthe opposing concenrration gradients of Na* and K+ ions acrossthe plasmamembrane.The human genome encodeshundreds of different types of transport proteins that use the energystored in the Na+ concentration

quent sections,we describe the structure and ooeration of specific examples of each class and show how members of families of homologous transport proteins have different 438

'

CHAPTER 11

|

2 Na*

ADP + P; K*

lons

Charqed polarmolecules

+++

Ti-

Plasma membrane

E t h an o l o

Lysine

Na*/K* pump

ATP K+ channel

Na+/lysine symporter

FIGURE 11-2 Multipletransportproteinsfunctiontogether in the plasmamembraneof metazoancells.Graotenrs are indicated by triangles with thetip pointing towardlower concentration TheNa+/K+ATPase in the plasma membrane uses energyreleased byATPhydrolysis to pumpNa+out of the celland K* inward;thiscreates gradient a concentration of Na+that is greater out thanin andoneof K+thatisgreater in thanout Movement of positively charged K* ionsout of thecellthrough membrane K+ channelproteins potential creates an electric across the plasma membrane-the cytosolic faceisnegative with respect to theextracellular face.A sodium-lysine transporter, a typical sodium-aminoacidtransporter,movesNa+ionstogetherwith lysine fromtheextracellular mediumintothecell."Uphill"movement of theaminoacidispowered by "downhill"movement of Na+ions, powered bothbytheout-greater-than-in Na+concentration gradient andbythe negative potential on the inside of thecellmembrane, whichattracts the positively charged Na* ionsTheultimate source of theenergy to poweraminoaciduptakecomesfromtheATp hydrolyzed bythe Na*/K+ATPase, sincethispumpcreates boththe Na- ionconcentration gradient and,viathe K+ channels, the membrane potential, powerinfluxof Na+ions whichtogether properties that enable different cell types to function appropriately. We also explain how subcellularmembranesencompass specificcombinations of transport proteins that enable cells to carry out essenrialphysiological processes,including the maintenanceof cytosolicpH, the accumulationof sucrose and salts in plant cell vacuoles,and the directed flow of water in both plants and animals. Epithelial cells, such as rhose lining the small intestine, transport ions, sugars, and other small moleculesand water from one side ro the other.'Wewill see how this understanding has led to the development of sports drinks and also therapiesfor cholera.

Overviewof MembraneTransport Many different proteins contribute to the transport of ions and small moleculesacross membranes.As we learn about different transport processesin this chapter,we will seehow different kinds of membrane-embeddedproteins accomplish the task of moving moleculesin different ways.

O n l y S m a l lH y d r o p h o b i cM o l e c u l e sC r o s s M e m b r a n e sb y S i m p l eD i f f u s i o n As we saw above,only gases,suchas 02 and CO2, and small uncharged polar molecules, such as urea and ethanol. can

T R A N S M E M B R A NTER A N s p o R To F r o N s A N D S M A L LM o L E c u L E S

readily move by simple diffusion across an artificial membrane composed of pure phospholipid or of phospholipid and cholesterol(seeFigure 11-1). Such moleculesalso can diffuse acrosscellular membraneswithout the aid of transport proteins. No metabolic energy is expended because movement is from a high to a low concentration of the molecule,down its chemical concentration gradient. As noted in Chapter2, suchtransportreactionsare spontaneousbecause they have a positive AS value (increasein entropy) and thus a negativeAG (decreasein free energy). The relative diffusion rate of any substanceacrossa pure phospholipid bilayer is proportional to its concentration gradient across the bilayer and to its hydrophobicity and size; the movement of charged moleculesis also affected by any electric potential across the membrane. tWhen a phospholipid bilayer separatestwo aqueous compartments, membrane permeability can be easily determined by adding a small amount of radioactive material to one compartment and measuring its rate of appearancein the other compartment. The greater the concentration gradient of the substance,the faster its rate of movement acrossa bilayer. The hydrophobicity of a substanceis measuredby its partition coefficientK, the equilibrium constant for its partition betweenoil and water. The higher a substance'spartition coefficient,the more lipid solubleit is. The first and rate-limiting step in transport by simple diffusion is movement of a molecule from the aqueoussolution into the hydrophobic interior of the phospholipid bilayer,which resemblesoil in its chemical properties.This is the reasonthat the more hydrophobic a moleculeis, the fasterit diffusesacrossa pure phospholipid bilayer. For example, diethylurea, with an ethyl group (CH3CH2-) attachedto eachnitrogen atom of urea, has a K of 0.01,whereasureahasa K of 0.0002 (seeFigure11-1).Diethylurea,which is 50 times (0.01/0.0002)more hydrophobic than urea, will thereforediffuse through phospholipid bilayer membranesabout 50 times faster than urea. Similarly, fatty

E ATP-powered pumps (100-103ions/s)

acids with longer hydrocarbon chains are more hydrophobic than those with shorter chains and will diffuse more rapidly acrossa pure phospholipid bilayer at all concentrations. If a transported substancecarries a net charge,its movement is influencedby both its concentrationgradient and the membrane potential, the electric potential (voltage) across the membrane. The combination of thesetwo forces, called the electrochemicalgradient, determinesthe energetically favorable direction of transport of a chargedmoleculeacrossa membrane.The electric potential that exists acrossmost cellular membranesresults from a small imbalance in the concentration of positively and negatively charged ions on the two sides of the membrane. \7e discusshow this ionic imbalance,and resulting potential' arise and are maintained in Sections17.4 and 1I.5.

MembraneProteinsMediate Transportof Most M o l e c u l e sa n d A l l l o n s A c r o s sB i o m e m b r a n e s As is evident from Figure 1'1'-L,very few molecules and no ions can cross a pure phospholipid bilayer at appreciable rates by simple diffusion. Thus transport of most molecules into and out of cells requires the assistanceof specialized membrane proteins. Even transport of moleculeswith relatively large partition coefficients(e'g., urea and certain gases such as CO2) is frequently acceleratedby specific proteins becausetheir transport by simple diffusion usually is not sufficiently rapid to meet cellular needs. All transport proteins are transmembraneproteins containing multiple membrane-spanningsegmentsthat generally are o.helices.By forming a protein-lined pathway across the membrane, transport proteins are thought to allow movement of hydrophilic substanceswithout their coming into contact with the hydrophobic interior of the membrane' Here we introduce the various types of transport proteins coveredin this chapter(Figure11-3).

a

E Transporters (02-104 molecules/s)

lon channels (1o7-108 ions/s)

Closed ATP ADP+P,

Open

1'l-3 Overviewof membranetransportproteins' FIGURE toward with thetip pointing bytriangles areindicated Gradients utilize potential, or both.E Pumps electrical lowerconcentration, specific power of movement to ATP hydrolysis released by energy the therrelectrochemical against or smallmolecules ions(redcircles) ions(orwater) permitmovement of specrfic gradientE Channels whichfallinto gradient Transporters, downtheirelectrochemical S or ions molecules small groups, of specific facilitate movement three

Uniporter

tr

Symporter

tr

aa Antiporter

q

downitsconcentratton a singletypeof molecule transport Uniporters (symporters, proteins EE, andantiporters, gradientEE.Cotransport againstitsconcentration of onemolecule the movement EE) catalyze of oneor moreions (blackcircles), drivenby movement gradient in the (red Differences gradient circles). .n electrochemical do*n of proteins by thesethreemajorclasses of transport mechanisms ratesof solutemovement for theirvarying account

O V E R V I E WO F M E M B R A N ET R A N S P O R T O

439

ATP-poweredpumps (or simply pumps) are ATpasesthat use the energy of AIP hydrolysis to move ions or small moleculesacrossa membraneagainsta chemicalconcentrationgradient, an electricpotential,or both. This process,referredto as activetransport, is an exampleof a coupledchemicalreaction (Chapter2). In this case,transport of ions or small molecules "uphill" against an electrochemicalgradient, which requires energy,is coupled to the hydrolysis of ATp, which releasesenergy. The overall reaction-ATP hydrolysis and the ,,uphill', movement of ions or small molecules-is energeticallyfavorable. The Na*/K* pump shown in our overview figure (see Figure 11-2) is an exampleof an Alp-powered pump. Channel proteins transport water, specific ions, or hydrophilic small moleculesdown their concentrationor electric potential gradients via facilitated transport (or facilitated diffusion), the protein-assistedmovement of a substance

PROPEBTY

SIMPLE DIFFUSION

In contrast,antiporters and symporterscouple the movement of one type of ion or moleculeagainst its concentrationgra, dient with the movement of one or more different ions doun its concentrationgradient, in the same (symporter) or different (antiporter) directions.Theseproteins ofren are calledcotransporters, referring to their ability to transporr rwo or more different solutes simultaneously.In Figure 11-2, lysine is moved into the cell via a Na+/lysine symporter. Like AIP pumps, cotransportersmediate coupled reactions in which an energeticallyunfavorable reaction (i.e., uphill movement of one type of molecule) is coupled to an energetically favorable reaction, the downhill movement of another. Note, however, rhat the nature of the energy-supplyingreaction driving active transport by these two classesof proteins differs. ATP pumps use energy from hydrolysis of ATp, whereas cotransportersuse the energy stored in an electrochemical gradient. This latter processsometimesis referred to as secondary activetransport. Table 11-1 summarizesthe four mechanisms by which small molecules and ions are transported across cellular membranes.Conformational changesare essentialto the function of all transport proteins. MP-powered pumps and transporters undergo a cycle of conformational change exposing a binding site (or sites)to one side of the membrane in one conformation and to the other side in a secondconformation. Becauseeach such cycle results in movement of only one (or a few) substrate molecules,these proteins are charactertzedby relatively slow rates of transport ranging from 100to 10aions or moleculesper secondlseefigure t i-:;. Ion channels shuttle between a closed state and an oDen state, but many ions can passthrough an open channel without any further conformational change. For this reason, channelsare characterizedby very fast rates of transport, up

FACITITATED TflANSPOBI

ACTIVE TRANSP()RT

Requires specific proteln

COTRANSP()RT+

Solute transported against its gradient

+

Coupled to ATp hydrolysis Driven by movement of a cotransported ion down its gradient

+

Examples of molecules transported

02, CO2, steroid hormones,many drugs

Glucose and amino acids (uniporters);ions and water (channels)

Ions,small hydrophilic molecules, lipids (ATP-powered pumps)

Also called secondary dctiue transport

440

CHAPTER 11

I

T R A N S M E M B R A NT E R A N S P O ROTF I O N SA N D S M A L LM O L E C U L E s

Glucose and amino acids (symporters); various ions and sucrose (antiporters)

to 10Eions per second.'Wewill begin with the simplesttransport proteins, the moleculesresponsiblefor the transport of glucose and water, before moving on to progressivelymore complex transport molecules.

Overview of Membrane Transport r The plasma membrane regulatesthe traffic of molecules into and out of the cell. r \With the exception of gases(e.g.,Oz and COz) and small hydrophobic molecules,most molecules cannot diffuse across a pure phospholipid bilayer at rates sufficient to meet cellularneeds.

Consequently,there is a maximum transport rate V^"* that is achievedwhen the concentration gradient acrossthe membrane is very large and each uniporter is working at its maximal rate. 4. Transport is specific.Each uniporter transports only a single speciesof molecule or a singlegroup of closely related molecules.A measureof the affinity of a transporter for its substrateis K-, which is the concentration of substrate at which transport is half maximal. Theseproperties also apply to transport mediated by the other classesof proteins depicted in Figure 11-3. One of the best-understood uniporters is the glucose

r Three classesof transmembraneproteins mediate transport of ions, sugars, amino acids, and other metabolites across cell membranes:ATP-powered pumps, channels' and transporters(seeFigure 11-3). r In active transport, a transport protein couples movement of a substrate against its concentration gradient to ATP hydrolysis. r In facilitated diffusion, a transport protein assistsin the movement of a specificsubstrate(moleculeor ion) down its concentration gradient. r In secondaryactive transport, or cotransport, a transport protein couples movement of a substrate against its concentration gradient to the movement of a secondsubstrate down its concentrationgradient(seeTable 11-1).

500 ot

UniportTransportof Glucose and Water Most animal cells utilize glucose as a source for ATP production; they employ a glucoseuniporter to take up glucose from the blood or other extracellular fluid down its concentration gradient. Many cellsutilize membranetransport proteinscalledaquaporinsto increasethe rate of water movement acrosstheir surface membranes.Here, we discussthe structure and function of theseand other uniport proteins.

o o

3 zso

GLUT2 (liver cellsl

0)

2

3

4 5 6 7 8 9 1011121314 Externalconcentrationof glucose(mM)

Km

SeveralFeaturesDistinguishUniport Transport from SimpleDiffusion The protein-mediated transport of glucose and other small hydrophilic moleculesacrossa membrane, a processknown as uniport, exhibits the following distinguishingproperties: l. The rate of facilitated diffusion by uniporters is far higher than simple diffusion through a pure phospholipid bilayer. 2. Becausethe transported moleculesnever enter the hydrophobic core of the phospholipid bilayer, the partition coefficient K is irrelevant. 3. Transport occurs via a limited number of uniporter moleculesrather than throughout the phospholipid bilayer.

11-4 Cellularuptakeof glucose FIGURE A EXPERIMENTAL proteins exhibitssimpleenzymekinetics GLUT by mediated and greatly exceedsthe calculatedrate of glucoseentry solely as uptake(measured by simplediffusion.Theinitialrateof glucose is seconds few first per in the permilliliter of cells hour) micromoles in the extracellular glucose concentration plottedagainst increasing in of glucose concentration initial the experiment, In this medium. and by erythrocytes, expressed zero BothGLUT1, the cellsisalways the rateof glucose by livercells,greatlyincrease expressed GLUT2, thatassociated with compared (burgundy andtancurves) uptake Like (blue concentrations external all at curve) diffusion with simple uptakeof glucose GLUT-facilitated reactions, enzyme-catalyzed at which rate(V'"^).TheK' istheconcentration a maximum exhibits K' of with a GLUT2, uptakeishalfmaximal the rateof glucose GLUT1, glucose than for affinity lower much has a about20 mM, with a K- of about1 5 mM AND WATER T F GLUCOSE U N I P O R TT R A N S P O R O

.

441

Exterior GLUTl

I Glucose

1l ll

Iu c o s e

Cytosol

Outward-facing conformation

Outward-facing conformation

A FIGURE 11-5 Modelof uniporttransportby GLUT1.In one conformation, theglucose-binding sitefacesoutward;in theother, the bindingsitefacesinwardBinding of glucose to theoutwardfacingsite(stepn) triggers a conformational changein thetransporter suchthatthe bindingsitenowfacesinwardtowardthecytosol (stepZ) Glucose thenisreleased to the insideof the cell(stepB).

Finally, thetransporter undergoes the reverse conformational change, regenerating the outward-facing bindingsite(stepE). lf theconcentration of glucose ishigherinsidethecellthanoutside, thecycle (step4 -+ step[), resulting willworkin reverse in netmovement of glucose frominsideto out Theactualconformational chanqes are probably smaller thanthosedepicted here.

enzyme-catalyzedreaction involving a single substrate.The kinetics of transport reactions mediated by other types of proteins are more complicatedthan for uniporters. Nonetheless,all protein-assistedtransport reactionsoccur faster than allowed by simple diffusion, are subsrrare-specific as reflected in lower K- values for some substratesthan others, and exhibit a maximal rate (V*"*).

where So,, - GLUT1 representsGLUT1 in the outwardfacing conformation with a bound glucose.This equation is similar to the one describing the path of a simple enzymecatalyzed reaction where the protein binds a single substrate and then transforms it into a different molecule. Here, however,no chemical modification occurs to the GLUT1-bound sugar; rather, it is moved across a cellular membrane. Nonetheless,the kinetics of this transport reaction are similar to those of simple enzyme-catalyzedreactions, and we can use the same derivation as that of the Michaelis-Menten equation in Chaprer 3 to derive the following expressionfor u, the initial transport rate for S into the cell catalyzed by GLUT1:

GLUT1UniporterTransportsGlucoseinto Most M a m m a l i a nC e l l s Most mammalian cells useblood glucoseas the malor source of cellular energy and express GLUT1. Since the glucose concentration usually is higher in the extracellular medium (blood, in the caseof erythrocytes)than in the cell, GLUT1 generally catalyzesthe net import of glucose from the extracellular medium into the cell. Under this condition, V_.* is achievedat high external glucoseconcentrations. Like other uniporters, GLUT1 alternates between two conformational states: in one, a glucose,binding site faces the outside of the membranel in the other, a glucose-binding site faces the inside. Figure 11-5 depicts the sequenceo?

The kinetics of the unidirectional transport of glucose from the outside of a cell inward via GLUT1 can be described by the same rype of equation used ro describe a simple enzyme-catalyzedchemical reaction. For simplicitS let's assumethat the substrateglucose, S, is present i"itiatiy only on the outside of the membrane.In this case.we can wnte

So,t+GLUT1

442

K:

C H A P T E R1 1

So,t-

|

V-u*

GLUT1 .-

Si, + GL1;rI1

V-"* v: __ u ^_

(11_t)

1*?

where C is the concentration of So,t (initiallg the concentration of Si" : 0). V-"*, the rate of transport when all molecules of GLUT1 contain a bound S, occurs at an infinitely high So",concentration.The lower the value of K-, the more tightly the substratebinds to the transporter and the greater the transport rate at a fixed concentration of substrate. Equation 11-1 describesthe curve for glucoseuptake by ery'1.'J.-4 throcytes shown in Figure as well as similar curves for other uniporters. For GLUT1 in the erythrocyte membrane, the K- for glucosetransport is 1.5 mM; at this concentration, roughly half the transporters with outward-facing binding iites would have a bound glucose and transport would occur at 50 percent of the maximal rate. Since blood glucoseis normally 5 mM, the erythrocyte glucose transporter usually is functioning at 77 percentof the maximal rate, as can be seen from Equation 11-1. GLUT1 and the very similar GLUT3 are expressedby erythrocytes and other cells that need to take up glucosefrom the blood continuously at high rates;

T R A N S M E M B R A NTER A N S P O R O T F I O N SA N D S M A L L M O L E C U L E S

the rate of glucose uptake by such cells will remain high regardlessof small changesin the concentration of blood glucose. In addition to glucose, the isomeric sugars D-mannose and n-galactose,which differ from l-glucose in the configuration at only one carbon atom, are transported by GLUT1 at measurablerates. However, the K- for glucose (1.5 mM) is much lower than the K- for D-mannose (20 mM) or ogalactose(30 mM). Thus GLUT1 is quite specific,having a much higher affinity (indicated by a lower K-) for the normal substrateo-glucosethan for other substrates. GLUT1 accounts for 2 percent of the protein in the plasma membrane of erythrocytes. After glucose is transported into the erythrocyte, it is rapidly phosphorylated, forming glucose 6-phosphate,which cannot leave the cell. Becausethis reaction, the first step in the metabolism of glucose (seeFigure 12-3), is rapid and occurs at a constant rate, the intracellular concentration of glucose is kept low even when glucose is imported from the medium. Consequently the concentration gradient of glucose outside greater than inside the cell is maintained at a sufficiently high ratio to support import of additional glucose molecules and maintain a constant rate of slucosemetabolism.

T h e H u m a nG e n o m eE n c o d e sa F a m i l yo f S u g a r TransportingGLUTProteins The human genome encodesat least 12 highly homologous GLUT proteins, GLUT1-GLUTI2, that are all thought to ct helices,suggestingthat they contain 12 membrane-spanning evolved from a single ancestraltransport protein. Although no three-dimensionalstructure of GLUT1 is available, detailed biochemical studies have shown that the amino acid residuesin the transmembranect helicesare predominantly hydrophobic; several helices, however, bear amino acid residues(e.g., serine, threonine, asparagine,and glutamine) whose side chains can form hydrogen bonds with the hydroxyl groups on glucose. These residues are thought to form the inward-facing and outward-facing glucose-binding sitesin the interior of the protein (seeFigure 11-5). The structuresof all GLUT isoforms are thought to be quite similar, and all transport sugars.Nonetheless,their differential expressionin various cell types and isoformspecificfunctional propertiesenabledifferent body cellsto regulate glucose metabolism independently and at the sametime maintain a constant concentrationof glucosein the blood. For instance,GLUT3 is found in neuronal cells of the brain. Neurons depend on a constant influx of glucose for metabolism, and the low K- of GLUT3 for glucose,like that of GLUT1, ensuresthat thesecells incorporate glucosefrom brain extracellular fluids at a high and constant rate. GLUT2, expressedin liver and the insulin-secreting B cellsof the pancreas,has a K- of :20 mM, about 13 times higher than the K^ of GLUT1. As a result, when blood glucoserisesfrom its basal level of 5 mM to 10 mM or so after a meal, the rate of glucose influx will almost double in

GlUT2-expressing cells, whereas it will increase only slightly in GLUT1-expressingcells (seeFigure 11-4).In liver, the "excess" glucose brought into the cell is stored as the polymer glycogen.In islet B cells, the rise in glucosetriggers secretionof the hormone insulin, which in turn lowers blood glucose by increasingglucose uptake and metabolism in muscle and by inhibiting glucose production in liver (see Figure 1,5-32). Another GLUT isoform, GLUT4, is expressedonly in fat and musclecells,the cellsthat respond to insulin by increasingtheir uptake of glucose,thereby removing glucose from the blood. In the absenceof insulin, GLUT4 is found in intracellular membranes,not on the plasma membrane, and is unable to facilitate glucoseuptake. By a processdetailed in Chapter 15, insulin causesthese GLUT4-rich internal membranesto fuse with the plasma membrane, increasing the number of GLUT4 molecules on the cell surface and thus the rate of glucose uptake. A defect in this process, one principal mechanism by which insulin lowers blood glucose,is one of the causesof adult onset, or type II, diabetes,a diseasemarked by continuously high blood glucose.

TransportProteinsCan Be EnrichedWithin Artificial Membranesand Cells Although transport proteins can be isolated from membranes and purified, the functional properties of these proteins can be studied only when they are associatedwith a membrane.Most cellular membranescontain many different types of transport proteins but a relatively low concentration of any particular one, making functional studies of a single protein difficult. To facilitate such studies, researchersuse two approachesfor enriching a transport protein of interest so that it predominatesin the membrane. In one common approach, a specifictransport protein is extracted and purified; the purified protein then is reincorporated into pure phospholipid bilayer membranes,such as liposomes (seeFigure 10-6). For example, all of the integral proteins of the erythrocyte membrane can be solubilized by a nonionic detergent, such as octylglucoside. The glucose uniporter GLUT1 can be purified by antibody affinity chromatography (Chapter 3) on a column containing a specificmonoclonal antibody and then incorporated into liposomes made of pure phospholipids. Alternatively, the geneencoding a specifictransport protein can be expressedat high levels in a cell type that normally does not expressit. The difference in transport of a substanceby the transfectedand by control nontransfected cells will be due to the expressedtransport protein. In these systems,the functional properties of the various membrane proteins can be examined without ambiguity. As an example, overexpressingGLUT1 in lines of cultured fibroblasts increasesseveralfoldtheir rate of uptake of glucose,and expressionof mutant GLUT1 proteins with specificamino acid alterations can identify residues important for substrate binding.

AND WATER T F GLUCOSE U N I P O R TT R A N S P O R O

443

OsmoticPressureCausesWater to Move Across Membranes Movement of water in and out of cells is an important feature of the life of both plants and animals. Aquaporins are a family of membrane proteins that allow water and a few other small uncharged molecules,such as glycerol, to cross biomembranes. But before discussingthese transport proteins, we need to review osmosis,the force that powers the movement of water. 'Water tends to move acrossa semioermeablemembrane from a solution of low solute concentiation to one of high concentration, a processtermed osmosis,or osmotic flow. In other words, since solutions with a high concentration of dissolvedsolute have a lower concentration of water, water will spontaneouslymove from a solution of high water concentration to one of lower. In effect, osmosisis equivalentto "diffusion" of water. Osmotic pressureis defined as the hydrostatic pressure required ro stop the net flow of water acrossa membrane separatingsolutions of different compositions (Figure t1-6).In this context, the "membrane" may be a layer of cellsor a plasmamembranethat is permeableto water but not to the solutes.The osmotic pressureis directly proportional to the difference in the concentration of the total number of solute molecules on each side of the membrane.For example,a 0.5 M NaCl solutionis actually0.5 M Na* ions and 0.5 M Cl- ions and has the same osmouc pressureas a 1 M solution of glucoseor sucrose. The movement of water across the plasma membrane also determinesthe volume of individuaf cells,which must be regulatedto avoid damageto the cell. Osmotic pressureis the force powering the movement of water in biological systems.

H y d r o s t a t i pc r e s s u r e Water-permeable r e q u r r e dt o p r e v e n l membrane n e t w a t e rf l o w I

* S o l u t i o nA CA

Solution B UB

A FIGURE 11-6 Osmoticpressure. Solutions A andB are separated by a membrane thatispermeable to waterbut impermeable to allsolutes. lf Cs(thetotalconcentration of solutes in solution B)isgreater thanC4,waterwilltendto flow across the membrane fromsolution A to solution B Theosmoticpressure n between thesolutions isthe hydrostatic pressure thatwouldhaveto be applied to solution B to prevent thiswaterflow.Fromthevan,t Hoffequation,osmoticpressure isgivenby n = ,q16',- Co),whereR rsthe gasconstant andf istheabsolute temperature 444

CHAPTER 11

I

In higher plants, water and minerals are absorbed from the soil by the roots and move up the plant through conducting tubes (the xylem); water loss from the plant, mainly by evaporation from the leaves,drives these movements of water. Unlike animal cells, plant, algal, fungal, and bacterial cells are surrounded by rigid cell walls, which resistthe expansionof the volume of the cell when the intracellular osmotic pressureincreases.Sfithout such a wall, animal cells expand when internal osmotic pressureincreases-if that pressurerises too much, the cells will burst like overextended balloons. Becauseof the cell wall in plants, the osmotic influx of water that occurs when such cells are placed in a hypotonic solution (even pure water) leads to an increasein intracellular pressurebut not in cell volume.In plant cells,the concentrationof solutes(e.g.,sugars and salts)usually is higher in the vacuole (seeFigure 9-7) than in the cytosol, which in turn has a higher solute concentration than the extracellular space. The osmotic pressure, called turgor pressure,generatedfrom the entry of water into the cytosol and then into the vacuole pushes the cytosol and the plasma membrane against the resistant cell wall. Plant cellscan harnessthis pressureto help them grow. Cell elongation during growth occurs by a hormone-induced localized loosening of a defined region of the cell wall, folIowed by influx of water into the vacuole, increasingits size and thus the size of the cell. I Although most protozoans (like animal cells) do not have a rigid cell wall, many contain a contractile vacuole that permits them to avoid osmotic lysis. A contractile vacuole takes up water from the cytosol and, unlike a plant vacuole, periodically discharges its contents through fusion with the plasma membrane. Thus even though water continuously enters the protozoan cell by osmotic floq the contractile vacuole prevents too much water from accumulating in the cell and swelling it to the bursting point.

Aquaporinslncreasethe Water Permeabilityof C e l lM e m b r a n e s Small changesin extracellular osmotic strength causemost animal cells to swell or shrink rapidly. N7henplaced in a hypotonic solution (i.e., one in which the concentration of solutes is lower than in the cytosol), animal cells swell owing to the osmotic flow of water inward. Converselg when placed in a hypertonic solution (i.e., one in which the concentration of solutes is higher than in the cytosol), animal cells shrink as cytosolic water leaves the cell by osmotic flow. Consequently,cultured animal cells must be maintained in an isotonic medium, which has a solute concentration and thus osmotic strength identical with that of the cell cytosol. In contrast, frog oocytesand eggsdo not swell when placed in pond water of very low osmotic strength, even though their internal salt (mainly KCI) concentrationis comparableto that of other cells (:159 mM KCI). Theseobservationswere what first led investigatorsto suspectthat the plasma membranesof

T R A N S M E M B R A NT E R A N S P O ROTF I O N SA N D S M A L LM O L E C U L E S

ZN

Video:Frog Oocyte Expressing AquaporinBurstsin HypotonicSolution 0 . 5m i n

1 . 5m i n

2 . 5m i n

3 . 5m i n

FIGURE 11-7 Expression A EXPERIMENTAL of aquaporinby frog oocytesincreasestheir permeabilityto water. Frogoocytes, an whichnormally areimpermeable to wateranddo notexpress protein, with mRNAencoding aquaporin weremicroinjected (bottomcellin Thesephotographs showcontroloocytes aquaporin (topcellin eachpanel) at the eachpanel)andmicroinjected oocytes (0 1 mM) indicated timesaftertransfer froman isotonic saltsolution (0 035 M) Thevolumeof the control saltsolution to a hvpotonic

to theyarepoorlypermeable because unchanged remained oocytes aquaporin expressing oocytes microinjected the water In contrast, of an osmoticinfluxof water, andthenburstbecause swelled protein[Courtesy of isa water-channel indicating thataquaporin of School Hopkins University Agre,Johns andPeter M Preston Gregory

erythrocytesand other cell types,but not frog oocytes,contain water-channel proteins that acceleratethe osmotic flow of water. The experimentalresultsshown in Figure 1L-7 demonstratethat an aquaporin in the erythrocyteplasma membrane functionsas a water channel. In its functional form, aquaporin is a tetramerof identical 28-kDa subunits (Figure 11-8a). Each subunit contains six membrane-spanningct helices that form a central pore through which water moves (Figure11-8b, c). At its center, gate, or pore, is only 0.28 the :2-nm-long water-selective nm in diameter-only slightly larger than the diameter of a water molecule. The molecular sieving properties of the constriction are determined by several conserved hydrophilic amino acid residueswhose side-chainand carbonyl groups extend into the middle of the channel.Several water moleculesmove simultaneouslythrough the channel, each of which sequentially forms specific hydrogen bonds and displacesanother water molecule downstream. The formation of hydrogen bonds between the oxygen atom of water and the amino groups of two amino acid side chains ensures that only water passesthrough the channel; even protons cannot passthrough, allowing ionic gradients to be maintained across membranes even when water is flowing across.

found in the kidney epithelial cells that resorb water from the urine, thus controlling the amount of water in the body. The activity of aquaporin2 is regulatedby vasopressin,also called antiduretic hormone. The regulation of the activity of aquaporin 2 in resting kidney cells resembles that of GLUT4 in fat and muscle in that when its activity is not required, when the cells are in their resting state and water is excreted to form urine, aquaporin 2 is localized to intraceland lular vesiclemembranes,not to the plasma membrane' '$7hen the into the cell. import water catalyze to so is unable polypeptide hormone vasopressinbinds to the cell-surface vasopressinreceptor, it activates a signaling pathway (detailed in Chapter 15) that causesthese aquaporin 2-containing vesiclesto fuse with the plasma membrane' increasing the rate of water uptake and its return into the circulation instead of the urine. Inactivating mutations in either the vasopressinor the aquaporin 2 gene causediabetes insipidus, a diseasemarked by excretion of large volumes of dilute urine. This finding establishesthe etiology of the diseaseand demonstratesthat the level of aquaporin 2 is rate limiting for water resorption from urine being formed by the kidney. I

Mammals express a family of aquaporins; 11 are known in humans.Aquaporin 1 is expressedin abundance in erythrocytes,and the homologous aquaporin 2 is

Medicine SeeL 5 King, D Kozono,and P Agre ,2004, Nat Rev.Mol Cell Biol 5:687-981

Other members of the aquaporin family transport hydroxyl-containing molecules such as glycerol rather than water. Human aquaporin 3, for instance,transports glycerol and is similar in amino acid sequenceand structure to the Escherichiacoli glyceroltransport protein G/pF. AND WATER T F GLUCOSE U N I P O R TT R A N S P O R O

445

Extracellular vestibule

Exterior Cytosol NHs* FIGURE 11-8 Structureof the water-channelprotein aquaporin.(a)Structural modelof thetetrameric proteincomprising four identical subunitsEachsubunitformsa waterchannel, asseen in thisviewlookingdownon the proteinfromtheexoplasmic side Oneof the monomers isshownwith a molecular surface in which the poreentrance canbe seen(b)Schematic diagram of the topology of a singleaquaporin subunitin relation to the membrane. Threepairsof homologous (A andA,,B transmembrane o helices andB',andC andC')areoriented in the opposite direction with respect to the membrane andareconnected bytwo hydrophilic loopscontaining shortnon-membrane-spanning helices and (N)residues. conserved asparagine Theloopsbendintothecavity formedbythe sixtransmembrane helices, meetinqin the middleto

Uniport Transport of Glucoseand Water r Protein-catalyzedtransport of a soluteacrossa membraneoccurs much faster than passivediffusion, exhibits a V^"* when the limited number of transporter moleculesare saturatedwith substrate,and is highly specificfor substrare(seeFigure 11-4). r Uniport proteins, such as the glucosetransporters (GLUTs), are thought to shuttle between two conformational states, one in which the substrate-bindingsite faces outward and one in which the binding site facesinward (seeFigure 11-5). r All membersof the GLUT protein family transporr sugars and have similar structures.Differencesin their K- values, expressionin different cell types, and substratespecificities are important for proper sugar metabolism in the body. r Two common experimental systems for studying the functions of transport proteins are liposomes containing a purified transport protein and cells transfected with the gene encoding a particular transport protein. 446

C H A P T E R1 1

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cooform partof the water-selective gate (c)Sideviewof the porein a singleaquaporin (red subunitin whichseveral watermolecules oxygens andwhitehydrogens) areseenwithinthe 2-nm-long watergatethat separates selective the waterfilledcytosolic and extracellular vestibules Thegatecontains highlyconserved arginine andhistidine residues, (blue) aswellasthetwo asparagine residues whosesidecharns formhydrogen bondswith transported waters. (Keygateresidues arehighlighted in blue) Transported watersalso formhydrogen bondsto the main-chain groupof a cysteine carbonyl residueThearrangement of thesehydrogen bondsandthe narrow porediameter (i.e.,HrO+)or passage of 0.28nm prevent of protons otherions lAfterH.Suietal, 2OO1, Nature 414.8]2SeealsoT.Zeuthen, 2001,Trends Biochem Sci.26:77, andK Murata etal, 2000,Nature 4O7:599 |

r Most biological membranesare semipermeable,more permeableto water than to ions or most other solutes. 'Water moves by osmosis across membranes from a solution of lower solute concentration to one of higher solute concentration. r The rigid cell wall surrounding plant cells prevents their swelling and leads to generation of turgor pressurein responseto the osmotic influx of water. r In responseto the entry of wate! protozoans maintain their normal cell volume by extruding water from contractile vacuoles. r Aquaporins are water-channel proteins that specifically increasethe permeability of biomembranesfor water (see F i g u r e1 1 - 8 ) . r Aquaporin 2 in the plasma membrane of certain kidney cells is essentialfor resorption of water from urine being formed; the absenceof aquaporin 2 leads to the medical condition diabetesinsipidus.

T R A N S M E M B R A NTER A N S P O R O T F I O N SA N D S M A L L M O L E C U L E S

ATP-Powered Pumpsand the lonicEnvironment lntracellular In previous sections,we focused on transport proteins that move molecules down their concentrations gradients. Here we focus our attention on a major class of proteins-the ATP-powered pumps-that use the energy releasedby hydrolysis of the terminal phosphoanhydride bond of ATP to transport ions and various small molecules across membranes against their concentration g.adietttt. All ATPpowered pumps are transmembrane proteins with one or more binding sites for ATP located on subunits or segments of the protein that face the cytosol. Although theseproteins commonly are called ATPases, they normally do not hydrolyze ATP into ADP and P1unlessions or other molecules are simultaneouslytransported.Becauseof this tight coupling between ATP hydrolysis and transport, the energy stored in the phosphoanhydridebond is not dissipatedbut rather used to move ions or other molecules uphill against

an electrochemicalgradient. As Figure 11-2 illustrates, these concentration gradients are used by other transport proteins to power uphill movement of yet other types of molecules.

Different Classesof PumpsExhibit Structuraland Functional Characteristic Properties The general structures of the four classesof ATP-powered pumps are depictedin Figure 11-9, with specificexamplesin each classlisted below the figure. Note that the membersof three of the classes(R R and V) only transport ions' whereas members of the ABC superfamily primarily transport small moleculessuch as amino acids and sugars. All P-classion pumps possesstwo identical catalytic o. subunits, each of which contains an MP-binding site. Most also have two smaller B subunits that usually have regulatory functions. During transport, at least one of the a

Exoplasmic face

vo

2Ht

Cytosolic face

ATP

ADP + P;

ADP + P;

ATP

P-classpumps

V-class proton pumps

F-classproton pumps

P l a s m am e m b r a n eo f p l a n t s f, u n g i , bacteria(H+pump)

V a c u o l a rm e m b r a n e si n plants,yeast,other fungi

Bacterialplasma membrane

P l a s m am e m b r a n eo f h i g h e r e u k a r y o t e (sN a + / Kp+u m p )

E n d o s o m aal n d l y s o s m a l m e m b r a n e si n a n i m a l cells

l n n e rm i t o c h o n d r i a l memDrane

A p i c a lp l a s m am e m b r a n eo f m a m m a l i a ns t o m a c h( H r / K rp u m p ) plasma membraneof all eukarvotic tr ta )ceils (La- pump,

P l a s m am e m b r a n eo f osteoclastsand some K l O n e YI U D U I eC e l l s

T h y l a k o i dm e m b r a n e of chloroplast

ABC superfamily Bacterialplasma m e m b r a n e s( a m i n oa c i d , sugar,and peptide transporters) M a m m a l i a np l a s m a membranes (transporters o f p h o s p h o l i p i d ss,m a l l l i p o p h i l i cd r u g s ,c h o l e s t e r o l , o t h e rs m a l l m o l e c u l e s )

S a r c o p l a s m i cr e t i c u l u m m e m b r a n e i n m u s c l e c e l l s ( C a 2 *p u m p )

11-9 The four classesof ATP-poweredtransport A FIGURE pumpsareindicated proteins.Thelocation beloweach of specific which pumpsarecomposed crsubunits, of two catalytic classP-class cycleTwoB aspartof thetransport becomephosphorylated present transport in someof thesepumps,mayregulate subunits, pumps V-class andF-class Onlyoneo andB subunitaredepicted. only andtransport intermediates do not formphosphoprotein proteins, protonsTheirstructures but andcontainsimilar aresimilar pumpsV-class arerelated to thoseof P-class noneof therrsubunits a pumpscoupleATPhydrolysis against to transport of protons pumpsnormally gradient, operatein whereas F-class concentration

or to utilizeenergyin a protonconcentratlon directron the reverse the large of members ATP All gradient synthesize to electrochemical (T)domains containtwo transmembrane of proteins ABCsuperfamily (A)domains, whichcoupleATP ATP-binding andtwo cytosolic as arepresent These coredomains to solutemovement. hydrolysis (depicted fused are here) but proteins ABC in some subunits separate andM T.Nishi in otherABCproteins[See intoa singlepolypeptide Forgac,2Oo2,Nature Rev.Mol CellBiol 3:94; C Toyoshimaet al , 2000, Nature405.64-/,D Mclntosh,2000, Nature Struc Biol 7:532; and T Elston, H W a n g ,a n d G O s t e r ,1 9 9 8 ,N a t u r e3 9 1 : 5 1 0 l

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447

subunitsis phosphorylated(hencethe name "P" class),and the transported ions move through the phosphorylated subunit. The amino acid sequencearound the phosphorylated residue is homologous in different pumps. This class includes the Na+/K+ ATPasein the plasma membrane, which generatesthe low cytosolic Na* and high cytosolic K* concentrationstypical of animal cells (SeeFigure 11-2). Certain Ca'- ATPasespump Ca'* ions out of the cytosol into the external medium; others pump Ca2* from the cytosol into the endoplasmicreticulum or into the specializedER called the sarcoplasmicreticulum, which is found in muscle cells. Another member of the P class,found in acid-secretingcells of the mammalian stomach,transportsprotons (H+ ions) out of and K* ions into the cell. The structures of V-classand F-classion pumps are similar to one another but unrelatedto, and more complicated than, P-classpumps. V- and F-classpumps contain several different transmembraneand cytosolicsubunits.All known V and F pumps transport only protons and do so in a processthat does not involve a phosphoproteinintermediate. V-classpumps generallyfunction to maintain the low pH of plant vacuoles and of lysosomesand other acidic vesiclesin animal cells by pumping protons from the cytosolic to the exoplasmicface of the membrane against a proton electrochemicalgradient.The H* pump that generates and maintains the plasma membrane electric potential in plant, fungal, and bacterialcellsalso belongsto this class. F-classpumps are found in bacterial plasma membranes and in mitochondria and chloroplasts.In contrasrto V-class pumps, they generally function as a kind of reverseproron pump, in which the energy releasedby the movement of protons from the exoplasmic to the cytosolic face of the membrane down the proton electrochemicalgradient is used to power the synthesisof ATP from ADp and pi. Becauseof their importance in ATP synthesisin chloroplastsand mitochondria, F-classproton pumps, commonly calledATP synthases,are treatedseparatelyin Chapter 12. The final class of ATP-powered pumps is a large family of multiple membersthat are more diversein function than those of the other classes.Referred to as the ABC (ATpbinding cassette)superfamily, this class includes several hundred different transporr proteins found in organisms ranging from bacteria to humans. As detailed below, some of these transporr proteins were first identified as multidrug-resistance proteins that, when overexpressed in canc e r c e l l s ,e x p o r t a n t i c a n c e d r r u g s a n d r e n d e ir u m o r s r e s i s t ant to their action. Each ABC protein is specificfor a single substrateor group of relatedsubsrrates, which may be ions, sugars,amino acids, phospholipids,cholesterol,peptides, polysaccharides,or even proteins. All ABC rransporr proteins share a structural organization consisting of four "core" domains:two transmembrane(T) domains,forming the passagewaythrough which transported moleculescross the membrane, and two cyrosolic ATp-binding (A) domains. In some ABC proteins,mostly those in bacteria,the core domains are present in four separarepolypeptides; in others,the core domains are fusedinto one or two multidomain polypeptides. 448

CHAPTER 11

I

ATP-Powered lon PumpsGenerateand Maintain l o n i cG r a d i e n t sA c r o s sC e l l u l a rM e m b r a n e s The specificionic composition of the cytosol usually differs greatly from that of the surrounding extracellular fluid. In virtually all cells-including microbial, plant, and animal cells-the cytosolic pH is kept near 7.2 regardlessof the extracellular pH. In the most extreme casethere is a millionfold difference in H+ concentration between the pH of the cytosol of the epithelial cells lining the stomach and the pH of the stomach lumen. Also, the cytosolic concentration of K* is much higher than that of Na*. In both invertebrates and vertebratesthe concentration of K+ is 20-40 times higher in cells than in the blood, while the concenrrationof Na* is 8-12 times lower in cells than in the blood (see Figure 11-2 and Table 11-2). Some Caz* in the cytosol is bound to the negatively charged groups in ATP and other

lON

(mM) CELL

(mM) Bt00D

SQUID AXON (INVERTEBRATE)-

K*

400

20

Na*

50

440

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40-150

560

caz*

o.ooo3

10

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5-10

MAMMALIAN CELL (VERTEBRATE)

K*

r39

4

Na-

12

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1.16

HCO3

1.2

29

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138

9

Mgt*

o.g

1.5

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< o.ooo2

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-The

large nerve axon of the squid has been widely used in studies of the mechanism of conduction of electric impulses. rX representsproteins, which have a net negative charge at the neutral pH of blood and cells.

T R A N S M E M B R A NT E R A N S P O ROTF I O N SA N D S M A L LM O L E C U L E s

molecules,but it is the concentration of free, unbound Ca"* that is critical to its functions in signaling pathways and muscle contraction. The concentration of free Ca2* in the cytosol is generallylessthan 0.2 micromolar (2 x 10-7 M), a thousand or more times lower than that in the blood. Plant cells and many microorganisms maintain similarly high cytosolic concentrations of K* and low concentrations of Caz* andNa* evenif the cells are cultured in very dilute salt solutions. The ion pumps discussedin this section are largely responsible for establishing and maintaining the usual ionic gradients acrossthe plasma and intracellular membranes.In carrying out this task, cells expend considerableenergy.For example, up to 25 percent of the ATP produced by nerve and kidney cells is used for ion transport, and human erythrocytes consume up to 50 percent of their available ATP for this purpose; in both cases, most of this ATP is used to power the Na+/K+ pump. The resultant Na+ and K* gradients in nerve cells are essentialfor their ability to conduct electric signals rapidly and efficiently, as we detail in Chapter 23. Certain enzymesrequired for protein synthesis in all cells require a high K+ concentration and are inhibited by high concentrations of Na*; these would ceaseto function without the operation of the Na*/K* pump. In cells treated with poisonsthat inhibit the production of ATP (e.g., 2,4-dinitrophenol in aerobic cells), the pumping stops and the ion concentrations inside the cell gradually approach those of the exterior environment as ions spontaneously move through channelsin the plasma membrane down their electrochemicalgradients.Eventually treated cells die: partly becauseprotein synthesisrequires a high concentration of K* ions and partly becausein the absenceof a Na* gradient acrossthe cell membrane, a cell cannot import certain nutrients such as amino acids. Studieson the effectsof such poisons provided early evidencefor the existenceand significance of ion pumps.

MuscteRelaxationDependson Ca2+ATPases That PumpCa2*from the Cytosolinto the S a r c o p l a s m iRce t i c u l u m In skeletal muscle cells, Ca2* ions are concentratedand stored in the sarcoplasmicreticulum (SR); releaseof stored Caz* ions from the SR lumen via ion channelsinto the cytosol causescontraction, as discussedin Chapter 17. A Pclass Ca2* AIPase located in the SR membrane of skeletal muscle pumps Ca2* from the cytosol back into the lumen of the SR, thereby inducing muscle relaxation. Becausethis muscle calcium pump constitutesmore than 80 percent of the integral protein in SR membranes,it is easily purified from other membrane proteins and has been studied extensively. Determination of the three-dimensionalstructure of this protein in severalconformational statesthat representdifferent steps in the pumping process has revealed much about its mechanismof action. In the cytosol of muscle cells, the free Ca2* concentra6 7 tion rangesfrom 10 M (restingcells)to more than 10 M

(contracting cells), whereas the total Ca2* concentration in the SR lumen can be as high as 10-2 M. TYo soluble proteins in the lumen of the SR vesiclesbind Ca"* and serveas a reservoir for intracellular Caz*, thereby reducing the concentration of free Ca2* ions in the SR vesiclesand consequently the energy needed to pump Ca2* ions into the SR fro- tit. cytosol.-Theactivity of the muscle Ca2+ AIPase increasesas the free Ca2* concentration in the cytosol rises.In skeletal muscle cells the calcium pump in the SR membrane works in concert with a similar Ca2* pump located in the plasma membraneto ensurethat the cytosolic concentration of free Ca2* in resting muscle remains below 1 pM. The current model for the mechanism of action of the Ca2* AIPase in the SR membrane involves multiple conformational states. For simplicity' we group these into E1 states,in which the two binding sitesfor Caz* ,located in the center of the membrane-spanningdomain, face the cytosol' andE2 states,in which thesebinding sitesface the exoplasmic face of the membrane,pointing into the lumen of the SR. Coupling of ATP hydrolysis with ion pumping involves several conformational changesin the protein that must occur in a defined order, as shown in Figure 11-10.'When the protein is in the E1 conformation, two Ca2* ions bind to two hieh-affinitv bindine sitesaccessiblefrom the cytosolic side; .r,".n thouei, the Ca)* concentration is very low (seeTable 11-2). calc]um ions still fill thesesites.Next, an ATP binds to a site on the cytosolic surface(step 1). The bound ATP is hydrolyzed to ADP in a reaction that requires Mgz* , and the liberated phosphate is transferred to a specific aspartate residue in the protein, forming the high-energy acyl phosphate bond denoted by E1 - P (step2).The protein then undergoesa conformational changethat generatesE2' in which the affinity of the two Ca2*-binding sitesis reduced (Figure 11.-1.1a\and in which these sites are now accessibleto the SR lumen (step 3). The free energy of hydrolysis of the aspartylphosphate bond in E1 - P is greater than that in E2-P, and this reduction in free energy of the aspartyl-phosphatebond can be said to power the E1 -+ E2 conformational change. The Ca2* ions spontaneouslydissociatefrom the low-affinity sites to enter thi SR lumen, because even though the Caz* concentration there is higher than in the cytosol, it is lower than the K6 for Ca2* binding in the low-affinity state (step

into the SR lumen. Much structural and biophysical evidencesupports the model depicted in Figure 11-10' For instance, the muscle calcium pump has been isolated with phosphate linked to the key aspartate residue, and spectroscopic studies have detected slight alterations in protein conformation during the E1 -+ E2 conversion. The two phosphorylated states can also be distinguishedbiochemically; addition of ADP to phosphorylated E1 results in synthesisof AIP, the reverseof it.p f i" Figure 11-10, whereas addition of ADP to phosphorylated E2 does not. Each principal conformational

O N I CE N V I R O N M E N T A T P - P O W E R EP DU M P SA N D T H E I N T R A C E L L U L AI R

449

SR lumen

Ca2t-bindrng

E1

c?'*.

\

"rl ',/

I

,\,

E1

I )

Cytosol \ Phosphorylat Y aspartate I ATPbinding stte

Conformational change Calcium rerease

<+-

E2 A FIGURE 11-10 Operationalmodel of the Ca2+ATpasein the SRmembraneof skeletalmusclecells.Onlyoneof the two catalytic o subunits of thisP-class pumpis depictedE1and E2are alternative conformations of the proteinin whichthe Ca2+-binding sitesareaccessible to the cytosolic and exoplasmic faces,respecrrvery An orderedsequence of stepslE), as diagrammed here,is essential for coupling ATPhydrolysis andthe transport of Ca2*ionsacross

state ofthe reaction cycle can also be characterizedby a different susceptibility to various proteolytic enzymessuch as trypsln. As seen in the three-dimensionalstructure of the Ca2* pump in the E1 state,the 10 membrane-spanningct helicesin the catalytic subunit form the passagewaythrough which Ca"- ions move, and amino acidsin four of thesehelicesform the two high-affinity E1 Ca2+-binding sites (Figure 11-11a, left). One site is formed out of negatively charged oxygen atoms from the carboxyl groups (COO-) of glutamate and aspartate side chains, as well as from water molecules.The other site is formed from side- and main-chain oxygen atoms. Thus most of the water moleculesthat normally surround a Ca2* ion in aqueous solution are replaced by oxygen atoms attachedto the protein. In contrast, in the E2 state '1.1-a, (Figure right), severalof thesebindine side chains have moved fractions of a nanometer and are Jnable to interact with Ca2* ions, accounting for the low affinity of the E2 state for Ca2* ions. The cytoplasmic part of the Ca2* pump consists of three domains that are well separatedfrom each other in the E1 state (Figure 11-11b). Each of thesedomains is connected to the membrane-spanning helices by short segments of amino acids, and movements of these cytosolic domains will causecorrespondingmovementsof attached membrane-spanning o' helices. The phosphorylated residue, Asp 351, is located on the p domain, and the adenosine moiety of ATp binds to the N domain. Follow450

'

cHAPTER 11

|

E2

E2

the membrane. In the figure,-P indicates a high-energy aspartyl phosphate bond;-P indicates a low-energy bond.Because the affinityof Ca2*for the cytosolic-facing bindingsitesin E1is a thougreater sandfold thanthe affinityof Ca2*for the exoplasmic-facing sitesin E2,thispumptransports Ca2+unidirectionally fromthe cytosol to the 5RlumenSeethe textand Figure11-11for moredetails. andG Inesi,2004, Ann Rev. Biochem [SeeC Toyoshima 73:269-92]

ing ATP and Ca2* binding, the N domain moves until rhe 1 phosphateof the bound ATP becomesadiacentro the aspartate on the P domain that is to receivethe phosphate. Although the details of these conformational changesare not yet clear, this movement is thought to be transmitted by scissorlike motions of the connecting segments to movements of several membrane-spanning a helices. These changesare especiallyapparent in the four helices that contain the two Ca2*-binding sites:the changesprevent the bound Ca2+ ions from dissociatingback rntothe cytosol but enable them to dissociateinto the exoplasmic medium. These changes also weaken the binding of the two Ca'- ions, as can be seenby comparing the structures in Figure 11-11a. This weakening enablesthe bound ions to dissociate into the exoplasmic space, here, the SR lumen. After the Caz* ions dissociate and the asoartvl phosphate is hydrolyzed, the protein reverts back io the E1 conformation. All P-classion pumps, regardlessof which ion they transport, are phosphorylated on a highly conserved aspartate residue during the transport process.Thus the operational model in Figure 11-11 is generally applicable to all of these ATP-powered ion pumps. In addition, the catalytic a subunits of all the P pumps examined to date have a similar molecular weight and, as deduced from their amino acid sequencesderived from cDNA clones. have a similar arrangement of transmembrane o. helices and cytosolf a c i n g A , P , a n d N d o m a i n s ( s e eF i g u r e 1 1 - 1 0 ) . T h e s e

T R A N s M E M B R ATNREA N s p o RoTF r o N sA N D s M A L LM o L E c u L E s

(a)

El state Ca2*-bound

state Ca2*-free

'-{

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I

(b) SR lumen

Membrane

Ca2*

Cytosol

ee

Actuator d o m a i n( A )

b i n d i n gd o m a i n( N ) 11-11Structureof the catalyticotsubunitof the A FIGURE (a)Ca2*-binding E2state sitesin the low-affinity muscleca2* ATPase. (flEtht), withoutboundions,andin the E1state(/eff),with two bound ions Sidechainsof keyamrnoacidsarewhite,andtheoxygen calcium andaspartate sidechainsarered.Inthe highatomson the glutamate 4, Ca2*ionsbindat two sitesbetweenhelices affinityE1conformation, Onesiteisformedout of negatively 5, 6, and8 insidethe membrane. sidechainsandof andaspartate oxygenatomsfromglutamate charged (notshown),andthe otherrsformedout of side-and watermolecules the Ca2*ion oxygenatomssurround oxygenatomsSeven main-chain modelof the proteinin the E1 in bothsites(b)Three-dimensional by x-raycrystallography determined statebasedon the structure fourof which(purple) o helices, Thereare10transmembrane in Ca2 bindingThecytosolic thatparticipate containresidues

domain the nucleotide-binding formsthreedomains: segment (green), the actuator and P (blue), domain phosphorylation the N two of the membrane-spanning thatconnects domainA (beige) helices(c)Modelsof the pumpin the E1state(left)andE2state the E1andE2statesin the (right).Notethe differences between these domains; andactuator of the nucleotide-binding conformations membraneof the changes power conformational the movements sites, (purple) the Ca'*-binding thatconstitute crhelices spanning sitesare themfromonein whichthe Ca2"-binding converting face(E1state)to one in whichthe now to the cytosolic accessible face(E2 to the exoplasmic ions boundcalcium areaccessible loosely Nature, Nomura,2002, and H Toyoshima from C state)[Adapted 73'269-92: Biochem 2004,Ann Rev. andG lnesi, 1;C Toyoshima 418:605-1 310:1461 2005, Science l andR MacKinnon, andE Gouaux

findings strongly suggestthat all such proteins evolved from a common precursor although they now transport different ions.

cellular responses.In order for Caz* to function in intracellular signaling,the concentration of Ca2* ions free in the cytosol usually must be kept below 0.1'-0.2 pM. Animal, yeast, and probably plant cells^expressplasma membrane bat* ATParesthat transport Ca2* out of the cell against its electrochemical gradient. The catalytic c subunit of these P-classpumps is similar in structure and sequenceto the c subunit of the muscle SR Ca2* PumP. The activity of plasma membrane Ca"- ATPasesis regulated by calmodulin, a cytosolic Ca'*-binding protein

C a l m o d u l i nR e g u l a t e st h e P l a s m aM e m b r a n e Ca2*PumpsThat Control CytosolicCa2+ Concentrations As we explain in Chapter 15, small increasesin the concentration of free Ca2* ions in the cytosol trigger a variety of

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(seeFigure 3-31). A rise in cytosolic Ca2* inducesthe binding of Ca2* ions to calmodulin, which triggers allosteric activation of the Ca2* ATPase.As a result, the export of Ca2* ions from the cell accelerates,quickly restoring the low concentration of free cytosolic Ca2* characteristicof the resting cell.

N a * / K + A T P a s eM a i n t a i n st h e I n t r a c e l l u t aN r a+ a n d K + C o n c e n t r a t i o nisn A n i m a l C e l l s A secondimportant P-classion pump present in the plasma membraneof all animal cellsis the Na+/K+ ATpase.This ion pump is a tetramer of subunit composition c2B2. (ClassicExperiment 11.1 describesthe discovery of this enzyme.)The small, glycosylatedB polypeptide helps newly synthesizedct subunits to fold properly in the endoplasmicreticulum but apparentlyis not involved directly in ion pumping. The amino acid sequenceand predicted tertiary structure of the catalytic cr subunit are very similar to those of the muscle SR Ca2+ MPase (seeFigure 11-11). In particular,the Na+/K+ ATpase has segmentson the cytosolic face that link the ATp-binding N domain, the phosphorylated aspartate on the p domain,

and the A domain to the membrane-embedded segment.The overall transport process moves three Na+ ions out of and two K- ions into the cell per ATP molecule hydrolyzed. The mechanism of action of the Na*/l(+ ATpase, out'1,1,-12, lined in Figure is similar to that of the musclecalcium pump, except that ions are pumped in both directions across the membrane, with each ion moving against its concentration gradient. In its E1 conformation, the Na*/K* ATpase has three high-affinity Na*-binding sites and two lowaffinity K*-binding sitesaccessiblero the cytosolic surfaceof the protein. The K- for binding of Na+ to these cytosolic sitesis 0.5 mM, a value considerablylower than the intracellular Na* concentration of :12 mM; as a result. Na* ions normally will fully occupy these sites. Conversely,the affinity of the cytosolic K+-binding sitesis low enough that K+ ions, transported inward through the protein, dissociatefrom E1 into the cytosol despitethe high intracellular K+ concentration. During the E1 -+ E2 transition, the three bound Na+ ions become accessibleto the exoplasmic face, and simultaneouslythe affinity of the three Na*-binding sitesdrops. The three Na+ ions, transported oufward through the protein and now bound to the low-affinity Na+ sitesexposedto the

OverviewAnimation:BiologicalEnergyInterconversions ffi I E2f

2K*

Dephosphorylation and conformational change

+

rj E2 A FIGURE 11-12Operationalmodelof the Na+/K*ATpase in the plasmamembrane.Onlyoneof thetwo catalytic o subunits of thisP-class pumpisdepictedlt isnot knownwhether justoneor bothsubunits in a srngle ATpase molecule transport ions.lonpumpingbythe Na+/K+ATpase phosphorylation, involves dephosphorylation, andconformational chanqes similar to those i n t h em u s c lC e a 2 *A T P a s( se e eF i q u r 1 e 1 - 1 0 )I .nt h i sc a s e .

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hydrolysis powersthe E2-+ E1 of the E2-Pintermediate conformational changeandconcomitant transport of two K* ionsinward.Na+ionsareindicated by redcircles; K+ ions,by purplesquares; high-energy acylphosphate bond,by -p; lowenergyphosphoester bond,by -P [SeeK Sweadner andC Donnet, 2001,Biochem I 356:6875, fordetails of thestructure of theo subunit I

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exoplasmicface, dissociateone at a time into the extracellular medium despitethe high extracellularNa* concentration. Transition to the E2 conformation also generatestwo highaffinity Kt sites accessibleto the exoplasmic face. Because the K- for K* binding to thesesites (0.2 mM) is lower than the extracellularK* concentration(4 mM), thesesiteswill fill with K+ ions as the Na* ions dissociate.Similarly,during the E2 -+ E1 transition, the two bound K* ions are transported inward and then releasedinto the cytosol. Certain drugs (e.g.,ouabain and digoxin) bind to the exoplasmic domain of the plasma membrane Na-/l(- ATPase and specificallyinhibit its ATPaseactivity. The resulting disruption in the Na*/I(* balanceof cellsis strong evidencefor the critical role of this ion pump in maintaining the normal K* and Na* ion concentration gradients.

V-ClassH* ATPasesMaintain the Acidity of Lysosomesand Vacuoles All V-classATPasestransport only H* ions. These proton pumps, presentin the membranesof lysosomes,endosomes, and plant vacuoles, function to acidify the lumen of these organelles.The pH of the lysosomallumen can be measured preciselyin living cells by use of particles labeledwith a pHsensitivefluorescent dye. ufhen these particles are added to the extracellular fluid, the cells engulf and internalize them (phagocytosis;seeChapter 17), ultimately transporting them into lysosomes.The lysosomal pH can be calculated from the spectrum of the fluorescenceemitted. Maintenance of the 10O-fold or more proton gradient betweenthe lysosomal lumen (pH :4.5-5.0) and the cytosol (pH :7.0) dependson a V-classATPaseand thus ATP production by the cell. The low lysosomal pH is necessaryfor optimal function of the many proteases,nucleases,and other hydrolytic enzymesin the lumen; on the other hand, a cytosolic pH of 5 would disrupt the functions of many proteins optimized to act at pH7 and lead to death of the cell. The ATP-powered proton pumps in lysosomal and vacuolar membraneshave been isolated, purified, and incorporated into liposomes.As shown in Figure11-9 (center),these V-class proton pumps contain two discrete domains: a cytosolic hydrophilic domain (V1) and a transmembrane domain (Ve) with multiple subunits forming each domain. Binding and hydrolysis of ATP by the B subunits in V1 provide the energy for pumping of H* ions through the protonconducting channel formed by the c and a subunits in Ve. Unlike P-class ion pumps, V-class proton pumps are not phosphorylated and dephosphorylatedduring proton transport. The structurally similar F-classproton pumps, which we describein Chapter 12, normally operatein the "reverse" direction to generateATP rather than pump protons; their mechanismof action is understood in great detail. Pumping of relatively few protons is required to acidify an intracellular vesicle.To understandwhy, recall that a solution of pH 4 has an H* ion concentration of 10-a moles per liter, or 10 7 molesof H* ions per milliliter. Sincethere are6.02 x 1023 atoms of H per mole (Avogadro'snumber), then a milliliter of a pH 4 solution contains6.02 x 1016H* ions. Thus at pH 4, a

primary sphericallysosomewith a volume of 4.18 x 10-15 ml (diameterof 0.2 pm) will contain just 252 protons. At pH 7, the sameorganellewould have only an averageof only 0.2 protons in its lumen, and thus pumping of only approximately 250 proteins is necessaryfor lysosomeacidification. By themselves,ATP-powered proton pumps cannot acidify the lumen of an organelle (or the extracellular space) becausethese pumps are electrogenic; that is, a net movement of electric charge occurs during transport. Pumping of just a few protons causesa buildup of positively charged H* ions on the exoplasmic (inside) face of the organelle membrane. For each H+ pumped across' a negative ion (e.g., OH- or Cl-) will be "left behind" on the cytosolic face, causing a buildup of negatively charged ions there. These oppositely charged ions attract each other on opposite faces of the membrane, generating a charge separation, or electric potential, across the membrane. As more and more protons are pumped, the excessof positive chargeson the exoplasmic face repels other H* ions, soon preventing pumping of additional protons long before a significant iransmembrane H* concentration gradient is established (Figure 11-13). In fact, this is the way that P-classH+

ATP

(a)

ADP + P1

Cytosol

@

NeutralpH

(b)

ATP

ADP + Pr

clH+ Acidic pH

H+

Cl H*

No electric potential

H* pumpson H11-13 Effectof V-class FIGURE concentrationgradientsand electricpotential gradientsacross only (a)lf an intracellular contains organelle cellularmembranes. potential across an electric pumps,protonpumpinggenerates V-class positive lumenal-side and negative facing side cytosolthe membrane, pH.(b)lf the organelle changein the intraluminal but no significant followthe anionspassively Cl- channels, alsocontains membrane of H+ andCl- ions in an accumulation resulting pumpedprotons, potential the across rnthe lumen(lowluminalpH)but no electric membrane.

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pumps generate a cytosol-negative potential across plant and yeastplasma membranes. In order for an organellelumen or an extracellular space (e.g.,the lumen of the stomach) to becomeacidic, movement of protons must be accompaniedeither by (1) movementof an equal number of anions (e.g.,Cl-) in the samedirection or by (2) movement of equal numbers of a different carion in the opposite direction. The first processoccurs in lysosomes and plant vacuoles, whose membranes contain V-class H+ ATPasesand anion channels through which accompanying Cl- ions move (Figure 11-13b). The secondprocessoccurs in the lining of the stomach,which contains a P-classH*/K* ATPasethat is not electrogenicand pumps one H+ outward and one K* inward. Operation of this pump is discussed later in the chapter.

BacterialPermeases Are ABC proteinsThat lmport a Varietyof Nutrientsfrom the Environment As noted earlier, all members of the very large and diverse ABC superfamily of transport proteins contain two transmembrane (T) domains and two cytosolic ATP-binding (A) domains (Figure 1.1-14).The T domains, each built of 10

Endoplasm

Membrane

Cytoplasm

A FfGURE11-14 Structureof the Escherichia coli BtuCD protein,an ABCtransportermediatingvitamin B12uptake.The complete transporter isassembled fromfoursubunits. two identical membrane-spanning (green), subunrts andtwo ATp-binding subunits (blue). Molecules of cyclotetravanadate, an analogof the phosphate groupsin ATP, arelocated in theATp-binding sitesandaredepicted in ball-and-stick Approxtmate boundaries of the membrane bilayer areindicated bythe grayshaded area,withtheexternal surface at the top andthe cytoplasm at the bottom [AfterK Locher et al, 2002. Sclence 295:1091-98 l

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membrane-spanningcr helices, form the pathway through which the transported substance (substrate)crossesthe membrane and determine the substrate specificity of each ABC protein. The sequencesof the A domains are approximately 30-40 percent homologous in all members of this superfamily,indicating a common evolutionary origin. Some ABC proteins also contain an additional exoplasmic substrate-bindingsubunit or regulatory subunit. The plasma membrane of many bacteria contains numerous permeases that belong to the ABC superfamily. Theseproteins use the energy releasedby hydrolysis of ATP to transport specificamino acids, sugars,vitamins, and even peptides into the cell. Since bacteria frequently grow in soil or pond water where the concentration of nutrients is low, these ABC transport proteins enable the cells to import nutrients against substantialconcentration gradients.Bacterial permeasesgenerally are inducible; that is, the quantity of a transport protein in the cell membrane is regulated by both the concentration of the nutrient in the medium and the metabolic needsof the cell. In the E. coli vitamin B12permease,a typical bacterial ABC protein whose structure is known in molecular detail (seeFigure 11-18), the two transmembranedomains and two cytosolic ATP-binding domains are formed by four separatesubunits.Gram-negativebacteria,such as E. coli, contain, besidesthe plasma membrane, an outer membrane also built of a phospholipid bilayer (seeFigure 1-2). This outer membrane contains porin proteins (seeFigure 10-18) that render it highly permeableto most small molecules, including amino acids and vitamins. Thus these moleculescan enter into the periplasmic spacein between the plasma and outer membranes(seeFigure 1-2). A soluble vitaminBl2-binding protein, located in the periplasmic space, binds vitamin B12 tightly and directs it to the T subunits of the permease, through which the vitamin crossesthe plasma membranepowered by ATP hydrolysis. Precisely how transport occurs is not known, but it is thought that B12 binding is signaledto the nucleotide hydrolysis sites where the affinity for ATP increases,a prerequisite for a productive transport cycle. The two ATPbinding domains then carry out a highly cooperarive ATP-binding and hydrolysis reaction that is concurrent with a substantial conformational change in the membrane-spanning segments;this change somehow allows the bound substrateto pass between the two membranespanning domains into the cell cytoplasm. Then the transporter returns to the resting state through the dissociation of ADP and inorganic phosphate. Mutant E. coli cells that are defectivein any of the 812 permease subunits or the soluble periplasmic-binding protein are unable to transport B12 into the cell but are able to transport other molecules such as amino acids whose uptake is facilitated by other transport proteins. Such genetic analysesprovide srrong evidencethat these permeasesfunction to transport specific solutes into bacterial cells.

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T h e A p p r o x i m a t e l y5 0 M a m m a l i a nA B C TransportersPlay Diverseand lmportant Roles i n C e l la n d O r g a n P h y s i o l o g y Discovery of the first eukaryotic ABC protein to be recognized came from studies on tumor cells and cultured cells that exhibited resistanceto several drugs with unrelated chemical structures.Such cells eventually were shown to express elevated levels of a multidrwg-resistance(MDR) transport protein known as MDR1. This protein usesthe energy derived from ATP hydrolysis to export a large variety of drugs from the cytosol to the extracellularmedium. The Mdrl gene is frequently amplified in multidrug-resistant cells,resulting in a large overproduction of the MDRl pfoteln. Most drugs transported by MDRl are small hydrophobic molecules that diffuse from the medium across the plasma membrane, unaided by transport proteins, into the cell cytosol, where they block various cellular functions. Two such drugs are colchicine and vinblastine, which block assemblyof microtubules (Chapter 18). ATP-poweredexport of such drugs by MDR1 reducestheir concentration in the cytosol. As a result, a much higher extracellular drug concentration is required to kill cells that express MDR1 than those that do not. That MDR1 is an AlP-powered small-moleculepump has beendemonstratedwith liposomes containing the purified protein. The AIPase activity of these liposomesis enhancedby different drugs in a dose-dependent manner corresponding to their ability to be transported by MDR1.

PR()TEIN

About 50 different mammalian ABC transport proteins are now recognized(Table 11-3)' Severalare expressedin abundance in the liver, intestines, and kidney-sites where natural toxic and waste products are removed from the body. Substratesfor these ABC proteins include sugars' amino acids, cholesterol' bile acids, phospholipids, peptides, proteins, toxins, and foreign substances.The normal function of MDR1 most likely is to transport various natural and metabolic toxins into the bile or intestinal lumen or to the urine being formed in the kidney. During the course of its evolution, MDRl appearsto have acquired the ability to transport drugs whose structures are similar to those of these endogenous toxins. Tumors derived from MDRexpressingcell types,such as hepatomas(liver cancers),frequently are resistant to virtually all chemotherapeutic agents and are thus difficult to treat' presumably because the tumors exhibit increasedexpressionof MDR1 or the related MDR2. Severalhuman geneticdiseasesare associatedwith defectiveABC proteins.The best studied is cystic fibrosis (CF), causedby a mutation in the geneencodingthe cysticfibrosistransmembraneregulator (CFTR)' This CI- transport protein is expressedin the apical plasma membranesof epithelial cellsin the lung, sweatglands' pancreas'and other tissues.For instance,CFTR protein is important for resorption of Cl- into cells of sweat glands' and babies with cystic fibrosis, if licked, often taste "salty." An increase in cyclic AMP (cAMP), a small intracellular signalingmolecule,causes

IUNCTION

EXPRESSION TISSUE

ABCB1(MDR1)

Adrenal,kidney,brain

Exportslipophilicdrugs

ABCB4 (MDR2)

Liver

Exportsphosphatidylcholine into bile

ABCB11

Ltver

Exportsbile saltsinto bile

CFTR

Exocrinetissue

TransportsCl ions

ABCDr

inperoxisoma' ::*ffn^1':':irr::ffitT;n.fl"H:ffi;'"'#

PROTTIN BYDETECTIVE CAUSEO DISEASE

Cysticfibrosis

(ADL) Adrenoreukodvstrophv

fatty acids p-Sitosterolemia

ABCG5/8

Liver,instestine

Exportscholesteroland other sterols

ABCA1

Ubiquitous

Exporrscholesteroland phospholipid Tangier'sdisease for uptakeinto high-densitY lipoprotein(HDL)

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phosphorylation of CFTR and stimulates Cl- transport by such cellsfrom normal individuals but not from CF individuals, who have a defectiveCFTR protein. (The role of cAMp in numerous signaling pathways is covered in Chapter 15.) The sequenceand predicted structure of the CFTR protein, basedon analysisof the cloned gene,are very similar to those of the MDR1 protein exceptfor the presenceof an additional domain, the regulatory (R) domain, on rhe cytosolic face. Moreover, the Cl--transporr acrivity of CFTR protein is enhanced by the binding of ATP, but it is uncertain whether or not CFTR is actually an ATP-powered Cl- pump.l

C e r t a i nA B CP r o t e i n s" F l i p " p h o s p h o l i p i d as n d O t h e r L i p i d - S o l u b lS e u b s t r a t e fsr o m O n e MembraneLeafletto the OppositeLeaflet The substratesof mammalian MDR1 are primarily planar, lipid-solublemoleculeswith one or more positive charges; they all compete with one another for transport by MDR1, suggestingthat they bind to the samesite or siteson the Drotein. In contrast to bacterial ABC proteins, all four domains of mammalian MDRl are fused into a single 170,000-M\fl protein. The recently determined three-dimensional structure of a homologous E. coli lipid-transport protein reveals that the molecule is V shaped, with the apex in the membrane and the arms containing the ATP-binding sites protruding into the cytosol (Figure11-15). Although the mechanism of transporr by MDR1 and similar ABC proteins has not been definitively demonstrated, a likely candidate is the flippase model depicted in Figure 11-15. According to rhis model, MDR1 ,,flips,' an amphiphilic subsrraremolecule from the cytosolic to the exoplasmic leaflet, an energericallyunfavorable reaction powered by the coupled ATPaseactivity of the protein. Support for the flippase model of transport by MDRl comes from ABCB4 (originally called MDR2), a homologous protein present in the region of the liver-cell plasma membrane that faces the bile duct. ABCB4 movei ohosphatidylcholinefrom the cytosolicto the exoplasmicleaflet of the plasma membrane for subsequentreleaseinto the bile in combination with cholesreroland bile acids, which themselvesare transported by other ABC family members. Severalother ABC superfamilymembersparticipatein the c e l l u l a re x p o r ro f v a r i o u sl i p i d s( s e ef a U t e t l - : t . ABCB4 was first suspectedof having phospholipidflippase activity becausemice with homozygous loss-of-function mutations in the ABCB4 geneexhibited defectsin rhe secretionof phosphatidylcholineinto bile. To determinedirectly if ABCB4 was in fact a flippase, researchersperformed experiments on a homogeneouspopulation of purified vesicleswith ABCB4 in the membrane and with the cytosolic face directed outward. These vesicleswere obtained by introducing cDNA encoding mammalian ABCB4 into a temperature-sensitiveyeasrsec mvtant. At lower, permissivetemperatureswhere the secproteinis functional, the ABCB4 protein expressedby the transfected

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ADP+ P, A FIGURE 11-15Flippase modelof transportby MDRl and similarABCproteins.Stepll: Thehydrophobic portion(black) of a substrate molecule movesspontaneously fromthecytosol intothe cytosolic-facing leaflet of the lipidbilayer, whilethecharged end(red) remains in thecytosolStepE: Thesubstrate diffuses laterally untilit encounters andbindsto a siteon the MDRlproteinwithinthe bilayer. StepS: Theproteinthen"flips"thecharged substrate molecule into theexoplasmic leaflet, an energetically unfavorable reaction involving conformational changes in the membrane-spanning domains thatare powered bythecoupled hydrolysis of ATpbythecytosolic domains Steps4 andE: Oncein theexoplasmic face,thesubstrate againcan diffuselaterally in the membrane andultimately movesintothe phaseon theoutside aqueous of thecell [Adapted fromp Borst, N Zelcer,and A van Helvoort,2000, Biochim Biophys Acta 14g5.12g,1

cellsmovesnormally through the secretorypathway to the cell surface(Chapter 1,4).At higher nonpermissiveremperarures, however,the secprotein is nonfunctionaland secretoryvesicles cannot fusewith the plasmamembrane,as they do in wild-type cells; so vesiclescontaining ABCB4 and other yeasrprorerns accumulatein the cells.After purifying thesesecretoryvesicles, investigatorslabeled them in vitro with a fluorescentphosphatidylcholine derivative. The fluorescence-quenchingassay outlined in Figure 11-16 was usedto demonstrarethat the vesicles containing ABCB4 exhibited an AlP-dependent flippase activity, whereasthose without ABCB4 did not.

ATP-PoweredPumps and the Intracellular lonic Environment r classesof transmembraneproteinscouple the energying hydrolysis of AIP with the energy-requiringtransport of substancesagainst their concentrationgradient: p-, V-, and F-classpumps and ABC proteins (seeFigure 11-9). r The combined action of P-classNa*/K* ATpasesin the plasma membrane and homologous Ca2* Mpases in the

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Externallabeledlipids: u nprotected,

ATPaSe

ABC84 (flippase)

u nprotected, a l l l a b e l e dl i p i d s quenched

Transmembrane domain

ADP

ADP

Add ATP

o q) c c) o 0) th o) u

T i m e( m i n ) 11-16ln vitro fluorescenceFIGURE a EXPERIMENTAL quenchingassaycan detect phospholipidflippaseactivity of population vesicles containing of secretory ABCB. A homogeneous with f roma yeastsecmutanttransfected ABC84proteinwaspurified phospholipids a gene containing Step Synthetic ABCB4 Ihe [: primarily modifiedheadgroup(blue)wereincorporated fluorescently vesicles StepZ: lf leaflets of the purified intotheouter,cytosolic of of ATPto the outside thenon addition ABC84actedasa flippase, labeled a smallfractionof the outward-facing thevesrcles, StepB: Flipping phospholipids wouldbe flippedto the insideleaflet. quenching by addinga membrane-impermeable wasdetected thevesicles to the mediumsurrounding calleddithionite compound its headgroup,destroying reacts with thefluorescent Dithionite

only (gray). of the quencher, Inthe presence abilityto fluoresce the Inner on protected environment in the phospholipid labeled to the additionof the quenching Subsequent leafletwillfluoresce at with timeuntilit plateaus decreases agent,thetotalfluorescence the quenched only and is fluorescence the pointat whichallexternal Theobservatton canbe detected. phospholipid fluorescence internal presence of ATPthan (less quenching) in the greater fluorescence of thatABC84hasflippedsomeof the labeled indicates in itsabsence to the phospholipid to the insideStep4: Addltionof detergent lipids accessible all fluorescent makes and generates micelles vesicles to baseline fluorescence the lowers and quenching agent to the Cell771071] andP Gros,1994, fromS Ruetz valueslAdapted

plasma membraneor sarcoplasmicreticulum createsthe usual ion milieu of animal cells: high K*, low Ca2*, and low Na* in the cytosol; low K+, high Ca2*, and high Na* in the extracellular fluid.

r V- and F-classATPases,which transport protons exclusively, are large, multisubunit complexes with a Protonconiucting channel in the transmembrane domain and ATP-binding sitesin the cytosolic domain. ss H* pumps in animal lysosomal and endosomal anes and plant vacuole membranesare responsible intaining a lower pH inside the organellesthan in the surroundingcytosol (seeFigure 11-13b).

r In P-classpumps, phosphorylation of the a (catalytic) subunit and a changein conformational statesare essential for coupling ATP hydrolysis to transport of H+, Na*, K*, or Ca2* ions (seeFigures 11-10, L1.-1.L,and 1'1-12).

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r AII members of the large and diverse ABC superfamily of transport proteins contain four core domains: two transmembrane domains, which form a pathway for solute movement and determinesubstratespecificity,and two cytosolic ATP-binding domains (see Figures 11-14 and 11-15). r The ABC superfamily includes bacterial amino acid and sugar permeasesand about 50 mammalian proteins (e.g., MDR1, ABCA1) that transport a wide array of substrates, including toxins, drugs, phospholipids, peptides, and proteins, into or out of the cell. r According to the flippasemodel of MDR activity,a substrate molecule diffuses into the cytosolic leaflet of the plasma membrane, then is flipped to the exoplasmic leaflet in an ATP-powered process,and finally diffuses from the membrane into the extracellularspace(seeFigure11-15). r Biochemical experiments directly demonstrate that ABCB4 (MDR2) possessesphospholipid flippase activity (seeFigure 11-16).

Nongatedlon Channels andthe RestingMembranePotential In addition to ATP-powered ion pumps, which transport ions againsl their concentration gradients, the plasma membranecontains channelproteins that allow the principal cellular ions (Na+, K+, Ca2+, and Cl-) to move through them at different rates down their concenrrarron gradients.Ion concentrationgradientsgeneratedby pumps and selectivemovementsof ions through channelsconstitute the principal mechanism by which a difference in voltage, or electric potential, is generatedacross the plasma membrane.In other words, ATp-powered ion pumps generate differencesin ion concentrationsacross the plasma membrane, and ion channels utilize these concentration gradients to generatean electric potential across the memb r a n e ( s e eF i g u r e I 1 - 2 ) . In all cells the magnitude of this electric potential generally is -70 millivolts (mV) with the inside of the cell membrane always negatiuewith respectto the outside.This value does not seemlike much until we consider rhat the thickness

The ionic gradients and electric potential across the plasma membrane play crucial roles in many biological processes.As noted previously,a rise in the cytosolic Ca2+ concentration is an important regulatory signal, initiating contraction in musclecells and triggering protein secretion 458

.

by many cells, such as that of digestive enzymes in exocrine pancreatic cells. In many animal cells, the combined force of the Na* concentration gradient and membrane electric potential drives the uptake of amino acids and other molecules against their concentration gradient by ion-linked symport and antiport proteins (seeSection 11.5). Furthermore,the conduction of action potentials by nerve cells depends on the opening and closing of ion channelsin responseto changesin the membranepotential ( C h a p t e r2 3 ) . Here, we discussthe origin of the membrane electric potential in resting cells, often called the cell's "resting potential"; how ion channels mediate the selectivemovement of ions acrossa membrane;and useful experimentaltechniques for characterizing the functional properties of channel protelns.

SelectiveMovement of lons Createsa Transmembrane ElectricPotentialDifference To help explain how an electric potential across the plasma membrane can arise, we first consider a set of simplified experimental systems in which a membrane separatesa 150 mM NaCl/15 mM KCI solution (similar to the extracellular medium surrounding metazoan cells) on the right from a 15 mM NaCl/150 mM KCI solution (similar to that of the cytosol) on the left. A potentiometer (voltmeter) is connectedto both solutions to measure any difference in electric potential across the membrane. If the membrane is impermeable to all ions, no ions will flow across it. There will be no difference in voltage, or electric potential gradient acrossthe membrane,as shown i n F i g u r e1 1 - 1 7 a . Now supposethat the membrane contains Na*-channel proteins that accommodateNa+ ions but exclude K+ and Cl- ions (Figure 11-17b). Na* ions then tend to move down their concentration gradient from the right side to the left, leaving an excessof negative Cl- ions compared with Na- ions on the right side and generating an excessof positive Na- ions compared with Cl- ions on the left side. The excessNa* on the left and Cl on the right remain near the respectivesurfacesof the membrane becausethe excesspositive chargeson one side of the membrane are attracted to the excessnegative chargeson the other side. The resulting separation of charge across the membrane constitutes an electric potential, or voltage, with the left (cytosolic) side of the membrane having excesspositive charge with respectto the right. As more and more Na* ions move through channels across the membrane, the magnitude of this charge differ-

point at which the two opposing factors that determine the

cHAprE1 R1 | T R A N S M E M B RTARNAEN s p o R o FT t o N sA N D S M A L LM O L E C U L E S

( a ) M e m b r a n ei m p e r m e a b l et o N a + ,K + ,a n d C l 0 Potentiometer-

Extracellular medium

Cell cytosol

Cytosolic face

1 5 0m M Na*Cl-

1 5m M Na'Cl1 5 0m M K+Cl-

1 5m M K*Cl-

Exoplasmic face

( b ) M e m b r a n ep e r m e a b l eo n l Y t o N a * 0 +60

Membrane electric Potential = +59 mV, cytosolic face of the membrane positive with respectto the exoplasmicface

IG < E X P E R I M E N TFA L U R1E1 - 1 7G e n e r a t i oonf a potential(voltage)dependson the electric transmembrane selectivemovementof ionsacrossa semipermeable a separates a membrane system, membrane.Inthisexperimental '1 mM NaCl/1 5 (/eft) 50 a from mM KCIsolution 15 mM NaCl/150 to those aresimilar (flqhf);theseionconcentrations mM KCIsolution the separating lf the membrane andblood,respectively in cytosol to allions(a),no ionscanmoveacross is impermeable two solutions on isregistered potential in electflc andno difference the membrane is membrane the lf solutions two the connecting the potentiometer (c), of diffusion (b) then K+ or to Na+ to permeable only selectively of charge leadsto a separation channels ionsthroughtheirrespective potential the membrane At equilibrium, the membrane across equalto the Nernst becomes bythechargeseparation caused Seethetextfor potentiometer on the Ex"or E6regtstered potential explanation further

movement of Na* ions-the membrane electric potential and the ion concentration gradient-balance each other out' At equilibrium, no net movement of Na- ions occurs across the membrane. Thus this semipermeablemembrane, like all

store positive chargeson one side and negative chargeson the other. Na* channel

chemistry: Chargeseparationacrossmembrane

EN':

RT. [Na,] l" zF tNurl

(1.1,-2)

( c ) M e m b r a n ep e r m e a b l eo n l Yt o K + 0

Membrane electric Potential = -59 mV, cytosolic face of the membrane negative with respectto the exoplasmicface.

K+channel

equation is written with the concentrationof ion in the extracellular solution (here the right side of the membrane) placed in the numerator and that of the cytosol in the denomrnator. At 20'C, Equation 11-2 reducesto

INu'] E r . r:, 0 . 0 5 t9o s ' o ; 5 u , l

(11-3)

C h a r g es e p a r a t i o na c r o s sm e m b r a n e M E M B R A N EP O T E N T I A L AND THE RESTING N O N G A T E DI O N C H A N N E L S

O

459

If [Na,]/[Na1] : 10, a tenfold ratio of concenrrarionsas in Figure 11-17b, rhen El.l. : +0.059 V (or +59 mV). with the left, cytosolic side positive with respectto the exoplasmic right side. If the membraneis permeableonly to K* ions and not to Na* or Cl- ions, then a similar equationdescribesthe potassium equilibrium potential E11:

Although restingK* channelsplay the dominant role in

ton pumps (seeFigure 11-1,3a).In aerobic bacterial cells the inside negarive potential is generated by outward pumping of protons during electron transporr, a process ( 1 1 - 4 ) similar to proton pumping in mitochondrial inner membranes that will be discussedin detail in Chapter 12 (see Figure 12-16). The magnitude of the membrane electric potential is the The potential acrossthe plasma membrane of large cells same (59 mV for a tenfold difference in i,on concenrrations), except that the left, cytosolic side is now negatrue can be measured with a microelectrode inserted inside the cell and a reference electrode placed in the extracellular with respect to the right (Figure 11-17b), opposite io the fluid. The two are connectedto a potentiometer capable of polarity obtained acrossa membraneselectiveiypermeable measuringsmall potential differences(Figure 11-1g). The to Na* ions. potential acrossthe surface membrane of most animal cells generally does not vary with time. In contrast, neurons and T h e M e m b r a n ep o t e n t i a li n A n i m a l C e l l s muscle cells-the principal types of electricallyactive cellsundergo controlled changesin their membrane potential that DependsLargelyon potassiumlon Movements we discussin Chapter 23. T h r o u g hO p e n R e s t i n gK + C h a n n e l s

:0.059loSroffi EN"

The plasma membranesof animal cellscontain many open K+ channelsbut few open Na*, Cl , or Ca2* channels.es

Potentiometer

ions through thesechannels,called resting K+ channels,is the major determinant of the inside-negativemembrane p o t e n t i a l . L i k e a l l c h a n n e l s ,t h e s e a l r e r n a t eb e t w e e n a n Microelectrode f i l l e dw i t h conducting s a l ts o l u t i o n

Quantitatively,the usual resting membranepotential of -70 mY is closero the potassiumequilibrium potential,cal_ culated from the Nernst equation and the K+ concentrations in cellsand surroundingmedia depictedin Table 11_2.Usu_ ally the potential is lower than ihat calculated from the

gradient that drives the flow of ions through resting K+ channelsis generatedby the Na+/K* ATpase describeJore_ viously (seeFigures 11-2 and 11-12). In the absenc. of thi, pump, or when it is inhibited, the K+ concenrrationgradient cannot be maintained, and eventually the magnitude of the membrane potential falls to zero.

460

C H A P T E R1 1

|

Referenceelectrode in contactwith b a t h i n gm e d i u m

Bathing medium +

+**++++

+++++++

Cytosol P l a s m am e m b r a n e

EXPERIMENTAL FIGURE 11-18The electricpotentialacross the plasmamembraneof living cellscan be measured.A microelectrode, constructed by fillinga glasstubeof extremely small d i a m e t ewr i t ha c o n d u c t i nf lgu i ds u c ha sa K C Is o l u t i o ni s, i n s e r t e d intoa cellin sucha waythatthe surface membrane sealsitself aroundthe tip of the electrode. A reference electrode ls placedin the bathingmedium. A potentiometer connectinq thetwo electrodes registers the potential, in thiscase-60 mV A potential difference is registered onlywhenthe microelectrode is inserted into the cell;no potential is registered if the microelectrode is in the b a t h i n fgl u i d

T R A N S M E M B R A NTER A N S P O R O T F | O N SA N D S M A L L M O L E C U L E S

( a ) S i n g l es u b u n i t

( b )T e t r a m e r i c h a n n e l filter

Exterior

Membrane

Cytosol

11-19Structureof restingK* channelfrom the FIGURE lividans.All K* channelproteinsare bacteriumStreptomyces two eachcontaining subunits, fouridentrcal comprising tetramers 55 cthelices, calledby convention membrane-spanning conserved (pink)(a)Oneof anda shorterB or poresegment and56 (yellow), features viewedfromtheside,with keystructural thesubunits, (b)Thecomplete viewedfromtheside channel tetrameric indicated. are (/eft)andthetop,or extracellular, end(nqht)ThePsegments the 55 and56 andconnect surface neartheexoplasmic located

"turret,"whichlinesthe upper of a nonhelical theyconsist a helices; loopthat protrudes extended an and partof the pore;a shorto helix; filter. ion-selectivity the pore forms and part of the intothe narrowest but nototherionsto pass'Below spheres) ThisfilterallowsK* (purple linedbythe inner,or 56 ct, thef ilteristhecentralcavityor vestibule whichopenandclosein gated K* channels, in helixesThesubunits helices transmembrane additional contain stimuli, to specific response etal, Y Zhou 23 in Chapter [See discussed are these here; shown not 414:43 2001,Nature l

l o n C h a n n e l sC o n t a i na S e l e c t i v i t yF i l t e r Formedfrom ConservedTransmembrane Segments

Severalrelated piecesof evidencesupport the role of P segmentsin ion selection.First, the amino acid sequenceof thI P segmentis highly homologousin all known K* chandifferent from that in other ion channels' Second' .r.1, "nJ

All ion channels exhibit specificity for particular ions: Kchannelsallow K* but not closely related Na* ions to enter' whereasNa* channelsadmit Na* but not K+. Determination of the three-dimensionalstructure of a bacterial K* channel first revealed how this exquisite ion selectivity is of other K+, Na+, achieved.Comparisonsof the sequences and Ca2* channelsestablishedthat all suchproteinssharea common structure and probably evolved from a single type of channelprotein. Like alf other K+ channels,bacterial K+ channelsare built of four identical subunits symmetrically arranged Each subunit contains around a central pore (Figure 1'1'-1'9)' ct helices(55 and 56) and a short P two membrane-spanning (pore domain) segmentthat partly penetratesthe membrane bilayer. In the tetrameric K* channel, the eight transmembrane a helices(two from each subunit) form an "inverted tepee," generating a water-filled cavity called the uestibule in the central portion of the channel that extends halfway through the membrane.Four extendedloops that are p^rt of the four P segmentsform the actual ion-selectiuityfilter in the narrow part of the pore near the exoplasmicsurface, above the vestibule.

other ions. Na* ions are smaller than K* ions. How, then' can a channel protein exclude Na* ions, yet allow passageof K*? The ability of the ion-selectivityfilter in K* channels to selectK* over Na* is due mainly to backbonecarbonyl oxygens on residues located in a Gly-Tyr-Gly sequence thaiis found in an analogousposition in the P segmentin

M E M B R A N EP O T E N T I A I SN D T H E R E S T I N G N O N G A T E DI O N C H A N N E L A

461

( a ) K * a n d N a +i o n s i n t h e p o r e o f a K +c h a n n e l( t o o v i e w ) K+in water

Na+in water

K* in K pore

Naiin K pore

( b ) K * i o n s i n t h e p o r e o f a K +c h a n n e l( s i d ev i e w )

oo Exoplasmic Tace

o o o o o

E

ot , t

| ..^' Carbonyl oxygens t '4j

Vestibule-.-

aK+

( c ) l o n m o v e m e n tt h r o u g hs e l e c t i v i t fyi l t e r

oooo

o

cooo

c State 1

C H A P T E R1 1

through the channel.A dehydratedNa* ion is too small to bind to all eight carbonyl oxygens that line the selectivity filter with the samegeometry as a Na+ ion surrounded by its normal eight warer molecules.As a result, Na* ions would "prefer" to remain in water rather than enter the

oooo

COO

462

< FIGURE 11-20Mechanism of ion selectivityand transportin (a)Schematic restingK+ channels. diagrams of K+ andNa+ions hydrated in solution andin the poreof a K+channelAs K+ ions passthroughthe selectivity filter,theylosetheirboundwater molecules andbecome coordinated instead to eightbackbone carbonyl oxygens, fourof whichareshown,thatarepartof the conserved aminoacidsin thechannel-lining loopof eachp segment Thesmaller Na* ionswith theirtightershellof watermolecules cannotperfectly coordinate with thechannel oxygenatomsand passthroughthechannel therefore onlyrarely(b)High-resolution electron density mapobtained fromx-raycrystallography showing K+ ions(purple passing spheres) throughtheselectivity filter.Only two of thediagonally opposed channel subunits areshown.Within theselectivity filtereachunhydrated K+ ionrnteracts with eight carbonyl oxygenatoms(redsticks) liningthe channel, two from eachof thefoursubunits, asif to mimictheeightwatersof hydration(c)Interpretation of the electron density mapshowing thetwo alternating statesbywhichK+ ionsmovethroughthe channelIn state1, movingfromtheexoplasmic sideof thechannel Inward, oneseesa hydrated K* ionwith itseightboundwater molecules, K- ionsat positions 1 and3 withintheselectivity filter, anda fullyhydrated K+ ionwithinthevestibule DuringK+ movement eachionin state1 movesonestepinward,formingstate 2 Thusin state2 the K+ ionon theexoplasmic sideof thechannel haslostfourof itseightwaters, the ionat position1 in statet has movedto posltion 2, andthe tonat position 3 in statet hasmoved to position 4 In goingfromstate2 to state1,the K+ at position 4 movesintothevestibule andpicksup eightwatermolecules, while anotherhydrated K* ionmovesintothechannel openingandthe otherK' ionsmovedownonestep.lpart(a)adapted fromC.Armstrong, parts 1998, (b)and(c)adapted Science280:56 fromy Zhouetal,2001. Nature414.43l

State 2

|

lnreracr properly with the O atoms in the selectivityfilter. Also, more energyis required to strip the waters of hydra_ tion from Ca2* than from K+. Recent x-ray crystallographicstudies reveal that both when open and closed, the channel contains K+ ions within the selectivityfilter; without theseions the channel probablv would collapse.The K+ ions are thought to be presenteither at positions 1 and 3 or at2 and 4, each surrounded by eight carbonyl oxygen atoms (Figure 1I-20b and c). Severalk* ions move simultaneouslythrough the channel such that when the ion on the exoplasmic face that has been partiallv stripped of its water of hydration moves into position 1, th! ion at position 2 jumps to position 3 and the one at position 4 exits the channel (Figure l1-20c). Although the amino acid sequencesof the p segmentin Nat and K* channels differ somewhat, they are similar

TRANSMEMBRAN TE R A N S P O ROTF I O N SA N D S M A L LM O L E C U L E S

11-21Currentflow through FIGURE > EXPERIMENTAL individualion channelscan be measuredby patch-clamping for measuring experimental arrangement technique.(a)Basic in the plasma ionchannels currentflowthroughindividual filledwith a currentof a livingcell.Thepatchelectrode, membrane to the with a slightsuction, isapplied, salinesolution, conducting a regionthat plasma tip covers The0 5pm-diameter membrane. is Thesecond electrode onlyoneor a few ionchannels. contains device intothe cytosolA recording throughthe membrane inserted in the patchof currentflow onlythroughthechannels measures plasma of the cellbodyof a membrane(b)Photomicrograph thecell neuronandthetip of a patchpipettetouching cultured (c)Different lsolated, patch-clamping configurations. membrane, theeffects for studying patches arethe bestconfigurations detached as solutes such and ion concentrations of different channels on (e g , messengers second hormones andintracellular extracellular (Nobel also (b)fromB Sakmann, lecture); 8:613 1992, Neuron cAMP). IPart Parl(c\ SciAm 266(3):44 published andB Sakmann,1992, inE Neher

Deviceto maintainconstant voltage acrossmembraneand to measurecurrentflow across membraneat tip of Patch electrode

Patchelectrodefilled with conductingsalt solution

adaptedfrom B Hille, 1992, lon Channelsof ExcitableMembranes,2d ed , S i n a u eA r s s o c i a t e sp, 8 9 l

enough to suggestthat the general structure of the ionselectivity filters are comparable in both types of channels. Presumablythe diameter of the filter in Na- channelsis small enough that it permits dehydratedNa* ions to bind to the backbone carbonyl oxygens but excludesthe larger K+ ions from entering,but as yet no three-dimensionalstructure of a Na* channelis available.

(c)

Tip of micropiPette

---/

PatchClampsPermit Measurementof lon M o v e m e n t sT h r o u g hS i n g l eC h a n n e l s The technique of patch clamping enablesworkers to investigate the opening,closing,regulation,and ion conductance of a single ion channel. In this technique, the inward or outward movement of ions across a patch of membrane is quantified from the amount of electric current needed to maintain the membranepotential at a particular "clamped" value (Figure 11'-21,aand b). To preserveelectroneutrality and to keep the membranepotential constant' the entry of each positive ion (e.g.,a Na* ion) into the cell through a channel in the patch of membraneis balancedby the addition of an electron into the cytosol through a microelectrode inserted into the cytosol; an electronic device measures the numbers of electrons (current) required to counterbalancethe inflow of ions through the membrane channels. Conversely,the exit of each positive ion from the cell (e.g., a K+ ion) is balancedby the withdrawal of an electron from the cytosol. The patch-clamping technique can be employed on whole cells or isolated membrane patches to measure the effects of different substancesand ion concentrations on ion flow (Figure 11-21c). The patch-clamp tracings in Figure 1'1-22 rllustratethe use of this techniqueto study the propertiesof voltage-gatedNachannelsin the plasma membraneof musclecells.As we discussin Chapter 23, thesechannelsnormally are closedin resting muscle cells and open following nervous stimulation.

lon channel

On-cellpatch measuresindirecteffectof extracellularsolutes on channelswithin membranepatch on intactcell Cytosolic face

Exoplasmic face :.:

Inside-outdetachedpatch measureseffectsof intrasnchannels c e l l u l asro l u t e o withinisolatedpatch

Outside-outdetachedpatch measureseffectsof extras nchannels c e l l u l asro l u t e o withinisolatedpatch

Patchesof muscle membrane, each containing one Na* channel, were clamped at a voltage slightly less than the resting membrane potential. Under these circumstances'translent pulsesof .ot..nt crossthe membraneas individual Na- chan.r.ls op.tt and then close. Each channel is either fully open or completelyclosed' From such tracings' it is possibleto determine the iime that a channel is open and the ion flux through it. For the channelsmeasuredin Figure 11-22,the flux is about 10 million Na* ions per channelper second'a typical value for ion channels. Replacement of the NaCl within the patch pipette (corresponding to the outside of the cell) with KCI or .ttttin. chloride abolishescurrent through the channels,confirming that they conduct only Na+ ions, not K* or other ions'

M E M B R A N EP O T E N T I A L AND THE RESTING N O N G A T E DI O N C H A N N E L S

O

463

Open

Closed

1 0m s

T 5.0 oA l

I

I

M i c r o i n i e cm t RNA encodingchannel protein of interest Plasma membrane

Twoinside-out patches of muscle plasma membrane wereclamped at a potential of slightly lessthanthatof the restinqmembrane potential. Thepatchelectrode contained NaCl.Thetransient pulses of electric currentin picoamperes (pA),recorded aslargedownward (bluearrows), deviations indicate theopeningof a Na* channel and movement of Na* ionsinwardacross the membrane Thesmaller deviations in currentrepresent background noiseTheaverage c u r r e n t t h r o ua gn ho p e nc h a n n ei sl 1 . 6p A ,o r 1 . 6x 1 0 1 2a m p e r e s . '1 Since ampere: 1 coulomb(C)of chargepersecond, thiscurrentis equivalent to the movement of about9900Na+ionsperchannel oer m i l l i s e c o n( 1 d. :6 x 1 0 ' 2 C / s ) ( 1 0 -s3/ m s ) (x6 1 0 2 3 m o l e c u l e s / m+o l ) 96,500C/mol [SeeF.J Sigworth andE Neher, 1980,Narure 287:44]l

p

lncubate24-48hfor svnthesisand m o v e m e n to f c h a n n e proteinto plasma memorane

M e a s u r ec h a n n e l -

Newly synthesized channel protein

Patchelectrode

E 3:?:f,',-:,:flJil"" t e c hn i o u e

N o v e ll o n C h a n n e l sC a n B e C h a r a c t e r i z e d by a Combinationof Oocyte Expressionand P a t c hC l a m p i n g

A EXPERIMENTAL FIGURE 11-23Oocyteexpression assayis usefulin comparingthe functionof normaland mutantforms of a channelprotein.A follicular frogoocyteisfirsttreatedwith collagenase to remove thesurrounding folliclecells,leaving a denuded oocyte, whichis microinjected with mRNAencoding the channelproteinunderstudy.lAdapted fromT.p Smith, 1988,Irends Neurosci 11:250 l

At the concentrationsof Na6 and Nao,, shown in Figure 11-24, which are typical for many mammalian cells, AG., the change in free energy due to the concentration gradient, is - 1.45 kcal for transporr of 1 mol of Na+ ions from outside to inside the cell, assumingthere is no membrane electric potential. Note the free energy is negative, indicating ,porrt"neous movement of Na+ into the cell. The free-energychange generated from the membrane electric potenrial is given by

N a + E n t r yi n t o M a m m a l i a nC e l l sH a sa N e g a t i v e Changein FreeEnergy(AG) As mentioned earlier, two forces govern the movement of rons across selectivelypermeablemembranes:the voltage and the ion concentration gradient across the membrane. The sum of theseforces,which may act in the samedirection or rn opposite directions,constitutesthe electrochemicalgra_ dient. To calculatethe free-energychangeAG corresponJng to the transport of any ion across a membrane, we need to consider the independentcontributions from each of the forces to the electrochemicalgradient. For example, when Na* moves from outside to inside the cell, the free-energychangegeneratedfrom the Na+ concentration gradient is given by

AG.:RThm 464

.

cHAprER 11

|

AG-: Pg

(r1-6)

where F is the Faraday constant and E is the membrane electric potential. lf E = -70 mV, then AG-, the free-energychangedue to the membranepotential,is - 1.61 kcal for transpoft of 1 mol of Na* ions from outside to inside the cell, assumingthere is no Na+ concentration gradient. Sinceboth forces do in fact act on Na- ions, the total AG is the sum of the two partial values: AG : AG. + AG- : (-1.45) + (-1.61):

-3.0d kcaVmole

In this example, the Na+ concentration gradient and the

( 11 - s )

T R A N S M E M B R A NTER A N S e o R To F t o N s A N D S M A L LM O L E C U L E S

EnergyInterconversions @ Ou"ruiewAnimation:Biological forcesactingon Na* ions'As 11-24Transmembrane > FIGURE the plasma of Na* ionsacross with all ions,the movement forces-the ion governed separate the sum of two is by membrane potentialAt the gradient electric andthe membrane concentration cells, typicalof mammalian Na+concentrations andexternal internal makingthe inward actin thesamedirection, theseforcesusually favorable. of Na* ionsenerqeticallv movement

Inside

Outside

lnside

Outside + +

1 4 5m M N a +

1 2m M N a *

-70 mV

Na+

LG" = -1.45 kcal/mol

or out of animal cells. The rapid, energetically favorable movement of Na+ ions through gated Na* channelsalso is critical in generating action potentials in nerve and muscle cells, as we discussin Chapter 23.

M e m b r a n ee l e c t r i c potential

lon concentration gradient

acm =

kcal/mol

Free-energychangeduring transport of Na* from outsideto inside Inside

Outside Na+ -70 mV

Nongated lon Channelsand the Resting Membrane Potential r An inside-negativeelectric potential (voltage) of 50-70 mV exists acrossthe plasma membrane of all cells. r In animal cells, the membrane potential is generatedprimarily by movement of cytosolic K* ions through resting K* channelsto the external medium. Unlike the more common gated ion channels,which open only in responseto various signals, these nongated K+ channels are usually open. r In plants and fungi, the membrane potential is maintained by the ATP-driven pumping of protons from the cytosol to the exterior of the cell. r K* channelsare assembledfrom four identicalsubunits, each of which has at least tlvo conservedmembrane-spanning cr helices and a nonhelical P segment that lines the ion pore and forms the selectivity filter (seeFigure 11-19). r The ion specificity of K* channel proteins is due mainly to coordination of the selectedion with the carbonyl oxygen atoms of specific amino acids in the P segments,thus lowering the activation energy for passageof the selected K* compared with other ions (seeFigure 1'1'-20).

AG = AGc+ AG. = -3.66 kcal/mol

and CotransPortbY SYmPorters Antiporters In previous sectionswe saw how ATP-powered pumps generate ion concentration gradients across cell membranesand how ion channel proteins use these gradients to establish an electric potential acrossthesemembranes.In this section we seehow cotransporters usethe energy stored in the electric potential and conientration gradients of Na+ or H* ions to power the uphill moYement of another substance,which may te a small organic moleculesuch as glucoseor an amino acid or a different ion (seeFigure 1'1,-2)-Fot instance' the ener-

r Patch-clamping techniques,which permit measurement of ion movementsthrough single channels,are used to determine the ion conductivity of a channel and the effect of various signalson its activity (seeFigure 11-21,). r Recombinant DNA techniquesand patch clamping allow the expressionand functional characterization of channel proteins in frog oocytes (seeFigure 11-23). r The electrochemical gradient across a semipermeable membrane determines the direction of ion movement through channel proteins. The two forces constituting the electrochemicalgradient, the membrane electric potential and the ion concentration gradient, may act in the same or opposite directions (seeFigure 11-24).

Cotransporters share common features with uniporters such as the GLUT proteins. The two types of transporters exhibit certain structural similarities' operate at equiva-

tration gradient. C O T R A N S P O RBTY S Y M P O R T E RASN D A N T I P O R T E R S

465

I7hen the transported molecule and cotransported ion move in the same direction, the process is called symport; when they move in opposite directions, the processis called antiport (seeFigure 11-3). Some cotransporrersrransporr only positive ions (cations), while others transport only negativeions (anions).An important example of a cation

plasma membrane.Yet other cotransportersmediatemovement of both cations and anions together. Cotransporters are presentin all organisms,including bacteria,plants, and animals, and in this section we describe the operation and function of several physiologically importanr symporrers and antiporters.

Na--LinkedSymporterslmport Amino Acids a n d G l u c o s ei n t o A n i m a l C e l l sA g a i n s tH i g h ConcentrationGradients Most body cells import glucose from the blood down the concentration gradient of glucose, utilizing GLUT proteins to facilitate this transport. However, certain cells, such as those lining the small intestine and the kidney tubules, need to import glucosefrom the intestinal lumen or forming urine againsta very large concentrationgradient. Such cells utilize a two-Na+ /one-glucosesymporter,;protein that couplesimport of one glucosemoleculeto the import of two Na* ions: 2 Na+o,. * glucoseout s

2 Na+;, * glucose1,

QuantitativelS the free-energychange for the symporr rransport of two Na* ions and one glucosemoleculecan be written

lslucose,.I

f Na,Il

lgtucoseo,,l

[NaJ",]

AG: RTln.fr

+ 2RTlnffi

+ zF E ( 11_7)

Thus the AG for the overall reaction is the sum of the free-energy changesgenerated by the glucoseconcentration gradient (1 molecule transported),the Na* concentrationgradient (2 Na+ ions transported),and the membrane potential (2 Na+ ions transported). As illustrated in Figure '1.'J.-24, the free energy released by movement of Na+ into mammalian cells down its electrochemical gradient has a free-energy change AG of about -3 kcal per mole of Na* transported.Thus the AG for transport of two moles of Na* inward is about -6 kcal. This negative free energy of sodium import is coupled to the uphill transport of glucose, a processwith a positive AG. 'We can calculate the glucose concentration gradient, inside greater than outside, againstwhich glucosecan be transported by realizing that at equilibrium for sodium-coupled glucose import, AG : 0. By substituting the values for sodium import into Equation 11-7 and sening AG : 0, we seethat

o : ^r,n_!4!!o'.,"L_ 6kcal Lglucoseourl

and we can calculatethat at equilibrium, the ratio of glucose;,/ glucoseo,,: :30,000. Thus the inward flow of two moles of Na* can generatean intracellular glucoseconcentration that is -30,000 times greater than the exterior concentration. If only one Na* ion were imported (AG of -3 kcal/mol) per glucose molecule, then the available energy could generate a glucose concentration gradient (inside > outside) of only about 1,70-fold.Thus by coupling the transport of two Na+ ions to the transport of one glucose, the two-Na*/one-glucose symporter permits cells to accumulate a very high concentration of glucoserelative to the external

Oveview Animation: BiologicalEnergyInterconversions Exrenol

2Na+o a

OGlucose

++ Inward-facing conformation

FIGURE 11-25 Operationalmodel for the two-Na+/one_ glucosesymporter.Simultaneous bindingof Na+andglucose to the conformation with outward-facing bindingsites(stepIl) causes a conformational changein the proteinsuchthatthe boundsubstrates aretransiently occluded, unableto dissociate intoeithermedium(stepZ) In stepEt the proteinassumes a

466

C H A P T E R1 1

I

Outward-facing conformation

thirdconformation with inward-facing sitesDissociation of the boundNa* andglucose (stepB) allowsthe protern intothecytosol to revertto itsoriginaloutward-facing (step[), ready conformation to transportadditional physiology substrate[SeeE Wrightet.at,2OO4, 19:370 for detailson the structureand function of thrs and related transportersl

T R A N S M E M B R A NTER A N S P O R O T F I O N SA N D S M A L L M O L E C U L E 5

concentration. This means that glucosepresent even at very low concentrations in the lumen of the intestine or in the forming urine can be efficiently transported into the lining cells and not lost from the body. The two-Na+/one-glucose symporter is thought to contain L4 transmembrane a helices with both its N- and Ctermini extending into the cytosol. A truncated recombinant protein consisting of only the five C-terminal transmembrane o helices can transport glucose independently of Na* across the plasma membrane, down its concentration gradient. This portion of the molecule thus functions as a glucose uniporter. The N-terminal portion of the protein, including helices 1-9, is required to couple Na+ binding and influx to the transport of glucoseagainst a concentration gradient. Figure 11-25 depicts the current model of transport by Na*/glucose symporters.This model entails conformational changesin the protein analogousto those that occur in uniport transporters, such as GLUT1, which do not require a

cotransportedion (seeFigure 11-5). Binding of all substrates to their sites on the extracellular domain is required before the protein undergoesthe conformational change that transitions the substrate-bindingsites from outward to inward facing; this ensures that inward transport of glucose and Na* ions are coupled.

BacterialSymporterStructureRevealsthe Mechanismof SubstrateBinding No three-dimensional structure of a mammalian sodium symporter has been determined' but the structuresof several homologous bacterial sodium-amino acid transporters have provided considerable information about symport function. The bacterial two-Na*/one-leucine symporter shown in Figure 1'1'-26aconsistsof 12 membrane-spanning a helices.Two of the helices (numbers 1, and 6l have nonhelical segmentsin the middle of the membrane that form part of the leucine-bindingsite.

fT

!l

Symporter eod.ust:The Two-Na+/one-Leucine

structureof the two-Na-11-26 Three-dimensional A FIGURE /one-feucinesymporterfrom the bacteriumAquifex aeolicus' (a)TheboundL-leucine, ionare two sodiumions,anda chloride The shownasCPKmodelsin yellow,purpleandgreen,respectively are cthelices thatbindthe Na*or leuctne threemembrane-spanning of thetwo sodiumions brown,blue,andorange(b)Binding colored

oxygens side-chain atomsor carboxyl oxygen main-chain to carbonyl (red)that arepartof helices1 (brown),6 (blue),or 8 (orange)lt is thatoneof thesodiumions(top)isalsoboundto the important A Yamashita leucine(yellow). [From groupof the transported carboxyl 431:811 Nature 2004, et al, Yernool D see also 437 :215: Nature 2005, et al, transporters l of thisandrelated andfunction onthestructure fordetails

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Amino acid residues involved in binding the leucine and the two sodium ions are located in the middle of the membrane-spanning segment (as depicted for the twoNa*/one-glucosesymporter in Figure 11-25) and are close together in three-dimensional space. This demonstrates that the coupling of substrate and ion transport in these transporters is the consequenceof direct or nearly direct physical interactions of the substrates.Indeed, one of the sodium ions (number 1 in Figure 11-26b) is bound to the carboxyl group of the transported leucine,indicating how binding of sodium and leucine are coupled. Each of the two sodium ions is bound to six oxyge., ,to-r. Sodium 1, for example, is also bound to carbonyl oxygens of several transporter amino acids as well as to carbonyl oxygens and the hydroxyl oxygen of one threonine. Equally importantly, there are no water moleculessurrounding either of the bound sodium atoms, as is the case for K+ ions in potassium channels (see Figure 11-20). Thus as the sodium ions lose their water of hydration in binding to the transporter,they bind to six oxygen atoms with a similar geometry. This reducesthe activation energy for binding of sodium ions and preventsother ions, such as porassium, from binding in place of sodium. One striking feature of the structure depicted in Figu r e 1 1 - 2 6 i s t h a t t h e b o u n d s o d i u m i o n s a n d l e u c i n ea r e occluded-that is, they cannot diffuse out of the protein to either the surrounding extracellular or cytoplasmic media. Apparently the process of crystallization of this protein with its bound substrateshas "trapped" it into an intermediate transport step (see Figure 11-25) in which the protein appears to be changing from a conformation with an exoplasmic- to one with a cytosolic-facing binding site.

Na+-LinkedCa2+Antiporter ExportsCa2*from C a r d i a cM u s c l eC e l l s In cardiac muscle cells a tbree-Na+/one-Ca2* anti\orter. rather than the plasma membrane Ca2* Alpase dis.ussej earlier, plays the principal role in maintaining a low concentration of Ca2* in the cytosol. The transporr reacrron mediated by this cation antiporter can be written 3 Na+or, * Ca2*;, ; -

3 Na+1. * C"'*o,.

Note that the movement of three Na+ ions is required to power the export of_oneCa2' ion from the .ytoto1, with a lcal*J of :2 x 10-7 M, ro the extracellularmedium, with a [C"t*] of 2 x 10-3 M, a gradientof some 10,000-fold.In all muscle cells, a rise in the cytosolic Ca2* concentration in cardiac muscle triggers conrraction; by lowering cytosolic Ca'-, operation of the Na+/Ca2+ 2lliporter reducesthe strengthof heart musclecontracrion. The Na*/K+ ATPasein the plasma membrane of cardiac muscle cells, as in other body cells, createsthe N a * concentration gradient necessaryfor export of Caz* 468

.

c H A p r E R1 1 |

by the Na*-linked Ca'* antiporter. As mentioned earlier, inhibition of the Na*/K* AIPase by the drugs ouabain and digoxin lowers the cytosolic K* concentration and, more relevant here, simultaneouslyincreasescytosolic Na*. The resulting reduced Na* electrochemicalgradient across the membranecausesthe Na+-linked Ca2* antiDorterto function less efficiently. As a result, fewer Ca21 ions are exported and the cytosolic Ca2* concentration increases, causing the muscle to contract more strongly. Becauseof their ability to increasethe force of heart muscle contractions, drugs such as ouabain and digoxin that inhibit the Na-/K- ATPase are widely used in the treatment of congestiveheart failure. I

SeveralCotransportersRegulateCytosolicpH The anaerobic metabolism of glucoseyields lactic acid, and aerobic metabolism yields CO2, which adds water to form carbonic acid (H2CO3).Theseweak acids dissociate,yielding H- ions (protons); if these excessprotons were not removed from cells, the cytosolic pH would drop precipitously, endangering cellular functions. Two types of cotransport proteins help remove some of the "excess" protons generatedduring metabolism in animal cells. One is a Na- HCO j- /Cl antiporter, which imports one Na+ ion together with one HCO3-, in exchangefor export of one Cl ion. The cytosolic enzymecarbonic anhydrasecatalyzesdissociation of the imported HCO3- ions into CO2 and an OH- (hydroxyl)ion: HCO.-

;-

CO, + OH

The OH- ions combine with intracellular protons, forming water, and the CO2 diffuses out of the cell. Thus the overall action of this transporter is to consume cytosolic H+ ions, thereby raising the cytosolic pH. Also importanr in raising cytosolic pH is a Na* /H* antiporter, which couplesentry of one Na* ion into the cell down its concenrrationgradient to the export of one H+ ron. Under certain circumstances,the cytosolic pH can rise beyond the normal range of 7 .2-7.5. To cope with the excess OH- ions associatedwith elevated pH, many animal cells utilize an anion antiporter that catalyzesthe one-for-one exchangeof HCO3- and Cl acrossthe plasma membrane.At high pH, this C/ /HCO3- antiporter exports HCO3(which can be viewed as a "complex" of OH and CO2) in exchangefor import of Cl-, thus lowering the cytosolic pH. The import of CI- down its concentration gradient (Cl -.air- ) Cl .r,oror)powers the transport. The activity of all three of these anriport proteins depends on pH, providing cells with a finely tuned mechanismfor controlling the cytosolic pH. The two antiporters that operateto increasecltosolic pH are activated when the pH of the cytosol falls. Similarly, a rise in pH above 7.2 stimulates the CI-iHCO3- antiporter, leading to a more rapid export of HCO3- and decreasein the cytosolic pH. In this manner,the cytosolic pH of growing cells is maintained very closeto pH7.4.

T R A N S M E M B R ATNREA N s p o RoTF t o N s A N D S M A L LM O L E C U L E S

A PutativeCation ExchangeProteinPlaysa Key R o l ei n E v o l u t i o no f H u m a nS k i nP i g m e n t a t i o n Sequencingof the human, mouse, and rat genomesindicates the presenceof hundreds of putative transport proteins, but the functions of most of these are as yet unknown. A particularly interesting human transporter called SLC24A5 emerged from a study of zebrafish that had abnormal skin color; in fish homozygous for the golden mutation, the eponymous black horizontal stripes were very pale (Figure 11-27a and b). Microscopy showed that the mutant fish had a much lower amount of the black pigment called melanin, and melanin vesicles,called melanosomes,were much smaller and paler than normal (Figure 11-27 c and d). Positional cloning of the golden gene demonstrated that it encodesa putative cation exchange protein termed SLC24A5. Immunofluorescence studies showed that the protein is found in intracellular

membranes,likely in the membrane of the melanosomeor its precursor,but the ions transported by SLC24A5 are not yet known. However, the amino acid sequence of the SLAC24A5 protein is closestto that of severalsodium/calcium antiporters, so the protein is likely a sodium/calcium antlporter. Most strikingly, investigators showed that the human version of SLC24A5 is highly similar in sequenceto the zebrafish protein; when the human protein is expressedin mutant golden zebrafish,it complementsthe mutant phenotype and the fish have normal black stripes. The most evolutionarily conservedform of the gene, or allele, most similar to the wild-type zebrafish gene predominates in dark-skinned African and East Asian human populations. In contrast. a version, or variant allele, of the SLC24A5 gene with a single amino acid change that is thought to encode a less active protein is found in virtually all people of European origin. Studies of the allele frequenciesin admixed populations indicate that different forms, or polymorphisms, in iust this cation transporter play a key role in determining the darkness of human skin color' Clearly much needsto be learned about the role of this transporter in cell physiology and how a single point mutation in this gene accountsfor the large differencesin skin pigmentation characteristic of individuals of European, African, and Asian origin.

t r o t e i n sE n a b l eP l a n t N u m e r o u sT r a n s p o r P Vacuolesto AccumulateMetabolitesand lons

11-27 Zebratishmutationsin the gene encodingthe A FfGURE causethe golden skin pigment cation exchangerSLC24A5 phenotype.Lateralviewsof adultwild-type(a)andgolden(b) (arrowheads) Scale bars,5 mm Insets showmelanophores zebrafish (inset, thatareon 0 5 mm) Goldenmutantshavemelanophores paler, thannormal andmoretransparent smaller, average fromwildmicrographs of skinmelanophores electron Transmission type(c) andgolden(d)larvaeshowthat goldenskinmelanophores (arrowheads fewer arethinnerandcontarn showedges) l c a lb e a r si n ( c ) a n d( d ) ,1 0 0 0n m l F r o m m e l a n o s o m e s t hnaonr m a S Scrence 310:1 782,1 etal, 2005, R L Lamason

The lumen of plant vacuolesis much more acidic (pH 3-6) than is the cytosol (pH 7.5). The acidity of vacuolesis maintained by a V-classAlP-powered proton pump (seeFigure 1,1,-9)and by a pyrophosphate-poweredpump that is unique to plants. Both of thesepumps, located in the vacuolar membrane, import H* ions into the vacuolar lumen against a concentration gradient. The vacuolar membrane also conchannels that transport these anions tains Cl- and NO: from the cytosol into the vacuole. Entry of these anions against their concentration gradients is driven by the insidepositive potential generated by the H* pumps. The comtined operation of these proton pumps and anion channels producesan inside-positiveelectricpotential of about 20 mV across the vacuolar membrane and also a substantial pH gradient (Figure 11-28). The proton electrochemical gradient across the plant vacuole membrane is usedin much the sameway as the Na* electrochemicalgradient acrossthe animal-cell plasma membrane: to power the selectiveuptake or extrusion of ions and small molecules by various antiporters. In the leaf, for example, excesssucrosegeneratedduring photosynthesisin the day is stored in the vacuole; during the night' the stored sucrose moves into the cytoplasm and is metabolized to CO2 and H2O with concomitant generation of ATP from ADP and P;. A proton/sucrose antiporter in the vacuolar membrane operatesto accumulatesucrosein plant vacuoles.The inward movement of sucrose is powered by the outward movement of H*, which is favored by its concentration C O T R A N S P O RBTY S Y M P O R T E RASN D A N T I P O R T E R S

469

H*-pumping proteins ADP+ P;

Cotransport by Symporters and Antiporters PPi

2Hlon-channel

r Cotransporters use the energy releasedby movement of an ion (usually Ht or Na*) down its electrochemicalgradient to power the import or export of a small molecule or different ion against its concenrrationgradient.

H' + +

P l a n tv a c u o l el u m e n ( p H= 3 - 6 ) Nat

H*

Ca2*

Sucrose

H+

Cytosol ( p H= 7 . 5 )

Protonantiportproteins A FIGURE 11-28 Concentration of ionsand sucroseby the plant vacuole.Thevacuolar membrane contains two typesof protonpumps(orange): (/eft)anda a V-class H* ATpase pyrophosphate-hydrolyzing protonpump(flght)thatdiffersfromall otheriontransport proteins andprobably isuniqueto plantsThese pumpsgenerate a low luminalpHaswellasan inside-positive potential electric across thevacuolar membrane owingto the inward pumpingof H* ions Theinside-positive potential powersthe movement of Cl- andNOr- fromthecytosol throughseparate (purple)Protonantiporters proteins channel (green), powered by the H + g r a d i e nat ,c c u m u l aNt ea + ,C a 2 +a, n ds u c r o si n e s i dteh ev a c u o l e . [ A f t e r P R e aa n d D S a n d e r s1, 9 8 7 ,p h y s i o t p l a n t 7 1 . t 3 1 ; J M M a a t h u i sa n d D S a n d e r s1, 9 9 2 ,C u r r O p i n C e l lB i o l . 4 . 6 6 1 ;a n d p A R e ae t a l , 1 9 9 2 . TrendsBiochem Sci. 17:348 l

gradient (lumen > cytosol) and by the cytosolic-negative potendal acro-ssthe vacuolar membrane (seeFigure 11-28). Uptake of Ca2* and Na+ into the vacuole from the cytosol against their concentration gradients is similarly mediated by proton antiporters. Understandingof the transport proteins in plant vacuolar membraneshas the potential for increasingagriculproduction in high-salt (NaCl) soils, which are found throughout the world. Becausemost agriculturally useful crops cannot grow in such saline soils, agricultural scientists have long sought to develop salt-tolerant plants by traditional breedingmerhods.\With the availability of the cloned gene encoding the vacuolar Na*/H* antiporter, researchers can now produce transgenicplants that overexpressthis transport protein, leading to increasedsequestrationof Na* in the vacuole. For insrance,rransgenlctomato plants that overexpressthe vacuolar Na*/H* antiporter can groq flower, and produce fruit in the presenceof soil NaCl concentrations that kill wild-type plants. Interestingly although the leavesof thesetransgenictomato plants accumulatelarge amounts of salt, the fruit has a very low salt content. I

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r The cells lining the small intestine and kidney tubules express symport proteins that couple the energetically favorable entry of Na+ to the import of glucose and amino acids against their concentration gradients (see Figure 11-25). r The molecular structure of a bacterial Na+-amino acid symporter revealshow binding of Na* and leucineare coupled and provides a snapshot of an occluded transport intermediate in which the bound substratescannot diffuse out of the protein. r In cardiac musclecells,the export of Ca2* is coupled to and powered by the import of Na* by a cation antiporter, which transports 3 Na* ions inward for each Ca2* ion exported. r As judged by mutations in zebrafish and polymor, phisms in humans, the presumed sodium/calcium cotransporter SLC24A5 plays a major role in forming melanin granules and in regulating the darkness of human skin pigmentation. r Two cotransportersthat are activated at low pH help maintain the cytosolic pH in animal cells very close to 7.4 despitemetabolic production of carbonic and lactic acids.One, a Na*/H* antiporter,exports excessprotons. The other, a Na*HCO3-lClcotransporrer, imports HCO3 , which dissociatesin the cytosol to yield pHraising OH- ions. r A CI-/HCO3 antiporter that is activated at high pH functions to export HCO3 when the cytosolic pH rises above normal and causesa decreasein pH. r Uptake of sucrose,Na*, Ca2*, and other substances into plant vacuoles is carried out by proton antiporters in the vacuolar membrane.Ion channelsand proton pumps in the membrane are critical in generatinga large enough proton concentration gradient to power accumulation of ions and metabolites in vacuoles by these proton antiporters (see Figure 11-28).

Transepithel iaI Transport Previous sections illustrated how several types of transporters function together to carry out important cell functions (seeFigure 11,-2).Here,we exrend this concept by focusing on the transport of several types of molecules and ions acrossthe sheetlikelayers of epithelial cells that cover

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most external and internal surfacesof body organs. Like all epithelial cells, an intestinal epithelial cell is said to be polarized becauseits plasma membrane is organized into at least two discreteregions.Typically, the surfacethat facesthe outside of the organism, here the lumen of the intestine,is called the apical, or top, surface, and the surface that faces the inside of the organism is called the basolateral surface (see Figure 19-9). Specializedregions of the epithelial-cellplasma membrane, called cell junctions, connect the cells and provide strength and rigidity to the cell sheet (seeFigure 1,9-9for details). One of these types of cell junctions-the tight junction-is of particular interesthere sincetight junctions prevent many water-solublesubstanceson one side of an epithelium from moving acrossto the other side through the extracellular space betweencells. For this reason, absorption of nutrients from the intestinal lumen into the blood occurs by the two-stage process called transcellwlar transport: import of moleculesthrough the plasma membrane on the apical side of intestinal epithelial cells and their export through the plasma membrane on the bloodfacing (basolateral,or serosal) side (Figure 11-29). The apical portion of the plasmamembrane,which facesthe intestinal lumen, is specialized for absorption of sugars, amino acids, and other moleculesthat are produced from food by various digestive enzymes.Numerous fingerlike projections (100 nm in diameter) called microvilli greatly increasethe area of the apical surfaceand so the number of

2 Na+/glucose symporter

GLUTz Glucose

Glucose

FI

t

2Na

Na

Glucose 2 Na'

Na+76+ ATPase

Apical membrane T i g h tj u n c t i o n Cytosol Low NaH i g hK *

11-29Transcellular transportof glucosefrom the A FIGURE in the intestinallumeninto the blood.TheNa*/K*ATPase generates membrane Nat andK* concentration surface basolateral (step1) Theoutwardmovement gradients of K+ ionsthrough (notshown)generates an inside-negative K* channels nongated gradient potentialBoththe Na* concentration and membrane from potential areusedto drivethe uptakeof glucose the membrane located symporter lumenbythetwo-Na'/one-glucose the intestinal (step2) Glucose leaves thecellvia membrane in the apicalsurface located uniporter a glucose catalyzed by GLUT2, facilitated diffusion (step3) membrane in the basolateral

transport proteins it can contain, enhancingthe cell's absorptive capacitY.

Multiple TransportProteinsAre Neededto Move Glucoseand Amino AcidsAcrossEpithelia Figure 11-29 depicts the proteins that mediate absorption of glucose from the intestinal lumen into the blood and illustrates the important concept that different types of proteins are localizedto the apical and basolateralmembranesof epithelial cells.In the first stageof this process'a two-Na*/oneglucosesymporter located in microvillar membranesimports glucose, against its concentration gradient' from the intestinal lumen acrossthe apical surfaceof the epithelial cells.As noted above, this symporter couples the energeticallyunfavorable inward movement of one glucosemoleculeto the en-

port, are pumped out across the basolateralmembrane, which facesthe underlying tissue.Thus the low intracellular Na* concentration is maintained. The Na*/K* ATPasethat accomplishesthis is found exclusivelyin the basolateral membraneof intestinalepithelialcells' The coordinatedoperation of thesetwo transport proteins allows uphill movement of glucoseand amino acids from the intestine into the cell. This first stage in transcellular transport ultimately is powered by ATP hydrolysis by the Na*/l(- ATPase. In the secondstage,glucoseand amino acidsconcentrated inside intestinal cells by symportersare exported down their concentrationgradientsinto the blood via uniport proteins in the basolateralmembrane.In the caseof glucose,this movement is mediatedby GLUT2 (seeFigure 11-29).As noted earlier, this GLUT isoform has a relatively low affinity for glucosebut increasesits rate of transport substantiallywhen the glucosegradient acrossthe membranerises(seeFigure 11-4). The net result of this two-stage processis movement of Na* ions, glucose,and amino acidsfrom the intestinallumen acrossthe intestinalepithelium into the extracellularmedium that surrounds the basolateralsurfaceof intestinal epithelial cells.Tight junctions betweenthe epithelialcellspreventthese moleculesfrom diffusing back into the intestinal lumen, and eventually they move into the blood. The increasedosmotic pressurecreated by transcellular transport of salt' glucose, and amino acids acrossthe intestinal epithelium draws water from the intestinal lumen into the extracellular medium that surrounds the basolateralsurface.In a sense,salts, glucose, and amino acids "carry" the water along with them.

S i m p l eR e h y d r a t i o nT h e r a p yD e p e n d so n t h e OsmoticGradientCreatedby Absorption of G l u c o s ea n d N a An understandingof osmosisand the intestinal absorption of salt and glucose forms the basis for a simple

TRANSEPITHELIT AR L ANSPORT

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therapy that savesmillions of lives each year, particularly in Iess-developedcountries. In these countries, cholera and other intestinal pathogens are major causesof death of young children. A toxin released by the bacteria acrivates chloride secretion by the intestinal epithelial cells into the lumen; water follows osmotically, and the resultant massive loss of water causesdiarrhea, dehydration,and ultimately death. A cure demands not only killing the bacteria with antibiotics but also rehydration-replacement of the water that is lost from the blood and other tissues. Simply drinking water does nor help, becauseit is excretedfrom the gastrointestinal tract almost as soon as it enters. Howeve! as we have just learned. the coordinated transport of glucoseand Na* acrossthe inrestinalepithelium creates a transepirhelial osmotic gradient, forcing movement of water from the intestinal lumen acrossthe cell layer and ultimately into the blood. Thus giving affected children a solution of sugarand salt to drink (but not sugar or salt alone)causesthe osmoticflow of water into the blood from the intestinallumen and leadsto rehydration.Similar sugar-saltsolu ns are the basisof popular drinks used by athletesto get gar as well as water into the body quickly and efficiently.

Cl /HCO3 antiporter at-

HC03-

Cl- channel

CI HCO3-

K+ channel

J.",oon'" anhvdrase I

coz Basolateral memorane

2

I + OH <-H2O

Tight junction

K" ATP ADP+P1 ----+ H+

K+ v+

H*/K* ATPase

Apical memDrane

Cytosol pH7 2

FIGURE 11-30Acidificationof the stomachlumenby parietalcellsin the gastriclining.Theapicalmembrane of parietal (a P-class pump)aswellasCl and cellscontains an H*/K* ATPase proteinsNotethecyclicK+transport K* channel across theapical membrane: K+ ionsarepumpedinwardbythe H+/K+ATpase and exitviaa K* channel. Thebasolateral membrane contains an anion antiporter thatexchanges HCO, andCl ions Thecombined proteins operation of thesefourdrfferent transport andcarbonic anhydrase acidifies thestomach lumenwhilemaintaininq the neutral pHandelectroneutrality of thecytosol

P a r i e t aC l e l l sA c i d i f yt h e S t o m a c hC o n t e n t s W h i l e M a i n t a i n i n ga N e u t r a lC y t o s o l i cp H The mammalian stomach conrainsa 0.1 M solution of hydrochloric acid (HCl). This strongly acidic medium kills many ingested pathogens and denatures many ingested proteins before they are degraded by proteolytic enzymes (e.g.,pepsin)that function at acidic pH. Hydrochloric acid is secretedinto the stomach by specializedepithelial cells calledparietal cells(alsoknown as oxyntic cells)in the gastric lining. These cells contain a H* /K+ ATpase in their

chondria in parietal cellsproduce abundant ATp for use by the H+/K* ATpase. If parietal cells simply exported H+ ions in exchange for K* ions, the loss of protons would lead to a rise in the concentrationof OH ions in the cytosol and thus a m a r k e d i n c r e a s ei n c y t o s o l i c p H . ( R e c a l l t h a t [ H + ] x t O H - ] a l w a y s i s a c o n s t a n t , 1 , 0 - 7 4M 2 . ) p a r i e t a l c e l l s avoid this rise in cytosolic pH in conjunction with acidification of the stomach lumen by using CI-/HCO3- antiporters in the basolateral membrane to export the "excess" OH ions from the cytosol to the blood. As noted earlieq this anion antiporter is activated at high cytosolic pH. The overall process by which parietal cells acidify the stomachlumen is illustratedin Figure 11-30. In a reacrroncatalyzed by carbonic anhydrase,the ,,excess',cytosolic OH 472

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combineswith CO2 that diffusesin from the blood, forming HCO3-. Catalyzedby the basolateralanion antiporter, this bicarbonateion is exported acrossthe basolateralmembrane (and ultimately into the blood) in exchangefor a Cl- ion. The Cl- ions then exit through Cl- channelsin the apical membrane, entering the stomach lumen. To preserveelectroneutrality, eachCl- ion that movesinto the stomachlumen across the apical membraneis accompaniedby a K* ion that moves ounvard through a separateK* channel.In this way, the excessK* ions pumped inward by the H*lK* AIPase are returned to the stomachlumen, thus maintaining the normal intracellular K+ concentration. The net result is secretion of equal amounts of H+ and Cl- ions (i.e.,HCI) into the stomach lumen, while the pH of the cytosol remains neutral and the excessOH ions, as HCO3 , are transported into the blood.

TransepithelialTransport r The apical and basolateralplasma membrane domains of epithelial cells contain different transport proteins and carry out quite different transport processes. r In the intestinal epithelial cell, the coordinated operation of Na+-linked symporters in the apical membrane with Nat/K* ATPasesand uniporters in the basolateral membrane mediates transcellular transport of amino acids and glucosefrom the intestinal lumen to the blood (seeFigure 17-29).

T R A N S M E M B R A NTER A N S P O ROTF I O N SA N D S M A L LM O L E C U L E s

r The combined action of carbonic anhydraseand four different transport proteins permits parietal cells in the stomach lining to secreteHCI into the lumen while maintaining their cytosolicpH near neutrality (seeFigure 11-30).

KeyTerms ABC superfamlly 448 active transport 440 antiport 466 aquapoins 444 corransport 465

In this chapter, we have explained the action of specific membranetransport proteins and their impact on certain aspectsof human physiology; such a molecular physiology approach has many medical applications. Even todaS specific inhibitors or activators of channels,pumps, and transporters constitute the largest single class of drugs. For instance, an inhibitor of the gastric H+/K+ ATPase that acidifies the stomach is the most widely used drug for treating stomach ulcers and gastric reflux syndrome. Inhibitors of channel proteins in the kidney are widely used to control hypertension (high blood pressure);by blocking resorption of water from forming urine into the blood, thesedrugs reduce blood volume and thus blood pressure.Calcium-channelblockers are widely employed to control the intensity of contraction of the heart. Drugs that inhibit a particular potassium channel in B islet cells enhancesecretionof insulin (seeFigure 1532) and are widely used to treat adult-onset (type II) diabetes. \7ith the completion of the human genome project, we are positioned to learn the sequencesof all human membrane transport proteins. Already we know that mutations in many of them causedisease-cystic fibrosis, due to mutations in CFTR, is one example. This exploding basic knowledge will enable researchersto identify new types of compounds that inhibit or activate just one of these membrane transport proteins and not its homologs.An important challenge,however,is to understandthe role of an individual transport protein in each of the severaltissuesin which it is expressed. Another major challenge is to understand how each channel, transporter, and pump is regulated to meet the needsof the cell. Like other cellular proteins, many of these proteins undergo reversiblephosphorylation, ubiquitination, and other covalent modifications that affect their activity, but in the vast majority of cases,we do not understand how this regulation affectscellular function. Many channels, transporters,and pumps normally reside on intracellular membranes,not on the plasma membrane, and move to the plasma membrane only when a particular hormone is present. The addition of insulin to muscle, for instance,causes the GLUT4 glucose transporter to move from intracellular membranesto the plasma membrane, increasingthe rate of glucose uptake. We noted earlier that the addition of vasopressin to certain kidney cells similarly causesan aquaporin to traffic to the plasma membrane,increasingthe rate of water transport. But despitemuch research,the underlying cellular mechanismsby which hormones stimulate the movement of transport proteins to and from the plasma membrane remain obscure.

electrochemical gradient 439 facilitated transport 440 F-classpumps 448 flippasemodel456 gated channel 440 GLUT proteins 443 hypertonic 444 hypotonic 444

isotonrc444 membranepotential439 microvrlli471 Na*/K* ATPase452 patchclamping463 P-classpumps448 restingKt channels460 simple diffusion439 symport466 tight junctions47L transcellularluansport471 uniport 44L V-classpumps448

Reviewthe Concepts 1. The basic structural unit of a biomembrane is the phospholipid bilayer.Acetic acid and ethanol are each composed of two carbons, hydrogen and oxygen, and both enter cells by passivediffusion. At pH 7' one is much more membrane permeable than the other' I7hich is more permeable and why? Predict how the permeability of each is altered when the pH is reducedto 1.0, a value typical of the stomach. 2. Uniporters and ion channels support facilitated diffusion across biomembranes.Although both are examples of facilitated diffusion, the rates of ion movement via an ion channel are roughly 10a- to 1Os-foldfaster than that of moleculesvia a uniporter. \7hat key mechanistic difference results in this large differencein transport rate? 3. Name the three classesof transporters.Explain which of theseclassesis able to move glucoseor bicarbonate(HCO3 ), for example, againstan electrochemicalgradient. In the case of bicarbonate, but not glucose, the AG of the transport process has two terms. What are these two terms' and why doesthe secondnot apply to glucose?'Whyare cotransporters often referredto as examplesof secondaryactivetransport? 4. GLUT1, found in the plasma membraneof erythrocytes, is a classic example of a uniporter. Design a set of experiments to prove that GLUT1 is indeed a glucose-specificuniporter rather than a galactose-or mannose-specificuniport.r. Glucose is a 6-carbon sugar while ribose is a s-carbon sugar.Despite this smaller size, ribose is not efficiently transported by GLUT1. How can this be explained? 5. Name the four classesof ATP-powered pumps that produce active transport of ions and molecules.Indicate which of theseclassestransport ions only and which transport primarily small molecules.The initial discovery of one classof these ATP-powered pumps came from studying not the transport of a natural substratebut rather artificial substrates used as cancer chemotherapy drugs. \fhat do investigators now think are common examplesof the natural substratesof this particular classof ATP-powered pumps? R E V I E WT H E C O N C E P T S .

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6. Genome sequencingprojects continue, and the complete genome sequencesfor an increasing number of organisms are known. How does this information allow us to state the total number of transporters or pumps of a given type in either mice or humans?Many of the sequence-identified transporters or pumps are "orphan" proteins, in the sensethat their natural substrate or physiological role is not known. How can this be, and how might one establishthe physiological role of an orphan protein? 7. As we saw in the section Perspectivesfor the Future, specific inhibitors or activators of channels,pumps, and transporters constitute the largest single classof drugs produced by the pharmaceutical industry. Skeletal muscle contraction is causedby elevation ofCa2* concentrarionin the cytosol. \fhat is the expectedeffect on musclecontraction of selectivedrug inhibition of sarcoplasmicreticulum (SR)P-classCa2* Alpase? 8. The membrane porential in animal cells, but not in plants, depends largely on resring K* channels.How do thesechannelscontribute to the restingpotential?Why are these channelsconsideredto be nongated channels?How do thesechannelsachieveselectivityfor K+ versusNa*? 9. Patch clamping can be used to measurethe conductance properties of individual ion channels. Describe how patch clamping can be used to determine whether or not the gene coding for a putative K* channel actually codesfor a K+ or Na- channel. 10. Plants use the proton electrochemicalgradient across the vacuole membrane to power the accumulation of salts and sugarsin the organelle.This createsa hypertonic situation. Why does thii not resuk in the planr iell bursting? How does the plasma membrane Na*/K* ATpase allow animal cells to avoid osmotic lysis even under isotonic conditions? 11. In the caseof the bacterialsodiumJeucinetransporter,what is the key distinguishing feature about the bound sodium ions that ensuresthat other ions, particularlyK+, do not bind? Describethe symporr processby which cells lining the small intestine import glucose. !7hat ion is responsiblefor the transport, and what two particular featuresfacilitate the energetically favored movement of this ion acrossthe plasma membrane? 12. Sequencingseveral genomes, including that of the zebrafish, has revealeda number of receptorsand transporters. In one case, positional cloning of the zebrafish golden gene identified a putative cation exchangeprotein calledSLC24A5. When the golden gene is mutated, the normally black horizontal stripes are pale or golden in appearancebecausethe amount of black pigment or melanin is greatly reduced.What is the name of the melanin-containingvesiclespresentin fish and humans?Design an experimentto identify where the protein encodedby the golden geneis expressedand localizedin the zebrafish.What is the term usedwhen a mutant geneand its phenotype,for insrancegolden in zebrafish,is rescuedby expressingan orthologousgenefrom another animal? 13. Movement of glucosefrom one side to the other side of the intestinal epithelium is a major example of transcellular transport. How doesthe Na*/I(* Alpase power the process?

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Why are tight junctions essentialfor the process?Rehydration supplementssuch as sport drinks include a sugar and a salt. Why are both important to rehydration?

Analyze the Data Imagine that you are investigating the transepithelial transport of radioactive glucose. Intestinal epithelial cells are grown in culture to form a complete sheet so that the fluid bathing the apical domain of the cells (the apical medium) is completely separatedfrom the fluid bathing the basolateral domain of the cells (the basolateral medium). Radioactive glucoseis addedto the apicalmedium, and the 114c-labeled; appearanceof radioactivity in the basolateralmedium is monitored in terms of counts per milliliter (cpm/ml), a measure of radioactivity per unit volume. Treatment 1: The apical and basolateralmedia each contain 150 mM Na+ (curve1). Treatment 2: The apical medium contains 1. mM Na+, and the basolateralmedium contains 150 mM Na+ (curve2). Treatment3: The apical medium contains 150 mM Na+, and the basolateralmedium contains L mM Na+ (curve 3). Radioactivity in BasolateralMedium

E 200

Treatments1, 3

a 150

E ,oo Hrn

i5 ""

fo T i m e( m i n )i n 1 a C - g l u c o s e a. What is a likely explanation for the different results obtained in treatments 1 and 3 versus treatment 2? In additional studies, the drug ouabain, which inhibits Na*/K* ATPases,is included as noted. Treatment 4: The apical and basolateralmedia contain 150 mM Na+ and the apical media contains ouabain ( c u r v e4 ) Treatment 5: The apical and basolateralmedia contain 150 mM Na- and the basolateral media contains ouabain (curve5) Radioactivity in BasolateralMedium

t zoo F

a 150

E ,oo c50 o on cx

2468 T i m e ( m i n )i n 1 a C - g l u c o s e

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b. Vhat is a likely explanation for the different resulrs ob, tained in treatment 4 versustreatment 5? c. A population of epithelialcellsusedin the abovestudieshas beenengineeredto expressGLUT1 rather than GLUT2 in their basolateralmembrane. These engineeredcells appear to be much lessrobust than the parental cellsand do not survive long in culture. \fhat is a reasonableexplanation for this findine?

References Uniport Transport of Glucose and Water Engel,A., Y. Fujiyoshi, and P. Agre. 2000. The importance of aquaporin water channel protein structures.EMBO /. 19:800-806. Hedfalk, K., et al. 2005. Aquaporin gating. Cwrr. Opinion Structural Biology 16:1-1 0. Hruz, P. W., and M. M. Mueckler.2001. Structuralanalysisof the GLUT1 facilitativeglucosetransporter (review).Mol. Memb. Biol. 18:183-193. King, L. S., D. Kozono, and P. Agre.2004. From structureto disease:the evolvingtale of aquaporin biology. Nat. Reu.Mol. Cell Biol. 5:687-598. Maurel, C. 1997. Aquaporins and water permeabilityof plant membranes.Ann. Reu.Plant Physiol. Plant Mol. Biol. 48:399430. Mueckler,M. 1.994.Facilitativeglucosetransporters.Eur. l. Biocbem. 219:713-725. Schafer,J. A. 2004. Renal water reabsorption:a physiologicretrospectivein a molecular era.Kidney Int. Swppl.9lS20-27. Schultz,S. G. 2001. Epithelialwater absorption:osmosisor cotransport?Proc. Nat'l. Acad. Sci.USA 98:3628-3630. 'Wang, Y., K. Schulten,and E. Tajkhorshid.2005.'What makes an aquaporin a glycerol channel?A comparativestudy of AqpZ and GlpF structure. Structure13 11107-1118. Verkman, A. S. 2005. Novel roles of aquaporinsrevealedby phenotypicanalysisof knockout mice. Rey.Physiol. Biochem.Pharmacol. 155:31.-55. ATP-Powered Pumps and the Intracellular lonic Environment Borst, P.,N. Zelcer,and A. van Helvoort. 2000. ABC transporters in lipid transport. Biochim. Biophys. Acta 1486:1,28-744. Guerini, D., L. Coletto, and E. Carafoli. 2005. Exporting calcium from cells. Cell Calcium 38:281,-289. Davidson,A. L., and J. Chen. 2004. ATP-bindingcassettetransporters in bacteria.Ann. Reu.Biochem. T3:241-268. Davies,J., F, Chen, and Y. Ioannou. 2000. Transmembranemolecular pump activity of Niemann-Pick C1 protein. Science290.2295J298. Gottesman,M. M. 2002. Mechanismsof cancerdrug resistance. Ann. Reu.Med. 53:615-627. Gottesman,M.M., and V. Ling. 2006.The molecularbasisof multidrug resistancein cancer:the early yearsof P-glycoproteinresearch.FEBS Lett. 580:998-1009. Inoue, T., S. Wilkens, and M. Forgac.2003. Subunit structure, function, and arrangementin the yeastand coatedvesicleVATPases.J. Bioenerg.Biomembr. 35:29'1,299. Jencks,!7. P. 1995. The mechanismof coupling chemicaland physicalreactionsby the calcium ATPaseof sarcoplasmicreticulum and other coupled vectorial systems.Biosci. Rept. 15:283-287. Kiihlbrandt, \7. 2004. Biology,structureand mechanismof p-type ATPases.Nature Reu.Mol. Cell Biol. 5:282-295. K. P. Locher,A. Lee, and D. C. Rees.2002. The E. coli BtuCD structure:a framework for ABC transDorterarchitectureand mechanism.Science296 109l.

K. Obara, et al. 2005. Structuralrole of countertransportrevealedin Ca'* pump crystal structurein the absenceof Ca'- PNAS. IO2:1.4489-74495. Ostedgaard,L. S., O. Baldursson,and M. J. I0elsh. 2001. Regulation of the cysticfibrosis transmembraneconductanceregulator Cl channelby its R domain.l. Biol. Chem.276:7689-7692. Raggers,R. J., et al. 2000. Lipid traffic: the ABC of transbilayer movement.Traffic l:226-234. Rea, P.A., et al. 1,992.Vacuolar H*-translocating pyrophosphatases:a new categoryof ion translocase.TrendsBiochem. Sci. 17:348-353. Riordan, J. 2005. Assemblyof functional CFTR chloride channels.Ann. Reu.Physiol. 67:701.-71.8. Tovoshima.C.. and G. Inesi.2004. Structuralbasisof ion of the sarcoplasmicreticulum. Ann. Reu. pumping by Ca2-ATPase Biochem.T3:269. Verkman, A. S., G. L. Lukacs, and L..|. Galietta.2005. CFTR chloride channeldrug discovery-inhibitors as antidiarrhealsand activatorsfor therapy of cystic fibrosis. Curr. Pharm. Des. 12:223511 14

Nongated lon Channels and the Resting Membrane Potential Clapham, D. L999. Unlocking family secrets:K* channeltransmembranedomains. Cell 97 :547-S 5 0. Cooper,E. C., and L. Y. Jan. 1999.lon channelgenesand human neurologicaldisease:recentprogress,prospects,and challenges. Proc. Nat'(. Acad. Sci. USA96:47594766. Dutzler, R., et al. 2002. X-ray structureof a CIC chloride channel at 3.0 A revealsthe molecularbasisof anion selectivity.Nature 415:287-294. Gouaux, 8., and R. Mackinnon. 2005. Principlesof selectiveion transport in channelsand pumps. Science310:146t-1.465. Hille, B. 2001. Ion Channelsof Excitable Membranes,3d ed. SinauerAssociates. Jentsch,T., M. Podt,J. Fuhrmann, and A. Zdebik 2005. Physiologicalfunctions of CLC Cl- channelsgleanedfrom human geneticdiseaseand mousemodels.Ann. Reu.Physiol. 67:779-807. K* Jiang, Y., et al. 2003. X-ray structureof a voltage-dependent channel.N ature 423 :33-41.. MacKinnon, R. 2004. Potassiumchannelsand the atomic basis of selectiveion conduction.Nobel Lecturereprinted rn Biosci. Rep. 24:7 5-1.00. Montell, C., L. Birnbaumer,and V. Flockerzi.2002. The TRP channels,a remarkably functional famrly.Cell 108:595-598. Neher,E. 1,992.lon channelsfor communicationbetweenand within cells.Nobel Lecturereprinted in Neuron 8:605-612 and Science256498-502. Neher,E., and B. Sakmann.1992. The patch clamp technique. Sci.Am. 266(3):28-35. Roux, B. 2005. Ion conduction and selectivityin K* channels. 2005. Ann. Reu.Biophys. Biomol. Struct. 34:153-t71. Zhou,Y., J. Morais-Cabral,A. Kaufman, and R' MacKinnon. 2001. Chemistryof ion coordination and hydration revealed by a K* channel-Fab complex at 2 A resolution. Nature 4t4:43-48. Cotransport by Symporters and Antiporters Alper, S. L., M. N. Chernova,and A. K. Stewart.2001. Regulation of Na+-independentCl-iHCO3- exchangersby pH. J. Pancreas2:1,7I-175 . Barkla, B., and O. Pantoja. 1996. Physiologyof ion transport acrossthe tonoplast of higher plants. Ann. Reu. Plant Physiol. Plant Mol. Biol. 47:1.59-184.

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Orlowski, J., and S. Grinstein.2004. Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Arch. 4472549-565. Ifakabayashi, S., M. Shigekawa,and J. Pouyssegur.1997. Molecular physiology of vertebrate Nat/lI* exchangers.Physiol. Reu. 77:51-74. 'Wright, E. M. 2001. Renal Na(*)-glucosecotransporters.Am. J. Physiol. Renal Physiol. 280:F10-F18. \7right, E. M., and D. D. Loo. 2000. Coupling befweenNa*, sugar,and water transport acrossthe intestine.Ann. NY Acad. Sci. 9L5:54-66. A. Yamashita,et al. 2005. Crystal structureof a bacterialhomologue of Na+/Cl--dependentneurorransmirtertransporters.Nature 437:215.

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Zhang, H-X., and E. Blumwald. 2001. Transgenicsalt-tolerant tomato plants accumulatesalt in foliage but not in fruit. Nature Biotech. l9:765-769.

Transepithelial Transport Furuse,M., and S. Tsukita. 2006. Claudins in occludingjunctions of humans and flies. TrendsCell Biol. l6:I8t-188. Goodenough,D. A. 1999. Pluggingthe leaks.Proc. Nat'(. Acad. Sci.USA 96:3t9. Mitic, L., and J. Anderson. 1,998.Molecular architectureof tight junctions.Ann. Reu.Physiol. 60:t21-1,42. Rao, M. 2004. OraI rehydration therapy: new explanations for an old remedy.Ann. Reu.Physiol. 66':385417.

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cLASSIC

EXPERIMENT

11

STUMBLING UPONACTIVETRANSPORT J. 5kou, 1957, Biochem. Biophys. Acta 23:394

In the mid-1950s Jens Skou was a young physician researchingthe effects of local anestheticson isolated lipid bilayers. He needed an easily assayed membrane-associated enzymeto use as a marker in his studies. lfhat he discovered was an enzyme critical to the maintenanceof membrane potential, the Na+/K+ ATPase, a molecular pump that catalyzesactive transport.

branes derived from squid axons contained a membrane-associatedenzyme that could hydrolyze ATP. Thinking that this would be an ideal enzyme for his purposes,Skou setout to isolatesuchan ATPasefrom a more readily available source,crab leg neurons.It was during his characterization of this enzyme that he discoveredthe protein's function.

The Experiment Background During the 1950s many researchers around the world were actively investigating the physiology of the cell membrane, which plays a role in a number of biological processes.It was well known that the concentration of many ions differs inside and outside the cell. For example, the cell maintains a lower intracellularsodium (Na*) concentration and higher intracellular potassium(K*) concentrationthan is found outside the cell. Somehow the membrane can regulate intracellular salt concentrations. Additionally, movement of ions across cell membranes had been observed, suggesting that somesort of transport is systemis present. To maintain normal intracellular Na* and K* concentrations,the transport systemcould not rely on passive diffusion becauseboth ions must move across the membrane against their concentration gradients. This energy-requiringprocesswas termed actrvetransport. At the time of Skou's experiments, the mechanismof active transport was still unclear.Surprisingly Skou had no intention of helping to clarify the field. He found the Na*/K* ATPase completely by accident in his search for an abundant, easily measuredenzyme activity associatedwith lipid membranes. A recent study had shown that mem-

Sincethe original goal of his study was to characterize the ATPase for use in subsequentstudies, Skou wanted to know under what experimental condition its activity was both robust and reproducible. As often is the casewith the characterization of a new eflzyme, this requires careful titration of the various components of the reaction. Before this can be done, one must be sure the system is free from outside sourcesof contamination. In order to study the influence of various cations, including three that are critical for the reaction-No-, K-, and Mg2*-Skou had to make sure that no contaminating ions were brought into the reaction from another source. Therefore all buffers used in the purification of the enzyme were prepared from salts that did not contain these cations. An additional s o u r c eo f c o n t a m i n a t i n gc a t i o n s w a s the ATP substrate, which contains three phosphate groups, giving it an overall negative charge. Becausestock solutions of ATP often included a cation to balance the charge, Skou converted the ATP used in his reactions to the acid form so that balancing cations would not affect the experiments. Once he had a well-controlled environment, he could characterizethe enzyme activity. These precautions were fundamental to his discovery.

Skou first showed that his enzyme could indeed catalyzethe cleavageof ATP into ADP and inorganic phosphate. He then moved on to look for the optimal conditions for this activity by varying the pH of the reaction, and the concentrations of salts and other cofactors, which bring cations into the reaction. He could easily determine a pH optimum as well as an optimal concentration of Mg'-, but optimizing Na* and K* proved to be more difficult. Regardlessof the amount of Kadded to the reaction, the enzymewas inactive without Na-. Similarly, without K+, Skou observed only a lowlevel ATPase activity that did not increase with increasing amounts of Na-. Theseresults suggestedthat the enzyme required both Na* and K* for optimal activity. To demonstrate that this was the case,Skou performed a series of experiments in which he measured the enzyme activity as he varied both the Na+ and K* concentratrons in the reaction (Figure 1). Although both cations clearly were required for significant activity, something interesting occurred at high concentrationsof each cation. At the optimal concentration of Na* and K-, the ATPaseactivity reacheda peak. Once at that peak, further increasing the concentration did not affectthe ATPaseactivity.Na' thus behavedlike a classicenzymesubstrate,with increasinginput leading to increased activity until a saturating concentration was achieved, at which t h e a c t i v i t y p l a t e a u e d .K * , o n t h e other hand, behaveddifferently.'Sfhen the K+ concentration was increased beyond the optimum, ATPase activity declined.Thus while K* was required for optimal activity, at high concentrations it inhibited the enzyme. Skou reasoned that the enzyme must have

S T U M B L I N GU P O N A C T I V ET R A N S P O R T

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(b)

40

Mg6mM/l

K120 mM/l

NaCl40 mMll

K3mM/l

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q, 20 a

N a C l1 0 m M , / l N a C l3 m M l l N a C l0 m M l l

0

20

40

60 KCImM/l

80

100

120

A FIGURE 1 Demonstration of the dependence of the Na*/K* ATPase activityon the concentration of eachion. Thegraphon the leftshowsthatincreasing K- leadsto an inhibition of theATPase activityThegraphon the rightshowsthatwith increasing Na-, the

separate binding sites for Na* and K*. For optimal ATPaseactivity, both must be filled. However, at high concentrations K- could comDetefor the ' N a - b i n d i n g s i t e . l e a d i n gr o e n z y m e inhibition. He hypothesized that this enzyme was involved in active transport, that is, the pumping of Na+ out of the cell, coupled ro the import of K* into the cell. Later studieswould prove that this enzymewas indeed the pump that catalyzed active transport. This finding was so exciting that Skou devoted his subsequentresearchto studyi n g t h e e n z y m e .n e v e r u s i n g i t a s a marker. as he initiallv intended.

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0

50

100 NaClmM,/l

150

200

enzyme activity increases up to a peakandthenlevels out.Thisgraph alsodemonstrates the dependence of theactivity on low levels of K+ [ndaptedfromJ Skou,1957,Biochem Biophys Acta23:3941

Discussion Skou's finding that a membrane ATPase used both Na* and K* as substrateswas the first step in understanding active transport on a molecular level. How did Skou know to test both Nao and K*? In his Nobel lecture in 1.997,he explained that in his first attempts at characterizing the ATPase, he took no precautions to avoid the use of buffers and ATP stock solutions that containedNa* and K*. Pondering the puzzling and unreproducible results that he obtained led to the realization that contaminating salts might be influencing the reaction. When he

repeated the experiments, this time avoiding contamination by Na and K at all stages,he obtained clear-cut, reproducibleresults. The discovery of the Na*/K* ATPasehad an enormous impacr on membrane biology, leading to a better understandingof the membranepotential. The generation and disruption of membrane potential forms the basis of many biological processes,including neurotransmissionand the coupling of chemical and electricalenergy.For this fundamental discovery, Skou was awarded the Nobel Prize for Chemistry rn 1997.

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CHAPTER

CELLULAR ENERGETICS lmmunofluorescence micrograph showingthe intertwined networko{ (red)in a cellfrom the ovaryof the Himalayan mitochondria Tahr mountaingoat Theunusualtwin nucleiin thiscellarestainedblue lcourtesyof M Davidson]

rom the growth and division of a cell to the beating of a heart to the electrical activity of a neuron that underlies thinking, life requires energy.Cells are complex systems in which a multitude of chemical reactions and transport processesare coordinately regulatedin time and space.Cells cannot generateand maintain their highly organized structures and conduct extensivemetabolism (e.g., carbohydrate synthesis)without material and energy from their environments. This chapter describesthe molecular mechanismsby which cells use sunlight or chemical nutrients as sourcesof energy,with a specialfocus on how cellsconvert theseexternal sourcesof energy into a biologically universal, intracellular, chemical energy carrier, adenosine triphosphate, or ATP (Figure 1.2-1.).ATP,found in all types of organismsand presumably present in the earliest life-forms, is generated from ADP and inorganic phosphate (HPO42-, often abbreviated as P1).Cells use the energy releasedduring hydrolysis of the terminal high-energyphosphoanhydridebond in ATP (seeFigure 2-31.)to power many otherwise energeticallyunfavorable processes.Examples include the synthesisof proteins from amino acids and of nucleic acids from nucleotides (Chapter 4), transport of moleculesagainst a concentration gradient by ATP-powered pumps (Chapter 11), contraction of muscle (Chapter 1.7),and beating of cilia (Chapter 18). The energy to drive ATP synthesisfrom ADP (AG : 7.3 kcallmol) is produced primarily by two processes:aerobic oxidation, which occurs in mitochondria in nearly all eukaryotic cells (Figure 1.2-1.,top), and photosynthesis,which occursin chloroplastsonly in leaf cellsof plants (Figure 12-1, bottoml and certain single-celled organisms, such as cyanobacteria.Two additional processes,glycolysis and the

citric acid cycle, are also important direct or indirect sources of ATP in both animal and plant cells. In aerobic oxidation, breakdown products of sugars (carbohydrates) and fatty acids (hydrocarbons)-both derived in animals from the digestion of food-are converted by oxidation with 02 to carbon dioxide and water. The energy releasedfrom this overall reaction is transformed into the chemical energy of phosphoanhydride bonds in ATP. This is analogous to burning wood (carbohydrates)or oil (hydrocarbons)to generateheat in furnacesor motion in automobile engines: both consume 02 and generate carbon dioxide and water. The key difference is that cells break the overall reaction down into many intermediate steps. This permits the amount of energy releasedin any given step to

OUTLINE 12.1

FirstStepsof Glucoseand FattyAcid Catabolism: and the CitricAcid Cycle Glycolysis

12"2 The ElectronTransportChainand Generation of the Proton-MotiveForce 12.3

Harnessingthe Proton-MotiveForcefor Processes Energy-Requiring

503

12.4

and Light-AbsorbingPigments 511 Photosynthesis

12.5

MolecularAnalysisof Photosystems

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CO2MetabolismDuring Photosynthesis

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Energy sou rce Stage lll Chemical bond Slucose \pyruvate) ATP NADH

oxidation FADH2 (citricacid cycle) (electron carriers) \ \

Electron -----) Proton--J ATP transport motive force Cj, H2O (H+gradient)

ATP

Photons ( s un l i gh t )

Energyabsorption + by pigmentsand direct transfer to electrons

Electron *

Stage 3 Proton----+ ATP

FIGURE 12-1 Overviewof aerobicoxidationand photosynthesis. Eukaryotic cellsusetwo fundamental mechanisms to convertexternal sources of energyintoATP(Top)ln aerobic o x i d a t i o n" ,f u e l "m o l e c u l e( p s r i m a r isl yu g a ras n df a t t ya c i d s ) p r e l i m i n apr yr o c e s s i ni ngt h e c y t o s oel , g , b r e a k d o wonf undergo glucose (stagel), and arethentransferred to pyruvate into m i t o c h o n d r iwah, e r et h e ya r ec o n v e r t ebdy o x i d a t i ow ni t h0 2 t o c a r b o nd i o x i d e a n dw a t e r( s t a g e sl l a n dl l l ) a n dA T Pi sg e n e r a t e d (stagelV).(Bottom)In photosynthesis, whichoccursin c h l o r o p l a stths e, r a d i a net n e r g yo f l i g h ti sa b s o r b ebdy s p e c i a l i z e d

(stage1);the absorbed pigments energyis usedto bothoxidize (stage2) necessary waterto 02 andestablish conditions for the generation of ATP(stage3) andcarbohydrates fromC02(carbon fixation,stage4) Bothmechanisms involve the production of (NADH,NADPH, reduced high-energy electron carriers FADHr) and movement potential of electrons downan electrical in an electron t r a n s p ocr th a i nt h r o u g hs p e c i a l i zm e de m b r a n eEsn e r gfyr o mt h e s e electrons is released andcaptured asa proton-motive force(proton gradient) electrochemical that isthenusedto driveATPsynthesis processes Bacteria utilizecomparable

match closely the amount of energyrequired for the next intermediate stage of the process. If there were not a close match, excessreleasedenergywould be lost as heat (which would be very inefficient) or not enough energy would be releasedto drive the next step in the process (which would be ineffective). In photosynthesis, the radiant energyof light is absorbed by pigments such as chlorophyll and used to make ATP and carbohydrates(primarily sucroseand starch).Unlike aerobic oxidation, which uses carbohydratesand 02 to generate CO2, photosynthesisusesCO2 as a substrateand generates O2 and carbohydratesas products. This reciprocal relationship betweenaerobic oxidation in mitochondria and photosynthesisin chloroplastsunderlies a profound symbiotic relationship between photosynthetic and nonphotosyntheticorganismsand is responsible for much of the life on earth. The oxygen generatedduring photosynthesisis the source of virtually all the oxygen in the air, and the carbohydrates produced are the ultimate sourceof energyfor virtually all nonphotosyntheticorganisms. (An exceptionis bacterialiving in deep oceanventsand the organismsthat feed on them-that obtain energy for converting COz into carbohydrates by oxidation of geoIogically generatedreduced inorganic compounds released by the vents.)

At first glance,it would seemthat the molecular mechanisms underlying the reciprocal processesof photosynthesis and aerobicoxidation have little in common. However. a revolutionary discoveryin cell biology establishedthat bacteria, mitochondria, and chloroplastsall use the same mechanism, known as chemiosmosis,to generateAIP from ADP and P1.In chemiosmosis(alsoknown as chemiosmoticcoupling), a proton electrochemicalgradient is generatedacrossa membrane, driven by energyreleasedas electronstravel through an electron transport chain. The energystoredin this gradient,called the proton-motive force, is useddirectly to power the synthesis of AIP and other energy-requiringprocesses(Figure12-2). In this chapter,we explore the molecular mechanismsof the tv'/oprocesses that sharethis central mechanism,focusingfirst on aerobicoxidation and then on photosynthesis.

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FirstStepsof Glucose and FattyAcid Catabolism: Glycolysis and the CitricAcid Cycle In an automobile engine, hydrocarbon fuel is oxidatively and explosively converted in an essentiallyone step process to mechanicalwork (i.e., driving a piston). The processis

Radiant energy (light)

H+

Low pH H+ H+ Positive electric potential

{

1,4 (chlorophyll)

Chemical b o n d si n carbohydrates andlipids

f

| NADH FADH2 ^

ADP+P; ATP

Exoplasmicface

H'

Membrane impermeable to H*

Synthesis of ATP

12-2 Proton-motive proton A FIGURE force.Transmembrane c o n c e n t r a t iaon de l e c t r i c(avl o l t a g eg)r a d i e n tcso, l l e c t i v eclayl l e d theproton-motive force,aregenerated duringaerobicoxidation (bacteria) andphotosynthesis in eukaryotes and prokaryotes H i g h - e n e r eg lye c t r o ngse n e r a t ebdy l i g h ta b s o r p t i obny p i g m e n t s ( e g , c h l o r o p h yol lr)h e l di n t h e r e d u c efdo r mo f e l e c t r ocna r r i e r s ( e g , N A D HF, A D H 2m) a d ed u r i n gt h e c a t a b o l i somf s u g a ras n d l i p i d sp a s sd o w na n e l e c t r otnr a n s p o cr th a i n( b l u ea r r o w s r) e , leasing energythroughout the processTheenergyis recovered by p r o t o nas c r o stsh e m e m b r a n(er e d c o u p l i n igt sr e l e a steo p u m p i n g generating arrows), the proton-motive force In chemiosmotic coupling, the energyreleased whenprotonsflow downthe gradientthroughATPsynthase drivesthe synthesis of ATPThe proton-motive forcecanalsopowertransport of metabolites across t h e m e m b r a naeg a i n st th e i rc o n c e n t r a t igorna d i e natn dr o t a t i o o nf b a c t e r i fal la g e l l a .

relatively inefficient in that both substantial amounts of the chemical energy stored in the fuel are wasted as they are convertedto unusedheat and substantialamounts of fuel are only partially oxidized and releasedas carbonaceous,sometimes toxic, exhaust.In the competition to survive,organisms cannot afford to squander their sometimes limited energy sources on an equivalently inefficient process. Cells have evolved incredibly efficient mechanisms for hydrocarbon (fatty acid) and carbohydrate (sugar)combustion coupled to ATP synthesis.That mechanism is aerobic oxidation. Each stage of fuel conversion to energy comprisesmultiple steps that are catalyzedor mediatedby specificproteins.This strategy providesthe following advantages: r By dividing the processinto multiple stepsthat generate severalenergy-carryingintermediates,bond energy is efficiently channeledinto the synthesisof ATP and energy lost as heat is reduced. r Different fuels are reducedto common intermediatesthat can then sharesubsequentpathwaysfor combustionand AIP synthesis.

r Sincetotal energystored in the bonds of the initial fuel moleculesis substantiallygreaterthan that required to drive the synthesisof a singleATP molecule (-7.3 kcal/mole), many ATP moleculesare produced. In our discussionof aerobic oxidation, we will be tracing the fate of the two main energy-producingdigestiveproducts of food: sugars (principally glucose) and fatty acids. Under certain conditions amino acids also feed into thesemetabolic pathways. The complete aerobic oxidation of each molecule of glucose yields 6 molecules of CO2 and the energy releasedis coupled to the synthesisof as many as 30 moleculesof ATP. The overall reaction is c6Hpo5 + 6 02 + 30 Pi2- + 30 ADP3- + 30 H+ --+ 6 CO2 + 30 ATP4- + 36 H2O, AG : 586 kcal/mol Glucose oxidation in eukaryotes takes place in four stages(seeFigure 12-1): I. Conversion in the cytosol of one 6-carbon glucosemolecule to two 3-carbon pyruvate molecules(glycolysis) II. Pyruvate oxidation to CO2 in the mitochondrion via a 2-carbon acetyl CoA intermediate (citric acid cycle) III. Electron transport to generatea proton-motive force IV. ATP synthesisin the mitochondrion (oxidative phosphorylation) In this section,we discussstagesI and II: the biochemical pathways that break down glucose and fatty acids to CO2, generating some ATP and high-energy electrons in the process; the fate of the releasedelectrons (stage III) is describedin the next section.

During Glycolysis(Stagel), CytosolicEnzymes ConvertGlucoseto Pyruvate Glycolysis occurs in the cytosol in both eukaryotes and prokaryotes and does not require molecular oxygen; thus it is called anaerobic glucose catabolism (biological breakdown of complex to simpler substances).A set of 10 watersoluble cytosolic enzymescatalyzethe reactionsconstituting the glycolytic pathway (glyco, "sweet"; /ysis, "split"), in which one molecule of glucose is converted to two moleculesof pyruvate (Figure 12-3). All the reaction intermediates produced by these enzymes are water soluble' phosphorylated compounds called metabolic intermediates.ln addition to chemicallyconvertingone glucosemoleculeinto theseintermediatesand the two pyruvates,these enzymatic reactions generatefour ATP molecules by phosphorylation of four ADPs (reactions7 and 10), a process called substrate-levelphosphorylation (to distinguish it from the oxidative phosphorylation that generatesATP in the third stageof aerobic oxidation). Unlike later stagesof

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E F T R SS T T E P SO F G L U C O S E A N D F A T T YA C I D C A T A B O L I S MG

481

> FIGURE 12-3 The glycolyticpathway.Glucose isdegraded to pyruvate, Tworeactions consume ATf formingADPand phosphorylated (red),two generate sugars ATPfromADPby (9reen), phosphorylation substrate-level andoneyieldsNADHby reduction of NAD*(yellow)Notethatallthe intermediates between glucose andpyruvate arephosphorylated compounds Reactions 1, 3, and 10,with singlearrows, (large areessentially irreversible n e g a t i vAeGv a l u e su)n d e or r d i n a rcyo n d i t i o ni nsc e l l s

,r

E

''

I

ArP

l\ noP V

- t J r^u^3^

k

Fructose1,6-bisphosphate

E

OH

2-

^6^ - u ru3

2

F

OHH

HO ill

OH

HO3PO-C C-C-H I HH

Dihydroxyacetone phosphate

tr

1L,roo.*r, , l l n-,' r o o , * , r . tv

tr ATP formation in mitochondria and chloroplasrs,a proronmotive force is not involved in substrate-levelphosphorylation. However, substrate-levelphosphorylation requires the addition (in reactions1 and 3) of two phosphatesfrom two ATPs. Thesecan be rhought of as "pump priming" reactions,which introduce a little energyup front in order to effectivelyrecover more energy downstream. Thus glycolysis yields a net of only two ATP molecules per glucose molecule. The balanced chemical equation for the conversion of glucose to pyruvate shows that four hydrogen atoms (four protons and four electrons)are also released:

1,3-Bisphosphoglycerate (2 molecules)

ll

Glucose

'''

Or*

4H++4e-

2ArP lL>

2H*

+ 4 e- -r 2 NAD+ --+2 NADH

Later we will seethat the energycarried by the electronsin NADH and an analogous carrier FADH2, the reduced form of flavin adeninedinucleotide (FAD), can be used to make additional ATPs via the electron transDort chain.

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CHAPTER 12

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CELLULAE RN E R G E T I C S

iltl

"l

HOO2OHH

- o - ci_l tc1_ c _ H

4l

E'',"""',],]',,i,]l! ll t 2-Phosphoglycerate {2 molecules)

tr

1l

t0

I

HO

OPO32-

OHH

ill '-o.Po

oH

ini)ilell

ll'>z u,o I

r ' " ,, , : 1 1 2 A D P ^ "'''" [ 2ATP

v

(For convenience,we show pyruvate here in its un-ionized form, pyruvic acid, although at physiologicalpH it would be largely dissociated.)All four electrons and two of the four protons are transferred (Figure 12-3, reaction 6) to two moleculesof the oxidized form of nicotinamide adeninedinucleotide (NAD*) to produce the reducedform, NADH (see F i g u r e2 - 3 3 ) :

OHH

U.TU-U-U-L

lrz^DP

tvPhosphoenolpyruvate (2 molecules)

Pvruvate

H

I HC- C -c-H I I H O oPo3'

4l

3-Phosphoglycerate {2 molecules)

oo C6H12O6-zCH.-J J

OH

Glyceraldehyde 3-phosphate (2 moleculesl

Pyruvate (2 molecules)

o

H

- o _ c _ c : c _ HI I ' o.Po ooH - o - c t_t cl _ c _ H I

H

The overall chemical ecuation for this first stage of glucosemetabolism is C 6 H 1 2 O 6+ 2 N A D + + 2 A D p 3 - + 2 p i 2 - - ) 2C3H4Or+2NADH+2ATP4After glycolysis, only a fraction of the energy available in glucose has been extracted and converted to ATP and NADH. The rest remains in the covalent bonds of the two pyruvate molecules.The ability to efficiently convert the energy in pyruvate to ATP dependson molecular oxygen. As

we will see,in the presenceof oxygen (aerobic conditions), energyconversionis highly efficient.In its absence(anaerobic conditions), the processis much lessefficient.

The Rateof Glycolysisls Adjustedto Meet the Cell'sNeedfor ATP Enzyme-catalyzed reactions and metabolic pathways are regulated by cells so as to produce the needed amounts of metabolites but not an excess.The primary function of the oxidation of glucoseto CO2 via the glycolytic pathway is to produce NADH and FADH2, whose oxidation in mitochondria generates ATP. Operation of the glycolytic pathway (stageI), as well as the citric acid cycle (stageII), is continuously regulated,primarily by allostericmechanisms,to meet the cell's need for AIP (seeChapter 3 for generalprinciples of allostericcontrol). Three allosterically controlled glycolytic enzymesplay a key role in regulating the entire glycolytic pathway (Figure 12-3). Hexokinase (step 0 ) is inhibited by its reaction product, glucose6-phosphate.Pyruuatekinase (step IO) is inhibited by ATP, so glycolysis slows down if too much ATP is present.The third enzyme,phosphofructokinase-l (step B), is the principal rate-limiting enzyme of the glycolytic pathway. Emblematic of its critical role in regulating the rate of glycolysis,this enzyme is allosterically controlled by several molecules(Figure12-4). For example, phosphofructokinase-1is allosterically inhibited by ATP and allosterically actiuated by AMP. As a result, the rate of glycolysisis very sensitiveto the cell's energy charge, reflected in the ATP:AMP ratio. The allosteric inhibition of phosphofructokinase-1by ATP may seem unusual, sinceATP is also a substrateof this enzyme.But the affinity of the substrate-bindingsite for ATP is much higher (has a lower K-) than that of the allosteric site. Thus at low

concentrations,ATP binds to the catalytic but not to the inhibitory allosteric site, and enzymatic catalysis proceedsat near maximal rates. At high concentrations,ATP also binds to the allosteric site, inducing a conformational changethat reduces the affinity of the enzyme for the other substrate, fructose 6-phosphate,and thus reducesthe rate of this reaction and the overall rate of glycolysis. Another important allosteric activator of phosphofructokinase-1 is frwctose 2,5-bisphospbate.This metabolite is formed from fructose 6-phosphate by an enzyme called pbosphofructokinase-2.Fructose 6-phosphate accelerates the formation of fructose 2,6-bisphosphate,which in turn activates phosphofructokinase-1.This type of control is known as feed-forward actiuation, in which the abundance of a metabolite (here,fructose 5-phosphate)induces an acceleration in its subsequent metabolism. Fructose 2,6-bisphosphateallostericallyactivatesphosphofructokinase-1in liver cellsby decreasingthe inhibitory effect of high ATP and by increasingthe affinity of phosphofructokinase-1 for one of its substrates,fructose 6-phosphate. The three glycolytic enzymesthat are regulated by allostery catalyzereactions with large negative AGo' values-reactions that are essentially irreversible under ordinary conditions. Theseenzymesthus are particularly suitablefor regulatingthe entire glycolytic pathway. Additional control is exerted by glyceraldehyde 3-phosphate dehydrogenase,which catalyzes the reduction of NAD+ to NADH (seeFigure 12-3, step 6). If cytosolic NADH builds up owing to a slowdown in mitochondrial oxidation, this reaction becomesthermodynamically lessfavorable. Glucosemetabolismis controlled differently in various mammalian tissues to meet the metabolic needs of the organism as a whole. During periods of carbohydratestarvation, for instance,it is necessaryfor the liver to release glucoseinto the bloodstream.To do this, the liver converts

High [ATP] gh [citrate]

High [AMP]

FruCtOSe

Phospho{ructo-

FTUCtOSe

Fructose 2,6-bisphosphate

12-4 Allostericregulationof glucosemetabolism. A FIGURE phosphofructokinase-1, is enzyme in glycolysis, Thekeyregulatory 2,6-bisphosphate, which activated byAMpandfructose allosterically is arelow.Theenzyme whenthecell'senergystores areelevated by ATp(whenenergystores arehigh)andcitrate,bothof inhibited qlucose to COr whenthe cellisactivelv oxidizinq whichareelevated

fromfructose 2,6-bisphosphate formsfructose activity kinase the reverse catalyzes activity anditsphosphatase 6-phosphate, whenblood bythe pancreas whichrsreleased Insulin, reaction. andthus kinase activity PFK2 glucose arehigh,promotes levels glucagon by isreleased glycolysis At low bloodglucose, stimulates in the liver, phosphatase activity PFK2 andpromotes the pancreas

Laterwe will seethat citrateis generatedduringstagell of glucose (PFK2)is a bifunctionalenzyme:its oxidation.Phosphofructokinase-2

indirectlyslowingdown glycolysis

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483

(a)

(b) (FERMENTATION) ANAEROBIC METABOLISM

Yeast CYTOSOL c6H1206 Glucose

AEROBIC IV]ETABOLISIV]

Muscle CYTOSOt

CYTOSOL

c6H1206

c 6 H1 2 0 6

Glucosc

Glucose

2ADP+2NAD++2P, Glycolysis

2 A D P + 2 N A D + + 2P ;

2ADP+2NAD++2P;

Glycolysis

2ATP+2NADH+2P; +2H2O

+2HrO

oo iltl

Glycolysis

oo iltl cH3-c-c-oH

cH3-c-c-oH Pyruyic acid

oo cH3-c

actld

hFuvic

2ATP+2NADH+2P; +2HrO

tl

c-oH

Pyruvic acid

I

Pyruvate I decarboxylase f.

,

l>co, v o tl

x2

"

l,-- NADH+ H+ LactateI

dehydrogenase N

cH3-cH

Acetaldchyde

| \> NAD* v oHo

MITOCHONDRION

ttl

cH3-cH-C-OH Lastlc acid

NADH + H+

coz

NAD'

x2

CoA-SH

Pyruvate d e h y dr o g e n a s e

NAD+ NADH

Overallreactionsof anaerobicmetabolism: G l u c o s+e 2 A D P+ 2 P , ----> 2 ethanol+ 2 CO2+2 ATP+ 2 H2O G l u c o s e + 2 A D P +P 2 , ---> 2 lactate + 2 ATP+ 2 H1O NADH

C i t r i ca c i d cycle

NAD+

Oxidative phosphorylation

-28 ADP + -28 Pi -28 AfP + -28 H2O

A FIGURE 12-5 Anaerobicversusaerobicmetabolismof glucose.The ultimatefateof pyruvate formedduringglycolysis depends on the presence or absence of oxygen. In theformation of pyruvate fromglucose, onemolecule (byaddition of NAD+isreduced of two electrons) to NADHfor eachmolecule of pyruvate formed(seeFigure12-3, reaction6) (a)In the absence of oxygen, two electrons aretransferred from eachNADHmolecule to an acceptor molecule to regenerate NAD+,whichis required for continued glycolysis. In yeasts(/eft),acetaldehyde isthe electronacceptorandethanolisthe product. Thisprocess iscalledalcoholic fermentationwhen oxygenis limitingin muscle cells(nght),NADHreduces pyruvate to formlacticacid,regenerating NAD+. (b) In the presence of oxygen,pyruvateistransported into mitochondriaFirstit isconverted by pyruvate dehydrogenase intoonemolecule of CO2andone of aceticacid,the latterlinkedto coenzyme A (CoA-SH) to form acetylCoA, concomitant with reduction of onemolecule of NAD+to NADH.Further metabolism of acetylcoA andNADHgenerates approximately an additional 28 molecules of ATPperglucose molecule oxidized. 484

C H A P T E R1 2

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2CO2 Overallreactionof aerobicmetabolism: Glucose+ 6 02 + -30 ADP + -30 Pi ----> 6 CO2+ 36 H2O+ -30 ATP

the polymer glycogen,a storageform of glucose(Chapter 2), directly to glucose 6-phosphate (without involvement of hexokinase,step [). Under theseconditions, there is a reduction in fructose 2,6-bisphosphatelevels and decreased phosphofructokinase-1activity (Figure 12-4). As a result, glucose6-phosphatederived from glycogenis not metabolized to pyruvate; rather, it is converted to glucose by a phosphataseand releasedinto the blood to nourish the brain and red blood cells,which dependprimarily on glucose as an energy fuel. In all cases,the activity of these regulated enzymesis controlled by the level of small-molecule metabolites, generally by allosteric interactions, or by hormone-mediated phosphorylation and dephosphorylation reactions (Chapter 15 gives a more detailed discussion of hormonal control of glucose metabolism in liver and muscle).

G l u c o s el s F e r m e n t e dU n d e r A n a e r o b i cC o n d i t i o n s Many eukaryotesare obligate aerobes:they grow only in the presenceof molecular oxygen and metabolizeglucose(or related sugars)completely to CO2, with the concomitant production of a large amount of ATP. Most eukaryotes, however, can generate some ATP by anaerobic metabolism. A few eukaryotesare facubatiue anaerobes:they grow in either the presenceor the absenceof oxygen. For example, annelids,mollusks,and someyeastscan live and grow for days without oxygen. In the absenceof oxygen, yeasts convert the pyruvate produced by glycolysisto one molecule each of ethanol and CO2; in these reactions two NADH molecules are oxidized to NAD* for each two pyruvates converted to ethanol, thereby regeneratingthe supply of NAD- (Figure 12-5a, left). This anaerobic degradation of glucose, called fermentation, is the basis of beer and wine production. Oxygen deprivation can also affect glucosemetabolism in animals. During prolonged contraction of mammalian skeletal muscle cells-for example, during exercise-oxygen within the muscle tissue can becomelimited and glucosecatabolism is limited to glycolysis(stageI). As a consequence, muscle cells convert the pyruvate from glycolysis to two molecules of lactic acid by a reduction reaction that also oxidizes two NADHs to NAD*s (Figure L2-5a, right). Although the lactic acid is releasedfrom the muscle into the blood, if the contractions are sufficiently rapid and strong, the lactic acid can transiently accumulate in that tissue and contribute to muscle and joint pain during exercise.Once it is secretedinto the blood, some of the lactic acid passesinto the liver, where it is reoxidized to pyruvate and either further metabolizedto C02 aerobically or converted back to glucose.Much lactate is metabolized to CO2 by the heart, which is highly perfused by blood and can continue aerobic metabolism at times when exercising, oxygen-poor skeletal muscles secretelactate. Lactic acid bacteria (the organisms that spoil milk) and other prokaryotes also generateATP by the fermentation of glucoseto lactate.

, itochondria U n d e rA e r o b i cC o n d i t i o n sM EfficientlyOxidizePyruvateand Generate ATP(Stagesll-lv) In the presenceof oxygen, pyruvate formed by glycolysis is transported into mitochondria, where it is oxidized by 02 to CO2 and H2O via a seriesof oxidation reactions. The overall process by which cells use 02 and produce COz is collectively termed cellular respiration (Figure 12-5b). Reactions in the mitochondria (stagesII-IV) generatean estimated 28 additional ATP molecules per original glucose molecule, far outstripping the ATP yield from anaerobic glucosemetabolism. Oxygen-producing photosynthetic cyanobacteria appeared about 2.7 billion years ago' The subsequentbuildup in the earth's atmosphere of sufficient oxygen during the next approximately billion years opened the way for organisms to evolve the very efficient aerobic oxidation pathway, which in turn permitted the evolution, especially during what is called the Cambrian explosion' of large and complex body forms and associatedmetabolic activities. In effect, mitochondria are ATP-generating factories, taking full advantageof this plentiful oxygen. We first describe their structure and then the reactions they employ to degrade pyruvate.

M i t o c h o n d r i aA r e D y n a m i cO r g a n e l l e s with Two Structurallyand Functionally D i s t i n c tM e m b r a n e s Mitochondria (Figure 12-6) are among the larger organelles in the cell. A mitochondrion is about the size of an E. coli bacterium, which is not surprising, becausebacteria are thought to be the evolutionary precursorsof mitochondria (see Chapter 6 and the discussionof endosymbiont hypothesis, below). Most eukaryotic cells contain many mitochondria, collectively occupying as much as 25 percent of the volume of the cytoplasm. The numbers of mitochondria in a cell, hundreds to thousandsin mammalian cells, are regulated to match the cell's requirements for ATP (e.g., stomach cells' which use a lot of ATP for acid secretion,have many mitochondria). Analysis of fluorescentlylabeled mitochondria in living cellshas shown that mitochondria are highly dynamic. They undergo frequent fusions and fissions that generate tubular, sometimesbranched networks (Figure 1'2-7),which may account for the wide variety of mitochondrial morphologiesseenin different types ofcells. Fusionsand fissions apparently play a functional role as well becausegeneticdisruptions in GTPasesuperfamily genesrequired for thesedynamic processescan disrupt function, such as maintenance of proper inner membrane electrical potential, and cause human disease,such as the neuromuscular diseaseCharcotMarie-Tooth subtype 2A. The details of mitochondrial structure can be observed with electron microscopy (seeFigure 9-8). Mitochondria have two distinct kinds of concentricallyrelated membranes. The outer membrane definesthe smooth outer perimeter of the mitochondrion. The inner membrane has numerous

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E A N D F A T T YA C I D C A T A B O L I S MG F T R SS T T E P SO F G L U C O S E

Video: Mitochondrion Reconstructedby ElectronTomography (b) F6F1complexes Intermembrane space C r i s t a ej u n c t i o n s

FIGURE 12-6 Internalstructureof a mitochondrion. ( a )S c h e m a tdi ica g r a m s h o w i ntgh ep r i n c i p m a le m b r a n a en sd compartments Thecristae formsheets andtubesby invagination o f t h ei n n e rm e m b r a naen dc o n n e ct o t t h ei n n e m r embrane throughrelatively smalluniformtubularstructures calledcnsta junctionsTheintermembrane spaceappears continuous with the lumenof eachcristaTheFeF,complexes (smallredspheres), whichsynthesize ATP, areintramembrane particles thatprotrude fromthecristae andinnermembrane intothe matrixThematrix contains the mitochondrial (small DNA(bluestrand), ribosomes

Video: MitochondrialFusionand Fission (d

E X P E R I M E N TFA L U RtE2 - 7 M i t o c h o n d r iuan d e r g o IG rapidfusionand fissionin living cells.Mitochondria labeled with a fluorescent proteinin a livingnormalmurineemoryontc fibroblast wereobserved usingtime-lapse fluorescence microscopy Several mitochondria undergoing fusion(top)or (bottom)areartificially jn blueandwith fission highllghted arrowslModified fromD C Chan, 2006,Cell125(])j241-12521

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(largeyellowspheres) (b)Computerbluespheres), andgranules generated modelof a section of a mitochondrion fromchicken brain. Thismodelisbased on a three-dimensional electron microscopic image calculated froma series of two-dimensional electron micrographs recorded at regularintervals Thistechnique isanalogous to a threedimensional x-raytomogram or CATscanusedin medical imaging (yellow-green), Notethetightlypacked cristae the innermembrane (lightblue),andtheoutermembrane (darkblue)[part(a)courtesy of T. Frey;part (b) from T. Freyand C Mannella,2000, TrendsBiochem Sci 25:319l

invaginationscalled cristae (seeFigure 12-6). These membranes topologically define two submitochondrial compartments: the intermembrane space, between the outer and inner membranes,and the matrix, or central compartment, which forms the lumen within the inner membrane.'When individual mitochondria fuse, each of their distinct comparrments intermixes (e.g.,matrix with matrix, inner membrane with inner membrane). Fractionation and purification of these membranes and compartments have made it possible to determine their protein, DNA, and phospholipid compositions and to localize each enzyme-catalyzedreaction to a specific membrane or comparrment. About 1000 polypeptides are required to make and maintain mitochondria and permit them to function. Only a small number of these-13 in humans-are encoded by mitochondrial DNA genes, while the remaining proteins are encoded by nuclear genes ( C h a p t e r6 ) . The most abundant protein in the outer membrane is mitochondrial porin, a transmembrane channel protein similar in structure to bacterial porins (seeFigure 10-1S). Ions and most small molecules(up to about 5000 Da) can

readily pass through these channel proteins when they are open. Although there may be metabolic regulation of the opening of mitochondrial porins and thus the flow of metabolites across the outer membrane, the inner membrane with its cristae are the major permeability barriers between the cytosol and the mitochondrial matrix, limiting the rate of mitochondrial oxidation. Protein constitutes 76 percent of the total weight of the inner membrane-a higher fraction than in any other cellular membrane. Many of these proteins are key participants in cellular respiration. They include ATP synthase, proteins responsiblefor electron transport, and a wide variety of transport proteins that permit the movement of metabolites between the cytosol and the mitochondrial matrix. The human genomeencodes48 membersof a family of mitochondrial transport proteins. One of these is called the ADP/ATP carrier, an antiporter that moves newly synthesizedATP out of the matrix and into the inner membrane space(and subsequentlythe cytosol) in ex'!flithout this changefor ADP originating from the cytosol. essential antiporter, the energy trapped in the chemical bonds in mitochondrial ATP would not be availableto the rest of the cell. The invaginating cristae greatly expand the surface area of the inner mitochondrial membrane (see Figure 12-6), enhancing its capacity to generateATP. In typical liver mitochondria, for example,the area of the inner membrane, including cristae, is about five times that of the outer membrane. In fact, the total area of all inner mitochondrial membranes in liver cells is about 1,7 times that of the plasma membrane. The mitochondria in heart and skeletal muscles contain three times as many cristae as are found in typical liver mitochondria-presumably reflecting the greater demand for AIP by muscle cells. Note that plants have mitochondria and perform cellular respiration as well. In plants, stored carbohydrates,mostly in the form of starch, are hydrolyzed to glucose.Glycolysis then produces pyruvate, which is transported into mitochondria, as in animal cells. Mitochondrial oxidation of pyruvate and concomitant formation of ATP occur in photosynthetic cells during dark periods when photosynthesisis not possible and in roots and other nonphotosynthetic tissuesat all times. The mitochondrial inner membrane, cristae, and matrix are the sites of most reactions involving the oxidation of pyruvate and fatty acids to CO2 and H2O and the coupled synthesisof ATP from ADP and P1, with each reaction occurring in a discretemembrane or spacein the mitochondrion (Figure12-8). The last three of the four stagesof glucoseoxidation are r StageII. Conversionof pyruvate to acetyl CoA, followed by oxidation to CO2 in the citric acid cycle.Theseoxidations are coupled to reduction of NAD* to NADH and of FAD to FADH2. (Fatty acid oxidation follows a similar route, with conversion of fatty acyl CoA to acetyl CoA.) Most of the reactionsoccur in or on the membrane facing the matrIX.

r StageIII. Electron transfer from NADH and FADH2 to 02 via an electron transport chain within the inner membrane, which generatesa proton-motive force acrossthat membrane. r StageIV. Harnessingthe energy of the proton-motive force for ATP synthesisin the mitochondrial inner membrane. StagesIII and IV are together called oxidative phosphorylation.

In Stagell, Pyruvatels Oxidizedto CO2 and High-EnergyElectronsStored in ReducedCoenzymes Pyruvate formed during glycolysis in stage I in the cytosol is transportedinto the mitochondrial matrix (Figure 12-8). StageII metabolism accomplishesthree things: (1) it converts the 3-carbon pyruvate to three moleculesof CO1' Q) it generates high-energy electron carriers (NADH and FADH2) that will be used for electrontransport (stageIII); and (3) it generatesa GTP molecule, which is then converted to ATP: GTP + ADP i-

GDP + AIP

StageII can be subdivided into two distinct parts: (1) the generationof acetyl CoA plus one molecule of COz and one NADH and (2\ the conversion of acetyl CoA to two molecules of CO2 and the high-energy intermediatesNADH (3 molecules),FADH2, and GTP. Generation of Acetyl CoA \il/ithin the mitochondrial matrix, pyruvate reactswith coenzymeA, forming CO2 and acetyl CoA and NADH (Figure 12-8). This reaction, catalyzedby pyruuate - 8.0 kcaUmol)and dehydrogena.sais highly exergonic(AG"' : essentiallyirreversible. Acetyl CoA (Figure 1'2-9) plays a central role in the oxidation of fatty acids and amino acids.In addition, it is an intermediate in numerous biosynthetic reactions' including transfer of an acetyl group to histone proteins and many mammalian proteins, and synthesisof lipids such as cholesterol. In respiring mitochondria, however' the acetyl group of acetyl CoA is almost always oxidized to CO2 via the citric acid cycle. Citric Acid Cycle Nine sequentialreactions operate in a cycle to oxidize acetyl CoA to CO2. The cycle is referred to by severalnames:the citric acid cycle, the tricarboxylic acid (or TCA) cycle, and the Krebs cycle. The net result is that for each acetyl group entering the cycle as acetyl CoA, two moleculesof CO2, three of NADH' and one each of FADH2 and GTP are produced. As shown in Figure 12-1'0,the cycle begins with condensation of the two-carbon acetyl group from acetyl CoA with the four-carbon molecule oxaloacetate to yield the six-carboncitric acid (hencethe name citric acid cycle).In both reactions 4 and 5, a CO2 molecule is releasedand NAD+ is reduced to NADH. Reduction of NAD- to NADH also occurs during reaction 9; thus three NADHs

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E F T R SS T T E p SO F G L U C o 5 EA N D F A T T YA C I D C A T A B O L I S MG

487

Outer mitochondrial membrane {permeable to metabolites)

coz

Intermembrane space

StageI Glucose

2 NAD*-J I 2 NADH.f" z efP J 2 Pyruvate -

Pyruvate

-----?2COt

Acetyl CoA

C i t r i ca c i d cycre

Mitochondrlal matrix

Succinate

2 e- + 2H* + !Or---+ Hrg Fumarate

Qz

Hzo

FoF,complex

A FIGURE 12-8 Summaryof aerobicoxidationof glucoseand fatty acids.Stagel: Inthecytosol, glucose isconverted to pyruvate (glycolysis) andfattyacidto fattyarylCoA hTruvate andfattyacylCoA thenmoveintothemitochondrion Mitochondrial porins maketheouter membrane permeable to thesemetabolites, butspecific rranspon (colored proteins ovals) in theinnermembrane arerequired to import pyruvate (yellow) andfattyacids(blue)intothematrixFattyacyl groupsaretransferred fromfattyarylCoAto an intermediate carriel transported across (blueoval),andthenreattached the innermembrane to CoAon thematrixsideStagell: Inthemitochondrial matrix, pyruvate andfattyacylCoAareconverted to acetylCoAandthen oxidized, releasing CO2Pyruvate isconverted to acetylCoAwiththe formation of NADHandCOr;two carbons fromfattyacylCoAare convefted to acetylCoAwiththeformation of FADH,andNADH Oxidation of acetylCoAin thecitricacidcycleqenerates NADHand

FADH2, GTBandCO2.Stagelll: Electron transport reduces oxygen to waterandgenerates (blue)from a proton-motive force.Electrons reduced coenzymes aretransferred viaelectron-transport complexes (blueboxes) to 02 concomitant withtransport of H* ions(red)fromthe matrixto theintermembrane generating space, theproton-motive force Electrons fromNADHflowdirectly fromcomplex I to complex lll, bypassing complex ll Electrons fromFADH, flowdirectly fromcomplex ll to complex lll,bypassing complex I StagelV:ATPsynthase, the FoFl (orange), complex harnesses the proton-motive forceto synthesize ATp (purpleandgreenovals) rnthematrix,Antiporter proteins transport ADp andP;intothematrixandexporlhydroxyl groupsandATpNADH generated in thecytosol isnottransported directly to thematnxoecause theinnermembrane rsimpermeable to NAD+andNADH;instead, a (red)transports shuttlesystem electrons fromcytosolic NADHto NAD+in the matrix02 diffuses intothematrix,andCOrdiffuses our

are generatedper turn of the cycle.In reaction 7, two electrons and two protons are transferredto FAD, yielding the reducedform of this coenzyme,FADH2. Reaction 7 is distinctive becauseit not only is an intrinsic part of the citric acid cycle (stageII), but also it is caralyzedty a membrane-

attached enzyme that is an intrinsic part of the electron transport chain (stageIII). In reaction 6, hydrolysis of the high-energythioester bond in succinyl CoA is coupled to synthesisof one GTP by subsrrate-levelphosphorylation. (BecauseGTP and ATP are interconvertible,this can be

Coenzyme A (CoA) a F I G U R E l 2 ' 9T h e s t r u c t u r e o fa c e t y lC o A . T h j s c o m p o u n d i s an importantintermediatein the aerobicoxidationof pyruvate,fatty

488

c H A P T E R1 2

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CELLULAE RN E R G E T T C S

a c i d s , a n d m a n y a m i n o a c il d t asl.s o c o n t r i b u t e s a c eg tr yolu p s i n many biosynthetic pathways

N A D H+ H HrO

coo I nu

tc-coo

HO-C-H

tl HC I

I

V /

QH, t-

coo-

iaa

vvv

crs-Aconitate

Malate

I Hro\tl

coo-

coo.1.,, UN

I CH"

t-

coo-

HC

I coo-

Z

Fumarate

//

,r Y

FADH,

t-

CH, I'

QH,

lC H "= FA

c:o I coo-

t-' coo

Succinate

H-C-COO

I nu

cooI

GDP + Pi + H2O

GTP + HSCoA

t

I

E

HO-C-H

/l

coolsocitrate

4-Keto-

glutarate C O r + N A D H+ H '

+ N A D H+ H -

12-10The citricacidcycle.AcetylCoAis metabolized a FIGURE In NADHandFADH2. electron carriers to C02andthe high-energy fromacetylCoAcondenses acetylresidue 1, a two-carbon reaction to formthe six-carbon molecule oxaloacetate with the four-carbon (2-9)eachmolecule of citrate reactions citrate.In the remaining losingtwo C02 backto oxaloacetate, converted iseventually In eachturn of the cycle,four pairsof in the process. molecules fromcarbonatoms,forminqthreemolecules areremoved electrons

of GTP andonemolecule of FADH2, of NADH,onemolecule Thetwo carbonatomsthatenterthe cyclewith acetylCoAare y lo A I n s u c c t n aat en d h i g h l i g h t ei ndb l u et h r o u g hs u c c i n C theycanno longer molecules, whicharesymmetric fumarate, haveshownthat studies lsotope-labeling denoted. be specifically thesecarbonatomsarenot lostin theturn of the cyclein which onewill be lostasC02duringthe next theyenter;on average, turns. the otherin subsequent and turn of the cycle

consideredan ATP-generatingstep.) Reaction 9 regenerates oxaloacetate,so the cycle can begin again. Note that molecular 02 does not participatein the citric acid cycle. Most enzymesand small moleculesinvolved in the citric acid cycle are soluble in the aqueousmitochondrial matrix. Theseinclude CoA, acetylCoA, succinylCoA, NAD*, and NADH, as well as most of the eight cycle enzymes. Succinate dehydrogenase(reaction 7), however, is a component of an integral membraneprotein in the inner membrane,with its active site facing the matrix. When mitochondria are disrupted by gentle ultrasonic vibration or osmotic lysis, non-membrane-boundenzymesin the citric acid cycle are releasedas very large multiprotein complexes.Within such complexes the reaction product of one enzyme is thought to pass directly to the next enzymewithout diffusing through the solution. However,much work is neededto determine the structuresof these large enzyme complexes as they exist in the cell. Since glycolysis of one glucose molecule generatestwo acetyl CoA molecules, the reactions in the glycolytic pathway and citric acid cycle produce six CO2 molecules, 10 NADH molecules, and two FADH2 molecules per glucose molecule(Table 12-1). Although thesereactionsalso generate four high-energyphosphoanhydridebonds in the form of

two ATP and two GTP molecules' this represents only a small fraction of the available energy releasedin the complete aerobic oxidation of glucose.The remaining energy is itored as high-energy electrons in the reduced coenzymes NADH and FADH2. The goal of stagesIII and IV is to recover this energy in the form of ATP.

T r a n s p o r t e risn t h e I n n e r M i t o c h o n d r i a l MembraneHelp Maintain AppropriateCytosolic and Matrix Concentrationsof NAD* and NADH In the cytosol NAD+ is required for step 6 of glycolysis (see Figure 12-3), and in the mitochondrial matrix NAD+ is reqrri..d for conversion of pyruvate to acetyl CoA and for th... tt.pt in the citric acid cycle (4, 5, and 9 in Figure 12-

FADH2 to FAD as it reduces02 to water and converts the energy stored in the high-energy electrons in the reduced

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E A N D F A T T YA C I D C A T A B O L I S MG F I R S Ts T E P SO F G L U C O S E

489

c02M0ltcljlts PBODUCED

fltAcTt0N

NAD+ Ml)LECULES FAD M{]TECULES REOUCED TONADH REDUCED T0tADH2

(OR ATP GTP)

L glucose molecule to 2 pyruvate molecules

0

2

0

z

2 pyruvates to 2 acetyl CoA molecules

a L

z

0

0

2 acetyl CoA to 4 CO2 molecules

4

t)

z

2

Total

5

10

2

+

forms of these molecules into a proton-motive force. Even though 02 is not involved in any reaction of the citric acid cycle, in the absenceof 02, this cycle soon stops operating as the intramitochondrial suppliesof NAD* and FAD dwindle due to the inability of the electron transport chain to oxidize NADH and FADH2. These observations raise the question of how a supply of NAD+ in the cytosol is regenerated. If the NADH from the cytosol could move into the mitochondrial matrix and be oxidized by the electron transport chain and if the NAD+ product could be transported back into the cytosol, regenerationof cytosolic NAD+ would be

Cytosol

,

simple. However, the inner mitochondrial membrane is impermeableto NADH. To bypassthis problem and permit the electrons from cytosolic NADH to be transfer red indirectly to 02 via the electron transport chain, cells use severalelectron shuttles to transfer electrons from cytosolic NADH to NAD+ in the matrix. Operation of the most widespreadshuttle-the malate-aspartateshwttle-is depicted in Figure 12-11. For every complete "turn" of the cycle,there is no overall change in the numbers of NADH and NAD* moleculesor the intermediatesaspartateor malate used by the shuttle.However, in the cytosol, NADH is oxidized to NAD+, which can be used

NADHcytosot NAD*cytosol

oxaroacetate \E/

Aspartate

, t",.,"

( \ - K e t olgu t a r a t eG l u t a m a t e Gluta

Mitochondrial inner membrane Glutamate

_->-_)

r r - K e t o g l u t ar a t e G l u t a m a t e

I Aspartate

\q/

FIGURE 12-11The malateshuttle.Thiscyclical series of reactions transfers electrons fromNADHin thecytosol (intermembrane space) a c r o stsh e i n n e rm i t o c h o n d r m i ael m b r a n w e ,h i c hi s i m p e r m e a b l e to NADHitself,to NAD* in the matrixThenet resultisthe replacement of cytosolic NADHwith NAD+and matrixNAD+ with NADHStepll: Cytosolic malatedehydrogenase transfers electrons fromcytosolic NADHto oxaloacetate, formingmalate. S t e pf , l : A n a n t i p o r t e( b r l u eo v a l )i n t h e i n n e rm i t o c h o n d r i a l m e m b r a nter a n s p o r m t sa l a t ei n t ot h e m a t r i xi n e x c h a n gf e or cr-ketogIutarate. StepS: MitochondriaI malatedehydrogenase converts malatebackto oxaloacetate, reducing NAD+in the matrix to NADHin the processStepE: Oxaloacetate. whichcannot CHAPTER 12

I

CELLULAR ENERGETICS

,,"nuY,1ll';,"."',

Oxaloacetate €-----------

Matrix

490

o - K e t o gI u t ar a t e

/E\

Malare

NADHmatrix NAD*m"tri"

directly crossthe innermembrane, isconverted to aspartate by additionof an aminogroupfromglutamateInthistransaminasecatalyzed reaction in the matrix,glutamate is converted to cr-ketoglutarate (redoval)exports Step[: A secondantrporter aspartate to the cytosolin exchange for glutamateStep@: A cytosoltc transamrnase converts aspartate to oxaloacetate and ct-ketoglutarate to glutamate, completing the cycleTheblue arrowsreflectthe movement of the cr-ketoglutarate, the red arrowsthe movement of glutamate, andthe blackarrowsthat of aspartate/malate lt is noteworthy that,asaspartate and malatecycleclockwise, glutamate andcr-ketoglutarate cyclein the oppositedirection

for glycolysis,and in the matrix, NAD* is reducedto NADH, which can be usedto generateATP via stagesIII and IV * NADH-.1'1" NADH.yrorot + NAD;".. * --+NAD.*y,oro1

MitochondrialOxidation of Fatty Acids GeneratesATP

o R-C

Up to now, we have focusedmainly on the oxidation of carbohydrates,namely glucose,for ATP generation.Fatty acids are another important source of cellular energy. Cells can take up either glucose or fatty acids from the extracellular spacewith the help of specifictransporter proteins (Chapter 11). Should a cell not needto immediatelyburn thesemolecules,it can store them as a polymer of glucosecalled glycogen (especiallyin muscleor liver) or as a trimer of fatty acids covalently linked to glycerol, called a triacylglycerol or triglyceride. In some cells, excessglucose is converted into fatty acids and then triacylglycerols for storage. However, unlike microorganisms, animals are unable to convert fatty acids to glucose.lWhen the cells need to burn these energy storesto make ATP (e.g.,when a resting muscle beginsto do work), enzymesbreak down glycogen to glucose or hydrolyze triacylglycerols to fatty acids, which are then oxidized to generateATP:

o cH3-(CH2)"-C- O-CH2 ^l

YI

* 3 Hro +

cH3-(cH2),-c-o-cH

oxidation. The differenceslie in the cytosolic stageI and the first part of the mitochondrial stageII. In stageI, fatty acids are convertedto a fatty acyl CoA in the cytosol in a reaction coupled to the hydrolysis of ATP to AMP and PPl (inorganic pyrophosphate)(seeFigure 12-8):

O- + HSCoA + ATP ------->

Fatty acid

o R-C-SCOA+AMP+PPI Fatty acyl CoA

Subsequenthydrolysis of PP1to two molecules of P; drives this reaction to completion. To transfer the fatty acyl group into the mitochondrial matrix, it is covalently transferred to a molecule called carnitine, moved across the inner mitochondrial membrane by an acylcarnitine transporter protein (seeFigure 12-8' blue oval), and then on th; matrix side, the fatty acyl group is released from carnitine and reattached to another CoA molecule' The activity of the acyl carnitine transporter is regulated to prevent oxidation of fatty acids when cells have adequate energy (ATP) supPlies. In the first part of stage II' each molecule of a fatty acyl CoA in the mitochondrion is oxidized in a cyclical sequence of four reactionsin which all the carbon atoms are converted two at a time to acetyl CoA with generation of FADH2 and NADH (Figure 1,2-1'2a).For example, mitochondrial oxidation of each molecule of the 18-carbon stearic acid'

?l

cH3- (cH2)"-c-o-cH2

HO-CH2

Triacylglycerol O

HO-CH

l

3 CH3-(CH2)"-C-OH + HO-CH2 Fatty acid

GlYcerol

Fatty acidsare the major energysourcefor many tissues, particularly adult heart muscle.In humans,in fact, the oxidation of fats is quantitatively more important than the oxidation of glucose as a source of ATP. The oxidation of 1 g of triacylglyceride to CO2 generatesabout six times as much AIP as does the oxidation of 1 g of hydrated glycogen. Thus triglycerides are more efficient than carbohydrates for storage of energy,in part becausethey are stored in anhydrous form and can yield more energy when oxidized and also becausethey are intrinsically more reduced (have more hydrogens)than carbohydrates.In mammals, the primary site of storageof triacylglyceridesis fat (adipose)tissue,whereasthe primary sitesfor glycogenstorage are muscle and the liver. Just as there are four stagesin the oxidation of glucose, there are four stagesin the oxidation of fatty acids.To optimize the efficiency of ATP generation,part of stageII (citric acid cycle oxidation of acetyl CoA) and all of stagesIII and IV of fatty acid oxidation are identical to those of glucose

section, the reduced NADH and FADH2 with their highenergyelectronsfrom stageII will be used in stageIII to generct; a proton-motive force that in turn is used in stageIV to power ATP synthesis.

PeroxisomalOxidation of Fatty Acids GeneratesNo ATP Mitochondrial oxidation of fatty acids is the major sourceof ATP in mammalian liver cells, and biochemistsat one time believedthis was true in all cell types. However, rats treated with clofibrate, a drug that affectsmany featuresof lipid metabolism, were found to exhibit an increasedrate of fatty acid oxidation and a large increasein the number of peroxisomes in their liver cells.This finding suggestedthat peroxisomes'as well as mitochondri a, can oxidize fatty acids' These small or-

carbonsin the fatty acyl chain, or (Cs)' medium-(C3-Crz),

: L Y C O L Y S IASN D T H E C I T R I CA C I D C Y C L E F I R S TS T E P SO F G L U C O s EA N D F A T T YA C I D C A T A B O L I S MG

491

> FIGURE12-12 Oxidation of fatty acids in mitochondria and peroxisomes.In both mitochondrial oxidation(a)and peroxisomal oxidation(b),fatty acidsare convertedto acetylCoA by a seriesof four enzyme-catalyzed reactions(showndown the centerof the fioure) A fatty acylCoA moleculeis convertedto u..tyl CoA and a fatty acylCoA shortenedby two carbonatoms Concomitantly, one FADmolecule is reducedto FADH2and one NAD* molecule is reducedto NADH The cycleis repeatedon the shortenedacylCoA untilfatty acidswith an evennumberof carbonatomsare completelv converted to acetylCoA In mitochondrra, electrons from FADH2and NADHenterthe electrontransport chainand ultimately are usedto generateATp;the acetylCoA generatedisoxidizedrn the citricacid cycle,resulting in release of CO2and ultimately the synthesis of additionalATP Becauseperoxisomes lackthe electrontransportcomplexescomposlng the electrontransportchainand the enzymesof the citricacidcycle,oxidationof fatty acidsin these organellesyieldsno ATPlAdapted fromD L Netson principlesof andl\,4M Cox,Lehninger Biochemistry,3d ed , 2000,WorthPublishers l

( a ) M I T O C H O N D R I AOLX t D A T | O N (b)PEROXTSOMAL OXtDATION

o R- CH2-CH2-CHr-C-SCoA Fatty acyl CoA

o

H r O+ ' t / z O ,

il

R- CH2-CH: CH-C-SCoA

HrO

NADH exportedfor reoxidation

R-CHr-C-SCoA I

o

Acyl CoA shortened by two carbon atoms +

o Citricacid +cycre

l

H3C-C-SCoA Acetyl CoA

Acetyl CoA exported

stead it is transported into the cytosol for use in the synthesis of cholesterol(Chapter10) and other metabolites.

oxidation of fatty acids, which is coupled to generation of ATP, peroxisomal oxidation of fatty acids is not linked to ATP formation, and energy is releasedas heat. The reaction pathway by which fatty acids are degraded to acetyl CoA in peroxisomesis similar to that used in mito_

dases, peroxisomes contain abundant catalase, which quickly decomposesthe H2O2, a highly cytotoxic metabo_ lite. NADH produced during oxidation of fatty acids is exported and reoxidized in the cytosol; there is no need for a malate/aspartateshuttle here. peroxisomesalso lack the cit_ ric acid cycle, so acetyl CoA generatedduring peroxisomal degradation of fatty acids cannot be oxidized further: in492

c H A P T E R1 2

|

cELLULAR ENERGETTCS

First Steps of Glucoseand Fatty Acid Catabolism: Glycolysisand the Citric Acid Cycle r In a processknown as aerobicoxidation, cellsconvert the energy releasedby the oxidation ("burning") of glucoseor fatty acidsinto the terminal phosphoanhydridebond of ATp. r The complete aerobic oxidation of each molecule of glucoseproduces six moleculesof CO2 and approximately 30 ATP molecules.The entire process,which starts in the cytosol and moves into the mitochondrion, can be divided into four stages:(I) glycolysis to pyruvate in the cytosol, (II) pyruvate oxidation to C02 in the mitochondrion. (III) electron transport to generatea proton-motive force together with conversion of molecular oxygen to wate! and (IV) ATP synthesis. r The mitochondrion hastwo distinctmembranes(outerand inner) and two distinct subcompartments(intermembrane

space between the two membranesand the matrix surrounded by the inner membrane).Aerobic oxidation occurs in the mitochondrial matrix and on the inner mitochondrial membrane. r Each turn of the citric acid cycle releasestwo molecules of CO2 and generatesthree NADH molecules,one FADH2 molecule. and one GTP. r In glycolysis (stageI), cytosolic enzymesconvert glucose to two moleculesof pyruvate and generatetwo molecules each of NADH and ATP. r The rate of glucoseoxidation via glycolysisand the citric acid cycle is regulated by the inhibition or stimulation of severalenzymes,dependingon the cell'sneed for ATP. Glucoseis stored (asglycogenor fat) when ATP is abundant. r Some of the energy releasedin the early stagesof oxidation is temporarily stored in the reducedcoenzymesNADH or FADH2, which carry high-energy electrons that subsequently drive the electron transport chain (stageIII). r In the absenceof oxygen (anaerobicconditions),cellscan metabolize pyruvate to lactate or (in the case of yeast) to ethanol and CO2, in the processconvertingNADH back to NAD*, which is necessaryfor continued glycolysis.In aerobic conditions (presenceof oxygen),pyruvate is transported into the mitochondrion, where stagesII through IV occur.

chain, also known as the respiratorychain into the protonmotive force.'We first describethe logic and componentsof the electron transport chain and the pumping of protons acrossthe mitochondrial inner membrane.'Weconcludethe sectionwith a discussion of the magnitude of the proton-motive force produced by electron transport and proton pumping. In the following section,we describestageIV, focusing on the structure of the AIP synthaseand how it usesthe proton-motive force to synthesizeATP.

StepwiseElectronTransportEfficientlyReleases t h e E n e r g yS t o r e di n N A D Ha n d F A D H 2 During electron transport' electrons are released from NADH and FADH2 and eventually transferred to 02, forming H2O according to the following overall reactions: NADH + H+ + 1/z02 --+ NAD* + H2O, LG: -52'6 kcal/mol FADH2 -r 1/z02 --+ FAD + H2O, L'G : -43.4 kcal/mol Recall that the conversion of 1 glucosemolecule to CO2 via the glycolytic pathway and citric acid cycle yields 10 NADH and-2 FADH2 molecules (seeTable 12-1). Oxidation of -613 kcal/mol thesereducedcoenzymeshas a total AGo'of

r In stage II, the three-carbon pyruvate molecule is first oxidized to generateone moleculeeach of CO2, NADH' and acetyl CoA. The acetyl CoA is then oxidized to CO2 by the citric acid cycle. r Neither glycolysis(stageI) nor the citric acid cycle (stage II) directly use molecularoxygen (02). r The malate/aspartateshuttle regeneratesthe supply of for continuedglycolysis. cytosolicNAD* necessary r Like glucoseoxidation, the oxidation of fatty acids takes place in four stages.In stageI, fatty acids are converted to fatty acyl CoA in the cytosol. In stageII, the fatty acyl CoA is first converted into multiple acetyl CoA moleculeswith generationof NADH and FADH2. Then, as in glucoseoxidation, the acetyl CoA entersthe citric acid cycle. StagesIII and IV are identical for fatty acid and glucoseoxidation. r In most eukaryotic cells, oxidation of short- to longchain fatty acids occurs in mitochondria with production of ATR whereas oxidation of very long chain fatty acids occurs primarily in peroxisomes and is not linked to ATP production; the releasedenergy is converted to heat.

Slfl The ElectronTransportchain and Generationof the Proton-MotiveForce Most of the energy releasedduring the oxidation of glucose and fatty acidsto CO2 (stagesI and II) is convertedinto highenergyelectronsin the reducedcoenzymesNADH and FADH2. \7e now turn to stageIII, in which the energytransiently stored in the coenzymesis converted by an electron transport

duction of FAD, which requires lessenergy. The energy carried in the reduced coenzymescan be released by oxidizing them. The biochemical challengefaced by the mitochondrion is to transfer,as efficiently as possible' the energy releasedby this oxidation into the energy in the terminal phosphoanhydridebond in ATP. P,t- * H* + ADP3- -+ATPa- + H2o, L,G : +7.3 kcal/mol A relatively simple one-to-onereaction in which reduction of one coenzyme molecule and synthesis of one ATP occurs would be terribly inefficient, becausethe AG'' for ATP generation from ADP and P1is substantially less than for the coenzymeoxidation and much energywould be lost as heat' To efficiently recover the energ5 the mitochondrion first converts the energy of coenzyme oxidation into a protonmotive force using a seriesof electron carriers' all but one of which are integral components of the inner membrane'

ElectronTransportin Mitochondrials Coupled t o P r o t o nP u m P i n g At severalsites during electron transport from NADH and FADH2 to C,2,protons from the mitochondrial matrix are 493

THEELECTRoNTRANSPORTcHA|NANDGENERAT|oNoFTHEPRoToN-MoT|VEFoRcE

pH electrode

O, solution

c

60

o

8= 40 +P

.=-

20

a)

(J n

0 Mitochondrion EXPERIMENTAL FIGURE 12-13Electrontransferfrom NADH to 02 is coupledto proton transportacrossthe mitochondrial membrane.lf NADHisaddedto a suspension of mitochondria depleted of 02, no NADHisoxidizedWhena smallamountof O, is (arrow), addedto thesystem thereisa sharprisein theconcentration of protonsin thesurrounding mediumoutside the mitochondria pumped acrossthe inner membrane;this generares proton concentration and electricalgradientsacrossthe inner membrane (seeFigure 12-2). This pumping causesthe pH of the mitochondrial matrix to becomehigher (i.e.,the H+ concentration is lower) than that of the intermembranespaceand cytosol.An electricpotential acrossthe inner membranealso resultsfrom the pumping of H* outward from the matrix, which becomes negative with respect to the intermembrane space.Thus free energy releasedduring the oxidation of NADH or FADH2 is storedboth as an electricpotential and a proton concentrarion gradient-collectivelS the proton-motive force-across the

major source of ATP in aerobic nonphotosyntheticcells. Much evidenceshows that in mitochondria and bacteriathis processof oxidative phosphorylation dependson generarion of a proton-motive force acrossthe inner membrane (mitochondria) or bacterial plasma membrane, with electron transport, proton pumping, and ATp formation occurring simultaneously.In the laboratory, for instance, addition o] 02 and an oxidizable substratesuch as pyruvate or succinare to isolated intact mitochondria results in a net synthesisof ATP if the inner mitochondrial membrane is intact. In the presence of minute amounts of detergents that make the membrane leaky, electron transport and the oxidation of these metabolites by 02 still occurs. However, under these conditions no ATP is made, becausethe proton leak prevents the maintenance of the transmembrane proton concentration gradientand the membraneelectricporential. The coupling between elecrron transporr from NADH (or FADH2) to 02 and proron rransporr aiross the inner mitochondrial membrane can be demonstrutedexperimentally 494

.

cHAprE1 R2 I c E L L U L AER NERGETtcs

60 120 180 E l a p s etdi m e( s )

240

300

(decrease in pH) Thustheoxidation of NADHby 02 iscoupled to the movement of protons out of the matrixOncethe 02 isdepleted, the protons excess slowlymovebackintothe mitochondria (powering the synthesis of ATP)andthe pHof the extracellular mediumreturns t o i t si n i t i avl a l u e .

with isolated, intact mitochondria (Figure 12-13). As soon as 02 is addedto a suspensionof mitochondria in an otherwise O2-free solution that contains NADH, the medium outside the mitochondria transiently becomesmore acidic (increasedproton concentration), becausethe mitochondrial outer membrane is freely permeableto protons. (Remember that malate/aspartateand other shuttles can convert the NADH in the solution into NADH in the matrix.) Once the 02 is depleted by its reduction, the excessprotons in the medium slowly leak back into the matrix. From analysisof the measuredpH changein such experiments,one can calculate that about 10 protons are transported out of the matrix for every electron pair transferred from NADH to 02. To obtain numbers for FADH2, the above experiment can be repeated,but with succinateinsteadof NADH as the substrate. (Recall that oxidation of succinateto fumarate in the citric acid cycle generatesFADH2; see Figure 12-10). The amount of succinateaddedcan be adiustedso that the amount of FADH2 generatedis equivalentto the amount of NADH in the first experiment. As in the first experiment, addition of oxygen causes the medium outside the mitochondria to becomeacidic, but lessso rhan with NADH. This is not surprising becauseelectronsin FADH2 havelesspotential energy (43.4 kcal/mol) than electrons in NADH (52.6 kcal/mole). and thus it drives the translocation of fewer protons from the matrix and a smaller changein pH.

ElectronsFlow from FADH2and NADHto 02 T h r o u g hF o u rM u l t i p r o t e i nC o m p l e x e s I7e now examine more closely the energetically favored movement of electronsfrom NADH and FADH2 to the final electron acceptor,02. For simplicity, we will focus our discussion on NADH. In respiring mitochondria, each NADH molecule releasestwo electronsto the electron transDort

chain; these electrons ultimately reduce one oxygen atom (half of an 02 molecule),forming one moleculeof water: NADH--+NAD* + H* + 2e t/rOr--HrO 2e- + 2H' + As electronsmove from NADH to 02, their potential declines by 1.14 V, which correspondsto 26.2 kcal/mol of electronstransferred,or :53 kcal/mol for a pair of electrons. As noted earlier,much of this energyis conservedin the proton-motive force generatedacross the inner mitochondrial membrane. There are four large multiprotein complexesin the electron transport chain that span the inner mitochondrial membrane: NADH-CoQ reductase(complex I, >40 subunits), swccinate-CoQreductase(complex II, 4 subunits), CoQH2-cytochromec reductdse(complexIII, 11 subunits), and cytochrome c oxidase (complex IV, 13 subunits). Electrons from NADH flow from complex I to III to IV, bypassing complex II; electronsfrom FADH2 flow from complex II to III to IV, bypassingcomplex I (seeFigure 12-8). Each complex contains several prosthetic groups that participate in moving electrons.These small nonpeptide organic moleculesor metal ions are tightly and specificallyassociatedwith the multiprotein complexes(Table 12-2).

COMP()NENT PROTEIN

GROUPSPR{]STHETIC

NADH-CoQ reductase (complex I)

FMN Fe-S

Succinate-CoQreductase (complex II)

FAD Fe-S

CoQH2-cytochrome c reductase (complex III)

Heme by Heme bp1 Fe-S Heme c1

Cytochrome c

Heme c

Cytochrome c oxidase (comPlex IV)

Crru2* Heme a LutHerne a3

*-Not included is coenzyme Q, an electron carrier that is not permanently bound to a protein complex. iou*.,.' J. \Xl.De Pierre and L. Ernster, L977, Ann. Reu. Biochem' 46:201..

Heme and the Cytochromes Severaltypesof heme,an ironcontaining prostheticgroup similar to that in hemoglobin and myoglobin (Figure 12-14a\, are tightly bound (covalently or noncovalently) to a set of mitochondrial proteins called cytochromes.Each cytochrome is designatedby a letter, such as A, b, c, or c1. Electron flow through the cytochromesoccurs by oxidation and reductionof the Fe atom in the centerof the hememolecule: Fe3* + e-

.

. --> Fe2*

Becausethe heme ring in cytochromesconsistsof alternating double- and single-bondedatoms, a large number of resonance hybrid forms exist. These allow the extra electron delivered to the cytochrome to be delocalizedthroughout the heme carbon and nitrogen atoms as well as the Fe ion. The various cytochromes have slightly different heme groups and surrounding atoms (called axial ligands), which

(b)

ta) HrC:CH II

9H.

Protein

tl

CH,

l--l o2c-cH2

H,C

H2c-co2

12-14 Hemeand iron-sulfurprostheticgroupsin A FIGURE bg the electrontransportchain.(a)Hemeportionof cytochromes c reductase of CoQHz-cytochrome andbs,whicharecomponents in allhemes (complex is present ring(yellow) lll) Thesameporphyrin porphyrin ring differin the to the attached substituents Thechemical

accept chain.All hemes transport in theelectron othercytochromes (Fe-S) (b) cluster iron-sulfur Dimeric time. at a oneelectron andrelease sulfur and inorganic are two atoms: four S to is bonded Fe atom Each proteinAll Fe-S of the associated sidechains two arein cysteine at a time. oneelectron acceptandrelease clusters

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generate different environments for the Fe ion. Therefore, each cytochrome has a different reduction potential, or tendency to accept an electron-an important property dictating the unidirectional "downhill" electron flow along the chain. Just as water spontaneously flows downhill from a higher to lower potential energy state-but not uphill-so too do electronsflow in only one direction from one heme (or other prosthetic group) to another due to their differing reduction potentials. All the cytochromes, excepr cytochrome c, are componentsof integral membranemultiprotein complexesin the inner mitochondrialmembrane. fron-Sulfur Clusters lron-sulfur clustersare nonheme,ironcontaining prosrheticgroups consistingof Fe atoms bonded both to inorganicS atomsand to S atomson cysteineresiduesin a protein (Figure I2-l4b). Some Fe aroms in the cluster bear a *2 charge;othershavea *3 charge.However,the net chargeof eachFe atom is actuallybetween+2 and *3, becauseelectrons in their outermost orbitals together with the extra electron delivered via the transport chain are dispersedamong the Fe atoms and move rapidly from one atom to another.Iron-sulfur clustersacceptand releaseelecffonsone at a time. Coenzyme Q (CoQ) Coenzyme e (Coe), also called ubiquinone, is the only small-moleculeelectron carrier in the chain that is not a protein-bound prosthetic group (Figure 12-15). It is a carrier of both protons and electrons.The oxidized quinone form of CoQ can accepr a single electron to

o (coo) ubiquinone ( o x i d i z feodr m )

H3co

cH, itt (CHr-CH:C-CH2)10-H

H3CO

o I e rl

J

os e m i q u i n o n e( c o e ; ) { f r e er a d i c a l )

H3cO

CH.

H3CO

(CHr-CH:J-CH2)10-H

9H'

form a semiquinone, a charged free radical denoted by CoQ .. Addition of a secondelectronand two protons (thus a total of two hydrogen atoms) to CoQ-. forms dihydroubiquinone (CoQH2), the fully reducedform. Both Coe and CoQH2 are solublein phospholipidsand diffuse freely in the hydrophobic centerof the inner mitochondrial membrane. This is how it participatesin the electron transport chaincarrying electronsand protons betweenthe complexes. As shown in Figure 12-16, CoQ acceptselectronsreleased from NADH-CoQ reductase(complex I) or succinate-Coe reductase(complex II) and donates them to CoeH2cytochrome c reductase(complex III). Importantly, reduction and oxidation of CoQ are coupled to pumping of protons. Ifhenever CoQ acceptselectrons,it does so at a binding site on the matrix (also called the cytosolic) face of the protein complex, always picking up protons from the medium there. \Thenever CoQH2 releasesits electrons,it doesso at a srteon the intermembranespace(also called the exoplasmic)side of the protein complex, releasingprotons into the intermembrane (or exoplasmic) fluid. Thus transport of each pair of electronsby CoQ is obligatorily coupled to movement of two protons from the matrix to the intermembranespacefluid. NADH-CoQ Reductase (Complex l) Electronsare transferred from NADH to CoQ by NADH-CoQ reducrase(Figure 12-16).In bacteriathe massof this complex is about 500 kDa (-14 subunits),whereasfor the L-shapedeukaryoticcomplex it is 1 MDa (14 centraland as many as 32 accessorysubunits). NAD- is exclusively a two-electron carrier: it accepts or releasesa pair of electronssimultaneously.In NADH-Coe reductase (complex I), electronsfirst flow from NADH to FMN (flavin mononucleotide), a cofactor related to FAD, then to an iron-sulfur clusteq and finally to CoQ. FMN, like FAD, can accept two electronsbut does so one electron at a trme. Each transported electron undergoesa drop in potential of =360 mV, equivalenrto a AG'' of -16.6kcallmol for the two electrons transported. Much of this releasedenergy is used to transport four protons across the inner membrane per molecule of NADH oxidized by the complex I. Those four protons are distinct from the two protons transferredto the CoQ in the chemical reaction shown above. The overall reaction catalyzedby this complex is

U.

zut +

"-

NADH + CoQ * 6H*1. --+

t.l

J

(Reduced) (Oxidized)

NAD- * Hti. + CoeH2 + 4H+o,t

OH

(Oxidized)

Dihydroquinone 1 999H,) (fully reduced form)

H3co

cH, ?*' (CHr-CH:C-CH2)10-H

H3CO OH

A FIGURE12-15 Oxidized and reduced forms of coenzyme e (CoQ),which can carry two protons and two electrons. Because of its long hydrocarbon "tail" of isopreneunits,Coe, alsocalled ubrquinone, is solublein the hydrophobic coreof phospholipid bilayers and is verymobile.Reductionof Coe to the fully reducedform, eH2 (dihydroquinone), occursin two stepswith a half-reduced free-radical intermediate , l l e ds e m i q u i n o n e ca 496

.

c H A p r E R1 2 |

C E L L U L AERN E R G E T t c s

(Reduced)

Succinate-CoQ Reductase (Complex ll) Succinatedehydrogenase,the enzyme that oxidizes a molecule of succinate to fumaratein the citric acid cycle(and in the processgenerares the reduced coenzymeFADH2), is one of the four subunits of complex II. In this way the citric acid cycle is physically as well as functionally linked to the electron transport chain. The two electronsreleasedin conversionof succinateto fumarate are transferredfirst to FAD in succinatedehydrogenase,then to iron-sulfur clusters-regenerating FAD-and finally to Coe,

llll+ Animation:ElectronTransport

at (b) Fromsuccinate

( a ) F r o mN A D H Intermembranespace (exoplasmic) 4 H+++

zn

Exoplasmic oO

Cytosolic

4HMatrix

(cytosolic) NADH

1120+ 22H O 2H* H* H z O

zn

NAD++ H+

NADH-CoOreductase { c o m p l e xl )

CoOH2-cytochrome c reductase (complex lll)

Complex lll

Gytochrome c oxidase (complex lV)

Succinate

Fumarate+2 H*

reductase Succinate-CoO (comPlexll)

FIGURE 12-16 Multiproteincomplexesand mobileelectron (bluearrows) carriersof the electrontransportchain.Electrons (l-lV) Electron flow throughfour majormultiprotein complexes rsmediated eitherbythe lipid-soluble movement between complexes form)or reduced form;CoQHz, molecule coenzyme Q (CoQ,oxidized proteincytochrome c (cytc),Themultipleprotein thewatersoluble frompassing electrons to pump usethe energyreleased complexes protons space(redarrows) fromthe matrixto the intramembrane (a)Pathway l, flowto complex fromNADHFromNADHelectrons perpairof aretranslocated thenlllandthenlV A totalof 10 protons intothe thatflowfromNADHto O, Theprotonsreleased electrons

I are of NADHby complex matrixspaceduringoxidation lV, by complex from 02 water of in formation the consumed fromthesereactions in no netprotontranslocation resulting (via flow fromsuccinate (b)Pathway Electrons fromsuccinate released lllandthenlV Electrons ll to complex in complex FADHz) ll areused in complex to fumarate of succinate duringoxidation additional without translocating to CoQHz CoQ reduce to fromCoQHz transport of electron protons, Theremainder proceeds bythe samepathwayasfor the NADHpathwayin (a). to 02, fromsuccinate transported Thusfor everypairof electrons lV lll and complexes by protons are translocated six

which binds to a cleft on the matrix side of the transmembrane portions of complex II (Figure 72-16). The overall reaction catalyzedby this complex is

Figure 12-12).There are severalfatty acyl-CoA dehydrogenase enzymeswith specificitiesfor fatty acyl chains of different lengths.Theseenzymesmediate the initial step in a four-step processthat removestwo carbons from the fatty acyl group by oxidizing the carbon in the B position of the fatty acyl chain (thus the entire processis often referred to as B-oxidation). Thesereactionsgenerateacetyl CoA, which in turn entersthe citric acid cycle. They also generatean FADH2 intermediate and NADH. The FADH2 generated remains bound to the enzymeduring the redox reaction,as is the casefor complex II. A water-soluble protein called electron transfer flauoprotein (ETF) transfersthe high-energy electrons from the FADH2 in the acyl-CoA dehydrogenaseto electron transfer flauoprotein:ubiquinoneoxidor eductase(ETF: QO), a membraneprotein that reducesCoQ to CoQH2 in the inner membrane.This CoQH2 intermixes in the membrane with the other CoQH2 moleculesgeneratedby complexesI and II.

Succinate+ CoQ --+fumarate + CoQH2 (Reduced)

(Oxidized) (Oxidized)

(Reduced)

Although the AGo' for this reactionis negative,the released energyis insufficientfor proton pumping in addition to reduction of CoQ to form CoQH2, which then dissociates from complex II. Thus no protons are translocateddirectly acrossthe membraneby the succinate-CoQreductasecomplex, and no proton-motive force is generatedin this part of the respiratorychain. Shortly we will seehow the protons and electronsin the CoQH2 moleculesgeneratedby complex I and complex II contribute to the generationof the proton-motive force. Complex II generatesCoQH2 from succinatevia FAD/ FADH2-mediatedredox reactions.Another setof proteinsin the matrix and inner mitochondrialmembraneperformsa comparedox reactionsto generate rable setof FAD/FADH2-mediated CoQH2 from fatty acyl CoA. Fatty acyl-CoA dehydrogenase, which is a water-solubleenzyme,catalyzesthe first stepof the oxidation of fatty acyl CoA in the mitochondrial matrix (see

CoQH2-Cytochrome c Reductase(Complex lll) A CoQHz generatedeither by complex I or complex II (or ETF:QO) donates two electronsto CoQH2-cytochrome c reductase(complex III), regeneratingoxidized CoQ. Concomitantlyit releases into the intermembranespacerwo protonspreviouslypickedup on the matrix face, generatingpart of the proton-motive force

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(Figure 12-16). Vithin complex III, the releasedelectronsfirst are transferredto an iron-sulfur cluster within complex III and then to cltochrome c1 or to two &-type cytochromes (by and bs, seeQ cycle below). Finally, the two electronsare transferred sequentially to two molecules of the oxidized form of cytochrome 6, a water-soluble peripheral protein that diffuses in the intermembranespace.For eachpair of electronstransferred, the overall reaction catalyzed by the CoQH2-cytochrome c reductasecomplex is CoQH2 + 2 Cytc3* *2 H+i. -+ Coe + 4 H+o,t+ 2 Cytc}+ (Reduced)

(Oxidized)

(Oxidized)

(Reduced)

The AG'' for this reaction is sufficiently negative that two protons in addition to those from CoQH2 are translocated from the mitochondrial matrix across the inner membrane for each pair of electronstransferred; this involves the proton-motive Q cycle, discussedlater. The heme protein cytochrome c and the small lipid-soluble molecule Coe play similar roles in the electron transport chain in that they both serveas mobile electron shuttles,transferring electrons(and thus energy)betweenthe complexesof the elecrronrransporr chain. Cytochrome c Oxidase (Complex lV) Cytochrome c, after being reduced by CoQH2-cytochrome c reductase(complex III), is reoxidized as it transports electrons,one at a time, to cytochrome c oxidase (complex IV) (Figure 12-16). Mitochondrial cytochrome c oxidases contain 13 different subunits, but the catalytic core of the enzyme consists of only

three subunits.The function of the remaining subunits is less well understood. Bacterial cytochrome c oxidases contain only the three catalyric subunits. Four moleculesof reduced cytochrome c bind, one at a time, to the oxidase.An electron is transferred from the heme of each cytochrome c, first to the pair of copper ions called Crr^2*,then to the heme in cytochrome a, and next to the Cub2+ and the heme in cytochrome a3 that together make up the oxygen reduction center. The electrons are finally passedto 02, the ultimate electron acceptor,yielding 4 H2O, which together with CO2 is one of the end products of the oxidation pathway. Proposed intermediatesin oxygen reduction include the peroxide anion (Ort-) and probably the hydroxyl radical iOH.), as well as unusual complexes of iron and oxygen atoms. These intermediateswould be harmful to the cell if they escaped from the reaction center, but they do so only rarely (seethe discussionof reactiveoxygen speciesbelow). During transport of four electrons through the cytochrome c oxidase complex, four protons from the matrix space are translocatedacrossthe membrane.However, the mechanism by which theseprotons are translocatedis not known. For each four electronstransferred, the overall reaction catalyzed by cytochrome c oxidase is 4 C y t c 2 * * 8 H + 1 , + 0 2 - + 4 C y t c 3 ++ 2 H z O + (Reduced)

4H+o,t

(Oxidized)

The poison cyanide, used as a chemical warfare agent, by spies to commit suicide when captured, in gas chambers to executeprisoners, and by the Nazis (Zyklon B

(a) C o m p l e xl l l d i m e r C o m p l e xl V ,-

#-

Supercomplexl/lll2llV gupspsomplexl/lll,

-

Complex I ATP synthase

-

C o m p l e xl l l d i m e r ( l l l 2 )

-

Complex lV

-

C o m p l e xl l

**-

EXPERIMENTAL FIGURE l2-17 Electrophoresis and electron microscopicimaging identifiesan electrontransportchain supercomplex containingcomplexesl, lll, and lV.(a)Membrane proteins in isolated bovineheartmitochondria weresolubilized wlth a detergent, andthe complexes andsupercomplexes wereseparated by gelelectrophoresis usingthe bluenative(BN)-PAGE method.Each blue-stained bandwithinthe gelrepresents protein the indicated complex or supercomplex, with ll12 representing a dimerof complex lll Intensity of the bluestainisapproximately proportional to theamount of complex or supercomplex present(b)Supercomplex l/lllrllV was

498

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CELLULAR ENERGETICS

extracted fromthe gel,andthe particles werenegatively stained with 1% uranylacetate andvisualized by transmission electron microscopy. lmages of 228 particles werecombined at a resolution o'f-3 4 nm to generate an averaged imageof the complex viewed fromthesidein the planeof the membrane Approximate locations of thecomplex llldimerandcomplex lV areindicated by dashed ovals; the outlineof complex I isalsoindicated by a dashed line (white).Scalebaris 10 nm. fAdapted fromE Schafer et al, 2006, ]. Biol. Chem 2A1Q2): 1537 O-1537 5 l

gas) for the mass murder of Jews and others, is toxic becauseit binds to the heme a3 in mitochondrial cytochrome c oxidase (complex IV), inhibiting cellular respiration and therefore production of ATP. Cyanide is one of many toxic small moleculesthat interfere with energy production in mitochondria. I

ReductionPotentialsof ElectronCarriersFavor ElectronFlow from NADHto 02

Electron Transport Supercomplexes Over 50 years ago Britton Chance proposed that electron transport complexes might assembleinto large supercomplexes.Doing so would bring the complexesinto closeand highly organized proximity, which might improve the speed and efficiency of the overall process.However, an alternativeview holding that the complexesbehaved as independententities diffusing freely in the inner membrane became the dominant paradigm. During the past several years, genetic, biochemical, and biophysical studies have provided very strong evidencefor the existenceof electron transport chain supercomplexes.These studies involved relatively new gel electrophoreticmethods called blue native (BN)-PAGE and colorless native (CN)-PAGE, which permit separation of very large macromolecular protein complexes, and electron microscopic analysisof their threedimensionalstructures. One such supercomplex contains one copy of complex I, a dimer of complex III (III2), and one or more copies of complex IV (Figure 1,2-1,7).The unique phospholipid cardiolipin (diphosphatidylglycerol)

is a measureof the equilibrium constant of that partial reaction. lfith the exception of the b cytochromes in the CoQH2-cytochrome c reductasecomplex, the standard reduction potential Eo' of the electron carriers in the mitochondrial respiratory chain increasessteadily from NADH to 02. For instance,for the partial reaction

Cardiolipin o

g6

+lta-o-[-o.-X-.o

do

As we saw in Chapter 2, the reduction potential E fot a partial reduction reaction

NAD* +H*

I

a-o-i-

appears to play an important role in the assembly and function of these supercomplexes.Generally not observed in other membranes of eukaryotic cells, cardiolipin has been observed to bind to integral membrane proteins of the inner membrane (e.g., complex II). Genetic and biochemical studies in yeast mutants in which cardiolipin synthesisis blocked have establishedthat cardiolipin contributes to the formation and activity of mitochondrial supercomplexes,and thus it has been called the glue that holds together the electron transport chain, though the precise mechanism remains to be defined. In addition, there is evidencethat cardiolipin may influence the inner membrane'sbinding and permeability to protons and consequentlythe proton-motive force.

+2C_

-

'NADH

-320 mV, the value of the standard reduction potential is for transfer + kcal/mol 14.8 AGo' of which is equivalentto a proceed to tends partial reaction Thus this of two electrons. toward the left, that is, toward the oxidation of NADH to NAD+. By contrast, the standard reduction potential for the partial reactron Cytochromeco*(Fe3*) + e

-

- cytochromecred(F.t*)

is +220 mV (AG"' : -5.1 kcal/mol)for transferof one electron. Thus this partial reaction tends to proceed toward the * right, that is, toward the reduction of cytochrome c (Fe3 ) to cytochrome c (Fe'-). The final reaction in the respiratory chain, the reduction of 02 to H2O 2H-

) HO< ) o

= reduced molecule

Oxidized molecule * e- -

+ 'lror+

2e- -+H2O

has a standard reduction potential of +816 mV (AG'' : -37.8 kcal/mol for transfer of two electrons),the most positive in the whole series;thus this reaction also tends to proceedtoward the right. As illustrated in Figure 12-1'8,the steady increasein Eo' values,and the correspondingdecreasein AGo' values' ofthe carriers in the electron transport chain favors the flow of electrons from NADH and FADH2 (generatedfrom succinate) to oxygen.

ExperimentU s s i n gP u r i f i e dC o m p l e x e s the Established Stoichiometryof Proton Pumping The multiprotein complexesresponsiblefor proton pumping coupled to electron transport have been identified by selectively extracting mitochondrial membranes with detergents, isolating each of the complexes in nearly pure form, and then preparing artificial phospholipid vesicles (liposomes)containing each complex. $7hen an appropriati electrondonor and electronacceptorare added to such

O F T H E P R O T O N - M O T I VFEO R C E T R A N S P O RC T HAINAND GENERATION THE ELECTRON

Redox potential tmV)

F r e ee n e r g y (kcal/mol)

60 -400 -

NADH-CoO reductase (complex l) NADH

NAD++ H+

\.v 2 e-

F u m a r a t e+ 2 H + 5U

-200

Succinate-CoO reductase(complexll) 40

H+

")

Fe-S

30

H* Cyt c.,

CoQHr-cytochrome c r e d u c t a s e( c o m p l e x l l l ) Cyt c cuu

I

20

t*

10

(complex lV)

800

2 e-

tlz Oz + 2H*

HzO

FIGURE 12-18Changesin redoxpotentialand free energy duringstepwiseflow of electronsthroughthe respiratory chain.Bluearrowsindicate electron flow;redarrows, translocation of protons across the innermitochondrial membraneElectrons pass throughthe multiprotein complexes fromthoseat a lowerreduction

potential to thosewith a higher(morepositive) (leftscale), potential with a corresponding reduction in freeenergy(rightscale)The energyreleased aselectrons flowthroughthreeof thecomplexes is sufficient to powerthe pumpingof H* ionsacross the membrane, establishing a proton-motive force

liposomes,a changein pH of the medium will occur if rhe embedded complex transporrs protons (Figure 12-19). Studies of this type indicate that NADH-Coe reductase (complex I) translocatesfour protons per pair of electrons transported, whereas cytochrome c oxidase (complex IV) t r a n s l o c a t e st w o p r o t o n s p e r e l e c t r o n p a i r t r a n s p o r t e d (or, equivalently,for every two moleculesof cytochrome c oxidized). Current evidencesuggeststhat a total of 10 protons are transportedfrom the matrix spaceacrossthe inner mitochondrial membranefor everyelectronpair that is transferredfrom NADH to 02 (seeFigure 12-1,6).Becausesuccinate-Coe reductase(complexII) doesnot transport protons and complex I is bypassedwhen the electronscome from succinatederived FADH2, only six prorons are rransported acrossthe

membrane for every electron pair that is transferred from this FADH2 to 02.

s00

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CELLULAE RN E R G E T T C S

The Q CycleIncreasesthe Numberof Protons Translocatedas ElectronsFlow Through C o m p l e xl l l Experiments such as the one depicted in Figure 12-1,9have shown that four protons are translocated across the membrane per electron pair transported from CoQH2 through CoQH2-cytochrome c reductase(complex III). Thus this complex transports two protons per electron transferred, whereas cytochrome c oxidase (complex IV) transports only one proton per electrontransferred.An evolutionarily conservedmechanism, called the Q cycle, accounts for the

(a)

-../

Phospholipid membrane

t02+ 2 Ht Hzo

(reduced)

into the intermembranespace,but one moleculeof CoQH2 is regeneratedfrom CoQ at the Q1 site (seeFigure 12-20, bottom).Thus the net result of the Q cycleis that four protons are translocatedto the intermembranespacefor every two electrons transported through the CoQH2-cytochrome c reductasecomplex and acceptedby two moleculesof cytochromec. The translocatedprotons are all derived from CoQH2, which

2Ht

lntermembrane space CoOH2 l2e I

K* Valinomycin-bound (b) Matrix

E a

q)

E -o-

GoOH2-cytochromec reductase(complex lll)

012 Elapsed time(min) FIGURE12-19 Electrontransfer from A EXPERIMENTAL reduced cytochrome c to 02 via cytochrome c oxidase (complex lV) is coupled to proton transport. The oxidasecomplexis c incorporated into liposomes with the bindingsitefor cytochrome positioned on the outersurface(a)When 02 and reducedcytochrome to 02 to form H2Oand protons c are added,electronsare transferred aretransportedfrom the insideto the mediumoutsideof the v e s i c l e sA d r u g c a l l e dv a l i n o m y c iwna s a d d e dt o t h e m e d i u mt o of H*, dissipatethe voltagegradientgeneratedby the translocation which would otherwisereducethe numberof protonsmovedacross the membrane(b) Monitoringof the medium'spH revealsa sharp drop in pH followingadditionof 02 As the reducedcytochromec and the becomesfully oxidized,protonsleakbackinto the vesicles, p H o f t h e m e d i u mr e t u r n st o i t s i n i t i avl a l u e M e a s u r e m e nst sh o w that two protonsare transportedper O atom reducedTwo electrons are neededto reduceone O atom, but cytochromec transfersonly of Cyt c'* are oxidizedfor eachO one electron;thus two molecules 1986,J Biol Chem26128254] reduced lAdaptedfrom B Reynafarleetal,

two-for-one transport of protons and electronsby complex I I I ( F i g u r e1 2 - 2 0 ) . The substratefor complex III, CoQH2, is generatedby severalenzymes,includingNADH-CoQ reductase(complex I) and succinate-CoQreductase(complex II), electron transfer flauoprotein:ubiquinone oxidoreductase(ETF:QO, during B-oxidation), and, as we shall see,by complex III itself. In one turn of the Q cycle,two moleculesof CoQH2 are oxidizedto CoQ at the Q. siteand releasea total of four protons

At Oo site: 2 CoOH2 + 2 Cyt C+ ---') ( 4 H + , z l e) 2 CoO+ 2 Cyt C+ +2 e +4 H+{outside)

(2e I At O; site: CoO + 2 e + 2 H+lmatrix -----+CoOH2 ";6sy ( 2 H ' ' 2 e \ Net O cycle (sum of reactions at Oo and O;): + CoOH2+ 2 Cyr C'+ 2 H+1661rix side) 12H*,'l t: I CoO + 2 Cyt C+ + 4 H+loutside) (2e ) t h r o u g hc o m p l e xl l l t o c y t o c h r o m ec , 4 H " P e r2 e t r a n s f e r r e d t o t h e i n t e r m e m b r a n sep a c e released A FIGURE12-20 The Q cycle.The Q cyclebeginswhen a molecule from the combinedpool of reducedCoQH, in the membranebinds to the Qo site on the intermembranespace(outel side of the portionof complexlll (steptr) There,CoQHz transmembrane space(stepEEI) and two protonsinto the intermembrane releases (stepB) One of the dissociate two electronsand the resultingCoQ protein and cytochromec1, iron-sulfur via an is transported, electrons that eachcytochromec directlyto cytochromec (stepEE) (Recall shuttlesone electronfrom complexlll to complexlV) The other b1 and bs and partiallyreduces electronmovesthroughcytochromes second,Qi, siteon the to the bound an oxidizedCoQ molecule matrix(inner)sideof the complex,forming a CoQ semiqutnone anion,Q ' (step4). The processis repeatedwith the bindingof a secondCoQH2at the Qo site(stepEt), proton release(stepEE), reductionof anothercytochromec (stepEEI),and additionof the other electronto the Q-'bound at the Qi site(stepZ) There,the additionof two protonsfrom the matrixyieldsa fully reducedCoQHz (stepsE and 9), moleculeat the Qrsite,which then dissociates (step I0) and begin freeingthe Q to bind a new moleculeof CoQ l99O, J Biol Chem B Trumpower from again the Q cycleover lAdapted BiochemSci26:4451 et al, 2001,Trends andE Darrouzet 265:11409,

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501

obtained its protons from the matrix, as a consequenceof the reduction of CoQ catalyzedby either NADH-CoQ re, ductase (complex I) or by CoQH2-cytochrome c reductase (complex III) (seeFigure 12-16). Although seeminglycumbersome, the Q cycle optimizes the numbers of protons pumped per pair of elecrronsmoving through complex III. The Q cycle is found in all plants and animals as well as in bacteria. Its formation at a very early stageof cellular evolution was likely essentialfor the successof all life-forms as a way of convertingthe potential energyin reducedcoenzymeQ into the maximum proton-motive force acrossa membrane. How are the two electronsreleasedfrom CoQH 2 at the Qo site directed to different acceptors, either to Fe-S, cytochrome c1 and then cytochrome c (upward in Figure 1220) or to cytochrome bL, cytochrome bs, and then CoQ at the Q1 site (downward in Figure 12-20)?The answer is simple and depends on a flexible hinge in the Fe-S-containing protein subunit of complex III. Initially the Fe-Scluster is close enough to the Qo site to pick up an electron from CoQH2 bound there. Once this happens, a segment of the protein containing this Fe-Scluster swings the cluster away from the Qo site to a position near enough to the heme on cytochrome c1 for electron transfer to occur. With the Fe-S subunit in this alternate conformation, the second electron releasedfrom CoQH2 bound to the Qo site cannot move to the Fe-Scluster-it is too far away, so it takes an alternative path open to it via a somewhat less thermodynamically favored route to cytochrome 61.

The Proton-MotiveForcein Mitochondria ls Due Largelyto a Voltage GradientAcross t h e I n n e rM e m b r a n e One result of the electron transport chain is the generation of the proton-motive force (pmf), which is the sum of a transmembraneproton concentration (pH) gradient and electricpotential, or voltage, gradient. It has beenpossibleto determine experimentally the relative contribution of the two componentsto the total pmf. The relative contributions depend on the permeability of the membrane to ions other than H+. A significant voltage gradient can develop only if the membrane is poorly permeable to other cations and to anions. Otherwise, anions would leak across from the matrix to the intermembrane spacealong with the protons and prevent a voltage gradient from forming. Similarly cations leaking acrossfrom the intermembrane spaceto the matrix (exchangeof like charge) would also short-circuit voltage gradient formation. Indeed, the inner mitochondrial membrane is poorly permeablero other ions. Thus proron pumping generatesa voltage gradient that makes it energetically difficult for additional prorons ro move across becauseof charge repulsion. As a consequence,proton pumping by the electron transport chain establishesa robust voltage gradient in the context of a rather small pH gradient. Becausemitochondria are much too small to be impaled with electrodes,the electricpotential and pH gradient across the inner mitochondrial membrane cannot be determined by direct measurement. Nevertheless,it has been possible to

502

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CELLULAE RN E R G E T I C S

develop methods to measureindirectly these critical values. The electricpotential can be measuredby adding radioactive 42K* ions and a trace amount of valinomycin to a suspension of respiring mitochondria. Although the inner membrane is normally impermeableto K*, valinomycin is an ionophore, a small lipid-soluble molecule that selectively binds a specific ion (in this case, K*) and carries it across otherwise impermeable membranes.In the presenceof valinomycin, a2K* equilibrates across the innir membrane of isolated mitochondria in accordancewith the electric potential: the more negativethe matrix side of the membrane, the more 42K* will be attracted to and accumulatein the matrix. At equilibrium, the measuredconcentration of radioactive K+ ions in the matrix, [K6], is about 500 times greater than that in the surrounding medium, [Ko"J. Substitution of this value into the Nernst equation (Chapter 11) shows that the electricpotential E (in mV) acrossthe inner membranein respiring mitochondria is - 160 mV, with the matrix (inside) negatlve: fK'-l E : - 5 9 l o e # i : - 5 e l o g 5 0 0: - 1 6 0 m V L N o u rl

Researcherscan measurethe matrix (inside)pH by trapping pH-sensitivefluorescent dyes inside vesiclesformed from the inner mitochondrial membrane, with the matrix side of the membrane facing inward. They also can measure the pH outside of the vesicles(equivalent to the intermembrane space)and thus determine the pH gradient (ApH), which turns out to be -1 pH unit. Since a differenceof one pH unit representsa tenfold differencein H+ concentrarion, according to the Nernst equation a pH gradient of one unit across a membrane is equivalent to an electric potential of 59 mV at 20 'C. Thus, knowing the voltage and pH gradients, we can calculatethe proton-motive force, pmf, as ^ T \ - (l R q/ - 59 ApH x Ap = -p m f : v ' /H I : \t where R is the gasconstantof 1.987 call(degree.mol),7 is the temperature (in degreesKelvin), F is the Faraday constant 123,062 call(V.mol)1, and V is the transmembraneelectric potential; V and pmf are measuredin millivolts. The electric potential V acrossthe inner membraneis - 160 mV (negative inside matrix) and ApH is equivalent to :60 mV. Thus the total pmf is -220 mV, with the transmembraneelectric potential responsiblefor about 73 percentof the total.

ToxicBy-productsof ElectronTransport C a n D a m a g eC e l l s About 1-2 percent of the oxygen metabolized by aerobic organisms, rather than being converted to water, is partially reducedto the superoxideanion radical (02 ). Superoxideis unstable in aqueous biological liquids, breaking down into especially toxic hydrogen peroxide (HzOz) and then hydroxyl radicals.Theseand other reactiue

oxygen species(ROS), which contribute to what is often called cellular oxidatiue stress, can be highly toxic, because they chemically modify proteins, DNA, and unsaturated fatty acyl groups in membrane lipids, thus interfering with normal function. Indeed, ROS are purposefully generated by body defensecells (e.g.,macrophages)to kill pathogens. In humans, excessiveor inappropriate generationof ROS has been implicated in many diverse diseases,including heart failure, neurodegenerativediseases,alcohol-induced liver disease,diabetes,and aging. Although ROS can be generatedby a number of metabolic pathways, the major source of ROS appearsto be the electron transport chain, in particular mechanismscoupled to complexes I and III. The semiquinone form of ubiquinone, CoQ-. (seeFigure 72-15), an intermediateform of CoQ generatedin the Q cycle, may play a particularly important role in superoxidegeneration. To help protect against ROS toxicity, mitochondria have evolved several defensemechanisms,including the use of enzymes that inactivate superoxide first by converting it to H2O2 (Mn-containing superoxide dismutase) a n d t h e n t o H 2 O ( g l u t a t h i o n e p e r o x i d a s e ,w h i c h a l s o detoxifies the lipid hydroperoxide products formed when ROS react with unsaturated fatty acyl groups). Cardiac mitochondria also have catalase(normally only found in p e r o x i s o m e s )t o h e l p b r e a k d o w n H z O z . T h i s i s n o t s u r p r i s i n g , b e c a u s et h e m o s t o x y g e n - c o n s u m i n go r g a n i n mammals is the heart. In addition, the small molecule antioxidants c-lipoic acid and vitamin E help protect the mitochondrion from ROS. f

ElectronTransport and Generation of the Proton-Motive Force r By the end of the citric acid cycle (stageII), much of the energy originally present in the covalent bonds of glucose and fatty acids is converted into high-energy electrons in the reduced coenzymesNADH and FADH2. The energy from theseelectronsis used to generatethe proton-motive force. r In the mitochondrion, the proton-motive force is generated by coupling electron flow (from NADH and FADH2 to 02 ) to the uphill transport of protons from the matrix across the inner membrane to the intermembrane space. This processtogether with the synthesisof ATP from ADP and P1driven by the proton-motive force is called oxidative phosphorylation. r The flow of electronsfrom FADH2 and NADH to 02 is directedthrough multiprotein complexes.The four major complexesareNADH-CoQ reductase(complexI), succinate-CoQ reductase(complex II), CoQH2-cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV). r Each complex contains one or more electron-carrying prostheticgroups: iron-sulfur clusters,flavins, hemegroups, and copper ions (seeTable 12-2). Cytochrome c, which contains heme, and coenzymeQ (CoQ), a lipid-soluble small

molecule,are mobile carriersthat shuttle electronsbetween the complexes. r ComplexesI, III, and IV pump protons from the matrix into the intermembrane space.Complexes I and II reduce CoQ to CoQH2, which carries protons and high-energy electrons to complex III. The heme protein cytochrome c carries electrons from complex III to complex IV, which usesthem to pump protons and reduce molecular oxygen to water. r The high-energyelectronsfrom NADH enter the electron transport chain through complex I, whereasthe high-energy electronsfrom FADH2 (derivedfrom succinatein the citric acid cycle) enter the electron transport chain through complex II. Additional electrons derived from FADH2 by the initial step of fatty acyl-CoA B-oxidation increasethe supply of CoQH 2 avallable for electron transport. r \(ithin the inner membrane, electron transport complexes assembleinto supercomplexesheld together by cardiolipin, a specialized phospholipid. Supercomplex formation may enhancethe speedand efficiency of generation of the proton-motive force. r Each electron carrier acceptsan electron or electron pair from a carrier with a lesspositive reduction potential and transfers the electron to a carrier with a more positive reduction potential. Thus the reduction potentials of electron carriers favor unidirectional electron flow from NADH and FADH2 to 02 (seeFigure 12-1'8). r The Q cycle allows four protons (rather than two) to be translocatedper pair of electronsmoving through complex III (seeFigure 12-20). r A total of 10 H+ ions are translocated from the matrix acrossthe inner membrane per electron pair flowing from NADH to 02 (seeFigure 12-1,6),whereas 6 H* ions are translocatedper electron pair flowing from FADH2 to 02. r The proton-motive force is due largely to a voltage gradient across the inner membrane produced by proton pumping; the pH gradient plays a quantitatively less important role. r Reactive oxygen species(ROS) are toxic by-products of the electron transport chain that can modify and damage proteins, DNA, and lipids. Specificenzymes(e.g.,glutathinone peroxidase, catalase)and small molecule antioxidants (e.g.,vitamin E) help protect againstROS-induced damaqe.

the Proton-Motive Harnessing Processes Forcefor Energy-Requiring The hypothesisthat a proton-motive force across the inner mitochondrial membrane is the immediate source of energy for ATP synthesiswas proposedin 1'961by PeterMitchell. Virtually all researchersstudying oxidative phosphorylation and photosynthesisinitially reiected his chemiosmotic hypothesis. They favored a mechanism similar to the then

EO R C EF O R E N E R G Y - R E Q U I R I N PG ROCESSES H A R N E S S I NT GH E p R O T O N - M O T I V F

'

503

well-elucidated substrate-levelphosphorylation in glycolysis, in which chemical transformation of a substrate molecule (i.e.,phosphoenolpyruvate) is directly coupled to ATP synthesis.Despite intenseefforts by a large number of investigators, however, compelling evidencefor such a direct mechanismwas neverobserved. Definitive evidencesupporting Mitchell's hypothesis depended on development of techniquesto purify and reconstitute organellemembranesand membrane proteins. The experiment with vesiclesmade from chloroplast thylakoid membranes(describedin detail below) that contain ATP synthase, outlined in Figure 12-21, was one of several demonstratingthat this protein is an ATP-generatingenzyme and that ATP generation is dependenton proton movement down an electrochemical gradient.It turns out that the protons actually move tbrougD the ATP synthaseas they rraversethe membrane!

troFr --1

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ATP H* li:i:li:la:l:li:illlia::iiilrrl:

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H+ pH 4.0

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EXPERIMENTAL FTGURE 12-21Synthesis of ATpby FeFl dependson a pH gradientacrossthe membrane.lsolated chloroplast thylakoid vesicles containing FoF,particles wereequllibrated in the darkwith a buffered solution at pH4.0 Whenthe pH in the thylakoid lumenbecame 4 0, thevesicles wererapidlymixedwith a solution at pH8 0 containing ADPandP, A burstof ATpsynthesis accompanied thetransmembrane movement of protons drivenbythe 1 0 , 0 0 0 - f oHl d* c o n c e n t r a t g i orna d i e n( ltO 4 M v e r s u1sO - 8M ) I n similar experiments using"inside-out" preparations of mitochondrial membrane vesicles, an artificially generated membrane electric potential alsoresulted in ATPsynthesis c H A P T E R1 2

|

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ADP + P;

Intermembrane space Stroma

Fl

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504

Outer membrane

CELLULAE RN E R G E T T C S

Inner memDrane T h v l a k o i dm e m b r a n e

A FIGURE 12-22Chemiosmosis in bacteria,mitochondria, and chloroplasts. Themembrane surface facinga shaded areaisa cytosolic face;thesurface facingan unshaded, whiteareaisan exoplasmic face,Notethatthecytosolic plasma faceof the bacterial membrane, the matrixfaceof the innermitochondrial membrane, andthestromal faceof thethylakoid membrane areallequivalent Duringelectron protons transport, arealwayspumpedfromthe cytosolic faceto theexoplasmic face,creating a protonconcentration (exoplasmic gradient face> cytosolic face)andan electric potential (negative cytosolic faceandpositive exoplasmic face)across the membraneDuringthe synthesis protons of ATP, flow in the reverse (downtheirelectrochemical direction gradient) throughATPsynthase (FoFr complex), whichprotrudes in a knobat the cytosolic facein all CASCS

As we shall see,the ATP synthaseis a multiprotein complex that can be subdivided into two subcomplexescalled Fe (containing the transmembraneportions of the complex) and F1 (containingthe globular portions of the complex that sit above the membrane and point toward the matrix spacein mitochondria). Thus the ATP synthaseis

ward what becamethe stromal spaceof the chloroplast (describedin detail below). In all cases,ATP synthaseis positioned with the globular F1 domain, which catalyzesATP synthesis,on the cytosolic face of the membrane, so ATP is always formed on the cytosolic face of the membrane (seeFigure 1'2-22).Protons always flow through ATP synthasefrom the exoplasmic to the cytosolic faceof the membrane, which in the mitochondrion is from the intermembrane to the matrix space.This flow is driven by the proton motive force. InvariablS the cytosolic face has a negativeelectric potential relative to the exoplasmic face. In addition to ATP synthesis,the proton-motive force acrossthe bacterial plasma membraneis usedto power other processes,including the uptake of nutrients such as sugars (using proton/sugar symporters) and the rotation of bacterial flagella. Chemiosmotic coupling thus illustrates an important principle introduced in our discussionof active transport in Chapter 1.1.:the membrane potential, the concentrdtion gradients of protons (and other ions) across a membrane, and the phosphoanhydride bonds in ATP are equiualent and interconuertibleforms of chemical potential energy.Indeed, AIP synthesisthrough ATP synthasecan be thought of as active transport ln reverse.

often also called the FeFl complex; we will use the terms interchangeably.

T h e M e c h a n i s mo f A T PS y n t h e s i sl s S h a r e d , itochondria, A m o n g B a c t e r i aM and Chloroplasts Although bacteria lack any internal membranes,aerobic bacteria nonethelesscarry out oxidative phosphorylation by the same processesthat occur in eukaryotic mitochondria. Enzymes that catalyze the reactions of both the glycolytic pathway and the citric acid cycle are presentin the cytosol of bacteria; enzymesthat oxidize NADH to NAD* and transfer the electrons to the ultimate acceptor 02 reside in the bacterialplasmamembrane. The movement of electronsthrough thesemembranecarriers is coupled to the pumping of protons out of the cell. The movement of protons back into the cell, down their concentration gradient, is coupled to the synthesisof ATP. This general processis similar for bacteria and eukaryotes (in both mitochondriaand chloroplasts)(Figuret2-22). The bacterial ATP synthases(F6F1complex) are essentiallyidentical in structure and function to the mitochondrial and chloroplast ATP synthasesbut are simpler to purify and study. A primitive aerobic bacterium was probably the progenitor of mitochondria in eukaryotic cells (Figure 12-23). hccording to this endosymbionthypothesis,the inner mitochondrial membrane would be derived from the bacterial plasma membrane with its cytosolic face pointing toward what became the matrix space of the mitochondrion. Similarly in plants the progenitor'splasma membranebecamethe chloroplast'sthylakoid membrane and its cytosolic face pointed to-

ATPSynthaseComprisesTwo Multiprotein ComplexesTermedFsand F1 With generalacceptanceof Mitchell's chemiosmoticmechanism, researchersturned their attention to the structure and operation of the F6F1complex. The FsFl complex, or AIP synthase,has two principal components, Fe and F1, both of which are

Eukarvotic o l a s m am e m b r a n e Endocytosisof bacterium caoableof oxidative phosphorylation

I Endocytosisof bacterium capableof photosynthesis

Bacterial p l a s m am e m b r a n e

B a c t e r i apl l a s m a m e m b r a n eb e c o m e s i n n e rm e m b r a n e of chloroplast

B a c t e r i apl l a s m a m e m b r a n eb e c o m e s i n n e rm e m b r a n e of mitochondrion

a(-- .{

,-#r..\

l n n e r m e m b r a n eb u d s off thylakoid vesicles

Thylakoid memorane

M i t o c h o n d r i am l atrix

12-23 Endosymbiont hypothesis for the evolutionary a FIGURE Endocytosis of a origin of mitochondriaand chloroplasts. eukaryotic cell(stepII) wouldgenerate bacterium by an ancestral with two membranes, derived an organelle theoutermembrane plasma membrane andthe inneronefrom fromtheeukaryotic (stepE) TheF1subunitof ATPsynthase, membrane the bactenal membrane, wouldthen to thecytosolic faceof the bacterial localized

(/eft)or chloroplast mitochondrion facethe matrixof the evolving membrane, (ngrht)Budding fromthe innerchloroplast of vesicles in contemporary chloroplasts of duringdevelopment suchasoccurs with the F1subunit membranes thethylakoid plants, wouldgenerate stroma(step face,facingthe chloroplast on the cytosolic remaining faces; areaarecytosolic facinga shaded surfaces B) Membrane faces areaareexoplasmic facinqan unshaded surfaces

H A R N E s s I N G T H E P R o T o N - M o T | V E F o R c E F o R E N E R G Y - R E Q U I R | N G P R o c E s 505 sES

Animation:ProtonTranslocating, RotaryF-ATPase{lttt 100nm ------------>l

l<--

ATP

ADP+ P;

fi static

Cytosolic medium

Rotates

Exoplasmic medium

Rotation of c ring

Proton half-channel

Proton bound to aspartate

multimericproteins(Figure12-24).The Fecomponenrcontains three rypesof integralmembraneproteins,designateda, b, and c. In bacteriaand in yeastmitochondriathe most common subunit compositionis a1b2c1e, but Fe complexesin animal mitochondria have 1,2c subunitsand thosein chloroplastshave 14. In all casesthe c subunits form a doughnut-shapedring in the plane of the membrane.The a and two b subunitsare rigidly linked to one another but not to the ring of c subunits,a critical feature of the protein to which we will return shortly. The F1 portion is a water-solublecomplex of five distinct polypeptideswith the composition ct3B3^yEe that is normally firmly bound to the F0 subcomplexat the surfaceof the membrane. The lower end of the rodlike ^ysubunit of the F1 subcomplex is a coiled coil that fits into the centerof the c-subunit ring of Fe and appearsrigidly attached to it. Thus when the c-subunit ring rotates, the rodlike "y subunit moves with it. The F1 e subunit is rigidly attachedto ^yand also forms tight contactswith severalof the c subunitsof F6.The o.and p subunits are responsiblefor the overall globular shapeof the F1 subcomplexand associatein alternatingorder to form a hexamer,ctBctBctB, or (ctB)3,which restsatop the singlelong n7subunit. The F1 6 subunit is permanentlylinked to one of the F1 cr subunits and also binds to the b subunit of F6. Thus the F6 a and b subunitsand the E subunit and (oB)3 hexamerof the F1 complex form a rigid structure anchored in the membrane. The rodlike b subunitsform a "srator" that preventsthe (ctB)3 hexamer from moving while it resrson the 1 subunit, whose rotation together with the c subunits of F6 plays an essential role in the ATP synthesismechanismdescribedbelow. 'Sfhen ATP synthaseis embeddedin a membrane, the F1 component forms a knob that protrudes from the cytosolic

s06

CHAPTER 12

I

< FIGURE 12-24 Structureand function of ATPsynthase(the FeFlcomplex)in the bacterialplasmamembrane.TheFe membrane-embedded portionof ATPsynthase isbuiltof three proteins: integral membrane onecopyof a, two copres of b, andon '1 average0 copies of c arranged in a ringin the planeof the membrane Twoprotonhalf-channels lieat the interface between thea subunitandthec ring Half-channel I allowsprotons to move oneat a timefromthe exoplasmic mediumandbindto aspartate-61 in thecenterof a c subunitnearthe middleof the membraneHalfchannel ll (afterrotation protons of thec ring)permits to dissociate fromtheaspartate andmoveintothecytosolic mediumTheF, portioncontains threecopies eachof subunits ctandB thatforma hexamer resting atopthesinglerod-shaped whichis 1 subunit, inserted intothec ringof F6.Thee subunitis rigidlyattached to the1 subunitandalsoto several of thec subunitsThe6 subunit permanently linksoneof theo subunits in the F1complex to the b s u b u no i tf F 6T h u st h eF ea a n db s u b u n i tasn dt h eF 16 s u b u n a i tn d (oB)3hexamer forma rigidstructure anchored in the membrane (orange)Duringprotonflow,the c ringandthe attached F1e and1 subunits rotateasa unit(green), causing conformation changes in the F1B subunits, leading to ATPsynthesis fromM J [Adapred Schnitzer, 2001,Nature 410:878, andP D Boyel1999,Nature 402:247 I

CELLULAE RN E R G E T I C S

(in the mitochondrion this is the matrix) face. BecauseF1 separatedfrom membranesis capable of catalyzingATP hydrolysis (ATP conversion to ADP plus Pi) in the absenceof the Fe component, it has beencalled the F1 ATPase;however, its function in the cells is the reverse,ro synthesizeATP. ATP hydrolysisis a spontaneousprocess(AG < 0); thus energyis required to drive the AIPase in "reverse" and generateATP.

Rotationof the F1^ySubunit,Driven by Proton MovementThrough Fs,PowersATPSynthesis Eachof thethreeB subunits in theglobularF1portionof the complete FsFl complex can bind ADP and P; and catalyze the endergonicsynthesisof ATP when coupled to the flow of protons from the exoplasmic (intermembrane space in the mitochondrion) medium to the cytosolic (matrix) medium. However, the coupling between proron flow and AIP synthesismust be indirect, sincethe nucleotide-bindingsites on the B subunitsof F1,where ATP synthesisoccurs,are 9-10 nm from the surface of the mitochondrial membrane. The most widely acceptedmodel for ATP synthesisby the F6F1 complex-th e binding-change mechanism-posits just such an indirect coupling (Figure 12-25). According to this mechanism,energy releasedby the "downhill" movement of protons through Fe directly powers rotation of the c-subunit ring together with its attached 1 and e subunits (seeFigure 12-24). The 1 subunit acs as a cam, or nonsymmetrical rotating shaft, whose rotation within center of the static (crB)3hexamer of F1 causesit to push sequentiallyagainsteach of the B subunits and thus causecyclical changesin their conformations between three

sts

ADP P

Rotation

Reaction (no rotation)

E

a

--+

--

Rotation

E

.v I A?P Pi

FIGURE 12-25The binding-change mechanism of ATP synthesisfrom ADPand P;.Thisviewislookingup at F1from (seeFigure12-24)Asthe1 subunitrotates the membrane surface by 120'inthecenter, eachof theotherwise identical F1B subunits (O,openwith oval alternates between threeconformational states representation of the bindingsite;L,loosewith a rectangular binding site;T,tightwith a triangular site)thatdifferin theirbindingaffinities for ATP, ADP,andP,.Thecyclebegins(upperleft)whenADP (here,arbitrarily andP,bindloosely to oneof thethreeB subunits designated siteisin the O (open) Br)whosenucleotide-binding conformation Protonfluxthroughthe Foportionof the protein powersa 120"rotationof the"ysubunit(relative to thefixedB (steptr) Thiscauses subunits) the rotating"ysubunit, whichis asymmetric, to pushdifferentially against the B subunits, resulting in a conformational changeandan increase in the bindingaffinity of the Br subunitfor ADPandP1(fromO -+ L),an increase in the bindingaffinityof the B3subunitfor ADPandP1thatwerepreviously

of in thebinding affinity bound(fromL -+ T ), anda decrease boundATP(fromT --+O),causing the B2subunitfor a previously rotationthe release of the boundATPStepZ: Withoutadditional formATBa reaction ADPandPlin theT site(herethe B3subunit) dueto the energy an inputof additional thatdoesnotrequire in the activesiteof theT stateAt thesame environment special O siteon B2 to the unoccupied timea newADPandP1bindloosely StepE: Protonf luxpowersanother120' rotationof the1 subunit, in the bindingsites(L-+T,O-> changes conformational consequent of ATPfromP3 Step4: Withoutadditional L,T+ O),andrelease rotation theADPandP,in theT siteof B1formATBandadditional O siteon B3 Theprocess ADPandP,bindto the unoccupied with rotation(stepE) andATPformation(step6) until continues for with threeATPshavingbeenproduced the cycleiscomplete, 1989, FASEB ].3:2164; fromP Boyer, every360"rotationof ry lAdapted andM Yoshida, AcadSciUSA94:10583; ProcNat'l. Y Zhouetal, 1997, Mol CellBiol2:669-677 2001,Nat Rev. andT,Hisabori, E Muneyuki, l

different states. As schematically depicted in a view of the bottom of the (ctB)3hexamer's globular structure in Figure 1.2-25,rotation of the 1 subunit relative to the fixed (aB)3 hexamer causesthe nucleotide-bindingsite of each B subunit to cycle through three conformational statesin the following order:

O state,thereby releasingATP and beginningthe cycle again. ATP or ADP also binds to regulatory or allostericsiteson the three ct subunits;this binding modifies the rate of ATP synthesis accordingto the level of AIP and ADP in the matrix, but is not directly involved in synthesisof ATP from ADP and P;. Severaltypes of evidencesupport the binding-change mechanism. First, biochemical studies showed that one of the three B subunits on isolated F1 particles can tightly bind ADP and P; and then form ATP, which remains tightly bound. The measuredAG for this reaction is near zero, indicating that once ADP and Pi are bound to the T state of a B subunit, they spontaneouslyform AIP. Importantly dissociation of the bound ATP from the B subunit on isolated F1 particles occurs extremely slowly. This finding suggested that dissociationof ATP would have to be powered by a conformational changein the B subunit, which in turn would be causedbv proton movement.

L An O (open) state that binds ATP very poorly and ADP and P; weakly 2. An L (loose)state that binds ADP and Pi more strongly but cannot bind ATP 3. A T (tight) state that binds ADP and Pl so tightly that they spontaneouslyreact and form ATP In the T state the ATP produced is bound so tightly that it cannot readily dissociatefrom the site-it is trapped until another rotation of the ^ysubunit returns that B subunit to the

PG ROCESSES H A R N E S S I NTGH E P R O T O N - M O T I VFEO R C EF O R E N E R G Y . R E Q U I R I N

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Video: Rotation of Actin Filament Bound to ATP 5 < EXPERf 12-26 lhe rysubunit of the F1 MENTALFIGURE complexrotatesrelativeto the (crg)ghexamer.F' complexes with an additional wereengineered thatcontained His6 B subunits whichcauses themto adhereto a glassplatecoated sequence, The1 subunitin the with a metalreagent thatbindspolyhistidine F1complexes waslinkedcovalently to a fluorescently engineered microscope, labeled actinfilament. Whenviewedin a fluorescence the actinfilaments wereseento rotatecounterclockwise in discrete 120"stepsin the presence of ATP,poweredby ATPhydrolysis by fromH Noliet al, 199-/ 386:299, the B subunitsfAdapted and , Nature R Yasuda etal. 1998.Cell93 1117I

X-ray crystallographicanalysis of the (oB)3 hexamer yielded a striking conclusion: although the three B subunits are identical in sequence and overall structure, the ADP/ATP-binding siteshave different conformations in each s u b u n i t . T h e m o s t r e a s o n a b l ec o n c l u s i o n w a s t h a t t h e three B subunits cycle in an energy-dependentreaction between three conformational states(O, L, T), in which the nucleotide-bindingsite has substantially different srrucrures. In other studies,intact F6F1complexeswere treated with chemical cross-linking agents that covalently linked the "y and e subunits and the c-subunit ring. The observation that such treated complexescould synthesizeATP or use ATP to power proton pumping indicates that the cross-linked proteins normally rotate together. Finally, rotation of the ^y subunit relative to the fixed (aB)3 hexamer, as proposed in the binding-changemechanism, was observeddirectly in the cleverexperimentdepicted in Figure 12-26.In one modification of this experimentin which tiny gold particles,rather than an actin filament, were attachedto the 1 subunit, rotation ratesof 134 revolutions per secondwere observed.Hydrolysis of 3 ATPs, which you recall is the reversereaction catalyzedby the sameenzyme,is thought to power one revolution; this result is close to the experimentally determined rate of ATP hydrolysis by F6F1 complexes:about 400 ATPs per second.In a related experiment, a ^ysubunit linked to an e subunit and a ring of c subunits was seento rotate relative to the fixed (cp)j hexamer. Rotation of the 1 subunit in these experimentswas powered by ATP hydrolysis,the reverseof the normal process in which proton movement through the Fs complex drives rotation of the ry subunit. These observationsestablished that the 1 subunit, along with the attachedc ring and e subunit, doesindeedrotate,therebydriving the conformational changesin the B subunits thar are required for binding of ADP and Pi, followed by synthesisand subsequentrelease of ATP. 508

c H A P T E R1 2

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CELLULAR ENERGETTCS

Number of Translocated Protons Required for ATP Synthesis A simple calculationindicatesthat the passageof more than one proton is required to synthesizeone molecule of ATP from ADP and P1.Although the AG for this reaction under standardconditions is * 7.3 kcaVmol,at the concentrations of reactantsin the mitochondrion, AG is probably higher (+10 to *12 kcaVmol).We can calculatethe amount of free energy releasedby the passageof 1 mol of protons down an electrochemicalgradient of 220 mY (0.22 V) from the Nernst equation, setting n : 1 and measuring AE in volts: A G ( c a l l m o l ): - n F L , E : - ( 2 3 , O 6 2 c a l . V - 1 . m o l : ( 2 3 , 0 5 2 c a 1 . V -r . m o l - 1 ; 1 o . z zV ; : -5074 callmol, or -5.1 kcal/mol

1)AE

Sincethe downhill movement of 1 mol of protons releasesjust over 5 kcal of free energy,the passageof at least rwo protons is required for synthesisof eachmoleculeof AT? from ADP and P1. Proton Movement Through F6 and Rotation of the c Ring Eachcopy of subunitc containstwo membrane-spanning cr helices that form a hairpin-like structure. An aspartate residue,Asp61, in the centerof one of thesehelicesis thought to participate in proton movement. Chemical modification of this aspartateby the poison dicyclohexylcarbodiimide or its mutation to alaninespecificallyblocks proton movement through F6. According to one current model, two proton half-channels,I and II, lie at the interface betweenthe a subunit and c ring (see Figure 12-24). Protons are thought to move one at a time through half-channel I from the exoplasmic medium and bind to the carboxylatesidechain on Asp61 of one c subunit.Binding of a proton to this aspartatewould result in a conformational changein the c subunit, causingit to move relative to the fixed a subunit or equivalentlyto rotate in the membraneplane. This rotation would bring the adjacent c subunit, with its ionized asparrylsidechain, into channelI, thereby allowing it to receive the next proton and subsequentlymove relative to the a subunit.Continuedrotation of the c ring, due to bindingof protons to additional c subunits,eventuallywould align the first c subunit containinga protonatedAsp61 with the secondhalfchannel (II), which is connectedto the cytosol. A positively chargedside chain of Arg210 in the a subunit has been proposed to interact with the negativelychargedAsp61 and facilitate movementof the c subunitsand proton translocation.Once

this occurs,the proton on the aspartylresiduecould dissociate (forming ionized aspartate) and move into the cytosolic medium. Sincethe 1 subunit of F1 is tighdy attachedto the c ring of Fe, rotation of the c ring associatedwith proton movemenr causesrotation of the 1 subunit. According to the bindingchangemechanism,a 720" rotation of 1 powers synthesisof one ATP (seeFigure 12-25). Thus complete rotation of the c ring by 360" would generatethree ATPs. In E. col1,where the Fo composition is a1b2c1s,movement of 10 protons drives one complete rotation and thus synthesisof three ATPs. This value is consistentwith experimentaldata on proton flux during ATP synthesis,providing indirect support for the model coupling proton movementto c-rlng rotatron depictedin Figure 12-24. The F6 from chloroplastscontains 14 c subunitsper ring, and movementof 14 protons would be neededfor synthesisof three ATPs. Why theseotherwise similar FsFl complexes have evolved to have different H*:ATP ratios is not clear.

ATP-ADPExchangeAcrossthe Inner M i t o c h o n d r i aM l e m b r a n el s P o w e r e d by the Proton-MotiveForce In addition to powering ATP synthesis,the proton-motive force acrossthe inner mitochondrial membranepowers the exchangeof ATP formed by oxidative phosphorylation inside the mitochondrion for ADP and P1in the cytosol.This exchange,which is requiredto supply ADP and Pi substrare for oxidative phosphorylationto continue, is mediated by two proteins ^in the inner membrane: a phosphate transporter (HPO4' /OH- antiporter) that mediatesthe import of one HPO42 coupled to the export of one OH- and an ATP/AD P antiporter (Figure12-27\. The ATP/ADP antiporter allows one moleculeof ADP to enter only if one molecule of ATP exits simultaneously.The MP/ADP antiporter, a dimer of two 30,000-Da subunits, makesup 10-15 percentof the protein in the inner membrane, so it is one of the more abundant mitochondrial proteins. Functioningof the two antiporterstogetherproducesan influx of one ADP3- and one P,2- and efflux of one ATPa- together with one OH . EachOH transportedoutr,vardcombineswith a proton, translocatedduring electrontransport to the intermembranespace,to form H2O. This drivesthe overallreaction in the direction of AIP export and ADP and P1import. Becausesome of the protons translocatedout of the mitochondrion during electron transport provide the power (by combining with the exported OH-) for the MP-ADP exchange,fewer protons are availablefor ATP synthesis.It is estimatedthat for every four protons translocatedout, three are used to synthesizeone ATP molecule and one is used to power the export of ATP from the mitochondrion in exchangefor ADP and P1.This expenditureof energyfrom the proton concentration gradient to export ATP from the mitochondrion in exchangefor ADP and Pi ensuresa high ratio of ATP to ADP in the cytosol, where hydrolysis of the highenergy phosphoanhydride bond of ATP is utilized to power reactlons. many energy-requlrlng

l n n e rm i t o c h o n d r i a l

* H concentration gradient Membrane electric potential

+

Matrix

T T

Translocationof Hd u r i n ge l e c t r o nt r a n s p o r t

"

.t '

aPPt

noet

fr' ATP4-(

- | Phosphatetransporter

^')

\

/

ATp/ADp antiportel

ATP4 ADP3-+ HPOo2

lntermembrane space

ATP4 + OH

FfGURE'12-27The phosphateand ATP/ADPtransport systemin the innermitochondrialmembrane.Thecoordinated (purple in the uptakeof andgreen)results actionof two antiporters for oneATPa-andone oneADP3-andoneHPOa2in exchange of oneproton powered by theoutwardtranslocation hydroxyl, chain,blue) (mediated transport of theelectron bythe proteins isnot shownhere transportTheoutermembrane duringelectron than5000Da smaller to molecules it ispermeable because Studiesof what turned out to be ATP/ADP antiporter activity were first recorded about 2000 years ago, when Dioscoridesdescribeda poisonousherb from the thistle Atractylis gummifera, found commonly in the Mediterranean region. The same agent is found in the traditional Zulu multipurposeherbal remedyimpila (Callilepislaureola). In Zulu impila means"health," although it has beenassociated with numerouspoisonings.In 1.962the activeagent in the herb, atractyloside,which inhibits the ATPiADP antiporter,was shown to inhibit oxidative phosphorylationof extramitochondrialADP but not intramitochondrial ADP. This demonstrated the importance of the ATP/ADP antiporter and has provided a powerful tool to study the mechanismby which this transporterfunctions. Dioscorides(-AD 40-90) lived near Tarsus'at the time a province of Rome in southeasternAsia Minor in what is now Turkey. His five-volume De Materia Medica (The Materials of Medicine) "on the preparation, properties' and testing of drugs" describedthe medicinalpropertiesof about 1000 natural products and 4740 medicinal usagesof them. For approximately 1600 yearsit was the basicreferencein medicine from northern Europe to the Indian Ocean,comparableto today'sPhysicians'Desk Referenceas a guide for using drugs' I

H A R N E S S I NTGH E P R O T O N . M O T I VFEO R C EF O R E N E R G Y - R E Q U I R IPNRGO C E S S E S

509

R a t eo f M i t o c h o n d r i aO l x i d a t i o nN o r m a l l y D e p e n d so n A D P L e v e l s If intact isolatedmitochondria are provided with NADH (or a source of FADH2 such as succinate)plus 02 and P1,but not ADP, the oxidation of NADH and the reduction of 02 rapidly cease,becausethe amount of endogenousADP is depletedby AIP formation. If ADP is then added,the oxidation of NADH is rapidly restored. Thus mitochondria can oxidize FADH2 and NADH only as long as there is a sourceof ADP and P; to generateAIP. This phenomenon,termed respiratory control, occursbecauseoxidation of NADH and succinate(FADH2) is obligatorily coupled to proton transport acrossthe inner mitochondrial membrane.If the resultingproton-motive force is not dissipatedduring the synthesisof AIP from ADP and P1 (or for some other purpose),both the transmembraneproton concentration gradient and the membrane electric potential will increaseto very high levels.At this point, pumping of additional protons acrossthe inner membranerequiresso much energy that it eventuallyceases,blocking the coupled oxidation of NADH and other substrates.

Brown-FatMitochondriaUsethe Proton-Motive Forceto GenerateHeat Broun-fat tissue,whosecolor is due to the presenceof abun, dant mitochondria, is specializedfor the gineration of heat. In contrast, tuhite-fat tissue is specializedfor the storage of fat and contains relatively few mitochondria. The inner membrane of brown-fat mitochondria contains thermogenin, a protein that functions as a natural uncoupler of oxidative phosphorylation and generationof a proton-motive force. Thermogenin, or UCP1.,is one of several uncoupling proteins (UCPs) found in most eukaryotes (but not in fermentative yeasts).Thermogenin dissipatesthe proton-motive force by rendering the inner mitochondrial membrane permeableto protons. As a consequencethe energy releasedby NADH oxidation in the electron transport chain is converted to heat. Thermogenin is a proton rransporter, not a proton channel, and shuttlesprotons acrossthe membrane at a rate that is a millionfold slower than that of typical ion channels(seeFigure 11-3).Thermogeninis similar in sequenceto the mitochondrial ATP/ADP transporte! as are many other mitochondrial transporter proteins that compose the ATP/ADP transporter family. Certain smallmolecule poisons also function as uncouplers by rendering the inner mitochondrial membrane permeableto protons. One example is the lipid-soluble chemical 2,4-dinitrophenol (DNP), which can reversiblybind to and releaseprotons and shuttle them acrossthe inner membrane from the intermembrane spaceinto the matrix. Environmental conditions regulate the amount of thermogenin in brown-fat mitochondria. For instance, during the adaptation of rats to cold, the ability of their tissuesto generateheat is increasedby the induction of thermogenin synthesis.In cold-adaptedanimals, thermogenin may constitute up to 15 percent of the total protein in the inner mitochondrial membrane. 510

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Adult humans have little brown fat, but human infants have a great deal. In the newborn, thermogenesisby brownfat mitochondria is vital to survival, as it also is in hibernating mammals.In fur sealsand other animalsnaturally acclimated to the cold, muscle-cellmitochondria contain thermogenin;as a result, much of the proton-motive force is usedfor generating heat, thereby maintaining body temperature.

Harnessingthe Proton-Motive Force for Energy-RequiringProcesses r Peter Mitchell proposed the chemiosmotic hypothesis that a proton-motive force acrossthe inner mitochondrial membrane is the direct sourceof energy for ATP synthesis. r Bacteria, mitochondria, and chloroplasts all use the samechemiosmoticmechanismand a similar ATP synthase to generate ATP. r ATP synthase(the FeFl complex) catalyzesATP synthesis as protons flow through the inner mitochondrial membrane (plasma membrane in bacteria)down their electrochemical proton gradient. r Fs contains a ring of 10-14 c subunits that is rigidly linked to the rod-shaped1 subunit and e subunit of F1. Together they rotate during ATP synthesis.Resting atop the T subunit is the hexameric knob of F1 [(ctB)3],which protrudesinto the mitochondrial matrix (cytosolin bacteria). The three B subunits are the sites of ATP synthesis (see Figure 12-24). r Movement of protons acrossthe membranevia two halfchannelsat the interface of the F6 a subunit and the c ring powers rotation of the c ring with its attached F1 e and 1 subunits. r Rotation of the F1 ^ysubunit, which is inserted in the centerof the nonrotating (cB)3 hexamerand operateslike a camshaft, leads to changesin the conformation of the nucleotide-bindingsites in the three F1 B subunits (see Figure 12-25). By means of this binding-changemechanism, the B subunits bind ADP and P1,condensethem to form ATP, and then releasethe ATP. Three ATPs are made for each revolution made by the assemblyof c, 1, and e subunits. r The proton-motive force also powers the uptake of P1 and ADP from the cytosol in exchangefor mitochondrial ATP and OH , thus reducing some of the energy available for ATP synthesis.The ATP/ADP antiporter that participates in this exchangeis one of the most abundant proteins in the inner mitochondrial membrane. r Continued mitochondrial oxidation of NADH and the reduction of 02 are dependent on sufficient ADP being present in the matrix. This phenomenon, termed respiratory control, is an important mechanism for coordinating oxidation and ATP synthesisin mitochondria. r In brown fat, the inner mitochondrial membrane contains the uncoupler protein thermogenin, a proton trans-

porter that dissipatesthe proton-motive force into heat. Certain chemicalsalso function as uncouplers (e.g.,DNP) and have the sameeffect, uncoupling oxidative phosphorylation from electron transDort.

Photosynthesis and Light-Absorbing Pigments We now shift our attention to photosynthesis,the second main process for synthesizingAIP. Photosynthesisin plants occursin chloroplasts,Iargeorganellesfound mainly in leaf cells.The principal end products generatedfrom carbon dioxide and water are two carbohydratesthat are polymers of hexose(six-carbon)sugars:sucrose,a glucose-fructose disaccharide(seeFigure2-19), and leaf starch, alarge insolubleglucose polymer that is the primary storage carbohydrate in higher plants (Figure 1.2-28).Leaf starch is synthesizedand stored in the chloroplast.Sucroseis synthesizedin the leaf cytosol from three-carbonprecursorsgeneratedin the chloroplast; it is transportedto nonphotosynthetic(nongreen)plant tissues(e.g.,roots and seeds),which metabolizesucrosefor energy by the pathways described in the previous sections. Photosynthesisin plants, as well as in eukaryotic single-celled algae and in several photosynthetic bacteria (e.g., the cyanobacteriaand prochlorophytes),also generatesoxygen. The overall reaction of oxygen-generatingphotosynthesis, 6 CO2 + 6 H2O -->6 02 + C6H12O5 is the reverseof the overall reaction by which carbohydrates are oxidized to CO2 and H2O. In effect, photosynthesisin chloroplastsproduces energy-richsugars that are broken down and harvestedfor energy by mitochondria during the processof cellular respiration. Although green and purple bacteria also carry out photosynthesis,they use a processthat does not generateoxygen. As discussedin Section 12.5, detailed analysisof the photosynthetic systemin thesebacteriahas provided insights about the first stagesin the more common processof oxygen-generatingphotosynthesis.In this section,we provide an overview of the stagesin oxygen-generatingphotosynthesis Glucose 6

cH2oH 5)-o H ,/.1.

H

\

H

oHOHHOH Starch Ipoly(c1+4 glucose]l

polymer FIGURE 12-28Structureof starch.Thislargeglucose (seeFigure sucrose 2-19)arethe principal end andthe disaccharide products Botharebuiltof six-carbon sugars of photosynthesis (hexoses)

and introduce the main components, including the chlorophylls, the principal light-absorbing pigments.I

T h y l a k o i dM e m b r a n e si n C h l o r o p l a s t s Are the Sitesof Photosynthesisin Plants Chloroplastsare lensshapedwith an approximatediameterof 5 pm and a width of -25 pm, bounded by two membranes, which do not contain chlorophyll and do not participate directly in photosynthesis(Figure 12-29). As in mitochondria, the outer membraneof chloroplastscontains porins and thus is permeableto metabolites of small molecular weight. The inner membrane forms a permeability barrier that contains transport proteins for regulating the movement of metabolites into and out of the organelle. Unlike mitochondria, chloroplasts contain a third membrane-the thylakoid membrane-on which photosynthesis occurs. The chloroplast thylakoid membrane is believed t
Threeof the Four Stagesin Photosynthesis O c c u rO n l y D u r i n gl l l u m i n a t i o n The photosyntheticprocessin plants can be divided into four stages(Figure 1.2-30),each localizedto a defined area of the chloroplast: (1) absorption of light, generation of a high energyelectron and formation of 02 from H2O; (2) electron transport leading to reduction of NADP- to NADPH, and generation of a proton-motive force; (3) synthesisof ATP; and (4) conversion of CO2 into carbohydrates,commonly referred to as carbon fixation. All four stagesof photosynthesisare tightly coupled and controlled so as to produce the amount of carbohydrate required by the plant. All the reactions in stages 1-3 are catalyzed by multiprotein complexes in the thylakoid membrane.The generationof a pmf and the useof the pmf to synthesizeAIP resemblestagesIII and IV of mitochondrial oxidative phosphorylation. The enzymesthat incorporate CO2 into chemical intermediatesand then convert them to starch are solubleconstituentsof the chloroplast stroma; the enzymesthat form sucrosefrom three-carbonintermediatesare in the cytosol. Stage 1: Absorption of Light The initial step in photosynthesisis the absorptionof light by chlorophyllsattached to proteins in the thylakoid membranes. Like the heme component of cytochromes,chlorophylls consist of a porphyrin ring attached to a long hydrocarbon side chain P H O T O S Y N T H E SAI N S D L I G H T - A B S O R B I NPGI G M E N T S

511

(Figure 12-37).In contrast to the hemes(seeFigure 12-14), chlorophylls contain a central Mg2* ion (rather than Fe) and have an additional five-memberedring. The energy of the absorbed light ultimately is used to remove electrons from a donor (water in greenplants), forming oxygen: Chloroplasts

2H2o!5or+4H++4e The electronsare transferredto a primary electron dcceptor, a quinone designatedQ, which is similar to CoQ in mitochondria. ln plants the oxidation of water takes place in a multiprotein complex calIedphotosystemII (PSII).

Mesophyll

Lower epidermis Cuticle

I n n e rm e m b r a n e : transportersfor p h o s p h a t ea n d sucroseprecursors

Chloroplast Stroma:enzymesthat catalyzeCO, fixation and starchsynthesis

T h y l a k o i dm e m b r a n e : a b s o r p t i o no f l i g h t b y c h l o r o p h y l ls, y n t h e s i s ofATP4 , NADPH,and I n t e r m e m b r a n e e l e c t r o nt r a n s p o r t space

Outer memDrane: p e r m e a b tl e o s m a l lm o l e c u l e s

Stage 2: Electron Transport and Generation of a Proton-Motive Force Electronsmove from the quinone primary electronacceptorthrough a seriesof electroncarriers until they reach the ultimate electron acceptor,usually the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+), reducing it to NADPH. NADP* is identical in structure with NAD* except for the presenceof an additional phosphategroup. Both moleculesgain and lose electronsin the sameway (seeFigure2-33).lnplants the reduction of NADP- takes place in a complex calledphotosystemI (PSI). The transport of electronsin the thylakoid membraneis coupled to the movementof protons from the stroma to the thylakoid lumen, forming a pH gradient acrossthe membrane(pHr,-.. ( pHr,ro-"). This processis analogousto generationof a proton-motiveforce acrossthe inner mitochondrial membraneand in bacterialmembranes during electrontransport (seeFigure 12-22). Thus the overall reaction of stages1, and 2 can be summarizedas 2H2O +2NADP+

Granum

Thylakoid memorane

'Jir FIGURE 12-29Cellularstructureof a leaf and chloroplast. L i k em i t o c h o n d r ipal ,a n tc h l o r o p l a satrseb o u n d e d b ya d o u b l e m e m b r a nsee p a r a t ebdy a n i n t e r m e m b r asnpea c ep h o t o s y n t h e s i s occuro s n a t h i r dm e m b r a n e t h, et h y l a k o im d embranw e ,h i c hi s s u r r o u n d ebdy t h e r n n e m r e m b r a naen df o r m sa s e r i eosf f l a t t e n e d v e s i c l e( tsh y l a k o i dt sh)a te n c l o sae s i n g l ei n t e r c o n n e c tl e ud minal s p a c eT h eg r e e nc o l o ro f p l a n t si sd u et o t h e g r e e nc o l o ro f c h l o r o p h yal ll,lo f w h i c hi s l o c a l i z et d o t h et h y l a k o im d embrane A g r a n u mi sa s t a c ko f a d j a c e nt ht y l a k o i dTs h es t r o m ai st h e s p a c e n c l o s ebdy t h e i n n e rm e m b r a naen ds u r r o u n d i nt hge thylakoidsIPhotomicrograph courtesy of Katherine Esau, University o f C a l i f o r n i aD, a v i sl 512

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riehtrtH*

+2NADPH+02

Stage 3: Synthesis of ATP Protons move down their concentration gradient from the thylakoid lumen to rhe stroma through the FeFl complex (ATP synthase),which couplesproton movement to the synthesisof ATP from ADP and P;. The chloroplastAIP synthaseworks similarly to the synthasesof mitochondriaand bacteria(seeFigure 12-25). Stage 4: Carbon Fixation The ATP and NADPH generated by the secondand third stagesof photosynthesisprovide the energyand the electronsto drive the synthesisof polymers of six-carbonsugarsfrom CO2 and H2O. The overall chemical equailon ls wrltten as 6 CO2 + 18 ATP4 + 12 NADPH + 12 H2O --> c6Hr2o6 + 1g ADp3 + 1g pi'z- + 12 NADP* + 6 H+ The reactions that generatethe ATP and NADPH used in carbon fixation are directly dependenton light energy;thus stages 1-3 are called the light reactions of photosynrhesis. The reactionsin stage4 are indirectly dependenton light energy; they are sometimescalled the dark reactions of photosynthesis becausethey can occur in the dark, utilizing the supplies of ATP and NADPH generatedby light energy.

Stage 4 Carbonfixation, carbohydratesynthesrs Sucrose

t

Cytosol outer

Stage 1

Stage 3

Stage 2

Light absorption, g e n e r a t i o no f h i g h energy electron, 02 formation

ATP synthesis ,

Electrontra nsport,formation of proton-motiveforce NADp+ H*

rDernbrane

nujln"'t"to'un"

6 COr+ 2 GlYceralde 3-phosphate (carbonfixation)

ADP + P1 ATP

s*"

/u+

HzO

Thylakoid membrane

2H++02

A FIGURE 12-30Overviewof the four stagesof photosynthesis. (LHC) In stage1, lightisabsorbed by light-harvesting complexes and reaction centerof photosystem ll (PSll)TheLHCstransfer the energyto the reaction absorbed centers, whichuseit, or theenergy froma photon,to oxidize absorbed by directly waterto molecular oxygenandgenerate high-energy In stage2, these electrons movedownan electron electrons transport chain,whichuseseither (Q/QHr) (plastocyanin, lipid-soluble or water-soluble PC)electron proteincomplexes to shuttleelectrons carriers betweenmultiple As electrons movedownthe chain,theyrelease energythatthe useto generate forceand,after complexes a proton-motive

lqrnen

of lightin photosystem by absorption energyisintroduced additional NADPHIn carrier electron the high-energy to synthesize | (PSl), proton-motive force powered proteins by the of stage 3, movement of ATPby an FoFrATPsynthaseStages1-3 in drivesthesynthesis ln membrane of thechloroplast plants takeplacein thethylakoid and storedin NADPH theenergy stroma, stage4, in thechloroplast molecules intothree-carbon C02initially ATPisusedto convert (glyceraldehyde knownascarbonfixation a process 3-phosphate), to thecytosolof the cellfor arethentransported Thesemolecules sugarsin theformof sucroseGlyceraldehyde to hexose conversion withinthechloroplast isalsousedto makestarch 3-phosphate However, the reactions in stage 4 are not confined to the dark; in fact, they occur primarily during illumination.

Chlorophyll a C H-,

ll cH H rlF

E a c hP h o t o no f L i g h t H a sa D e f i n e dA m o u n t of Energy

,;1 (cg

Quantum mechanicsestablishedthat light, a form of electromagnetic radiation, has propertiesof both waves and particles.\7hen light interactswith matter, it behavesas discrete

o-c I

o

cH.

cH.

PhytolI llCHr-CH: C- CH2- (CH2-CH2-CH-CHr)3H

12-31Structureof chlorophylla, the principal < FIGURE among aredelocalized pigmentthat trapslight energy.Electrons andtheatoms a'sfourcentralrings(yellow) threeof chlorophylls a Mg'* ion,ratherthanthe them.In chlorophyll, that interconnect ringand Fe3*ionfoundin heme,sitsat the centerof the porphyrin (blue) present; the otherwise, ts ring iive-membered an additional to thatof heme,foundin molecules issimilar of chlorophyll structure (seeFigure12-14a)Thehydroandcytochromes suchashemoglobin to hydrophobic bindingof chlorophyll carbonphytol"tail"facilitates is proteinsTheCH3group(9reen) of chlorophyll-binding regions (CHO) group b. in chlorophyll a formaldehyde by replaced N IGG M E N T S . PHOTOSYNTHEA SN I SD L I G H T - A B S O R B I P

513

packets of energy (quanta) calledphotozs. The energy of a photon, e, is proportional to the frequency of the light wave: , : h ^ 1w , h e r eb i s P l a n c k ' sc o n s t a n t( 1 . 5 8 x 1 0 - 3 4c a l . s ,o r 6.63 x t0 34;.s1and 1 is the frequencyof the light wave. It is customaryin biology to refer to the wavelengthof the light wave, tr, rather than to its frequency1. The two are relatedby the simple equation 'l : c + \, where c is the velocity of light (3 x 1010cm/s in a vacuum). Note that photons of shorter wavelengthhave bigher energies.Also, the energyin 1 mol of photons can be denoted by E : Ne, where N is Avogadro's number (6.02 x 1023moleculesor photons/mol).Thus E:

Action spectrum of photosynthesis C h l o r o p h y lal

o c

-o

o

60

o

o @

Nhc N D ^' iv_

o

The energy of light is considerable,as we can calculate for light with a wavelengthof 550 nm (550 x 10 7 cm), typical 500 600 of sunlight: (nm) Wavelength ( 6 . 0 2x 1 0 2 3 p h o t o n s / m o l ) ( 1x. 5180 3 a c a l . s ) (x3 1 0 1 0 c m / s )A EXPERIMENTAL FIGURE 12-32The rate of photosynthesis is E-greatestat wavelengthsof light absorbedby three pigments. 550x 10 7cm : 5 1 , 8 8 1callmol Theactionspectrum of photosynthesis in plants(theabilityof light or about 52 kcal/mol. This is enough energy to synthesize severalmoles of ATP from ADP and Pi if all the energywere used for this purpose.

PhotosystemsComprisea ReactionCenter and AssociatedLight-HarvestingComplexes The absorption of light energyand its conversioninto chemical energy occurs in multiprotein complexes called photosystems.Found in all photosynthetic organisms, both eukaryotic and prokaryotic, photosystems consist of two closely linked components: a reaction center,where the primary events of photosynthesisoccur, and an antenna complex consistingof numerous protein complexes,including internal antenna within the photosystem proper and external antenna made up of specializedproteins termed lightharuesting complexes (LHCs), which capture light energy and transmit it to the reaction center (seeFigure 12-30). Both reaction centers and antennascontain tightly bound light-absorbing pigment molecules.Chlorophyll a is the principal pigment involved in photosynthesis,being present in both reaction centersand antennas.In addition to chlorophyll a, antennascontain other light-absorbing pigments: chlorophyll b in vascular plants and carotenoids in both plants and photosynthetic bacteria.Carotenoidsconsistoflong branchedhydrocarbon chains with alternating single and double bonds; they are similar in structure to the visual pigment retinal, which absorbs light in the eye. The presenceof various antenna pigments, which absorb light at different wavelengths,greatly extendsthe rangeof light that can be absorbedand usedfor photosynthesis. One of the strongestpiecesof evidencefor the involvement of chlorophylls and carotenoids in photosynthesisis that the absorption spectrum of thesepigments is similar to the action spectrum of photosynthesis(Figure 72-32). The latter is a measureof the relative ability of light of different wavelengthsto support photosynthesis. 514

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of different wavelengths to supportphotosynthesis) isshownin blackTheenergyfromlightcanbe converted intoATPonlyif it canbe absorbed by pigments in thechloroplast. Absorption spectra (showing howwelllightof different wavelengths isabsorbed) for pigments present threephotosynthetic in the antennas of plant photosystems areshownin colorComparison of the actionspectrum with the individual absorption spectra suggests that photosynthesis at 680 nm isprimarily dueto lightabsorbed by chlorophyll a, at 650nm,to liqhtabsorbed by chlorophyll b, andat shorter wavelengths, to lightabsorbed by chlorophylls a andb andby carotenoid pigments, including B-carotene When chlorophyll a (or any other molecule)absorbsvisible light, the absorbedlight energyraiseselectronsin the chlorophylla to a higher-energy(excited)state.This state differs from the ground (unexcited)state largely in the distribution of the electronsaround the C and N atoms of the porphyrin ring. Excited statesare unstable,and the electronsreturn to the ground stateby one of severalcompetingprocesses. For chlorophyll a moleculesdissolvedin organic solventssuch as ethanol, the principal reactionsthat dissipatethe excited-stateenergy are the emissionof light (fluorescenceand phosphorescence) and thermal emission(heat).Ifhen the samechlorophyll a is bound in the unique protein environment of the reaction center,dissipation of excited-stateenergy occurs by a quite different processthat is the key to photosynthesis.

PhotoelectronTransportf rom Energized Reaction-Center Chlorophylla Produces a C h a r g eS e p a r a t i o n The absorption of a photon of light of wavelength =680 nm by one of the two "special-pair" chlorophyll a moleculesin the reaction center increasesits energy by 42 kcal/mol (the first excited state).Such an energizedchlorophyll a molecule in a plant reaction center rapidly donates an electron to an intermediate acceptor,and the electron is rapidly passedon

llil+ Animation:Photosynthesis P r i m a r ye l e c t r o n Light

Reaction centel

S t r oonnggr e d u c i n g a g e nntt( e l e c t r o nro o n o r

ge

rkoid brane -umen C h l o r o p h y l la

Strong o> x i ddiizziinngg aq g e nntt( e 3 l et c t r o na c c e p t o r )

12-33 PhotoelectrontransPort,the primaryevent in < FIGURE of a photonof light,oneof the photosynthesis. Afterabsorption center in the reaction pairof chlorophyll a molecules excited special (/eft)donatesviaseveral intermediates an electronto a looselybound surface of the molecule, the quinoneQ, on the stromal acceptor charge irreversible an essentially membrane, creating thylakoid (righi fhe electron cannoteasily across the membrane separation the positively centerto neutralize returnthroughthe reaction of waterto molecular a. In plantstheoxidation chlorophyll charged ll. calledphotosystem place complex in a multiprotein oxygen takes photoelectron transport photosystem I usesa similar Thecomplex the electron water,it reduces pathway,but insteadof oxidizing c a r r i eNr A D P - .

to the primary electron acceptor,quinone Q, near the stromal surfaceof the thylakoid membrane (Figure i2-33). This light-driven electron transfer,called photoelectron transport, dependson the unique environment of both the chlorophylls and the acceptor within the reaction center. Photoelectron transport, which occurs nearly every time a photon is absorbed, leavesa positive charge on the chlorophyll a closeto the luminal surface of the thylakoid membrane (opposite side from the stroma) and generatesa reduced, negatively chargedacceptor(Q-) near the stromal surface. The Q- produced by photoelectron transport is a powerful reducing agent with a strong tendencyto transfer an electron to another molecule, ultimately to NADP+. The positively charged chlorophyll a*, a strong oxidizing agent, attracts an electron from an electron donor on the luminal surface to regeneratethe original chlorophyll a. In plants, the oxidizing power of four chlorophyll a* moleculesis used,by way of intermediates,to remove four electronsfrom 2 H2O moleculesbound to a site on the luminal surfaceto form 02: 2HzO * 4 chlorophylla* --+4 H* + C2+ 4 chlorophyll a These potent biological reductants and oxidants provide all the energy neededto drive all subsequentreactions of photosynthesis:electron transport (stage 2), ATP synthesis (stage3), and CO2 fixation (stage4). Chlorophyll a also absorbs light at discretewavelengths shorter than 680 nm (see Figure 12-32). Such absorption raisesthe molecule into one of severalexcited states,whose energiesare higher than that of the first excited state describedabove,which decayby releasingenergywithin 10-12 seconds(1 picosecond,ps) to the lower-energyfirst excited state with loss of the extra energy as heat. Becausephotoelectron transport and the resulting charge separationoccur only from the first excited state of the reactron-center chlorophyll a, the quantum yield-the amount of photosynthesisper absorbedphoton-is the samefor all wavelengths of visible light shorter (and therefore of higher energy)than 680 nm. How closely the wavelength of light matches the absorption spectraof the pigmentswill determinehow likely it is that the photon will be absorbed. Once absorbed, the

photon's exact wavelength is not critical, provided it is energetic enough to push the chlorophyll into the first excited state.

I n t e r n a lA n t e n n aa n d L i g h t - H a r v e s t i n g ComplexesIncreasethe Efficiency of Photosynthesis Although chlorophyll a moleculeswithin a reaction center that are involved directly with charge separation and electron transfer are capableof directly absorbing light and initiating photosynthesis,they most commonly are energizedindirectly by energy transferred to them from other light-absorbing and energy-transferringpigments. These other pigments, which include many other chlorophyll molecules, are involved with absorption of photons and passingthe energyto the chlorophyll a molecules in the reaction center. Some are bound to protein subunitsthat are consideredto be intrinsic componentsof the photosystemand thus are called internal antennas;others are bound to proteinscomplexesthat bind to but are distinct from the photosystemcore proteins and are called light-harvesting complexes(LHCs). Even at the maximum light intensity encountered by photosynthetic organisms (tropical noontime sunlight),eachreaction-centerchlorophyll a moleculeabsorbs only about one photon per second' which is not enough to support photosynthesis sufficient for the needs of the plant. The involvement of internal antenna and LHCs greatly increasesthe efficiency of photosynthesis,especiallyat more typical light intensities,by increasingabsorption of 680-nm light and by extending the range of wavelengths of light that can be absorbedby other antennapigments. Photonscan be absorbedby any of the pigment molecules in internal antennasor an LHC. The absorbedenergyis then rapidly transferred(in <10-e seconds)to one of the two "special-pair" chlorophyll a moleculesin the associatedreaction center, where it promotes the primary photosynthetic charge separation(Figure 12-33). Photosystemcore protetns and LHC proteins maintain the pigment moleculesin the preciseorientation and position optimal for light absorption and energy transfer,thereby maximizing the very rapid and efficient resonancetransfer of energy from antenna pigments to A INSD L I G H T - A B S O R B I P N IGG M E N T S . PHOTOSYNTHES

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(a)

Bridging Reaction c h l o r o p h y l l center

L i gh t

E n e r g yr e s o n a n c e tra nsfer

Stroma

(b)

recial-pair cn orophylls

LHC

d ne

(c) Bridging chlorophyll

vF

ight

2\ 4--'-e-

E:nergy n e r g yresonance transfer .f\

Reaction center

Special-pair chlorophylls

A FIGURE 12-34Light-harvesting complexesand photosystems in cyanobacteria and plants.(a)Diagram of the membrane of a cyanobacterium, in whichthe multiprotein light(LHC) harvesting complex contains (green) 90 chlorophyll molecules and31 othersmallmolecules, allheldin a specific Aeometric arrangement for optimallightabsorption andenergytransferOf the sixchlorophyll molecules in the reaction center, two constitute the (ovals, special-pair chlorophylls darkgreen) thatcaninitiate photoelectron (bluearrow)Resonance transport whenexcited transfer of energy(redarrows) rapidly funnelsenergyf romabsorbed

(squares, lightto oneof two "bridging"chlorophylls darkgreen)and (b)Three-dimensional thenceto chlorophylls in the reaction center. organization of the photosystem I (PSl) with its LHCsof Pisumsativum (garden pea)asdetermined by x-raycrystallography andasseenfrom the planeof the membrane Onlythechlorophylls together withthe reaction centerelectron carriers areshown(c)Expanded viewof the reaction centerfrom(b)rotated90' abouta vertical axislpart(a)adapted fromW KUhlbrandt, 2001,Nature 411:896, andPJordan etal. 2001,Narure (bandc)based 411:909Parts onthestructural determination bvA Ben-Sham etal, 2003,Nature426:6301

reaction-centerchlorophylls. Resonanceenergy transfer does not involve the transfer of an electron. Studieson one of the two photosystemsin cyanobacteria,which are similar to those in higher planrs, suggestthat energy from absorbed light is funneled first to a "bridging" chlorophyll in each LHC and then to the specialpair of reaction-centerchlorophylls (Figurel2-34a). Surprisingly,however,the molecular structuresof LHCs from plants and cyanobacteriaare completely different from those in green and purple bacteria, even though both types contain carotenoids and chlorophylls in a clustered arrangement within the membrane. Figure 12-346 shows the distribution of the chlorophyll pigments in the plant photosystem I from Pisum satiuum (gardenpea)togetherwith its peripheralLHCI antenna.The large number of internal and LHC antenna chlorophylls surround the core reaction center to permit efficient transfer of absorbedlight energyto the specialchlorophyllsin the reactlon center. Although LHC antenna chlorophylls can transfer light energyabsorbedfrom a photon, they cannot releasean electron. As we've seenalready,this function residesin the two reaction-centerchlorophylls. To understand their electronreleasingability, we examine the structure and function of

the reaction center in bacterial and plant photosystemsin the next sectlon.

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Photosynthetic Stages and Light-Absorbing Pigments r The principal end products of photosynthesisin plants are molecular oxygen and polymers of six-carbon sugars (starchand sucrose). r The light-capturing and ATP-generatingreactions of photosynthesisoccur in the thylakoid membrane located within chloroplasts. The permeable outer membrane and inner membrane surrounding chloroplasts do not participate directly in photosynthesis(seeFigure 12-29). r There are four stagesin photosynthesis:(1) absorption of light, generationof a high energyelectron and formation of 02 from H2O; (2) electron transport leading to reduction of NADP* to NADPH, and to generationof a protonmotive force; (3) synthesisof ATP; and (4) conversion of CO2 into carbohydrates(carbonfixation). r In stage 1 of photosynthesis,light energy is absorbed by one of two "special-pair" chlorophyll a molecules bound

to reaction-centerproteins in the thylakoid membrane.The energizedchlorophylls donate via intermediatesan electron to a quinone on the opposite side of the membrane, creating a charge separation(seeFigure 12-33). In green plants, the positively charged chlorophylls then remove electrons from water, forming molecular oxygen (02). r In stage 2, electrons are transported from the reduced quinone via carriers in the thylakoid membrane until they reach the ultimate electron acceptor,usually NADP*, reducing it to NADPH. Electron transport is coupled to movement of protons across the membrane from the stroma to the thylakoid lumen, forming a pH gradient (proton-motiveforce) acrossthe thylakoid membrane. r In stage 3, movement of protons down their electrochemical gradient through FeFl complexes (ATP synthase) powers the synthesisof ATP from ADP and P,.

The reaction centerof purple bacteriacontains three protein subunits(L, M, and H) locatedin the plasmamembrane (Figure 12-35). Bound to these proteins are the prosthetic groups that absorb light and transport electrons during photosynthesis. The prosthetic groups include a "special pair" of bacteriochlorophyll a molecules equivalent to the reaction-centerchlorophyll a moleculesin plants, as well as severalother pigmentsand two quinones,termed Qa and Qs' that are structurally similar to mitochondrial ubiquinone. Initial Charge Separation The mechanismof chargeseparation in the photosystemof purple bacteria is identical with that in plants outlined earlier; that is, energy from absorbed light is used to strip an electron from a reaction-centerbacteriochlorophyll a molecule and transfer it, via several different pigments, to the primary electron acceptor Qs, which is loosely bound to a site on the cytosolic membrane face'

r In stage4, the NADPH and ATP generatedin stages2 and 3 provide the energyand the electronsto drive the fixation of CO2, which resultsin the synthesisof carbohydrates.These reactionsoccur in the thylakoid stroma and cytosol. r Associatedwith eachreaction centerare multiple internal antenna and light-harvestingcomplexes (LHCs), which contain chlorophylls a and b, carotenoids, and other pigmentsthat absorb light at multiple wavelengths.Energy,but not an electron,is transferredfrom the internal antennaand LHC chlorophyll moleculesto reaction-centerchlorophylls by resonanceenergytransfer (seeFigure 12-34).

MolecularAnalysis of Photosystems As noted in the previous section,photosynthesisin the green and purple bacteria does not generateoxygen, whereasphotosynthesisin cyanobacteria,algae,and higher plants does." This differenceis attributable to the presenceof two types of photosystem (PS) in the latter organisms: PSI reduces NADP* to NADPH, and PSII forms 02 from H2O. In contrast, the green and purple bacteria have only one type of photosystem,which cannot form 02. !7e first discussthe simpler photosystem of purple bacteria and then consider the more complicatedphotosyntheticmachinery in chloroplasts. Pheophytin

The SinglePhotosystemof PurpleBacteria Generatesa Proton-MotiveForcebut No 02 The three-dimensionalstructuresof the photosynthetic reaction centers have been determined, permitting scientiststo trace in detail the paths of electronsduring and after the absorption of light. Similar proteins and pigments compose photosystemsI and II of plants and photosynthetic bacteria. *A very different type of bacterial photosynthesis, which occurs only in certain archaebacteria, is not discussed here because it is very different from photosynthesis in higher plants. In this type of photosynthesis, the plasma-membrane protein bacteriorhodopsin pumps one proton from the cytosol to the extracellular space for every quantum of light absorbed.

Accessory chlorophyll Special-pair chlorophyll

structureof the 12-35Three-dimensional FIGURE photosyntheticreactioncenterfrom the purplebacterium (Top)IheL subunit(yellow) andM subunit spheroides. Rhodobacter andhavea verysimilar crhelices (gray) eachformfivetransmembrane to themembrane the H subunit(lightblue)isanchored overall; structure (notshown) isa A fourthsubunit a helix. transmembrane bya single of theother segments proteinthat bindsto theexoplasmic peripheral distincenter, but not easily Withineachreaction subunits(Bottom) a pairof bacteriochlorophyll isa special guished in thetop image, photoelectron two transport; (green), of initiatinq capable molecules (darkblue),andtwo (purple); turopheophytins chlorophylls accessory acceptor electron quinones, Qeistheprimary QaandQe(orange). etal, 1997,Science2T6:8121 duringphotosynthesis. lAfterM H Stowell M O L E C U L A RA N A L Y S I SO F P H O T O S Y S T E M S '

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The chlorophyll thereby acquires a positive charge, and Qg acquires a negative charge. To determine the pathway traversed by electronsthrough the bacterial reacrion center, researchersexploited the fact rhat eachpigment absorbslight of only certain wavelengths,and its absorptron specrrum changeswhen it possesses an extra electron.Becausethese electronmovementsare completedin lessthan 1 millisecond (ms), a specialtechnique calledpicosecondabsorption spectroscopy is required to monitor the changesin the absorption spectraof the various pigmentsas a function of time shortly after the absorptionof a light photon. \7hen a preparation of bacterial membrane vesiclesis exposedto an intensepulse of laser light lasting less than 1 ps,eachreactioncenterabsorbsone photon (Figure12-36). l-ight absorbedby the chlorophylla moleculesin each reaction center convertsthem to the excited state, and the subsequent electron transfer processesare synchronizedin all reactioncentersin the experimentalsample.\Tithin 4 x 10 12 seconds(4 ps), an electronmoves,possiblyvia the accessory bacterial chlorophyll as an intermediare,to the pheophytin molecules(Ph),leaving a positivechargeon the chlorophyll a. It takes200 ps for the electronto move ro Qe, and then, in the slowest step, 200 ps for it to move to Qn. This pathway of electronflow is traced in the left part of Figore 12-36. Subsequent Electron Flow and Coupled Proton Movement After the primary electronacceptor,Qe, in the bacterial reactioncenteracceptsone electron,forming Qs-., it

accepts a second electron from the same reaction-center chlorophyll following its re-excitation (e.g.,by absorption of a second photon or transfer of energy from antenna molecules). The quinone then binds two protons from the cytosol, forming the reducedquinone (QHz), which is released from the reaction center (Figure 12-36). QH2 diffuseswithin the bacterial membraneto the Qo site on the exoplasmicface of a cytochrome bc1 electron transport complex similar in structure to complex III in mitochondria. There it releasesits tvvo protons into the periplasmicspace(the spacebetweenthe plasma membrane and the bacterial cell wall). This process movesprotons from the cytosol to the outsideof the cell, generating a proton-motive force acrossthe plasma membrane. Simultaneously,QHr releasesits two electrons,which move through the cytochrome bcl complex exacrly as depicted for the mitochondrial complex III (CoQH2-cytochrome c reductase) in Figure 12-20. The Q cycle in the bacterial reaction center, like the Q cycle in mitochondria, pumps additional protons from the cytosol to the intermembrane space, thereby increasingthe proton-motive force. The acceptor for electrons transferred through the cytochrome bc1complex is a solublecytochrome,a one-electron carrier, in the periplasmic space,which is reduced from the Fe3* to the Fe2* state.The reducedcytochrome(analogousto cytochromec in mitochondria) then diffusesto a reactloncenter,where it releasesits electronto a positivelychargedchlorophyll a-, returning that chlorophyll to the unchargedground stateand the cytochromero the Fe3* state.This cyclicelectron

O c y c l e :a d d i t i o n a l proton transport H_

Pi + ADP

ATP

Cytosol

Plasma membrane 2 photons

++++ Periplasmic space

Bacterial reaction

FIGURE 12-36Cyclicelectronflow in the single photosystemof purplebacteria.Cyclic electron flow generates a proton-motive forcebut no O, Bluearrowsindicate flow of electrons; redarrowsindicate protonmovement(left)Energy absorbed directly fromlightor funneled froman associated LHC (notillustrated here)energizes oneof the special-pair chlorophylls in the reaction centerPhotoelectron transport fromtheenergized chlorophyll, viaaccessory (ph),andquinone chlorophyll, pheophytin e ( Q sf)o r m st h es e m i q u i n oen-e a n dl e a v eas A ( Q i , t o q u i n o nB positive chargeon the chlorophyll Following absorption of a second photonandtransfer of a second electron to the semiquinone, the quinonerapidlypicksup two protons fromthe cytosol to formeH2, 518

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FoFt complex

(Cente)Afterdiffusingthroughthe membrane andbindingto the faceof thecytochrome Qositeon theexoplasmic bc1complex, eH, donates two electrons givesup two protons andsimultaneously to theexternal mediumin the periplasmic generating space, a proton (proton-motive gradient electrochemical force)Electrons are transported backto the reaction-center chlorophyll viaa soluble cytochrome, whichdiffuses in the periplasmic spaceNotethecyclic path(blue)of electrons Operation of a Q cyclein thecytochrome pumpsadditional protons bcl complex across the membrane to the external medium, asin mitochondria. from.]Deisenhofer and fAdapted H M i c h a e l ,1 9 9 1, A n n R e v . C e lBl i o l . 7 : 1I

flow generatesno oxygen and no reducedcoenzymes,but it has generateda proton-motive force. Electrons also can flow through the single photosystem purple bacteria via a linear (noncyclic) pathway. In this of instead of the electron removed from reactron-center case, chlorophylls by light moving to the cytochrome bcl complex and then cycling back again via the water-soluble cytochrome to the reaction center.the electron removed from reaction-centerchlorophyll is eventually transferred to NAD* (rather than NADP+ as in plants),forming NADH. As a consequence,an electron from a different source must be returned to the special-pairchlorophylls if additional light energyis to be harvestedby photoelectrontransport. In bacteriausing a linear pathway, the electronsto do this come from the oxidation of either hydrogen sulfide (H2S) H2S--+S+2H+ *2eto form elementalsulfur (S)or hydrogengas (H2): H2--+2H- *2eThese electrons are used to reduce a cytochrome, which in turn passesan electron to the special-pairchlorophylls in the reaction center to bring the oxidized reaction-centerchlorophyll a back to its ground state. Overall, the linear pathway resultsin the light-mediatedoxidation of H2S (or H2) and the reduction of NAD- to NADH. SinceH2O is not the electron donor, no 02 is formed. Both the cyclic and linear pathways of electron flow in the bacterial photosystem generate a proton-motive force. As in other systems,this proton-motive force is used by the FeFl complex located in the bacterial plasma membrane to synthesizeAIP and also to transport molecules across the membrane against a concentration gradient.

y nd C h l o r o p l a s tC s o n t a i nT w o F u n c t i o n a l l a Distinct Photosystems Spatially In the 1940s, biophysicist R. Emerson discoveredthat the rate of plant photosynthesisgeneratedby light of wavelength 700 nm can be greatly enhanced by adding light of shorter wavelength (higher energy).He found that a combination of light at, say, 600 and 700 nm supports a greater rate of photosynthesisthan the sum of the rates for the two separatewavelengths.This so-calledEmerson effect led re' searchersto conclude that photosynthesisin plants involves the interaction of two separatephotosystems,referred to as PSI and PSII. PSI is driven by light of wavelength 700 nm light (<680 nm). or less;PSII,only by shorter-wavelength In chloroplasts, the special-pair reaction-centerchlorophylls that initiate photoelectron transport in PSI and PSII differ in their light-absorptionmaxima becauseof differencesin their protein environments. For this reason, these chlorophylls are often denoted P680(PSII) and PTes(PSI). Like a bacterial reaction center, each chloroplast reaction center is associatedwith multiple internal antenna and lightharvesting complexes (LHCs); the LHCs associatedwith PSII and PSI contain different proteins.

The two photosystems also are distributed differently in thylakoid membranes:PSIIprimarily in stackedregions(grana, seeFigure 12-29) and PSIprimarily in unstackedregions.The stacking of the thylakoid membranesmay be due to the binding properties of the proteins in PSII. Evidence for this distribution came from studiesin which thylakoid membraneswere gently fragmented into vesiclesby ultrasound' Stacked and unstacked thylakoid vesicles were then fractionated by density-gradientcentrifugation. The stacked fractions contained primarily PSII protein and the unstackedfraction PSI. Finally, and most importantly, the two chloroplast photosystems differ significantly in their functions (Figure 12-37): only PSII splits water to form oxygen' whereas only PSI transfers electronsto the final electron acceptor,NADP*. Photosynthesisin chloroplastscan follow a linear or cyclic pathway, again like green and purple bacteria. The linear pathwaS which we discussfirst, can support carbon fixation as well as ATP synthesis.In contrast, the cyclic pathway supports only ATP synthesisand generatesno reducedNADPH for use in carbon fixation. Photosyntheticalgaeand cyanobacteriacontain two photosystemsanalogousto those in chloroplasts.

LinearElectronFlow Through Both Plant PSlland PSl,Generates Photosystems, a Proton-MotiveForce,02, and NADPH Linear electron flow in chloroplastsinvolves PSII and PSI in an obligate series in which electrons are transferred from H2O to NADP+. The processbegins with absorption of a photon by PSII, causing an electron to move from a P53s chlorophyll a to an acceptorplastoquinone(Qs) on the stromal surface (Figure 12-37). The resulting oxidized P5ssstrips one electron from the relatively unwilling donor H2O, forming an intermediate in 02 formation and a proton' which remains in the thylakoid lumen and contributesto the proton-motive force. After P5s6absorbsa secondphoton, the semiquinoneQ-' acceptsa secondelectron and picks up tvvo protons from the stromal space,generatingQH2. After diffusing in the membrane, QHz binds to the Q" site on a cytochrome b/complex (analogousto bacterialcytochrome bc1 complex and mitochondrial complex III). As in thesesystems' a Q cycle operates, thereby increasing the proton-motive force generatedby electron transport. After the cytochrome bf complex accepts electrons from QH2' it transfers them' one at a time, to the Cu2+ form of the solubleelectroncarrier plastocyanin(analogousto bacterial cytochrome c), reducing it to the Cu* form. Reducedplastocyaninthen diffusesin the thylakoid lumen, carrying the electron to PSI. Absorption of a photon by PSI leads to removal of an electron from the reaction-centerchlorophyll a,P7ss (Figure 1,2-37).The resulting oxidized P706* is reduced by an electron passedfrom the PSII reaction centervia the cytochrome bf complex and plastocyanin' Again, this is analogousto situation in mitochondria, where cytochrome c acts as a single electron shuttle from complex III to complex IV (seeFigure 12-1.6).The electron taken up at the luminal surfaceby Pzoo energizedby photon absorption moveswithin PSIvia several carriers to the stromal surface of the thylakoid membrane, A N A L Y S I SO F P H O T O S Y S T E M S O MOLECULAR

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Ferrr loxi) Fe-S

Stroma AU+

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photons

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O2-evolving comprex

o ' 680

Plastocya ocvanrn D

chlorophyll HzO

| 700

cn orophyll 2 H'+ 1lz

PSll reaction center

Cytochrome bf complex

PSI reaction center

FsFt complex

A FIGURE 12-37Linearelectronflow in plants,which requires both chloroplastphotosystems PSIand PSll.Bluearrowsindicate flow of electrons; protonmovementLHCsare redarrowsindicate not shown(Left)ln the PSllreaction center, two sequential lightinduced excitations of thesameP6s6 chlorophylls resultin reduction of the primary electron acceptor side Qato eHz Onthe luminal of PSll, electrons removed fromH2Oin thethylakoid lumenare transferred to P6se+, restoring the reactron-center chlorophylls to the groundstateandgenerating Oz Genter)Thecytochrome bf complex thenaccepts electrons fromQH2,coupled to the release of two protonsintothe lumenOperation of a e cyclein the

cytochrome bf complex translocates protons additional across the membrane to thethylakoid lumen,increasing the proton-motive f orce (Right)In the PSIreactioncenter,eachelectronreleased f rom light-excited P76s chlorophylls movesviaa series of carriers in the reaction centerto thestromal (an surface, wheresoluble ferredoxin Fe-Sprotein) transfers the electron to ferredoxin-NADP+ reductase (FNR)Thisenzyme usesthe prosthetic groupflavinadenine dinu(FAD) cleotide anda protonto reduce NADP+, formingNADPHpToo+ isrestored to itsgroundstateby addition of an electron carried from PSllviathecytochrome bf complex andplastocyanin, a soluble electron carrier.

where it is acceptedby ferredoxin, an iron-sulfur (Fe-S)protein. Electrons excited in PSI can be transferred from ferredoxin via the enzymeferredoxin-NADP+ reductase(FNR). This enzyme uses the prosrhetic group FAD as an electron carrierto reduceNADP*, forming, togetherwith one proton picked up from the stroma, the reduced molecule NADPH. FeFl complexesin the thylakoid membraneuse the proton-motive force generatedduring linear electron flow to synthesizeATP on the stromal side of membrane.Thus this pathway exploits the energy from multiple photons absorbed by both PSII and PSI and their anrennasro generare both NADPH and ATP in the stroma of the chloroDlast. where they are utilized for CO2 fixation.

'S7hen PSII absorbs a photon with a wavelength of <680 nm, it triggersthe loss of an electronfrom a P5semolecule, €leneratingP.so*. As in photosyntheticpurple bacteria,the electron is transported rapidly, possibly via an accessory chlorophyll, to a pheophytin, then to a quinone (Qa), and then to the primary electron acceptor, Q", or the outer (stromal)surfaceof the thylakoid membrane(Figures12-37 and 12-38). The photochemically oxidized reaction-centerchlorophyll of PSII, P5s0*, is the strongesl biological oxidant known. The reduction potential of P6s6* is more positive than that of water, and thus it can oxidize water to generate 02 and H* ions. Photosyntheticbacteria cannot oxidize water becausethe excited chlorophyll a* in the bacterial reaction center is not a sufficiently strong oxidant. (As noted earlier,purple bacteria useH2S and H2 as electron donors to reduce chlorophyll a* in Iinear electron flow.) The splitting of H2O, which provides the electrons for reduction of P5se* in PSII, is catalyzedby a three-protein complex, the oxygen-euoluingcomplex,located on the luminal surfaceof the thylakoid membrane.The oxygen-evolving complex contains four manganese(Mn) ions as well as bound Cl and Ca2* ions (Figure 12-38);this is one of the very few casesin which Mn plays a role in a biological system. These Mn ions together with the three extrinsic proteins can be removed from the reaction center by treatment with solutions of concentrated salts; this abolishes ()2 formation but does not affect light absorption or the initial stagesof electron transport.

A n O x y g e n - E v o l v i nC g o m p l e xl s L o c a t e do n t h e L u m i n a lS u r f a c eo f t h e p S l lR e a c t i o nC e n t e r Somewhat surprisingly,the structure of the pSII reaction center,which removeselectronsfrom H2O to form 02, r€semblesthat of the reactioncenrerof photosyntheticpurple bacteria,which does not form ()2. Like the bacterial reaction center,the PSII reactioncentercontainstwo molecules of chlorophyll a (P5s6),as well as two other accessory chlorophylls,two pheophytins,rwo quinones(e6 and es), and one nonheme iron atom. These small molecules are bound to two proteinsin PSII,calledD1 andD2, whose sequencesare remarkably similar to the sequencesof the L and M subunitsof the bacterialreactioncenrer,attestingto their common evolutionary origins (see Figure I2-35). 520

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Fe

Special-pair chlorophylls

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O2-evolving comprex

2H2O

4H*+02

FIGURE12-38 Electronflow and 02 evolution in chloroplast P5ll.The PSllreactioncenter,comprisingtwo integralproteins,D1 (Poeo), and other electroncarriers,is chlorophylls and D2, special-pair wrth an oxygen-evolving complexon the luminalsurface associated Boundto the threeextrinsicproteins(33,23, and 17 kDa)of the complexare four manganeseions(Mn, red),a Ca2* oxygen-evolving ion (blue),and a Cl ion (yellow)Thesebound ionsfunctionin the for high splittingof H2Oand maintainthe environmentessential 1 1 6 1 ) o ft h e D 1 p o l y p e p t i d e r a t e so f 0 2 e v o l u t i o nT y r o s i n e - 1 6( Y conductselectronsfrom the Mn ionsto the oxidizedreaction-center from reducingit to the groundstateP6s6[Adapted chlorophyll(Poeo-), 277:1953I andG Babcock, 199-l, Science C Hoganson The oxidation of two molecules of H2O to form 02 requires the removal of four electrons, but absorption of each photon by PSII results in the transfer of iust one electron. A simple experiment, described in Figure 12-39, resolved

!

whether the formation of 02 depends on a single PSII or multiple ones acting in concert. The results indicated that a single PSII must lose an electron and then oxidize the oxygen-evolvingcomplex four times in a row for an 02 molecule to be formed. Manganeseis known to exist in multiple oxidation states with from two to five positive charges.Indeed, spectroscopic studiesshowed that the bound Mn ions in the oxygen-evolving complex cycle through five different oxidation states' Se-Sa.In this S cycle, a total of two H2O moleculesare split into four protons, four electrons'and one 02 molecule. The electronsreleasedfrom H2O are transferred, one at a time, via the Mn ions and a nearby tyrosineside chain on the D1 subunit to rthereaction-centerPnro*, where they regenerate the reducedlchlorophyll, P536ground state. The protons releasedfrom H2O remain in the thylakoid lumen.

a

o o_ n 0)

0)

123456789101112 F l a s hn u m b e r A E X P E R I M E N T AFLI G U R E1 2 - 3 9 A s i n g l e P S l la b s o r b s a photon and transfers an electron four times to generate o n e 0 2 . D a r k - a d a p t e cdh l o r o p l a s tws e r e e x p o s e dt o a s e r i e so f c l o s e l ys p a c e d s, h o r t( 5 p s ) p u l s e so f l i g h t t h a t a c t i v a t e dv i r t u a l l y a l l t h e P 5 l l si n t h e p r e p a r a t i o nT h e p e a k si n O 2 e v o l u t i o no c c u r r e d a f t e r e v e r yf o u r t h p u l s e ,i n d i c a t i n gt h a t a b s o r p t i o no f f o u r p h o t o n sb y o n e P S l li s r e q u i r e dt o g e n e r a t ee a c h0 2 m o l e c u l e the dark-adapted c h l o r o p l a s tws e r e i n i t i a l l yi n a p a r t i a l l y Because r e d u c e ds t a t e ,t h e p e a k si n 0 2 e v o l u t i o no c c u r r e da f t e r f l a s h e s3 , 7 , a n d 1 1 l F r o mJ B e r ge t a l , 2 0 0 2 ,E r o c h e m i s t r y ,e5dt h, W H F r e e m aann dC o m p a nl y

Herb:icidesthat inhibit photosynthesisnot only are very limportant in agriculture but also have proved useful in dissectingthe pathway of photoelectron transport in plants. One such classof herbicides,the s-triazines (e.g., atrazine),binds specificallyto the D1 subunit in the PSII reaction center, thus inhibiting binding of oxidized Qs to its site on the stromal surfaceof the thylakoid membrane. \7hr:n added to illuminated chloroplasts,s-triazines causeall downstreamelectroncarriersto accumulatein the oxidized form, sinceno electronscan be releasedfrom PSII. mutants, a singleamino acid changein In atrazine-resistant D1 rendersit unable to bind the herbicide,so photosynthesis proceeds at normal rates. Such resistant weeds are prevalentand presenta major agricultural problem. I

C e l l sU s eM u l t i p l e M e c h a n i s mtso P r o t e c t Against Damagefrom ReactiveOxygen Species During PhotoelectronTransport As we saw earlier in the caseof ROS generationby the mitochondrion, ATP generation via the electron transport chain brings with it potential deleteriousside effects.The same is true for the chloroplast. Even though the PSI and PSII photosystemswith their associatedlight-harvestingcomplexesare remarkably efficient at converting radiant energy to useful chemical energy in the form of ATP and NADPH, they are not perfect. Depending on the intensity of the light and the physiologicconditions of the cells,a relativelysmall-but sigof energy absorbed by chlorophylls in the .tifi."ttt-u-ount light-harvestingantennasand reactionscentersresultsin the chlorophyll being converted to an activated state called "triplet" chlorophyll. In this state' the chlorophyll can transfer some of its energyto molecular oxygen (02), converting it from its normal, relatiuely unreactive ground state, called triplet oxygen (3Oz) to a very highly reactive (ROS) singlet 1Or. If the 1O2 is not quickly quenchedby reactstate form, tOr "scau.nger molecules," it will react ing with specialized with and usually damagenearby molecules'This damagecan suppressthe efficiencyof thylakoid activity and is calledpDotoiihibitior. Carotenoids (polymers of unsaturatedisoprene groups, including beta-carotene,which gives carrots their M O L E C U L A RA N A L Y S I SO F P H O T O S Y S T E M S .

521

Photoinhibition (2400pE m-1 s 1)

Recovery ( 2 0p E m - 1 s 1 )

< EXPERIMENTAT FIGURE 12-40ThechaperoneHSP7OB helps PSllrecoverfrom photoinhibitionafter exposureto intense fight. Theunicellular greenalgaChlamydomonas reinhardtiiwas genetically manipulated sothatit hadabnormally highor low levels proteinHSP7OB. of thechaperone Thehigh,low,andnormalstrains werethenexposed to high-intensity light(2400pE m 2 s-1)for 60 minutes to inducephotoinhibition followedbyexposure to low light(20pE m-2 s-1)forup to 150minutes. Theeffects of photoinhibition bythe high-intensity lightandtheabilityof pSilto recover fromthe photoinhibition weremeasured usingfluorescence spectroscopy to determine PSllactivityTheabilityof the cellsto recover PSllactivitydepends on the levels of HSP7OB-the more HSP7OB available, the morerapidthe recovery-dueto HSp7OB protection of the PSllreaction centers that hadwithstood1Or-induced D1subunit damage. Schroda etal, 1999, Plant Cetl 1l:t 165] [From

o

binds to the damagedPSII and helps prevent loss of the other components of the complex as the D1 subunit is replaced. The extent of photoinhibition can depend on the amounr of HSP7OBavailableto the chloroplasts.

a

CyclicElectronFlow Through PSIGenerates a Proton-MotiveForcebut No NADPHor 02

-60

-30

0

30 60 T i m e( m i n )

90

120

150

orange color) and o-tocopherol (a form of vitamin E) are hydrophobic small moleculesthat play important roles as 1O2 quenchersto protect plants. For example,inhibition of tocopherol synthesis in the unicellular green alga Chlamydomonas reinhardtii by the herbicidepyrazolynatecan result in greaterlight-inducedphotoinhibition. To help further limit the potential damagein light-harvestingantennas,carotenoid molecules siphon off energy from the dangerous triplet chlorophyll, thus preventing 102 formation. Under intenseillumination, photosystempSII is especially prone to generating tO2, whereas PSI will produce other ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals. The D1 subunit in the pSII reacion cenrer (seeFigure 12-38) is, even under low light conditions, subjected to almost constant 1O2-mediateddamage. The damaged reaction center moves from the grana to the unstacked regionsof the thylakoid, where the D1 subunit is degradedby a proteaseand replaced by newly synthesizedD1 proteins in what is called the D1 protein damage-repaircycle.The rapid replacementof damagedD 1, which requiresa high rate of b 1 synthesis,helps the PSII recover from photoinactivation and maintain sufficient activity. The experiment in Figure 12-40 showsthat an important component in the damage-repaircycle is the chaperoneprotein HSP7OB(seeChapter 3), which

522

CHAPTER 12

I

CELLULAR ENERGETICS

As we've seen,electronsfrom reduced ferredoxin in PSI are transferred to NADP* during linear electron flow, resulting in production of NADPH (seeFigure 12-37).In some circumstancescells must generaterelative amounts of ATP and NADPH that differ from those produced by linear electron flow (e.g., greater ATP-Io-NADPH ratio). To do this, they photosyntheticallyproduce ATP from PSI without concomitant NADPH production. This is accomplishedusing a PSIIindependent process called cyclic ph otop h osph orylation. In this processelectronscycle between PSI, ferredoxin, plastoquinone (Q), and the cytochrome b/complex (Figure 1.2-41); thus no net NADPH is generated,and there is no need to oxidize water and produce 02. There are two distinct cyclic electron flow pathways: the NAD(P)H dehydrogenase(Ndh)dependent (shown in Figure 1,2-41) and Ndh-independent pathways. Ndh is an enzyme complex very similar to the mitochondrial complex I (see Figure 12-16) that oxidizes NADPH or NADH while reducing Q to QH2 and contributing to the proton motive force by transporting protons. During cyclic electron flow, the substrate for the Ndh is the NADPH generated by light absorption by PSI, ferredoxin, and ferredoxin-NADP reductase (FNR). The QH2 formed by Ndh then diffusesthrough the thylakoid membraneto the Q" binding site on the luminal surfaceof the cytoch rome bf complex. There it releasestwo electronsto the cytochrome b/ complex and two protons to the thylakoid lumen, generatrng a proton-motive force. As in linear electron flow, these electronsreturn to PSI via plastocyanin. This cyclic electron flow is similar to the cyclic processthat occurs in the single photosystemofpurple bacteria (seeFigure 12-36). A e cycle operatesin the cytochrome bf complex during cyclic electron flow, leading to transport of two additional protons into the lumen for each pair of electrons transported and a greater proton-motive force.

NADP+ H'

/Ferredoxin\ +

NADPH NADP*+Ht

NADPH

O c y c l e :a d d i t i o n a l protontransport Hi

s9,/

P i+ ADP

Ferre oxtn ="-S_,,

Stroma

Thylakoid membrane

++++ Lumen

photon

Plastocyanin AU+

D | 700 chlorophyll

NAD(P)Hdehydrogenase (Ndh)

Cytochrome bf complex

PSI reaction center

F6F1complex

12-41 Cyclicelectronflow in plants,which generates A FIGURE a proton-motiveforce and ATPbut no oxygen or net NADPH. (Ndh)-dependent pathway for cyclic ln the NAD(P)H-dehydrogenase electrons In a flow,lightenergyisusedby PSIto transport electron forceandATPwithoutoxidizing a proton-motive cycleto generate of formedviathe PS|/ferredoxin/FNR-instead waterTheNADPH

electrons by Ndh Thereleased beingusedto f ixcarbon-isoxidized to generate (Q)withinthe membrane to plastoquinone aretransferred bf complex, to the cytochrome the electrons QH2,whichthentransfers for the case the as is PSl, to back finally and plastocyanin, thento f low pathway(seeFigure12-37) linearelectron

In Ndh-independent cyclic electron floq the mechanism of which has not yet beencompletely defined, electronsfrom the ferredoxin are used to reduceQ, either via a hypothetical ferredoxin:plastoquinoneoxidoreducmembrane-associated tase (FQR) or via the Q1site, which is part of the Q cycle in

to electron flow. The distribution of LHCII betweenPSI and

the cytochromeb/complex.

RelativeActivitiesof PhotosystemsI and ll Are Regulated In order for PSII, which is preferentially located in the stacked grana)and PSI,which is preferentially located in the unstacked thylakoid membranes,to act in sequenceduring linear electron flow, the amount of light energy deliveredto the two reaction centersmust be controlled so that eachcenter activates the same number of electrons. This balanced condition is called state 1 (Figure 12-42).If the two photosystems are not equally excited, then cyclic electron flow occurs in PSI and PSII becomeslessactive (state2). Variations in the wavelengthsand intensitiesof ambient light (as a consequentof the time of day, clouds, etc.) can changethe relative activation of the two photosystems'potentially upsetting the appropriate relative amounts of linear and cyclic electron flow necessaryfor production of optimal ratios of ATP and NADPH. One mechanismfor regulating the relative contributions of PSI and PSII,in responseto varying lighting conditions and thus the relative amounts of linear and cyclic electron flow, entails redistributing the light-harvestingcomplex LHCII between the two photosystems.The more LHCII associated with a particular photosystem,the more efficiently that system will be activatedby light and the greater its contribution

flow in state2 (Figure 1'2-42). Regulating the supramolecular organization of the photoryst.-s in plants thus has the effect of directing th.- to*atd ATP production (state 2) or toward genera-

chapter.

Molecular Analysis of Photosystems In the single photosystemof purple bacteria, cyclic elecon flow fiom light-excited, special-pairchlorophyll a moleculesin the reaction center generatesa proton-motive O M O L E C U L A RA N A L Y S I SO F P H O T O S Y S T E M S

523

State 1, linear electron flow

P S Im e m b r a n ed o m a i n s (unstacked)

P S l lm e m b r a n ed o m a i n s ( s t a c k e )d T h y l a k o i dm e m b r a n e

NADPH e

ADP + P;

Plastocyanin

ATP svnthase State 2, cyclic electron flow Thylakoid membrane

Stroma Lumen

0

A FIGURE 12-42Phosphorylation of LHCIIand the regulationof finearversuscyclicelectronflow.(Top)ln pSIand normalsunlight, PSllareequally activated, andthephotosystems areorganized in state 1 ln thisarrangement, light-harvesting complex ll (LHCll) isnot pirosphorylated andistightlyassociated withthe psrrreaction centerin pSllandpSlcanfunctionin parallel thegranaAsa result, in linear eleclronflow (Bottom) when lightexcitation of thetwo photosvstems force, which is used mainly to power ATp synthesisby the FeF, complex in the plasmamembrane(seeFigure 12-36). r Plants contain two photosystems,pSI and pSII, which have diff'erentfunctions and are physically separatedin the thylakoid membrane. PSII splits H2O into 02, and pSI reduces NADP+ to NADPH. Cyanobacteria have two analogousphotosystems. In chloroplasts,light energyabsorbedby light-harvesting rnplexes(t.HCs) is transferredto chlorophyll a moleculei the reactioncenters(Pos6in pSII and pr,16in pSI). r Electronsflow through PSII via the samecarriersthat are presentin the bacterialphotosystem.In contrastto the bac_ rerial system,photochemicallyoxidized p680+in pSII is re€eneratedro P6ssby electronsderived from the splitting rrf FI2O with evolution of 02 (seeFigure 72-37, left). r [n linear electronflow, photochemicallyoxidized proo* in I)Sl is reduced, regeneratingpzoo, by electrons transferred frorn PSll via the cytochrome b/ complex and soluble plas_ tocyanin. Electronsreleasedfrom pTeefollowing excitation of PSI are transported via severalcarriers u[imately to NADP' , generaringNADPH (seeFigure 72-37 rigbt). , -f r he absorption of light by pigmentsin the chloroplastcan generirtetoxic reacive oxygen species(ROS), including sin_ glet crxygen,rOr, and hydrogenperoxide, HzOz.Small mol_

524

c H A P T E R1 2

|

cELLULAR ENERGETTCS

ATP

(eg , too muchviapsll),LHCII isunbalanced becomes phosphorylated, drssociates fromPSll, anddiffuses intotheunstacked membranes, whereit associates with PSIanditspermanently associated LHCI. In thisalternative supramolecular (state organtzation 2),mostof the absorbed lightenergyistransferred to psl,supporting cyclic electron flowandATPproduction butno formation of NADPH andthusno CO2 fixatron. fromFA.Wollman, [Adapted 2OOj, EMBO J.20:3623.] ecule scavengersand antioxidant enzymeshelp to protect againstROS-induceddamage;however,singletoxygendamageto the D1 subunit of PSIIstill occurs,causingphotoinhibition. An HSP70 chaperone helps PSII recover from the damage. r In contrast to linear electron flow, which requires both PSII and PSI, cyclic electron flow in plants involves only PSI.In this pathwaS neither ner NADPH nor 02 is formed although a proton-motive force is generated. r Reversiblephosphorylation and dephosphorylationof the light-harvesting complex II control the functional organization of the photosynthetic apparatus in thylakoid membranes. State 1 favors linear electron flow, whereas state2 favors cyclic elecron flow (seeFigure 12-42).

CO2MetabolismDuring Photosynthesis Chloroplasts perform many metabolic reactions in green leaves.In addition to CO2 fixation-incorporation of gaseousCO2 into small organic moleculesand then sugars-the synrhesisof almost all amino acids. all fattv acids and carotenes, all pyrimidines, and probably ail

cH"-o- Po"H cHr-o-Po3H I O:C:O

c:o I

H-C-OH

o cHr-Oill

PO3H-

HrO

____!___>

o c-c-oH c:o

I H-C-OH

I c:o I oT

H-C-OH

H-C-OH

I cH2-o- Po3H

cH2-O-PO3H

-

I

c:o l

H-C-OH

I

cHr-o-Po3H

co'

l,'3i'.l,ii."on"r"

intermediate tnzvme'bound

ir-i:Tltl:$[:T""

a F I G U R E l 2 - 4T3h e i n i t i a rl e a c t i o n o rf u b i s c o t h a t f i x e s c o 2 1,5by ribulose catalyzed into organiccompounds.In thisreaction, (rubisco), with the CO2condenses carboxylase bisphosphate

ductsaretwo f i v e - c a r b o n s u g a r r i b u l o s e l , 5 - b i s p hTohs e pp h raot e of 3-phosphoglycerate molecules

purines occurs in chloroplasts.However, the synthesisof ,,rg"r, from CO2 is the most extensivelystudied biosynthetic p"Ih*"y in plani cells. !7e first considerthe unique pathway, known as the Calvin cycle (after discovererMeivin Calvini, that fixes CO2 into thr..-."rbon compounds, powered by energy ,el."rJ during ATP hydrolysis and oxidation of NADPH.

10 molecules(30 C atoms) are convertedto 6 moleculesof ribulose 1,5-bisphosphate(Figure 12-44, top) ' The fixation of six CO2 molecules and the net formation of two glyceraldehyde3-phosphatemoleculesrequire the consumption of 18 ATPs and1,2 NADPHs, generatedby the light-requiring of photosynthesis. processes

RubiscoFixesCO2in the ChloroplastStroma

Synthesisof SucroseUsing FixedCO2 ls Completedin the Cytosol

After its formation in the chloroplast stroma, glyceraldehyde The enzyme ribulose 1,5-bisphosphatecarboxylase, or rusubseare that precursor molecules into CO2 fixes bisco, quently convertedinto carbohydrates.Rubisco is locatedin the stromal spaceof the chloroplast.This enzymeadds CO2 to form to the five-carbonsugar ribulose 1,5-bisphosphate two moleculesof the three-carbon-containing3-phosphoglycerate (Figure 72-43). Rubisco is a large enzyme (:500 kDa) composed of eight identical large and eight identical small subunits.One subunit is encodedin chloroplastDNA; the other, in nuclear DNA. Becausethe catalytic rate of rubisco is quite low, many copies of the enzyme are neededtcl fix sufficient CO2. Indeed, this enzyme makes up almost 50 percentof the chloroplastprotein and is believedto be the most abundantprotein on earth. When photosynthetic algae are exposed to a brief pulse 1ac-labeledCO2 and the cellsare then quickly disrupted, of is radiolabeledmost rapidly, and all the 3-phosphoglycerate Liqht and Rubisco Activase Stimulate radioactivityis found in the carboxyl group. Because(iO:, rs C5, Lv f ixation initially incorporated into a three-carboncompound, the Calvin cycleis also calledthe C3 pathway of carbe'nfixatron (Figure 12-44). The fate of 3-phosphoglycerateformcd by rubisco is complex: some is converted to hexosesincorporated into starch or sucrose,but some is used to regenerateribulose protons are transportedfrom Calvin cycleenzymes..Because 1,5-bisphosphate.At least nine enzymesare required to photoelectrotl from 3-ph<-rspLoglycer- the stroma into the thvlakoid lumen during ribulose1,5-bisphosphate regenerare increascs stroma the transport (seeFigure 1)'37),the pL1of-fhe atl euantitatively fo, .u.ry 12 moleculer-o63-phosphoactivity =8 increased in the ligirt' fr.r,,, =7 in the ri"rrii t
D U R I N GP H O T O S Y N T H E S I S CO, METABOLISM

525

CO, O:C:O

o c-o-

cHroH I

c:o

I

<----ry 6ADP

co2 FtxATtoN (cALVtNCYCLEI

6 ATp___/f

ofififfi",":sc

\r

+ P <--{

cH2- oPo323-Phosphoglycerate

12ADP

i7

I

Ribulose 5-phosphate

c-oPo"2t-

1 2N A D P *

I

H-C-OH cH2-oPO32-

o

12NADPH

N-r .,0 Glyceraldehyde: '" 3C 3-phosphate

H-C-OH

I

1 2 1 , 3 - B i s p h o s p h o g l y c e r -a t 3 eC

enzymes

I

H-C-OH

12 ATP

H-C-OH

cH2-oPO32I

t^

cH2- oPo3'-

.'.' G l y c e r a l d e h y d-e r u 3-phosphate

c:o

,- G l y c e r a l d e h y d e 2 ^ 3-phosphate

I

I

H-C-OH I

2P,

cH2- oPo32Stroma

Phosphatetriosephosphate antiportprotein

c:o

1,3-Bisphosphoglycerate I H-C-OH I H H-C-OH I cH2-oPO32Ribulose 1,5-bisphosphate

Glyceraldehyde3-phosphate

Inner chloroplast membrane Cytosol 2P,

=3c ,3.',#::1f:lyd" ''

J 6c f3-Tl;?".ohat"= cH2-o-PO32I

J"

c:o

, f3-Tl;?",ohat": 6G

I

HO-C-H

I

,r-7

H-C-OH

I

H-C-OH

I

' fliil','o"h.,": u" I

cH2-o-Po32Fructose 1,6-bisphosphate

sucRor

.. f-'J,i315n..":

,| 1 5':;:oss;at" : 6c OH 2

OH

1 UDP-glucose

Glucose 1-phosphate

OH Fructose 6-phosphate

: 1r. ' 3-l"il:iin",u

t

'l'-> P' : 12C Sucrose

526

o

c H A p r E R1 2 |

C E L L U L AERN E R G E T t c s

OH

OH

Sucrose6-phosphate

and Mg2* concentrations' The activating reaction entails covalent addition of CO2 to the side-chainamino group of lysine in the active site, forming a carbamate group that then binds a Mg2* ion required for activity. Under normal conditions' however, with ambient levels of CO2, the reaction is slow and usually requires catalysis by rubisco actiuase,an enzyme that simultaneouslyhydrolyzesATP and usesthe energyto attach a an activatCO2 to the lysine.Rubiscoactivasealso accelerates (inactive-closed to rubisco in ing conformational change by activase rubisco of regulation The u.iiu.-op.n.d state). thioredoxin is, at leastin part in some species,responsiblefor rubisco'slight/redox sensitivity'Furthermore, rubisco activase's activity is sensitiveto the ratio of ATP:ADP. If that ratio is low (relatively high ADP), then the activase will not activate rubisco (and so the cell will expend less of its scarceATP to fix carbon).Giventhe key role of rubiscoin controlling energyutilization and carbon flux-both in an individual chloroplast and, in a sense,throughout the entire biosphere-it is not surprising that its activity is tightly regulated.

12-tt4The pathwayof carbonduring < FIGURE intotwo photosynthesis.(Top)Sixmolecules of C02areconverted which Thesereactions, 3-phosphate. of glyceraldehyde molecules Via occurin the stromaof thechloroplast theCalvincycle, constitute 3someglyceraldehyde antiporter, the phosphate/triosephosphate for phosphate phosphate to thecytosolin exchange istransported (Bottom)In the cytosol,an exergonic converts seriesof reactions Two 1,6-bisphosphate. to fructose glyceraldehyde 3-phosphate oneof areusedto synthesize 1,6-bisphosphate of f ructose molecules (not 3-phosphate sucroseSomeglyceraldehyde the disaccharide to aminoacidsandfats,compounds shownhere)isalsoconverted growth plant for essential A stromal protein called thioredoxin (Tx) also plays a role in controlling some Calvin cycle enzymes.In the dark, thioredoxin contains a disulfide bond; in the light, electrons are transferred from PSI, via ferredoxin, to thioredoxin, reducing its disulfide bond: PSI

z--\s

(Tx)l \__As

Which Competeswith Photorespiration, ls Reducedin PlantsThat Photosynthesis, Fix CO2by the C4PathwaY

sH SH

As noted above, rubisco catalyzesthe incorporation of CO2

Reducedthioredoxin then activatesseveralCalvin cycle enzymesby reducing disulfide bonds in them. In the dark, when thioredoxin becomesreoxidized,theseenzymesare reoxidized and so inactivated. Thus these enzymesare sensitiveto the redox stateof the stroma, which in turn is light sensitive-an elegantmechanismfor regulatingenzymaticactivity by light. Rubisco is one such light/redox-sensitiveenzyme,although its regulation is very complex and not yet fully understood. Rubisco is spontaneouslyactivatedin the presenceof high CO2

of the two-carbon compound phosphoglycolate' The first (carbon-fixing) reaction is favored when the ambient CO2 concentration is relatively high' whereasthe secondis favored when

o ---> ---> Sugars

-->

| H-C-OH

n u - n pv , n vr ,2

2 v3

o

3-Phosphoglycerate

CH,OH Glycolate 3-Phosphoglycerate

Phosphoglycolate

FfGURE12-45 C}2fixation and photorespiration.These 1,5-bisphosphate pathways by ribulose arebothinitiated competing C02 (rubisco), 1,5-bisphosphate. andbothutilizeribulose carboxylase pathwayIl, isfavored by highc02 andlow02 pressures; fixation, pathway[, occurs at lowc02 anorrgno, photorespiration, (thatis,undernormalatmospheric conditions) pressures

that take viaa complexsetof reactions is recycled Phosphoglycolate The as chloroplasts well as mitochondria, and placein feroxisomes by formed phosphoglycolate of molecules two for every result: net of 3-phosphoglycerate (fourc atoms), onemolecule photorespiration of c02 islost' andonemolecule andrecycled isultimatelyformed

c o 2 M E T A B O L I S MD U R I N GP H O T O S Y N T H E S I S

o

527

CO2 is low and 02 relatively high. The pathway initiated by the second reaction with 02 is called photorespiration-a processthat takes place in light, consumes02, and converts

react and form distincr products with the same initial enzyme/ribulose 1,5-bisphosphate intermediate. Excessivephororespiration could become a problem for plants in a hot, dry environment, becausethey must keep the gas-exchangepores (stomata) in their leavesclosed much of the time to prevent excessiveloss of moisture. As a consequence,the CO2 level inside the leaf can fall below the K- of

< FfGURE'12-46Leatanatomy of Coplantsand the Co pathway.(a)In C+plants, bundlesheath cellslinethevascular bundles containing thexylemandphloemMesophyll cells, whichareadjacent to the substomal airspaces, canassimilate CO, intofour-carbon molecules at low ambientC02anddeliver it to the interiorbundle sheathcells.Bundlesheathcellscontainabundant chloroplasts and arethesitesof photosynthesis andsucrose synthesis Sucrose is carried to the restof the plantviathe phloemIn C3plants, which lackbundlesheathcells,theCalvincycleoperates in the mesophyll cellsto fix COz(b)Thekeyenzyme in the Capathwayis phosphoenolpyruvate carboxylase, whichassimilates CO,to formoxaloacetatein mesophyll cellsDecarboxylation of malateor otherCo intermediates in bundlesheathcellsreleases COr,whichenters thestandard Calvincycle(seeFigure12-44,top)

V a s c u l a rb u n d l e ( x y l e m ,p h l o e m )

a' ra

o! e

Mesophyll cells I

rl

rubisco for CO2. Under theseconditions, the rate of photosynthesisis slowed, photorespiration is greatly favored, and the plant might be in danger of fixing inadequateamounts of CO2. Corn, sugarcane,crabgrass,and other plants that can grow in hot, dry environmentshave evolved a way to avoid this problem by utilizing a two-step pathway of CO2 fixation in which a CO2-hoarding stepprecedesthe Calvin cycle. The pathway has been named the Ca pathway because [14C]CO2 labeling showed that the first iadioactive moleculesformed during photosynthesisin this pathway are fourcarbon compounds, such as oxaloacetateand malate, rather than the three-carbonmoleculesthat initiate the Calvin cvcle (C3 pathway). The Ca pathway involves two types of cells: mesophyll cells, which are adjacent to the air spacesin the leaf interior, and bundle sheath cells, which surround the vascular tissue and are sequesteredaway from the high oxygen levelsto which mesophyllcellsare exposed(Figure 12-46a).In the mesophyll cellsof Ca plants,phosphoenolpyruvate, a three-carbonmolecule derived from pyruvate, reacts with CO2 to generate

Bundle sheath cells

n

Epidermis Chloroplast

Mesophyll cell

o

lreoen

f;

+H+

c-o

NADP+

I

l \ / 1 ---------\--z------

9H"

|

c

o co

.,

I CH,

9H,

1'

o

H-C-OH

c-o lltt

c_o-

H-C-OH

I

I

Og

co2

Malate

9Ht

il .- C |

^ O-PO"'--

Pyruvatephosphate dikinase

.-^t-----__---Z---| u -u

528

/ oYt Phosphoe?otpyruvate

\ olt

PP,

P,

CHAPTER 12

I

co, +

c"'y:l|

o

Oxaloacetate

co2+

Bundle sheath cell

CELLULAR ENERGETICS

nu ;"3 v-v

Malate

cH. I' c:o I

c_o

c-o

d

o

Pyruvate

Pyruvate

NADP-

N A D P H+ H -

in this regulation as does the regulation of the activity of oxaloacetate,a four-carbon compound (Figure 12-46b). The rubisco by rubisco activase. p h ate n, h o sp o enolpyruu this reactio that catalyzes enzyme plants and unin Ca is found almost exclusively carboxylase, r ln C3 plants, a substantial fraction of the COz fixed by Iike rubisco is insensitiveto 02. The overall reaction from the Calvin cycle can be lost as the result of photorespirapyruvate to oxaloacetateinvolves the hydrolysis of one ATP tion, a wasteful reaction catalyzed by rubisco that is faand has a negativeAG. Therefore,CO2 fixation will proceed vored at low CO2 and high 02levels (seeFigure 1'2-45). even when the CO2 concentration is low. The oxaloacetate r In Ca plants, CO2 is fixed initially in the outer mesophyll formed in mesophyll cells is reduced to malate, which is cells by reaction with phosphoenolpyruvate.The fourtransferred by a special transporter to the bundle sheath carbon moleculesso generatedare shuttled to the interior cells, where the CO2 releasedby decarboxylation entersthe bundle sheath cells, where the CO2 is releasedand then Calvin cycle (Figure12-46b). used in the Calvin cycle.The rate of photorespiration in Ca Becauseof the transport of CO2 from mesophyllcells, plants is much lower than in C3 plants' the CO2 concentration in the bundle sheathcellsof Ca plants is much higher than it is in the normal atmosphere.Bundle sheathcellsare also unusualin that they lack PSIIand carry out only cyclic electron flow catalyzedby PSI, so no ()2 is evolved.The high CO2 and reduced02 concentrationsin the Although the overall processesof photosynthesisand mitobundle sheath cells favor the fixation of CO2 by rubisco to chondrial oxidation are well understood, many important form 3-phosphoglycerateand inhibit the utilization of ribudetails remain to be uncoveredby a new generationof scienin photorespiration. lose 1,5-bisphosphate For example,little is known about how complexesI and In contrast, the high 02 concentrationin the atmosphere tists. IV in mitochondria couple proton and electronmovementsto favors photorespiration in the mesophyll cells of C3 plants createa proton-mottve force. SimilarlS although the binding(pathway2 in Figure 1,2-45\;as a result,as much as 50 percent changemechanismfor ATP synthesisby the F6F1complex is of the carbon fixed by rubiscomay be reoxidizedto CO2 in C-3 now generally accepted,we do not understand how conforplants. Ca plants are superior to C3 plants in utilizing the mational changesin each B subunit are coupled to the cycliavailableCO2, sincethe Ca enzymephosphoenolpyruvatecarcal binding of ADP and P;,formation of ATP, and then release boxylasehas a higher affinity for CO2 than doesrubisco in the of ATP. In addition, many questionsremain about the precise Calvin cycle.However, one ATP is consumedin the cyclic Ca mechanismof action of transport proteins in the inner mitoprocess (to generate phosphoenolpyruvatefrom pyruvate); chondrial and chloroplast membranesthat play key roles in thus the overall efficiencyof the photosyntheticproduction of oxidative phosphorylation and photosynthesis. sugarsfrom NADPH and ATP is lower than it is in C3 plants' \fe now know that releaseof cytochrome c and other which use only the Calvin cycle for CO2 fixation. Nonetheless,the net rates of photosynthesisfor Ca $rasses,such as corn or sugarcane,can be two to three times the ratesfor otherwise similar C3 grasses,such as wheat, rice, or oats, owing to the elimination of lossesfrom photorespiration. Of the two carbohydrate products of photosynthesis, connectionsbetween energy metabolism and mechanisms starch remains in the mesophyll cells of C3 plants and the bundle sheafcellsin Ca plants. In thesecells,starch is subjected to glycolysis, mainly in the dark, forming ATR NADH, and small moleculesthat are usedas building blocks for the synthesisof amino acids, lipids, and other cellular constituents.Sucrose,in contrast, is exported from the photosynthetic cells and transported throughout the plant.

CO2Metabolism During Photosynthesis r In the Calvin cycle,CO2 is fixed into organic moleculesin a seriesof reactionsthat occur in the chloroplaststroma.The initial reaction, catalyzedby rubisco, forms a three-carbon intermediate. Some of the glyceraldehyde3-phosphate generatedin the cycle is transported to the cytosol and converted to sucrose(seeFigure 72-44). r The light-dependent activation of several Calvin cycle enzymesand other mechanismsincreasesfixation of COz in the light. The redox state of the stroma plays a key role

and inexpensivefood to all who need it.

KeyTerms aerobic oxidation 479 ATP synthase504

chemiosmosis480 chlorophylls511

Calvin cycle525 carbon fixation 511 cellularrespiration485

chloroplasts479 citric acid cycle479

Ca pathway 528

cytochromes495 electron carriers487 K E YT E R M S

529

electron transport chain 481 endosymbiont hypothesis485 fermentation 485 FeFl complex 505 glycolysis481 mitochondria 479 oxidative phosphorylation 481 photoelectron transport 51 5 photorespiration 528

photosynthesis479 photosystems514 proton-motiv e force 48 0 Q cycle500 reactive oxygen species502 respiratory control 51 0 rubisco 525 substrate-level phosphorylation 481 thylakoids511 uncouplers5-10

Reviewthe Concepts 1. The proton-motive force (pmf) is essentialfor both mitochondrial and chloroplast function. \fhat produces the pmf, and what is its relationship to ATp? 2. The mitochondrial inner membrane exhibits all of the fundamental characteristicsof a typical cell membrane, but it also has severalunique characteristicsthat are closely associatedwith its role in oxidative phosphorylation. What are these unique characteristics?How does each contribute to the function of the inner membrane? 3. Maximal production of ATP from glucose involves the reactions of glycolysis,the citric acid cycle, and the electron transport chain. \fhich of these reactions requires 02, and why? \Which, in certain organisms or physiological conditions, can proceed in the absenceof 02? 4. Describe how the elecrronsproduced by glycolysis are deliveredto the electron transport chain. What would be the consequencefor overall ATp yield per glucosemolecule if a mutation inactivated this delivery system?rVhat would be the,longer-termconsequencefor the activity of the glycolytic pathway? 5. Mitochondrial oxidation of fatty acids is a major source of ATP, yet fatty acids can be oxidized elsewhere.Sfhat organelle, besidesthe mitochondrion, can oxidize fatty acids? \What is the fundamental difference between oxidaiion occurring in this organelleand mitochondrial oxidation? 6. Each of the cytochromes in the mitochondria contains prosthetic groups. \fhat is a prosthetic group? Vhich type of prosthetic group is associatedwith the cytochromes? 'What property of the various cytochromesensuresunidirectional electron flow along the electron transport chain? 7. It is estimatedthat eachelecron pair donated bv NADH leadsto the synthesisof approximateiy three AIp molecules, whereas each electron pair donated by FADH2 leads to the synthesisof approximately two ATp molecules.\7hat is the underlying reason for the difference in yield for electrons donated by FADH2 versusNADH? 8. Much of our understanding of ATp synthaseis derived from researchon aerobic bacteria.\fhat makes theseorgan_ isms useful for this research?Where do the reactions of gly_ colysis,the citric acid cycle, and the electron t.urrrpo.t .li"i., 530

.

cHAprE1 R2 | C E L L U L E AN RE R G E T t c s

occur in theseorganisms?Where is the pmf generatedin aerobic bacteria?rVhat other cellular processesdepend on the pmf in theseorganisms? 9. An important function of the mitochondrial inner membrane is to provide a selectivelypermeable barrier to the movement of water-solublemoleculesand thus generatedifferent chemical environments on either side of the membrane. However, many of the substratesand products of oxidativephosphorylarionare water solubleunJ rnurt crossthe inner membrane. How does this transport occur? 10. The Q cycle plays a major role in the elecrron ffansport chain of mitochondria, chloroplasts, and bacteria. What is the function of the Q cycle, and how does it carry out this function? Sfhat electron transport componentsparticipate in the e cycle in mitochondria, in purple bacteria, and in chloroplasts? 11. \frite the overall reaction of oxygen-generatingphotosynthesis.Explain the following statement:the 02 generated by photosynthesisis simply a by-product of the pathway's generationof carbohydratesand ATP. 12. Photosynthesiscan be divided into multiple stages. 'What are the stagesof photosynthesis,and where does each occur within the chloroplast?Vhere is the sucroseproduced by photosynthesisgenerated? 13. The photosystemsresponsiblefor absorption of light energy are composed of two linked components, the reaction center and an antenna complex. What is the pigment composition and role of each in the processof light absorption? IThat evidenceexists that the pigments found in thesecomponents are involved in photosynthesis? 14. Photosynthesisin green and purple bacteria does not produce 02. 'Why?How can theseorganismsstill usephoto'S7hat synthesisto produce ATP? moleculesserveas electron donors in theseorganisms? 15. Chloroplasts conrain two photosystems.Sfhat is the function of each?For linear electron flow, diagram the flow of electrons from photon absorption to NADPH formation. Ifhat does the energy stored in the form of NADPH synthesize? 16. The Calvin cycle reactionsthar fix CO2 do not function in the dark. What are the likely reasonsfor this? How are thesereactionsregulated by light? 17. Rubisco,which may be the most abundant protein on earth, plays a key role in the synthesisof carbohydrates in organismsthat usephotosynthesis.What is rubisco. where is it located, and what function does it serve?

Analyzethe Data A proton gradient can be analyzed with fluorescent dyes whose emission-intensityprofiles depend on pH. One of the most useful dyes for measuring the pH gradient acrossmltochondrial membranes is the membrane-impermeant, water-solublefluorophore 2',7' -bis-(2-carboxyethyl)-5(6)carboxyfluorescein(BCECF). The effect of pH on the emission intensity of BCECE,excited at 505 nm. is shown in the accompanyingfigure. In one studg sealedvesiclescontaining this compound were prepared by mixing unsealed,isolated

inner mitochondrial membraneswith BCECF; after resealing of the membranes,the vesicleswere collected by centrifugation and then resuspendedin nonfluorescentmedium. a. When thesevesicleswere incubatedin a physiological buffer containingNADH, ADR Pi, and 02, the fluorescenceof BCECF trapped insidegradually decreasedin intensity. What does this decreasein fluorescentintensity suggestabout this vesicularpreparation? b. How would you expect the concentrationsof ADP, P1,and 02 to changeduring the courseof the experiment describedin oart a? Whv?

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c. After the vesicleswere incubated in buffer containing ADP, P;, and 02 for a period of time, addition of dinitrophenol caused an increasein BCECF fluorescence.In contrast, addition of valinomycin produced only a small transient effect. Explain thesefindings. d. Predict the outcome of an experiment performed as describedin part a if brown-fat tissuewas used as a source of unsealed,isolated inner mitochondrial membranes. Explain your answer.

References First Steps of Glucose and Fatty Acid Catabolism: Glycolysis and the Citric Add Cycle Berg,J., J. Tymoczko, and L. Stryer.2002. Biochemistry,Sth ed. 'W. H. Freemanand Company,chaps.16 and 1,7. Canfield,D. E. 2005. The early history of atmosphericoxygen: homageto Robert M. Garrels.Ann. Reu.Earth Planet.Sci.33:1.-36. Chan, D.C. 2005. Mitochondria: dynamic organellesin disease, 52. aging, and development.Cell 125(7l:'1.241-1.2 Depre, C., M. Rider, and L. Hue. 1998. Mechanismsof control of heart glycolysis.Eur. J. Biochem.258:277:290. Eaton, S., K. Bartlett, and M. Pourfarzam.1.996.Mammalian mitochondrial beta-oxidation. Biochem./. 320( Part 2\:345-S57. Fersht,A. 1999. Structureand Mechanismin Protein Science:A Guide to Enzyme Catalysisand Protein Folding. W. H. Freemanand Comoanv.

Fothergill-Gilmore,L. A., and P. A. Michels. 1993. Evolution of glycolysis.Prog. Biophys. Mol. Biol. 59:105-135. Guest,J. R., and G. C. Russell.1'992.Complexesand complexities of the citric acid cycle in Escherichia coli. Curr. Top. Cell Reg. t t

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Energy-Requiring Processes Aksimentiev, A., L A. Balabin.R. I{. Fillingame.and K. Schulten.2004. Insightsinro the molecularmelhanisrnof rotation in the F6 sectorof ATP synthase.Bxtphys.J.86(3)t1332-1344. Biancher,M. A., J. Hullihen,P. Pedersen, and M. Arnzel.1998 The 2.8 A structureof rat liver F1-ATPase: confieurationof a critical irrtermediate in ATP synrhesis/hydrolysis. Proc. Nat'1.Acad. Sci. U S A9 . 5 : 1 1 0 6 5 -110 7 0 . Bover,P.D.1997. The AT'Psynrhase-asplendidrrolecularmachine.Ann. Reu.Biochem.66:7L7-749. Capaldi,R., and R. Aggeler.2002. Mechanismof the FeF,-typeATp synthase-a biologicalrotary motor.TrendsBiochem.Sci.27:1 54-160. Elsron,T., lL Wang,and G. Osrer.1998.Energytransductionin ATP synthase. Nature 391:-510-512. Hinkle, P. C. 2005. P/O ratiosof mitochondrialoxidativephosphorylation. Biochim. Biophys.Acta. 1706(1-2):1,-11. Kinosita,K., et al. 1998.Fr-ATPase: a rotary motor madeof a s i n g l em o l e c u l eC. e l l 9 3 : 2 1 - 2 4 . Klingenberg,M., and S. Huang. 1999.Structureand functionof the uncoupling protein from brown adipose rissue.Biochim. Bktp h y s .A c t a l 4 l 5 : 2 7l - 2 9 a . Nury, H., et al.. 2006. Relationsberweenstructureand function of the mitochondrialADP/ATP carrter.Ann. Reu.Biocbem.T5:713-741. Tsunoda,S.,R. Aggeler,M. Yoshrda,and R. Capaldi.2001. Rotationof the c subunitoligomerin fully functirtnil FoF1ATp synthase.Proc. Nat'1.Acad. Sci.USA 98:898-902. Vercesi,A. 8., et al. 2006. Plant uncoupling mitochondrial proteins.Ann. Reu.Plant Biol. 57:383404. Yasuda,R., et al. 2001. Resolutionof distinctrotarionalsubstens by submillisecond kineticanalysisof F1-ATPase. Nature 4lO:898-904.

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MolecularAnalysisof Photosystems Allen, J. F. 2002. Photosynthesisof A fp-electrons! proron pumps, rotors, and poise.Cell 110:273-276. _ Aro, E. M., I. Virgin, and B. Andersson.1993.photoinhibirion ot pbotosystemII: Ltactivation, protein damage,and turnover. Biochim.Biophys.Acta Lt43:1|3-1 34.

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CHAPTER 12

I

CELLULAR ENERGETICS

Deisenhofer,J., and H. Michel. 1989. The photosyntheticreaction centerfrom the purple bacteriumRbodopseudomonasuiridis. Science245t1463-1473. (Nobel Prize Lecture.) Deisenhofer,I., and H. Michel. 1991. Structuresof bacterial photosynthetic reactioncenters.Ann. Reu.Cell Biol.7:1-23. Dekker,J. P., and E. J. Boekema.2005. Supramolecularorganizarion of thylakoid membraneproteins in green plants.Biochim. -21:1,2-39. B iophys. A cta. 1706('1. Finazzi,G. 2005. The centralrole of the greenalga Chlamydomondsreinhardtii in revealingthe mechanismof statetransitions J. Exp. Bot. 56(411):383-388. Haldrup, A., P.Jensen,C. Lunde, and H. Schelle r. 2001..Balance of power: a view of the mechanismof photosyntheticstaterransitions. TrendsPlant Sci.6:301-30.5. Hankamer,B., J. Barber,and E. Boekema.1.997.Structureand membraneorganizationof photosystemII from green plants.Ann. Reu.Plant Physiol. Plant Mol. Biol.48:641-672. Heathcote,P.,P. Fyfe, and M. Jones.2002. Reactioncentres:the structureand evolution of biological solar power. TrendsBiochem. Sci.27:79-87. Horton, P.,A. Ruban, and R. Walters.1,996.Regulationof light harvestingin green plants.Ann. Reu.Plant Physiol. Plant Mol. Biol. 47:655-684. Joliot, P.,and A. Joliot. 2005. Quantificarion of cyclic and linear flows in plants.Proc. Natl. Acad. Sci.USA 102(131:49134918. Jordan, P.,et al. 2001 Three-dimensionalstructureof cyanobacterialph()tosystem I at 2.5 A resolution.Nature 411:909-917. KUhlbrandr,\7. 2001. Chlorophyllsgalore.Nature 411:896-898. Martin, J. L., and M. H. Vos. 1992. Femtosecondbiology.Ann. Reu.Biophys. Biomol. Struc.2l:'1,99-222. Penner-Hahn,J. 1998. Structural characterizationof the Mn site in the photosynthericoxygen-evolvingcomplex. Struc. Bonding 90:'1,-36. Tommos, C., and G. Babcock.1998. Oxygen production in nature: a light-drivenmetalloradicalenzymeprocess.Acc. Chem. Res. 3l:18-25.

CO2Metabolism DuringPhotosynthesis Buchanan,B. B. 1991,.Regulationof CO2 assimilationin oxygenic photosynthesis:the ferredoxin/thioredoxinsystem.Perspective on its discovery,presentstatus,and future development.Arch. Biochem. Biophys.288: | -9. Gutteridge,S., and J. Pierce.2006. A unified rheory for the basis of the limitations of the primary reacrionof photosyntheticCO2 fixation: was Dr. Panp;loss right?Proc. Natl. Acad. Sci.USA 103:7203-7204. Portis,A. 1992. Regulationof ribulose 1,S-bisphosphate car' boxylase/oxygenase activity.Ann. Reu.Plant Pbysiol.Plant Mol. Biol.43:415437. Rawsthorne,5.1992. Towardsan understanding of C3-Caphotosynthesis. Essays Biocbem.27:135-146. Rokka, A.,I. Zhang, and E.-M. Aro. 2001. Rubrscoactrvase: an enzymewith a remperature-dependent dual function? Plant l. 25:463-472. Sage,R., and J. Colemana.2001,. Effectsof low atmosphericCO,on plants:more than a thing of the past.TrendsPlant Sci.Z:18-24. Schneider,G., Y. Lindqvisr, and C. I. Branden.1992. Rubisco: structureand mechanism.Ann. Reu.Biophys. Biomol. Struc. 2l:119-153. Tcherkez.,G. G., G. D. Farquhar,and T. J. Andrews. 2006.Despite slow catalysisand confusedsubstrarespecificity,all ribulose bisphosphatecarboxylasesmay be nearly perfectly optimized..proc. Natl. Acad. Sci.USA 103(19):7246-7251. 'Wolosiuk, R. A., M. A. Ballicora,and K. Hagelin.1993.The reductivepentosephosphatecycle for photosyr-rthetic CO2 assimilation: enzymemodulation.FASEB[. 7:622-637.

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SECRETORY PATHWAY FIGURE 13-1 Overviewof major protein-sorting pathwaysin eukaryotes. All nuclear-encoded mRNAs are translated on cytosolic ribosomesRight(nonsecretory pathways). Synthesis of proteins lacking an ERsignalsequence iscompleted (step[) Thoseproteins on freeribosomes thatcontainno targeting sequence arereleased intothe cytosol andrematn tnere (stepE). Proteins with an organelle-specific targeting sequence (pink)firstarereleased (stepZ) butthenare intothecytosol imported intomitochondria, chloroplasts, peroxisomes, or the (stepsB-E) Mitochondrial nucleus andchloroplast proteins passthroughtheouterandinnermembranes typically to enter

the matrixor stromal space, respectively Otherproteins aresortedto othersubcompartments of theseorganelles by additional sorting proteins stepsNuclear enterandexitthroughvisible poresin the nuclearenvelopeLeft(secretory pathway):Ribosomes synthesizing proteins nascent pathwayaredirected in the secretory to the rough (ER) endoplasmic (pink;steps[, reticulum byan ERsignalsequence iscompleted on the ER,theseproteins Z) Aftertranslation can moveviatransport (stepB) Further vesicles to the Golgicomplex proteins sortingdelivers eitherto the plasma membrane or to (stepsEE, @ ) Theprocesses lysosomes underlying the secretory pathway(stepsB, El, shadedbox)arediscussed in Chapter'l4

well as in the plasma membrane. Targeting to the ER generally involves nascent proteins still in the process of being synthesized.Once translocatedacross the ER membrane, proteins are assembledinto their native conformation by protein-folding catalystspresenrin the lumen of the ER. This process is monitored carefullS and only after their folding

and assemblyis complete are proteins permitted to be transported out of the ER to other organelles.Proteins are also modified in various ways after translocation into the ER. These modifications can include addition of carbohydrate groups, stabilization of protein structure through disulfide bond formation, and specificproteolytic cleavages.Proteins

534

CHAPTER 13

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M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

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l N V U S l A l t A U l l H t S S O U ) VS N E I O U d , l . U O l _ : l U ) tJSO N O t J _ V ) O ' l S N V U l

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SRP

Signal sequence

SRPreceptor

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Translocon (closed)

Translocon (open)

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FIGURE 13-6 Cotranslational translocation. polypeptide Steps[, [: chainelongates, it chainStepE: Asthe polypeptide Oncethe ERsignalsequence emerges fromthe ribosome, passes it isbound intothe ERlumen,where throughthetranslocon channel (SRP). by a signal-recognition particle StepS: TheSRpdelivers the thesignalsequence iscleaved by signalpeptidase andisrapidly ribosome/nascent polypeptide complex to the SRpreceptor in the ER degradedStep6: Thepeptide chaincontinues to elongate as membrane. Thisinteraction isstrengthened by bindingof GTpto the mRNAistranslated towardthe 3' end Because the ribosome boththe SRP anditsreceptor. Step4: Transfer of the ribosome/nascent isattached to thetranslocon, thegrowingchainisextruded polypeptide to thetranslocon leadsto openingof thistranslocation throughthetranslocon intothe ERlumenStepsfl, S: Once channel andinsertion of the signalsequence andadjacent segment iscomplete, is released, translation the ribosome the remainder of the growingpolypeptide intothe centralpore Boththe SRpand of the proteinisdrawnintothe ERlumen,thetranslocon closes, SRPreceptor, oncedissociated fromthetranslocon, hydrolyze their andthe oroteinassumes itsnativefoldedconformation. boundGTPandthenarereadvto initiate theinsertion of another

SRP releasethe nascent chain, allowing elongation to continue at the normal rate. Thus the SRPand SRPreceptor not only help mediate interaction of a nascentsecretoryprotein with the ER membrane but also act together to permit elongation and synthesis of complete proteins only when ER membranesare present,

Passageof Growing PolypeptidesThrough the Transloconls Driven by EnergyReleased D u r i n gT r a n s l a t i o n Once the SRP and its receptor have targeted a ribosome synthesizinga secretoryprotein to the ER membrane, the ribosome and nascentchain are rapidly transferredto the translocon, a protein-lined channel within the membrane. As translation continues, the elongating chain passesdi-

rectly from the large ribosomal subunit into the central pore of the translocon. The 50S ribosomal subunit is aligned with the pore of the translocon in such a way that the growing chain is never exposed to the cytoplasm and is prevented from folding until it reaches the ER lumen ( F i g u r e1 3 - 5 ) . The translocon was first identified by mutations in the yeast gene encoding Sec61o,which causeda block in the translocation of secretoryproteins into the lumen of the ER. SubsequentlSthree proteins called the Sec61complex were found to form the mammalian translocon: Sec51cr, an integral membrane protein with 10 membrane-spanning ct helices,and two smaller proteins, termed Sec61B and Sec51"y.Chemical cross-linking experiments demonstrated that the translocating polypeptide chain comes into contact with the Sec61a protein in both yeast and

T R A N S L O C A T I OO NF S E C R E T O RPYR O T E I N A S C R O s sT H E E R M E M B R A N E

539

ArtificialmRNA

Cytosol

Sec61o Microsomal membrane

Crosslinking agent Nascent protein

Microsomal lumen

NH:

EXPERIMENTAL FIGURE 13-7 Sec61cis a translocon component.Cross-linking experiments showthatSec6lcrisa proteinsas translocon component that contactsnascentsecretory theypassintothe ERlumen.An mRNAencoding the N-terminal wastranslated in 70 aminoacidsof the secreted oroteinorolactin (seeFigure13-4b). The a cell-free system containing microsomes mRNAlackeda chain-termination onelysine codonandcontained a codon,nearthe middleof the sequence. Thereactions contained chemically modifiedlysyl-tRNA in whicha light-activated crosslinkingreagent wasattached to the lysine sidechainAlthoughthe polypeptide entiremRNAwastranslated, couldnot the completed fromthe ribosome be released withouta chain-termination codon andthusbecame"stuck"crossing the ERmembrane. Thereaction mixtures thenwereexposed to an intense light,causing the nascent proteins chainto become werenear covalently boundto whatever it in thetranslocon. Whentheexperiment wasperformed using microsomes frommammalian cells,the nascent chainbecame linkedto Sec61oDifferent covalently versions of the prolactin mRNA werecreated sothatthe modified lysine residue wouldbe placedat different distances fromthe ribosome; to Sec61c was cross-linking observed onlywhenthe modifiedlysine waspositioned withinthe translocation channel[Adapted 1992,Science fromT.A Rapoport, 258:931, andD Gorlich andT.A Rapoport, 1993, Cell75:615]1

mammalian cells, confirming its identity as a translocon component (Figure 1 3-7). lfhen microsomes in the cell-free translocation system were replaced with reconstituted phospholipid vesiclescontaining only the SRP receptor and Sec61complex, nascent secretory protein was translocated from its SRP/ribosome complex into the vesicles.This finding indicates that the SRP receptor and the Sec61 complex are the only ERmembrane proteins absolutely required for translocation. Becauseneither of these can hydrolyze ATP or otherwise provide energyto drive the translocation, the energyderived from chain elongation at the ribosome appears to be sufficient to push the polypeptide chain acrossthe membrane in one direction.

540

CHAPTER 13

I

The translocon must be able to allow passageof a wide variety of polypeptide sequenceswhile remaining sealedto small moleculessuch as ATP and amino acids. Furthermore, to maintain the permeability barrier of the ER membrane in the absenceof a translocating polypeptide, there must be some way to regulate the translocon so that it is closed in its default state, opening only when a ribosome-nascentchain complex is bound. A high-resolution structure of the Sec61 complex from the archaebacteriumMethanococcusiannaschii was recently determined by x-ray crystallograph5 which suggestshow the translocon preservesthe integrity of the membrane(Figure13-8).The 10 transmembranehelices of Sec6lcr form a central channel through which the translocating peptide chain passes.A constriction in the middle of the central pore is lined with hydrophobic isoleucine residues that may form a gasket around the translocating peptide. In addition, the structural model of the Sec61 complex (which was isolated without a translocating peptide and therefore is presumed to be in a closed conformation) reveals a short helical peptide plugging the central channel. Biochemical studies of the Sec51complex have shown that the peptide that forms the plug undergoes a significant conformational change during active translocation, and researchersthink that once a translocating peptide enters the channel, the plug peptide swings away to allow translocation to proceed. As the growing polypeptide chain entersthe lumen of the ER, the signal sequenceis cleavedby signal peptidase,which is a transmembraneER protein associatedwith the translocon (seeFigure 13-6). Signalpeptidaserecognizesa sequence on the C-terminal side of the hydrophobic core of the signal peptide and cleavesthe chain specifically at this sequence once it has emergedinto the luminal spaceof the ER. After the signal sequencehas been cleaved,the growing polypeptide moves through the translocon into the ER lumen. The translocon remains open until translation is completed and the entire polypeptide chain has moved into the ER lumen. Electron microscopy of the Sec61complex isolated from the ER of eukaryotic cells reveals that three or four copies of Sec51acoassemblein the plane of the membrane.The functional significanceof this association between translocon channels is not understood at this time. but the oligomerization of translocon channels may facilitate the association between the translocon, signal peptidase, and other luminal protein complexes that participate in the translocation process.

ATPHydrolysisPowersPost-translational Translocationof SomeSecretoryProteins in Yeast In most eukaryotes,secretoryproteins enter the ER by cotranslationaltranslocation.In yeast,however,some secretory proteins enter the ER lumen after translation has been completed. In such post-translational translocation, the translocating protein passesthrough the sameSec61translocon that is used in cotranslational translocation. However, the SRP

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

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(b) Top view

P o r er i n g

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EXPERIMENTAL FIGURE 13-8 Structureof a bacterialSec61 complex.Thestructure of thedetergent-solubilized Sec61 complex fromthearchaebacterium (alsoknownasthe Secy M jannaschl complex) wasdetermined (a)A sideview byx-raycrystallography showsthe hourglass-shaped channel throughthe centerof the pore A ringof isoleucine residues at theconstricted waistof the poremay forma gasketthat keepsthe channel sealed to smallmolecules even polypeptide asa translocating passes throughthechannelWhenno peptideispresent, translocating thechannel isclosedby a short plug,Thisplugisthoughtto moveout of thechannel helical during translocation Inthisviewthefronthalfof proteinhasbeenremoved to bettershowthe pore.(b)A viewlookingthroughthe centerof the channel showsa region(onthe leftside)wherehelices mayseparate allowinglateralpassage of a hydrophobic transmembrane domain intothe lipidbilayer. fromA R Osborne etal, 2005.Ann Rev. [Adapted CellDev.Biology 21:529l

and SRP receptor are not involved in post-translational translocation, and in such casesa direct interaction between the translocon and the signal sequenceof the completedprotein appears to be sufficient for targeting to the ER membrane. In addition, the driving force for unidirectional translocation acrossthe ER membraneis provided by an additional protein complex known as rhe Se;63 complex and a member of the Hsc70 family of molecular chaperonesknown as BiP.The tetrameric Sec63complex is embeddedin the ER membrane in the vicinity of the translocon. whereas BiP is

within the ER lumen. Like other members of the Hsc70 famlIy, BiP has a peptide-binding domain and an ATPasedomain. These chaperonesbind and stabilize unfolded or partially folded proteins(seeFigure3-15). The current model for post-translationaltranslocation of a protein into the ER is outlined in Figure 13-9. Once the Nterminal segmentof the protein enters the ER lumen, signal peptidase cleavesthe signal sequencejust as in cotranslational translocation (step [). Interaction of BiP.ATP with the luminal portion of the Sec53complex causeshydrolysis of the bound ATR producing a conformational change in BiP that promotes its binding to an exposed polypeptide chain (step [). Sincethe Sec63complex is located near the translocon, BiP is thus activated at sites where nascent polypeptidescan enter the ER. Certain experimentssuggest that in the absenceof binding to BiP, an unfolded polypeptide slides back and forth within the translocon channel. Such random sliding motions rarely result in the entire polypeptide'scrossingthe ER membrane. Binding of a molecule of BiP.ADP to the luminal portion of the polypeptide prevents backsliding of the polypeptide out of the ER. As further inward random sliding exposesmore of the polypeptide on the luminal side of the ER membrane. successive binding of BiP.ADP moleculesto the polypeptide chain acts as a ratchet, ultimately drawing the entire polypeptide into the ER within a few seconds(stepsB and 4). O" a slower time scale,the BiP molecules spontaneouslyexchangetheir bound ADP for ATP, leading to releaseof the polypeptide, which can then fold into its native conformation (steps E and 6). The recycled BiP.ATP then is ready for another interaction with Sec53.BiP and the Sec53complex are also required for cotranslational translocation. The details of their role in this process are not well understood, but they are thought to act at an early stageof the processsuch as threading the signal peptide into the pore of the translocon. The overall reaction carried out by BiP is an important example of how the chemical energy releasedby the hydrolysis of ATP can power the mechanical movement of a protein across a membrane. Bacterial cells also use an ATPdriven process for translocating completed proteins across the plasma membrane. In bacteria the driving force for translocation comes from a cytosolic ATPaseknown as the SecA protein. SecA binds to the cytoplasmic side of the translocon and hydrolyzes cytosolic AIP. By a mechanism that is not well understood, the SecA protein pushes segments of the polypeptide through the membrane in a mechanical cvcle coupled to the hvdrolvsis of ATP.

Translocation of Secretory Proteins Across t h e E RM e m b r a n e r Synthesisof secretedproteins, integral plasma-membrane proteins, and proteins destinedfor the ER, Golgi complex, or lysosome begins on cytosolic ribosomes,which become attachedto the membraneof the ER, forming the rough ER (seeFigure 13-1, left).

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541

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Translocon

ER lumen NHs* Cleaved s i gn a l sequenc

BiP (bound to ATP)

FIGURE 13-9 Post-translational Thismechanism translocation. isfairlycommonin yeastandprobably in higher occurs occasionally random eukaryotes Smallarrowsinside thetranslocon represent polypeptide slidingof thetranslocating inwardandoutwardSuccessive prevents bindingof BiP.ADP to entering segments of the polypeptide the chainfromslidingout towardthecytosol[See K E Matlack etal, 1997 277:938 . Science I

r The ER signal sequenceon a nascent secretory protein consistsof a segmentof hydrophobic amino acids, generally locatedat the N-terminus. r In cotranslational translocation, the signal-recognition particle (SRP) first recognizesand binds the ER signal sequenceon a nascent secretoryprotein and in turn is bound by an SRP receptor on the ER membrane, thereby targeting the ribosome/nascentchain complex to the ER. r The SRP and SRP receptor then mediate insertion of the nascent secretoryprotein into the translocon (Sec61complex). Hydrolysis of two moleculesof GTP by the SRP and its receptor drive this docking processand causethe dissociation of SRP(seeFigures13-5 and 13-6).As the ribosome attached to the translocon continues translation, the unfolded protein chain is extruded into the ER lumen. No additional energy is required for translocation. r The translocon contains a central channel lined with hydrophobic residues that allows transit of an unfolded protein chain while remaining sealedto ions and small hydrophilic molecules.In addition, the channel is gated so that it only is open when a polypeptide is being translocated.

542

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r In post-translational translocation, a completed secretory protein is targetedto the ER membrane by interaction of the signal sequencewith the translocon. The polypeptide chain is then pulled into the ER by a ratcheting mechanism that requires ATP hydrolysis by the chaperone BiP, which stabilizesthe enteringpolypeptide (seeFigure 13-9). In bacteria, the driving force for post-translational translocation comes from SecA,a cytosolic ATPasethat pushespolypeptides through the translocon channel. r In both cotranslational and post-translationaltranslocation, a signal peptidasein the ER membranecleavesthe ER signal sequencefrom a secretoryprotein soon after the Nterminus entersthe lumen.

lnsertionof Proteinsinto the ERMembrane In previous chapters we have encountered many of the vast array of integral (transmembrane)proteins that are present throughout the cell. Each such protein has a unique orientation with respectto the membrane'sphospholipid bilayer.

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Integral membrane proteins located in the ER, Golgi, and lysosomes and also proteins in the plasma membrane, which are all synthesizedon the rough ER, remain embedded in the membrane in their unique orientation as they move to their final destinationsalong the same pathway followed by soluble secretory proteins (see Figure 13-1, left).During this transport, the orientation of a membrane protein is preserved;that is, the samesegmentsof the protein always face the cytosol, whereas other segments always face in the opposite direction. Thus the final orientation of these membrane proteins is establishedduring their biosynthesison the ER membrane.In this secrion,we first seehow integral proteins can interact with membranes and then examine how severaltypes of sequences,collectively known as topogenic sequences,direct the membrane insertion and orientation of various classesof integral proteins. Theseprocessesoccur via modifications of the basic mechanism used to translocatesoluble secretoryproteins acrossthe ER membrane.

SeveralTopologicalClassesof Integral MembraneProteinsAre Synthesized on the ER The topology of a membraneprotein refers to the number of times that its polypeptide chain spansthe membrane and the orientation of these membrane-spanning segmentswithin the membrane.The key elementsof a protein that determine

coo

Cytosol

its topology are membrane-spanningsegmentsthemselves, which usually are ct-helicescontaining 20-25 hydrophobic amino acids that contribute to energeticallyfavorable interactions within the hydrophobic interior of the phospholipid bilayer. Most integral membrane proteins fall into one of the four topological classesillustrated in Figure 13-10. Topological classesI, II, and III comprise single-passproteins, which have only one membrane-spanninga-helical segment. Type I proteins have a cleaved N-terminal ER signal sequenceand are anchored in the membrane with their hydrophilic N-terminal region on the luminal face (also known as the exoplasmic face) and their hydrophilic Cterminal region on the cytosolic face. Type II proteins do not contain a cleavableER signal sequenceand are oriented with their hydrophilic N-terminal region on the cytosolic face and their hydrophilic C-terminal region on the exoplasmic face (i.e., opposite to type I proteins). Type III proteins have the same orientation as type I proteins but do not contain a cleavablesignal sequence.These different topologies reflect distinct mechanismsused by the cell to establish the membrane orientation of transmembrane segments,as discussedin the next section. The proteins forming topological classIV contain two or more membrane-spanning segments and are sometimes called multipass proteins. For example, many of the membrane transport proteins discussedin Chapter 11 and the numerous G protein-coupled receptors covered in Chapter 15 belong to this class. A final type of membrane protein

NHst

ii,,ltla itirir Exoplasmic space (ERor Golgi lumen; cell exterior)

i Cleaved s i gn aI sequence

NHs'

Type I LDL receptor InfluenzaHA orotein Insulinreceoto'. Growth hormone receptor

Type ll Asialoglycoprotein receptor

Type lll

Type lV

Cytochromep450

Transferrinreceptor Golgi galactosyltransferase Golgi sialyltransferase

A FIGURE 13-10ERmembraneproteins.Fourtopological classes proteins of integral membrane aresynthesized on the roughERas wellasa fifthtypetethered to the membrane by a phospholipid proteins anchor. Membrane areclassified bytheirorientation in the membrane andthetypesof signals theycontainto directthemthere In classes l-lv the hydrophobic segments of the proteinchainformo helices embedded in the membrane bilayer; the regions outside the membrane arehvdrophilic andfold intovarious conformations. All

G protein-coupledreceptors Glucosetransporters Voltage-gatedCa2+channels A B C s m a l l m o l e c u l ep u m p s CFTR(Cl-) channel

N Hg*

GPI-linkedprotein Plasminogen activator receptor Fasciclinll

Sec61 proteins a helicesThetypelV havemultiple transmembrane typelV to thatof G protein-coupled herecorresponds topologydepicted on theexoplasmic sideof receptors: the N-terminus seveno helices, side.Othertype on the cytosolic the membrane, andthe C-terminus numberof helices andvarious lV proteins mayhavea different E Hartmann etal, andC-terminus. orientations of the N-terminus [See andS,D,Black, andC.A Brown 1989, ProcNat'lAcad.SciUSA86:5786, 1989,JBiol Chem254:44421 I N S E R T I OO N F P R O T E I NISN T OT H E E R M E M B R A N E

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lacks a hydrophobic membrane-spanningsegment altogether; instead, these proteins are linked to an amphipathic phospholipid anchor that is embedded in the membrane (Figure 13-10, right).

Internal Stop-Transfer and Signal-Anchor S e q u e n c eD s e t e r m i n eT o p o l o g y o f S i n g l e - P a sPsr o t e i n s \Ve begin our discussionof how membraneprotein topology is determined with the membrane insertion of integral proteins that contain a single,hydrophobic membrane-spanning segment.Two sequencesare involved in targeting and orienting type I proteins in the ER membrane,whereastype II and type III proteins contain a single,internal topogenicsequence. As we will see,there are three main types of topogenic sequencesthat are used to direct proteins to the ER membrane and to orient them within it. We have alreadybeenintroduced to one, the N-terminal ER signalsequence.The other two, introduced here, are internal sequencesknown as stop-transfer anchor sequencesand signal-anchorsequences. Type I Proteins All type I transmembraneproteins possess an N-terminal signal sequencethat targetsthem to the ER as well as an internal hydrophobic sequencethat becomesthe membrane-spanninga helix. The N-terminal signal sequence

on a nascenttype I protein, like that of a secretoryprotein, initiates cotranslationaltranslocationof the protein through the combined action of the SRP and SRP receptor.Once the Nterminus of the growing polypeptide entersthe lumen of the ER, the signalsequenceis cleaved,and the growing chain continues to be extruded across the ER membrane. However, unlike the casewith secretoryproteins,when the sequenceof approximately 22 hydrophobic amino acidsthat will become a transmembrane domain of the nascent chain enters the translocon,it stopstransferofthe protein through the channel (Figure 13-11). The structure of the Sec61complex suggests that the channelmay be able to open like a clamshell,allowing the hydrophobic transmembranesegmentof the translocating peptide to move laterally betweenthe protein domains constituting the translocon wall (seeFigure 13-8). \Whenthe peptide exits the translocon in this manner, it becomesanchored in the phospholipid bilayer of the membrane.Because of the dual function of sucha sequenceto both stop passageof the polypeptidechain through the transloconand to becomea hydrophobic transmembranesegment in the membrane bilayer,it is called a stop-transferanchor sequence. Once translocation is interrupted, translation continues at the ribosome, which is still anchored to the now unoccupied and closedtranslocon. As the C-terminus of the protein chain is synthesized,it loops out on the cytosolic side of the membrane.When translationis completed,the ribosome is

Cytosol

Open translocon

'l,.il ji;i i lr,!t rl

'fr " Signal peptidase

Nascent polypeptide chain

Stop-transfer ancnor sequence

Cleaved s i gn aI sequence

ER lumen

NHs NH:

proteins. FIGURE 13-11Positioning type lsingle-pass Step[: Afterthe ribosome/nascent chaincomplex becomes associated with a translocon in the ERmembrane, the N-terminal signal sequence iscleaved Thisprocess occurs bythesamemechanism (seeFigure astheonefor soluble proteins secretory 13-6)Steps2, untilthe hydrophobic stop-transfer anchor B: Thechainiselongated sequence issynthesized andentersthe translocon, whereit prevents the nascent chainfromextruding fartherintothe ERlumen Step@: Thestop-transfer anchorsequence moveslaterally between 544

CHAPTER 13

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NHs

in the phospholipid the translocon subunits andbecomes anchored probably bilayer. At thistime,thetranslocon closesStepS: As synthesis continues, theelongating chainmayloopout intothe andtranslocon cytosolthroughthe smallspacebetweenthe ribosome rscomplete, Step6: Whensynthesis the ribosomal subunits are released intothe cytosol, leaving the proteinfreeto diffusein the m e m b r a n[eS e e HD o e t a,l1 9 9 6C, e l8l 5 : 3 6 a9 n, d WM o t h e s e t,a1l9 9 7 , Cell89:523l

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

releasedfrom the translocon and the C-terminus of the newly synthesizedtype I protein remains in the cytosol. Support for this mechanism has come from studies in which cDNAs encoding various mutant receptorsfor human growth hormone (HGH) are expressedin cultured mammalian cells. The wild-type HGH receptor, a typical type I protein, is transported normally to the plasma membrane. However, a mutant receptor that has charged residues inserted into the single a-helical membrane-spanningsegment or that is missingmost of this segmentis translocatedentirely into the ER lumen and is eventually secretedfrom the cell as a soluble protein. These kinds of experimentsestablishthat the hydrophobic membrane-spanningo helix of the HGH receptor and of other type I proteins functions both as a stoptransfer sequenceand a membrane anchor that preventsthe C-terminus of the protein from crossingthe ER membrane. Type ll and Type lll Proteins Unlike type I proteins, type II and type III proteins lack a cleavableN-rerminal ER signalsequence.Instead, both possessa single internal hydrophobic signal-anchorsequencethat functions as both an ER signal sequenceand membrane-anchorsequence.Recall that type II

and type III proteins have opposite orientations in the membrane (seeFigure 13-10); this differencedependson the orientation that their respectivesignal-anchorsequencesassume within the translocon.The internal signal-anchorsequencein type II proteins directs insertion of the nascentchain into the ER membrane so that the N-terminus of the chain facesthe cytosol, using the sameSRP-dependentmechanismdescribed for signal sequences(Figure 13-l2a). However, the internal signal-anchorsequenceis not cleayedand moves laterally between the protein domains of the translocon wall into the phospholipid bilayer, where it functions as a membrane anchor.As elongationcontinues,the C-terminal region of the growing chain is extruded through the transloconinto the ER lumen by cotranslationaltranslocation. In the case of type III proteins, the signal-anchor sequence, which is located near the N-terminus, inserts the nascent chain into the ER membrane with its N-terminus facing the lumen, in the opposite orientation of the signal anchor in type II proteins. The signal-anchor sequenceof type III proteins also functions like a stop-transfer sequence and prevents further extrusion of the nascentchain into the ER lumen (Figure 13-12b). Continued elongation of the

(a)

(b)

E Nascent polypeptide chain NHs* NH:*

Cposol E

E

mRNA

coo + + +

ilttffi

i,ri:j;ii:l:,1

1ru,|i NH : *

SignalER lumen a n c n o r sequence

coo FIGURE 13-12Positioningtype ll and type ilt single-pass proteins.(a)Typell proteinsStep[: Afterthe internal signalanchorsequence issynthesized on a cytosolic ribosome, it isbound by an SRP(notshown), whichdirects the ribosome/nascent chain complex to the ERmembrane Thisissimilar to targeting of soluble proteins secretory exceptthatthe hydrophobic signalsequence is not located at the N-terminus andis notsubsequently cleavedThe nascent chainbecomes oriented in thetranslocon with itsN-terminal portiontowardthecytosolThisorientation isbelieved to be mediated by the positively charged residues shownN-terminal to thesignalanchorsequence StepE:As the chaintselongated andextruded i n t ot h el u m e nt,h ei n t e r n asli g n a l - a n c hmoorv e lsa t e r a lol yu to f t h e

bilayer. translocon andanchors the chainin the phospholipid iscompleted, theC-terminus of the StepB: Onceproteinsynthesis polypeptide subunits is released intothe lumen,andthe ribosomal Step[: Assembly arereleased intothe cytosol(b)Typelll proteins. pathway to thatof typell proteins exceptthatpositively isby a similar sequence charged residues on theC-terminal sideof thesignal-anchor withinthetranslocon segment to be oriented causethetransmembrane portionoriented to the cytosol andthe N-terminal with itsC-terminal s i d eo f t h ep r o t e i inn t h eE Rl u m e nS t e p [s, B : C h a i ne l o n g a t i o n portionof the proteiniscompleted in the cytosol, of theC-terminal arereleased. M Spiess andH F Lodish, andribosomal subunits [See 1986, Cell 44:177, and H Do et al , 1996, Ce//85:369 l

I N S E R T I OO N F P R O T E I NISN T OT H E E R M E M B R A N E

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chain C-terminal to the signal-anchor/stop-transfer sequenceproceeds as it does for type I proteins, with the hydrophobic sequence moving laterally between the translocon subunits to anchor the polypeptide in the ER membrane(seeFigure 13-11). One of the features of signal-anchor sequencesthat appears to determine their insertion orientation is a high density of positively chargedamino acids adjacentto one end of the hydrophobic segment.For reasonsthat are not well understood,thesepositively chargedresiduestend to remain on the cytosolic side of the membrane, not traversing the membrane into the ER lumen. Thus the position of the charged residuesdictates the orientation of the signal-anchorsequencewithin the translocon as well as whether or not the rest of the polypeptide chain continues to pass into the ER lumen: type II proteins tend to have positively charged residues on the N-terminal side of their signal-anchor sequence,orienting the N-terminus in the cytosol and allowing passageof the C-terminal side into the ER (Figure 1.3-L2a), whereas type III proteins tend to have positively charged residues on the C-terminal side of their signal-anchor sequence,inserting the N-terminus into the translocon and restricting the C-terminus to the cytosol (Figure 13-1,2b). A striking experimental demonstration of the importance of the flanking charge in determining membrane ori-

entation is provided by neuraminidase,a type II protein in the surface coat of influenza virus. Three arginine residues are located just N-terminal to the internal signal-anchorsequencein neuraminidase.Mutation of thesethree positively charged residues to negatively charged glutamate residues causes neuraminidase to acquire the reverse orientation. Similar experimentshave shown that other proteins, with either type II or type III orientation, can be made to "flip" their orientation in the ER membrane by mutating charged residuesthat flank the internal signal-anchorsegment.

M u l t i p a s sP r o t e i n sH a v eM u l t i p l eI n t e r n a l TopogenicSequences Figure 13-13 summarizesthe arrangementsof topogenic sequencesin single-passand multipass transmembrane proteins. In multipass (type IV) proteins, each of the membrane-spanninga helices acts as a topogenic sequence in the ways that we have already discussed:they can act to direct the protein to the ER, to anchor the protein in the ER membrane, or to stop transfer of the protein through the membrane. Multipass proteins fall into one of two types depending on whether the N-terminus extends into the cytosol or the exoplasmicspace(e.g.,the ER lumen, cell exterior). This N-terminal topology usually is determined

STA= Internalstop-transferanchorsequence SA = Internalsignal-anchorsequence

(a) Type I

( b ) T y p el l

N H 3 +Signal sequence

NH:*

(c) Type lll

NHs*

(d)Type lV-A

NHs*

coo STA

+++

coo SA

coo

+++ SA Cvtosol

Lumen COO

STA

SA

Lumen

(e) Type lV-B

Cytosol

C H A P T E R1 3

Cytosol

Lumen

+++

I

STA

SA

Cytosol

Lumen

NHqt

STA

546

sequences determine < FIGURE 13-13Topogenic orientationof ERmembraneproteins.Topogenic hydrophilic sequences areshownin red;soluble, portions sequences form in blue Theinternal topogenic or cthelices thatanchorthe proteins transmembrane of proteinsin the membrane(a)TypeI proteins segments anda singleinternal signalsequence containa cleaved (b, c)Typell andtypelll stop-transfer anchor(STA). (SA) proteins signal-anchor containa singleinternal in theorientation of these Thedifference sequence. proteins depends largely on whetherthereisa high charged aminoacids(+ + +) on density of positively (typell)or on sideof the SAsequence the N-termrnal (typelll).(d,e) SA sequence sideof the the C-terminal proteins lacka cleavable signal Nearly all multipass in theexamples shownhere. asdepicted sequence, facesthe cytosol, TypelV-Aproteins, whoseN-terminus typell S sequences andSTA containalternating TypelV-Bproteins, whoseN-terminus faces sequences followedby the lumen,beginwith a typelllSAsequence Proteins typell SAand STAsequences of alternating (oddor eachtypewith differentnumbersof ct helices even)areknown

Lumen

COO

+++ SA

STA

MOVINGPROTEINS INTO MEMBRANES AND ORGANELLES

Cytosol

SA

STA

by the hydrophobic segment closest to the N-terminus and the charge of the sequencesflanking it. lf a type IV protein has an euen number of transmembrane cr helices, both its N-terminus and C-terminus will be oriented toward the same side of the membrane (Figure 13-13d). Conversely, if a type IV protein has an odd number of a helices, its two ends will have opposite orientations ( F i g u r e1 3 - 1 3 e ) . Type lV Proteins with N-Terminus in Cytosol Among the multipass proteins whose N-terminus extends into the cytosol are the various glucose transporters (GLUTs) and most ion-channel proteins, discussedin Chapter 11. In these proteins, the hydrophobic segmentclosestto the N-terminus initiates insertion of the nascent chain into the ER membrane with the N-terminus oriented toward the cytosol; thus this a-helical segment functions like the internal signalanchor sequenceof atype II protein (seeFigure 13-12a).As the nascent chain following the first a helix elongates, it moves through the translocon until the secondhydrophobic a helix is formed. This helix prevents further extrusion of the nascentchain through the translocon;thus its function is similar to that of the stop-transferanchor sequencein a type I p r o t e i n ( s e eF i g u r e1 3 - 1 1 ) . After synthesisof the first two transmembranecl helices, both ends of the nascentchain face the cytosol and the loop between them extends into the ER lumen. The C-terminus of the nascentchain then continues ro grow into the cytosol, as it does in synthesis of type I and type III proteins. According to this mechanism,the third a helix acts as another type II signal-anchor sequenceand the fourth as another stop-transferanchor sequence(Figure 13-13d). Apparently, once the first topogenic sequenceof a multipass polypeptide initiates associationwith the translocon, the ribosome remains attached to the translocon, and topogenic sequences that subsequentlyemerge from the ribosome are threaded into the translocon without the need for the SRP and the SRP receptor. Experiments that use recombinant DNA techniques to exchangehydrophobic cr heliceshave provided insight into the functioning of the topogenic sequencesin type IV-A multipass proteins. Theseexperimentsindicate that the order of the hydrophobic a helicesrelative to each other in the growing chain largely determineswhether a given helix functions as a signal-anchor sequenceor stop-transfer anchor sequence. Other than its hydrophobicitS the specific amino acid sequenceof a particular helix has little bearing on its function. Thus the first N-terminal a helix and the subsequent odd-numbered ones function as signal-anchor sequences,whereas the intervening even-numberedhelices function as stop-transferanchor sequences. Type lV Proteins with N-Terminus in the Exoplasmic Space The large family of G protein-coupled receptors,all of which contain seventransmembranect helices,constitute the most numerous type IV-B proteins, whose N-terminus extends into the exoplasmic space.In theseproteins, the hy-

drophobic ct helix closestto the N-terminus often is followed by a cluster of positively charged amino acids, similar to a As a retype III signal-anchorsequence(seeFigure 1,3-1,2b)'. sult, the first a helix inserts the nascent chain into the translocon with the N-terminus extending into the lumen (seeFigure 13-13e).As the chain is elongated,it is inserted into the ER membrane by alternating type II signal-anchor sequencesand stop-transfer sequences,as just describedfor type IV-A proteins.

A PhospholipidAnchor TethersSomeCellSurfaceProteinsto the Membrane Somecell-surfaceproteins are anchored to the phospholipid bilayer not by a sequenceof hydrophobic amino acids but by a covalently attached amphipathic molecule, glycosylphosphatidylinositol (GPI) (Figure 1.3-1.4aand Chapter 10). These proteins are synthesizedand initially anchored to the ER membrane exactly like type I transmembrane proteins, with a cleavedN-terminal signal sequenceand internal stoptransfer anchor sequencedirecting the process (see Figure 13-11).However,a short sequenceof amino acidsin the luminal domain, adjacent to the membrane-spanningdomain, is recognizedby a transamidaselocated within the ER membrane. This enzymesimultaneously cleavesoff the original stop-transferanchor sequenceand transfersthe luminal portion of the protein to a preformed GPI anchor in the membrane (Figure 1.3-'1.4b). \X/hy change one type of membrane anchor for another? Attachment of the GPI anchor, which results in removal of the cytosol-facing hydrophilic domain from the Proteinswith GPI protein, can have severalconsequences. anchors, for example, can diffuse relatively rapidly in the plane of the phospholipid bilayer membrane. In contrast, many proteins anchored by membrane-spanningct helices are impeded from moving laterally in the membrane becausetheir cytosol-facing segments interact with the cytoskeleton. In addition, the GPI anchor targets the attachedprotein to the apical domain of the plasma membrane in certain polarized epithelial cells, as we discussin C h a p t e r1 4 .

The Topologyof a MembraneProteinOften Can Be Deducedfrom lts Sequence As we have seen,various topogenic sequencesin integral membrane proteins synthesizedon the ER govern interac'When sciention of the nascent chain with the translocon. tists begin to study a protein of unknown function, the identification of potential topogenic sequenceswithin the corresponding gene sequencecan provide important clues about the protein's topological class and function. Suppose, for example, that the gene for a protein known to be required for a cell-to-cell signaling pathway contains nucleotide sequencesthat encodean apparent N-terminal signal sequence and an internal hydrophobic sequence.These findings suggest that the protein is a type I integral membrane protein

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drophilic amino acids negative values. Although different scalesfor the hydropathic index exist, all assignthe most positive valuesto amino acids with side chains made up of = tO Mannose mostly hydrocarbon residues(e.g.,phenylalanineand me= NH, P1-'o.01''o"thanolamine P o o* thionine) and the most negativevalues to charged amino acids (e.g., arginine and aspartate).The second step is to identify longer segmentsof sufficient overall hydrophobic% L POo PO4-+ NH3+ Fattyacyl chains ity to be N-terminal signal sequencesor internal stopTo accomtransfer sequences and signal-anchorsequences. plish this, the total hydropathic index for each successive segmentof 20 consecutiveamino acids is calculatedalong Hvdrophobic NHg* Polar J the entire length of the protein. Plots of these calculated values againstposition in the amino acid sequenceyield a hydropathy profile. (b) Figure 13-15 shows the hydropathy profiles for three GPI different membrane proteins. The prominent peaks in such transamidase Cytosol coo plots identify probable topogenicsequences as well as their position and approximate length. For example, the hydropathy profile of the human growth hormone receptor revealsthe presenceof both a hydrophobic signal sequence NHs* . at the extreme N-terminus of the protein and an internal hydrophobic stop-transfersequence(Figure 13-15a). On the Preformed G P Ia n c h o r basis of this profile, we can deduce, correctly, that the hugrowth hormone receptor is a type I integral memman Precu rsor protein. The hydropathy profile of the asialoglycobrane NlH3* protein NHs* protein receptor, a cell-surface protein that mediates Mature GPI-linked ER lumen removal of abnormal extracellular glycoproteins, reveals a protein prominent internal hydrophobic signal-anchor sequence FIGURE 13-14GP|-anchored proteins.(a)Structure of a glygives no indication of a hydrophobic N-terminal signal but (GPl)fromyeast. cosylphosphatidylinositol portion Thehydrophobic sequence(Figure 13-15b). Thus we can predict that the of the molecule iscomposed of fattyacylchains, whereas the polar (hydrophilic) asialoglycoproteinreceptor is a type II or type III membrane portionof the molecule iscomposed of carbohydrate protein. The distribution of charged residueson either side residues groupsIn otherorganisms, andphosphate boththe length of theacylchains andthe carbohydrate moieties mayvarysomewhat of the signal-anchorsequenceoften can differentiate befromthe structure shown(b)Formation proteins of GPI-anchored in tween these possibilities since positively charged amino the ERmembrane Theproteinissynthesized andinitially inserted acids flanking a membrane-spanning segment usually are i n t ot h eE Rm e m b r a naess h o w ni n F i g u r 1e 3 - 11 .A s p e c i f ti cr a n s a m i - oriented toward the cytosolic face of the membrane. For dasesimultaneously proteinwithinthe cleaves the precursor instance,in the caseof the asialoglycoproteinreceptor,exexoplasmic-facing domain,nearthe stop-transfer anchorsequence amination of the residuesflanking the signal-anchor se(red),andtransfers groupof the newC-terminus the carboxyl to the quence reveals that the residues on the N-terminal side terminal amrnogroupof a preformed GPIanchor[See C Abeijon and carry a net positive charge, thus correctly predicting that C B Hirschberg,1992,TrendsBioch Sec m i 1 7 : 3 2 , a n d K K o d u k u l ae t a l , this is a type II protein. 1992, Proc Nat'|.Acad Sci USA89:49821 The hydropathy profile of the GLUT1 glucose transporter, a multipass membrane protein, shows the presence of many segmentsthat are sufficiently hydrophobic to be membrane-spanninghelices(Figure 13-15c).The complexand therefore may be a cell-surfacereceptor for an extracelity of this profile illustrates the difficulty both in unambigulular ligand. ously identifying all the membrane-spanningsegmentsin a Identification of topogenic sequences requiresa way to multipass protein and in predicting the topology of individscan sequencedatabasesfor segmentsthat are sufficiently ual signal-anchorand stop-transfer sequences.More sohydrophobic to be either a signal sequenceor a transmemphisticated computer algorithms have been developed that brane anchor sequence.Topogenicsequencescan often be take into account the presenceof positively charged amino identified with the aid of computer programs that generare acids adjacent to hydrophobic segmentsas well as the a hydropathy profile for the protein of inrerest. The first length of and spacing between segments.Using all this instep is to assign a value known as the bydropathic index to formation, the best algorithms can predict the complex each amino acid in the protein. By convention, hydrophotopology of multipass proteins with an accuracy of greater bic amino acids are assigned positive values and hythan 7 5 percent. (a)

548

O = Inositol I = Glucosamine

CHAPTER 13

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M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

( a ) H u m a ng r o w t h h o r m o n er e c e p t o r( t y p el ) A

3 z

1 0 -1 -2 -3 N-terminus

100

200

( b )A s i a l o g l y c o p r o t e ri ne c e p t o r( t y p el l )

4

S i g n a l - a n c h osre q u e n c e

2 1 0 -1

400

300

C-terminus

( c ) G L U T(tt y p el V ) 4 z

r'i;.:'1+i i

...,,

-z

1 0 -1 -z

100

200

100

A E X P E R I M E N TFA IG L U R1E3 - 1 5H y d r o p a t h p yrofiles. profilescanidentifylikelytopogenic Hydropathy sequences in proteinsTheyaregenerated integral membrane by plottingthetotal hydrophobicity of eachsegment of 20 contiguous aminoacidsalong the lengthof a proteinPositive valuesindicate relatively hydrophobic

Finally, sequencehomology to a known protein may permit accurateprediction of the topology ol a newly discovered multipass protein. For example, the genomesof multicellularorganismsencodea very large number of multipass proteins with seventransmembranecr helices.The similaritiesbetweenthe sequences of theseproteinsstrongly suggestthat all have the sametopology as the well-studied G protein-coupled receptors,which have the N-terminus orientedto the exoplasmicsideand the C-terminusoriented to the cytosolicside of the membrane.

Insertion of Proteins into the ER Membrane r Integralmembraneproteinssynthesized on the rough ER fall into four topological classesas well as a lipid-linked t y p e ( s e eF i g u r e1 3 - 1 0 ) . r Topogenic sequences-N-terminal signal sequences,internal stop-transferanchor sequences, and internal signalanchor sequences-direct the insertion and orientation of nascentproteinswithin the ER membrane.This orientarion is retained during transport of the completedmembrane p r o r e i nt o i r s f i n a l d e s t i n a t i o n . r Single-passmembrane protelns contarn one or two topogenic sequences. In multipassmembraneproteins, each ct-helicalsegmentcan function as an internal topogenic sequence,depending on its location in the polypeptide

200

400

polarportions portions; relatively of the protein values, negative profiles for Thecomplex aremarked. sequences Probable topogenic (typelV)proteins, in part(c),oftenmustbe suchasGLUT1 multipass of to determrne the topology with otheranalyses supplemented theseoroteins

chain and the presenceof adjacent positively charged r e s i d u e s( s e eF i g u r e1 3 - 1 3 ) . r Somecell-surfaceproteins are initially synthesizedas type I proteins on the ER and then are cleavedwith their luminal domain transferredto a GPI anchor (seeFigure t3-14). r The topology of membraneproteinscan often be correctly predicted by computer programs that identify hydrophobic topogenicsegmentswithin the amino acid sequenceand generatehydropathyprofiles(seeFigure13-15).

Folding, ProteinModifications, and QualityControlin the ER Membrane and soluble secretoryproteins synthesizedon the rough ER undergo four principal modifications before they reachtheir final destinations:(1) covalentaddition and processing of carbohydtates (glycosylation) in the ER and Golgi, (2) formation of disulfidebondsin the ER, (3) proper folding of polypeptide chains and assemblyof multisubunit proteins in the ER, and (4) specific proteolytic cleavagesin the ER, Golgi, and secretory vesicles.Generally speaking, these modifications promote folding of secretory proteins into their native structures and add structural stability to proteins exposed to the extracellular environment. Modifications such as glycosylation also allow the cell to produce a vast array of chemically distinct moleculesat the cell surface

IN THEER PROTEIN M O D I F I C A T I O NFSO . L D I N GA, N D Q U A L I T YC O N T R O L

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that are the basis of specific molecular interactions used in cell-to-cell adhesionand communication. One or more carbohydrate chains are added to the vast majority of proteinsthat are synthesizedon the rough ER; indeed, glycosylationis the principal chemicalmodification to most of these proteins. Proteins with attached carbohydrates are known as glycoproteins. Carbohydrate chains in glycoproteins may be attached to the hydroxyl group in serineand threonine residuesor to the amide nitrogen of asparagine.Theseare referred to as Olinked oligosaccharidesand Nlinked oligosaccharides,respectively.The various types of O-linked oligosaccharides include the mucin-type O-linked chains (named after abundantglycoproteinsfound in mucus)and the carbohydrate modifications on proteoglycansdescribedin Chapter 19. OIinked chains typically contain only one to four sugar residues,which are added to proteins by enzymesknown as glycosyltransferases, located in the lumen of the Golgi complex. The more common Nlinked oligosaccharides are larger and more complex,containingseveralbranchesin mammalian cells. In this section we focus on N-linked oligosaccharides, whose initial synthesisoccurs in the ER. After the initial N-glycosylationof a protein in the ER, the oligosaccharidechain is modified in the ER and commonly in the Golgi as well. Disulfide bond formation, protein folding, and assembly of multimeric proteins, which take place exclusively in the rough ER, also are discussedin this section. Only properly folded and assembledproteins are transported from the rough ER to the Golgi complex and ultimately to the cell surface or other final destination. Unfolded, misfolded, or partly folded and assembledproteins are selectivelyretained in the rough ER. Sile consider several features of such "quality control" in the latter part of this section. As discussedpreviously,N-terminal ER signal sequences are cleaved from secretory proteins and type I membrane proteins in the ER. Someproteins also undergo other specific proteolytic cleavagesin the Golgi complex or secretoryvesicles. We cover thesecleavages,as well as carbohydrate modifications that occur primarily or exclusively in the Golgi complex, in the next chapter.

A PreformedA/-LinkedOligosaccharide ls Added t o M a n y P r o t e i n si n t h e R o u g hE R Biosynthesisof all N-linked oligosaccharidesbeginsin the rough ER with addition of a preformed oligosaccharide precursorcontaining 14 residues(Figure 13-t6). The structure of this precursor is the same in plants, animals, and single-celledeukaryotes-a branchedoligosaccharide,containing three glucose(Glc), nine mannose(Man), and two N-acetylglucosamine(GlcNAc) molecules, which can be written as GIcaMane(GlcNAc)2. Once added to a protein, this branchedcarbohydrate srructureis modified by addition or removal of monosaccharidesin the ER and Golgi compartments. The modifications to N-linked chains differ from one glycoprotein to another and differ among different organisms,but a core of 5 of the 14 residuesis conservedin the structures of all N-linked oligosaccharideson secretoryand membrane proteins. 550

CHAPTER 13

I

Glc

I I

Glc

G l c N A c= N - A c e t y l g l u c o s a m i n e Man = Mannose Glc= Glucose = Conserved = Variable

Glc

Man

I I

Man

Man

II

II

Man

Man

Man

Ma n

Man

*in GlcNAc

GlcNAc

II

N H s *. . - X - A s n - X - ( S e r / T h r ) - ' . . C O O A FIGURE 13-16Commonprecursorof N-linkedoligosacprecursor charides. This14-residue of tV-linked oligosaccharides is proteins addedto nascent in the roughERSubsequent removal and in somecases addition of specific sugarresidues occurin the ERand GolgicomplexThecoreregion, composed of fiveresidues highlightedin purple,is retained in allN-linked oligosaccharides The (Asn)residues precursor canbe linkedonlyto asparagine thatare (Ser) (Thr) separated by oneaminoacid(X)froma serine or threonine on the carboxyl side.

Prior to transfer to a nascent chain in the lumen of the ER, the precursor oligosaccharideis assembledon a membrane-attached anchor called dolichol phosphate, a Iongchain polyisoprenoid lipid (Chapter 10). After the first sugar, GlcNAc, is attached to the dolichol phosphate by u pyrophosphate bond, the other sugarsare added by glycosidic bonds in a complex set of reactions catalyzed by enzymes attached to the cytosolic or luminal faces of the rough ER membrane (Figure L3-1.7).The final dolichol pyrophosphoryl oligosaccharideis oriented so that the oligosaccharide portion facesthe ER lumen. The entire 14-residueprecursor is transferred from the dolichol carrier to an asparagine residue on a nascent polypeptide as it emergesinto the ER lumen (Figure 13-18, step E). Only asparagineresidues in the tripeptide sequencesAsn-X-Ser and Asn-X-Thr (where X is any amino acid except proline) are substratesfor oligosaccharyltransferase, the enzyme that catalyzes this reaction. Two of the three subunits of this enzyme are ER membrane proteins whose cytosol-facingdomains bind to the ribosome,localizing a third subunit of the transferase,the catalytic subunit, near the growing polypeptide chain in the ER lumen. Not all

M O V T N GP R O T E | NtSN T O M E M B R A N E A SN D O R G A N E L L E S

Gytosol 5 GDP

a 4GDP 'flin

a-

e

I\

3 UDP l 3 UDP

4 GDP O

^.

,/

t\l

I

Dolichol phospha I = N-Acetylglucosamine

Completed precursor

O = Mannose A - Glucose

ER lumen

FIGURE 13-17Biosynthesis precursor. of the oligosaccharide phosphate Dolichol isa strongly hydrophobic lipid,containing 75-95 c a r b o na t o m st,h a t i s e m b e d d eidn t h e E Rm e m b r a n e T.w o (GlcNAc) tV-acetylglucosamine andfivemannose residues areadded phosphate oneat a timeto a dolichol on the cytosolic faceof the ER (stepsIl-B) Thenucleotide-sugar membrane donorsin theseand laterreactions aresynthesized in thecytosolNotethatthefirstsugar residue isattached pyrophosphate to dolichol by a high-energy linkageTunicamycin, whichblocks thefirstenzyme in thispathway, inhibits thesynthesis of allN-linked oligosaccharides in cellsAfter

pyrophosphoryl isflipped intermedrate dolichol theseven-resrdue andall four mannose face(stepZl),the remaining to the luminal areaddedoneat a time(steps5, 6). In the residues threeglucose froma laterreactions, thesugarto be addedisfirsttransferred phosphate face on thecytosolic to a carrier dolichol nucleotide-sugar face,wherethe of the ER;thecarrieristhenflippedto the luminal afterwhichthe o|gosaccharide, to the growrng sugaristransferred "empty"carrier face.[After C Abeijon isflippedbackto the cytosolic Sci17:32]1 1992,Trends Biochem andC B Hirschberg,

Asn-X-Ser/Thr sequencesbecome glycosylated,and it is not possible to predict from the amino acid sequencealone which potential N-linked glycosylation sites will be modified; for instance, rapid folding of a segment of a protein containing an Asn-X-Ser/Thr sequencemay prevent transfer precursorto it. of the oligosaccharide

Immediately after the entire precursor, GlcaMane (GlcNAc)2, is transferred to a nascent polypeptide, three different enzymes, called glycosidases,remove all three glucoseresiduesand one particular mannoseresidue (Fig, hich u r e 1 3 - 1 8 ,s t e p sZ - 4 ) . T h e t h r e eg l u c o s er e s i d u e sw are the last residuesadded during synthesisof the precursor

Dol

E

To ctsGolgi

(Man)a(GlcNAc)z

ER lumen Dol = Dolichol

o = Mannose

I = N-Acetylglucosamine

a = Glucose

FIGURE 13-18Additionand initial processing of N-linked Inthe roughERof vertebrate oligosaccharides. cells,the precursor GlcrMann(GlcNAc)2 istransferred fromthe dolichol carrier proteinassoonas to a susceptible asparagine residue on a nascent the asparagine crosses to the luminal sideof the ER(step[) In (stepEl),thentwo reactions, threeseparate firstoneglucose residue

(step4) (stepB), andfinallyonemannose residue glucose residues (stepl[) playsa residue of oneglucose Re-addition areremoved. in the ER,asdiscussed foldingof manyproteins rolein the correct 45:631, and Rev. Biochem 1985,Ann S Kornfeld, R Kornfeldand later[See M S o u s a a n d AJ P a r o d i1, 9 9 5 , E M B O l 1 4 i 4 1 9 6 )

, N D Q U A L I T YC O N T R O LI N T H E E R P R O T E I NM O D I F I C A T I O N SF,O L D I N G A

551

The oligosaccharidesattachedto glycoproteinsservevarious functions. For example, some proteins require N-linked oligosaccharidesin order to fold properly in the ER. This function has been demonstrated in studies with the antibiotic tunicamycin, which blocks the first step in the formation of the dolichol-linked oligosaccharideprecursor and therefore inhibits synthesisof all N-linked oligosaccharidesin cells (seeFigure 13-17).In the presenceof tunicamycin,the hemagglutinin precursor polypeptide (HAe) is synthesized, but it cannot fold properly and form a normal trimer; in this case,the protein remains,misfolded, in the rough ER. Moreover, mutation of a particular asparaginein the HA sequence to a glutamine residue prevents addition of an N-linked oligosaccharideto that site and causesthe protern to accumulate in the ER in an unfolded state. In addition to promoting proper folding, N-linked oligosaccharides also confer stability on many secretedglycoproteins.Many secretoryproteins fold properly and are transported to their final destination even if the addition of all N-linked oligosaccharides is blocked,for example,by tunicamycin. However, such nonglycosylatedproteins have been shown to be lessstablethan their glycosylatedforms. For instance,glycosylatedfibronectin, a normal component of the extracellularmatrix, is degradedmuch more slowly by tissueproteasesthan is nonglycosylatedfibronectin. Oligosaccharideson certain cell-surfaceglycoproteins also play a role in cell-cell adhesion. For example, the plasma membrane of white blood cells (leukocytes)contains cell-adhesionmolecules(CAMs) that are extensively glycosylated.The oligosaccharidesin these moleculesinteract with a sugar-binding domain in certain CAMs found on endothelial cells lining blood vessels.This interaction tethers the leukocytesto the endothelium and assists in their movement into tissuesduring an inflammat o r y r e s p o n s et o i n f e c t i o n ( s e e F i g u r e 1 , 9 - 3 6 ) .O t h e r c e l l - s u r f a c eg l y c o p r o t e i n s p o s s e s so l i g o s a c c h a r i d es i d e chains that can induce an immune response.A common example is the A, B, O blood-group antigens,which are O-linked oligosaccharidesattached to glycoproteins and glycolipids on the surface of erythrocytes and other cell t y p e s ( s e eF i g u r e 1 0 - 2 0 ) .

groups (-SH), also known as thiol groups, on two cysteine residuesin the same or different polypeptide chains. This reaction can proceed spontaneouslyonly when a suitable oxidant is present.In eukaryotic cells, disulfide bonds are formed only in the lumen of the rough ER; in bacterial cells, disulfide bonds are formed in the periplasmic space between the inner and outer membranes.Thus disulfide bonds are found only in secretoryproteins and in the exoplasmic domains of membraneproteins. Cytosolic proteins and organelleproteins synthesizedon free ribosomeslack disulfide bonds and depend on other interactionsto stabilize their structures. The efficient formation of disulfide bonds in the lumen of the ER dependson the enzymeprotein disulfide isomerase (PDI), which is presentin all eukaryotic cells.This enzymeis especiallyabundant in the ER of secretorycells in such organs as the liver and pancreas,where large quantities of proteins that contain disulfide bonds are produced. As shown in Figure t3-1.9a, the disulfide bond in the active site of PDI can be readily transferred to a protein by two sequential thiol-disulfide transfer reactions.The reducedPDI generated by this reaction is returned to an oxidized form by the action of an ER-residentprotein, called Ero1, which carriesa disulfide bond that can be transferredto PDI. Erol itself becomes oxidized by reaction with molecular oxygen that has diffused into the ER. In proteins that contain more than one disulfide bond, the proper pairing of cysteineresiduesis essentialfor normal structure and activity. Disulfide bonds commonly are formed between cysteinesthat occur sequentially in the amino acid sequencewhile a polypeptide is still growing on the ribosome. Such sequential formation, however, sometimesyields disulfide bonds between the wrong cysteines.For example,proinsulin, a precursor to the peptide hormone insulin, has three disulfide bonds that link cysteines 1 and 4, 2 and 6, and 3 and 5. In this case,a disulfide bond that initially formed sequentially(e.g., between cysteinesI and 2) would have to be rearranged for the protein to achieveits proper folded conformation. In cells, the rearrangementof disulfide bonds also is acceleratedby PDI, which acts on a broad range of protein substrates, allowing them to reach their thermodynamicallymost stable conformations (Figure 13-19b). Disulfide bonds generally form in a specificorder, first stabilizingsmall domains of a polypeptide, then stabilizing the interactions of more distant segments;this phenomenon is illustrated by the folding of influenza HA protein, discussedin the next sectron.

D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e d b y P r o t e i n si n t h e E RL u m e n

C h a p e r o n eas n d O t h e r E RP r o t e i n sF a c i l i t a t e F o l d i n ga n d A s s e m b l yo f P r o t e i n s

In Chapter 3 we learned that both intramolecular and intermolecular disulfide bonds (-S-S-) help stabilize the tertiary and quaternary structure of many proteins. These covalent bonds form by the oxidative linkage of sulfhydryl

Although many denatured proteins can spontaneouslyrefold into their native state in vitro, such refolding usually requires hours to reach completion. Yet new soluble and membraneproteins produced in the ER generallyfold into

on the dolichol carrier, appear to acr as a signal that the oligosaccharideis complete and ready to be transferredto a protern.

O l i g o s a c c h a r i dSei d eC h a i n sM a y P r o m o t e F o l d i n ga n d S t a b i l i t yo f G l y c o p r o t e i n s

552

CHAPTER 13

|

M O V T N Gp R O T E t N |SN T O M E M B R A N E A SN D O R G A N E L L E S

( a ) F o r m a t i o no f a d i s u l f i d eb o n d

Reduced substrate prorern

Oxidized substrate protein

( b ) R e a r r a n g e m e notf d i s u l f i d eb o n d s . -qr.l 'Reduced t "' ' i PDI ic

zSH \Q.

"" Reduced PDI -SH

-

$z t"

,n.o,.,.i.',oj"i il rHi'J o""o,

e_

.",.,."L':[,l,ilXto"o.

(PDl).PDI FIGURE 13-19Actionof proteindisulfideisomerase formsandrearranges disulf idebondsviaan activesitewithtwo closely spaced cysteine residues interconverted thatareeasily between the reduced dithiolformandtheoxidized disulfide form Numbered redarrowsindicate thesequence of electron transfers Yellowbars represent disulfide bonds(a)Intheformation of disulfide bonds,the (-S )form of a cysteine proteinreacts ionized thiolin thesubstrate (9-S) bondin oxidized withthedisulfide PDIto forma disulfideproteinintermediate bondedPDI-substrate A second ionized thiolin

forminga disulfide withthisintermediate, thenreacts thesubstrate proteinandreleasing PDIPDl,in reduced bondwithinthesubstrate bondin the luminalprotein to a disulfide electrons turn,transfers PDI formof PDl.(b)Reduced theoxidized regenerating Ero1,thereby bondsby formeddisulfide of improperly rearrangement cancatalyze PDIboth reactions. Inthiscase,reduced transfer similar thiol-disulfide Thesereactions in the reactionpathway. initiates andis regenerated of the proteinis untilthe moststableconformation arerepeated F.Gilbert, 1991 l achieved ,Biochemistry30t6l9 [SeeMM LylesandH

their proper conformation within minutes after their synthesis. The rapid folding of these newly synthesizedproteins in cells dependson the sequentialaction of several proteins present within the ER lumen. We have already seen how the molecular chaperone BiP can drive posttranslationaltranslocationin yeastby binding fully synthesized polypeptidesas they enter the ER (seeFigure 13-9). BiP can also bind transiently to nascentchains as they enter the ER during cotranslationaltranslocation.Bound BiP is thought to preventsegmentsof a nascentchain from misfolding or forming aggregates,thereby promoting folding of the entire polypeptide into the proper conformation. P r o t e i n d i s u l f i d e i s o m e r a s e( P D I ) a l s o c o n t r i b u t e s t o proper folding, becausecorrect 3-D conformation is stabil i z e db y d i s u l f i d eb o n d si n m a n y p r o t e i n s . As illustrated in Figure 1,3-20,two other ER proteins, the homologous lectins (carbohydrate-binding proteins)

cdlnex,in and calreticulin, bind selectively to certain Nlinked oligosaccharideson growing nascent chains. The ligand for thesetwo lectins, which contains a single glucose residue, is generatedby a specific glucosyltransferasein the ER lumen (seeFigure 1,3-18,step Ed). This enzyme acts only on polypeptide chains that are unfolded or misfolded, acts as one of the and in this respectthe glucosyltransferase primary surveillance mechanismsto ensure quality control of protein folding in the ER. Binding of calnexin and calreticulin to unfolded nascent chains marked with glucosylated N-linked oligosaccharidesprevents aggregation of adjacent segmentsof a protein as it is being made on the ER. Thus calnexin and calreticulin, like BiP' help prevent premature, incorrect folding of segmentsof a newly made protern. Other important protein-folding catalysts in the ER Iumen are peptidyl-prolyl isomerases,a family of enzymesthat

IN THEER PROTEIN M O D I F I C A T I O NFSO , L D I N GA, N D Q U A L I T YC O N T R O L

553

(al

Oligosaccharyl transferase Dolichol oligosaccharide

M e m b r a n e - s p anni n g s helix

Cytosol

Luminal crhelix

I

ER lumen

HAotrimer Completed H A sm o n o m e r

(b) > FIGURE 13-20Hemagglutinin folding and assembly. (a)Mechanism of (HAo) trimerassembly Transtent bindingof the chaperone BiP(stepIE) to the nascent chainandof two lectins, (steplE) calnexin andcalreticulin, to certainoligosaccharide chains promotes properfoldingof adjacent segments. A totalof seven N-linked oligosaccharide portionof the chains areaddedto theluminal nascent chainduringcotranslational translocation, andPDIcatalyzes theformation of sixdisulfide bondspermonomerCompleted HA6 monomers areanchored in the membrane by a singlemembranes p a n n i nagh e l i xw i t ht h e i rN - t e r m i n u i nst h el u m e n( s t e p[ ) Interaction of threeHAechains with oneanother, initially viatheir transmembrane crhelices, apparently triggers formation of a long stemcontaining onecthelixfromthe luminalpartof eachHAs polypeptide Finally, interactions between thethreeglobular heads occur,generating a stableHA6trimer(stepB) (b)Electron micrograph of a complete influenza virionshowing trimers of HAprotein protruding (a) asspikes fromthesurface of theviralmembrane [part SeeU Tatuetal, 1995, EMBO J 14:1340, andD Hebert elal, 1997, J Cell Biol.139:613 Part(b) ChrisBjornberg/PhotoResearchers, Inc l

acceleratethe rotation about peptidyl-prolyl bonds at proline residuesin unfolded segmentsof a polypeptide:

Rotation about peptide bond

-

2, Prolyl

o*^

t11

.U

,/I

lzu\ O' NH

\

crs

trans

Such isomerizations sometimesare the rate-limiting step in the folding of protein domains. Many peptidyl-prolyl isomerasescan catalyzethe rotation of exposedpeptidyl-prolyl bonds indiscriminately in numerous proteins, but some have very specificprotein substrates. 554

CHAPTER 13

I

Many important secretory and membrane proteins synthesizedon the ER are built of two or more polypeptide subunits. In all cases,the assemblyof subunits constituring these multisubunit (multimeric) proteins occurs in the ER. An important class of multimeric secretedproteins is the immunoglobulins, which contain two heavy (H) and two light (L) chains,all linked by intrachaindisulfidebonds. Hemagglutinin (HA) is another multimeric protein that provides a good illustration of folding and subunit assembly(Figure 1320). This trimeric protein forms the spikes that protrude from the surface of an influenza virus particle. The HA trimer is formed within the ER of an infected host cell from

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

response'lre1,a 13-21The unfolded-protein < FIGURE hasa bindingsite proteinin the ERmembrane, transmembrane c o n t a i nas d o ; ec y t o s o l i c m a i n f o r B i Po n i t sl u m i n adlo m a i nt h unfolded Accumulating Step RNAendonuclease. Il: specific themfrom releasing proteins in the ERlumenbindBiPmolecules, its of lrel thenactivates lrel Dimerization monomeric mRNA activityStepsE, B: Theunspliced endonuclease by is cleaved Hacl factor thetranscription precursor encoding joined functional form to are exons two dimericlre1,andthe thatthisprocessing indicates evidence Hacl mRNACurrent generally processing althoughpre-mRNA occurstn thecytosol, Hacl into is translated occursin the nucleusStep4: Hacl activates and nucleus into the back protein, whichthenmoves catalysts' protein-folding several of genesencoding transcription 2000' etal' Bertolotti Cell'lO7:103;A 2001, etal, Ruegsegger U [See

Hacl

Spliced Hacl mRNA

E n d o n u c l e a s e - cH ua t cl Unspliced Hacl mRNA

andP Walter,1997,Ceil CellBiol 2:326;andC Sidrauski Nature 9 0 : 1 0 3l1

lrel dimer

Unfoldedproteins

Unfoldedproteins w i t h B i Pb o u n d

three copiesof a precursor protein termed HA6, which has a single membrane-spanningct helix. In the Golgi complex, each of the three HAe proteins is cleaved to form two polypeptides, HA1 and HA2; thus each HA molecule that eventually resideson the viral surfacecontains three copies of HAi and threeof HA2 (seeFigure3-10).The trimer is stabilized by interactions between the large exoplasmic domains of the constituent polypeptides,which extend into the ER lumen; after HA is transported to the cell surface,these domains extend into the extracellular space' Interactions between the smaller cytosolic and membrane-spanning portions of the HA subunits also help stabilize the trimeric protein. Studieshave shown that it takesjust 10 minutesfor the HAs polypeptidesto fold and assembleinto their proper trimeric conformation.

l m p r o p e r l yF o l d e dP r o t e i n si n t h e E RI n d u c e Expressionof Protein-FoldingCatalysts Vild-type proteins that are synthesizedon the rough ER cannot exit this compartment until they achievetheir completely folded conformation. Likewise, almost any mutation ih"t p..u..tts proper folding of a protein in the ER also blocks movement of the polypeptide from the ER lumen or membrane to the Golgi complex. The mechanismsfor retaining unfolded or incompletely folded proteins within the ER probably increase the overall efficiency of folding by keeping intermediateforms in proximity to folding catalysts, which are most abundant in the ER. Improperly folded proteins retained within the ER generallyare seenbound to the ER chaperonesBiP and calnexin. Thus theseluminal folding catalvsts perform two related functions: assisting in the

folding of normal proteins by preventing their aggregation and binding to irreversibly misfolded proteins' Both mammalian cellsand yeastsrespond to the presence

proteins that assistin protein folding' . Mammalian cells contain an additional regulatory pathway that operatesin responseto unfolded proteins in in. En. In this pathway, accumulation of unfolded pro-

F i g u r e s1 6 - 3 6 a n d 1 6 - 3 8 ) . A hereditaryform of emphysemaillustratesthe detrimental effects that can result from misfolding of proteins in the ER. This disease is caused by a point -rrt"tion in ct1-antitrypsin,which normally is secretedby

IN THEER M O D I F I C A T I O NFSO , L D I N GA, N D Q U A L I T YC O N T R O L PROTEIN

O

555

U n a s s e m b l eodr M i s f o l d e dp r o t e i n si n t h e ERAre Often Transportedto the Cytosol for Degradation

the cell altogether by degradation in the proteasome(see Figure 3-29).

Protein Modifications, Folding, and euality Control in the ER

proteases were never found. More recent studies have shown that misfolded membrane and secretoryprorelns are recognized by specific ER membrane proteins and are tar_ geted for transport from the ER lumen into the cytosol, by a processknown as dislocation or letrotrlnslocat.ion. The dislocationof misfoldedproteins out of the ER de_ pends on a set of proteins located in the ER membrane and in the cytosol that perform three basic functions. The first

r All N-linked oligosaccharides,which are bound to as_ paragineresidues,contain a core of three mannoseand two N-acetylglucosamineresidues and usually have several branches (seeFigure 13-16). O-linked olisosaccharides. which are bound to serineor threonine resid-ues, g.rr.r".. ally short, often containing only one to four ,rlg"r..ridrl... r Formarion of all N-linked oligosaccharidesbegins with assemblyof a conserved14-residuehigh-mannose precur_ sor on dolichol, a lipid in the membrane of the rough ER (seeFigure 73-17). After this preformed oligosaccharideis transferred to specific asparagine residues of nascent polypeptide chains in the ER lumen, three glucoseresidues and one mannoseresidueare removed(seeFigure 13-1g). r Oligosaccharideside chains may assist in the proper folding of glycoproteins, help protect the mature p.ot"irrc from proteolysis,participate in cell-celladhesion, fr.rrr._ ".rd tion as antigens. r Disulfide bonds are added to many secretory proteins and the exoplasmic domain of membrane proteins in the ER. Protein disulfide isomerase(pDI), presentin the ER lu_ men, catalyzesboth the formation and the rearrangement of disulfide bonds (seeFigure 13-19). r The chaperoneBiP, the lectins calnexin and calreticulin, and peptidyl-prolyl isomeraseswork together ro ensure proper folding of newly made secreroryand membrane proteins in the ER. The subunits of multimeric proteins also assemblein the ER. r Only properly folded proteins and assembledsubunits are transported from the rough ER to the Golgi complex in vesicles. r The accumularion of abnormally folded proteins and unassembledsubunits in the ER can induce increasedex_ pression of ER protein-folding catalystsvia the unfolded_ protern response(seeFigure 13-21). r Unassembledor misfolded proteins in the ER often are transported back to the cytosol, where they are degradedin the ubiquitin/proteasomepathway.

556

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M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Sortingof Proteinsto and Chloroplasts Mitochondria In the remainder of this chapter, we examine how proteins synthesizedon cytosolic ribosomes are sorted to mitochondria, chloroplasts, peroxisomes,and the nucleus (seeFigure 13-1).In both mitochondriaand chloroplastsan internallumen called the matrix is surrounded by a double membrane, and internal subcompartments exist within the matrix. In contrast, peroxisomesare bounded by a single membrane and have a single luminal matrix compartment. Becauseof these and other differences,we consider peroxisomesseparately in the next section. Likewise, the mechanism of protein transport into and out of the nucleus differs considerably from sorting to mitochondria and chloroplasts; this is d i s c u s s eidn t h e l a s t s e c t i o n . In addition to being bounded by two membranes'mitochondria and chloroplastsshare similar types of electron transport proteins and use an F-classATPase to synthesize ATP (seeFigure 12-2). Remarkably,gram-negativebacteria also exhibit thesecharacteristics.Also like bacterial cells' mitochondria and chloroplastscontain their own DNA, which encodesorganellerRNAs, tRNAs, and someproteins (Chapter 6). Moreover, growth and division of mitochondria and chloroplasts are not coupled to nuclear division. Rather, these organellesgrow by the incorporation of cellular proteins and lipids, and new organellesform by division of preexisting organelles.The numerous similarities of free-living bacterialcellswith mitochondria and chloroplastshave led scientiststo hypothesize that these organelles arose by the incorporation of bacteria into ancestraleukaryotic cells,

ORGANETTE TARGTT

Proteins encoded by mitochondrial DNA or chloroplast DNA are synthesizedon ribosomes within the organelles and directed to the correct subcompartmentimmediately af' ter synthesis.The majority of proteins located in mitochondria and chloroplasts, however, are encoded by genesin the nucleus and are imported into the organellesafter their synthesis in the cytosol' Apparently, over aeons of evolution' much of the geneticinformation from the ancestralbacterial DNA in these endosymbiotic organellesmoved' by an unknown mechanism, to the nucleus' Precursor proteins synthesizedin the cytosol that are destinedfor the matrix of mitochondria or the equivalent space in chloroplasts, the stroma, usually contain specificN-terminal uptake-targeting sequencesthat specifybinding to receptor proteins on the organelle surface. Generally, this sequenceis cleaved once it i.a.hes the matrix or stroma. CIearlS theseuptake-targeting sequencesare similar in their location and general function to ihe signal sequencesthat direct nascentproteins to the ER lumen. Although the three types of signalsshare some common sequencefeatures, their specific sequencesdiffer considerably,as summarizedin Table 13-1. In both mitochondria and chloroplasts, protein import requiresenergy and occurs at points where the outer and innei organellemembranesare in closecontact' Becausemitochondiia and chloroplasts contain multiple membranesand

OFSEOUEI|CE BEM()VAL PROTTIN WITHIN OFSEOUENCE LOCATII]N

OTSEOUEIICE NATURE

Endoplasmic reticulum (lumen)

N-terminus

Yes

Core of 6-12 hydrophobicamino acids, often precededby one or more basicamino acids (Arg, Lys)

Mitochondrion (matrix)

N-terminus

Yes

Amphipathichelix, 20-50 residuesin length, with Arg and Lys residueson one sideand hydrophobicresidueson the other

Chloroplast (stroma)

N-terminus

Yes

No commonmotifs;generallyrich in Ser' Thr, and smallhydrophobicresiduesand poor in Glu and AsP

Peroxisome (matrix)

C-terminus(mostproteins) N-terminus(few proteins)

No

at extreme PTSl signal(Ser-Lys-Leu) C-terminus;PTS2signalat N-terminus

Nucleus (nucleoplasm)

Varies

No

Multipledifferentkindsla commonmotif includesa short segmentrich in Lys and Arg residues

subcompartments' "Different or additional sequencestarget proteins to organelle membranes and AND CHLOROPLASTS SO M I T O C H O N D R I A S O R T I N GO F P R O T E I N T

557

> EXPERIMENTAL FIGURE 13-22lmport of mitochondrialprecursorproteinsis assayedin a cell-freesystem.Inside mitochondria, proteins areprotected from the actionof proteases suchastrypsinWhen no mitochondria arepresent, mitochondrial proteins synthesized in thecytosol are degraded by addedproteaseproteinuptake occurs onlywith energized (respiring) mitochondria, whichhavea proton electrochem icalgradient (proton-motive force)across the innermembrane The imported proteinmustcontainan appropriate uptake-targeting sequence Uptakealso requires ATPanda cytosolic extractcontaininq chaperone proteins thatmarntain the precursor proterns in an unfolded conformation Thisassay hasbeenusedto studytargeting sequences andotherfeatures of thetranslocation process

Uptaketargeting sequence

h_\{L/ t

Mitochondrial prolern

Add energized yeasr mitochondria

Yeast mitochondrial proteinsmade by cytoplasmicribosomes in a cell-freesystem

Proteintaken up into mitochondria; uptake-ta rgeting sequenceremoved and degraded

Proteinssequestered within mitochondria are resistantto trypsin ..a

Trypsin

Uptake-targeting sequenceano mitochondrial protein degraded

membrane-limited spaces,sorting of many proteins to their correct location often requires the sequentialaction of two targetlng sequencesand two membrane-boundtranslocation

A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c e s D i r e c tP r o t e i n st o t h e M i t o c h o n d r i aM l atrix

. .a. , & 't' '.e ' j .r'' r t'. a' a. 'a -..3

-

pathicity of matrix-targeting sequencesis critical to their function. The cell-free assay outlined in Figure 13-22 has been widely used in studies on the import of mitochondrial precursor proteins. In this system, respiring (energized)mito_ chondria extractedfrom cellscan incorporate mitochondrial precursor proteins carrying appropriate uptake-targetingse_ quencesthat have been synthesizedin the absenceof mitochondria. Successfulincorporation of the precursor into the

translocation of secretoryproteins into the ER, which gener_ ally occurs only when microsomal (ER-derived)-.rnbn"rr., are present during synthesis(seeFigure 13-4).

M i t o c h o n d r i aP l r o t e i nl m p o r t R e q u i r e s Outer-MembraneReceptorsand Translocons in Both Membranes Mitochondrial matrix-targeting sequencesare thought to assume an a,helical conformation in which positively charged amino acids predominate on one side ofthe helix

558

'

c H A p r E R1 3 I

Figure 13-23 presentsan overview of protein import from the cytosol into the mitochondrial matrix, the route into the mitochondrion followed by most imported proteins. \Wewill discussin detail each step of protein transport into the matrix and then consider how some proteins subsequentlyare ' targeted to other compartments of the mitochondiion. After synthesisin the cytosol, the soluble precursors of mitochondrial proteins (including hydrophobic integral membrane proteins) inreract directly with the mitochondrial membrane. In general, only unfolded proteins can be im_ ported into the mitochondrion. Chaperoneproteins such as

M o v r N Gp R o r E r NrsN T oM E M B R A N A EN s Do R G A N E L L E '

coo

ATP A D P+ P ; Cytosolic Hsc70

Matrix-targeting sequence

-'.'./

NHs*

General i m p o r tp o r e (Tom40)

lmport recepror (Tom 20122\ Cytosol

+\

Outer membrane

13-23Proteinimport intothe < FIGURE proteins mitochondrialmatrix.Precursor are ribosomes on cytosolic synthesized folded or partially in an unfolded maintained Hsc70 as such chaperones, stateby bound proteinbindsto (steptr) Aftera precursor neara siteof contactwith an importreceptor (stepZ), it istransferred the innermembrane importpore(stepB) The intothe general protein thenmovesthroughthls translocating c h a n n ealn da n a d j a c e nc th a n n ei nl t h ei n n e r (stepsZl, E). Notethat membrane at rare"contactsites" occurs translocation outermembranes and inner at whichthe of thetranslocating appearto touch.Binding Hsc70and proteinbythe matrixchaperone by Hsc70helps ATPhydrolysis subsequent driveimportintothe matrixOncethe by a is removed sequence uptake-targeting from andHsc70isreleased matrixprotease protein(step6), it folds the newlyimported within activeconformation intothe mature, proteins (step of some Folding the matrix Z) [See on matrixchaperonins depends and J BiolChem271:31763, 1996, G Schatz, el al , 1997,Ann Rev.CellDevelBiol N Pfanner 13:25l

Matrix Hsc70 ADP + P; Matrix processlng protease

Cleaved targeting seq uence

cytosolic Hsc70 keep nascentand newly made proteins in an unfolded stateso that they can be taken up by mitochondria' This processrequiresATP hydrolysis' Import of an unfolded mitochondrial precursor is initiated by the binding of a mitochondrial targeting sequenceto an import receptor in the outer mitochondrial membrane. These receptors were first identified by experimentsin which antibodiesto specificproteins of the outer mitochondrial membranewere shown to inhibit protein import into isolated mitochondria. Subsequent genetic experiments, in which the genes for specific mitochondrial outer-membraneproteins were mutated, showed that specificreceptorproteinswere responsiblefor the import of different classesof mitochondrial proteins. For example, N-terminal matrix-targeting sequencesare recognized by Tom20 and Tom22. (Proteinsin the outer mitochondrial membrane involved in targeting and import are designated Tom oroteins for /ranslocon of the outet membtane.)

The import receptorssubsequentlytransfer the precursor proteins to an import channel in the outer membrane' This ch".tn.l, composed mainly of the Tom40 protein, is known ^s the general import pore becauseall known mitochondrial compartments precursor proteins gain accessto the interior 'Sfhen purified channel' this through -itt.hondrion ft tn. transmema forms Tom40 liposomes, into incorporated and brane channel with a pore wide enough to accommodatean unfolded polypeptideihain. The general import pore forms a largely p"tti". channel through the outer mitochondrial and the driving force for unidirectional transport -.*L.".t., into mitochondria comesfrom within the mitochondrion' In the caseof precursorsdestinedfor the mitochondrial matrix, transfer through the outer membrane occurs simultaneously with transfer through an inner-membranechannel composed of the Tim23 and1iml7 proteins. \Tim standsfor translocon of the lnner membrane.)Translocation into the matrix

O SO M I T O C H O N D R I A N D C H L O R O P L A S T S S O R T I N GO F P R O T E I N T

559

thus occurs at "contact sites" where the outer and inner membranesare in close proximitv.

drial membraneby interactingwith transmembraneprotein Tim44. This interaction stimulatesATp hydrolysis by matrix Hsc70, and together these two proteins are thought to power translocationof proteinsinto the matnx. Some imported proteins can fold into their final, active conformation without further assistance.Final folding of many matrrx proteins, however, requires a chaperonin. As discussedin Chapter 3, chaperoninproteins aciively facilitate protein folding in a processthat dependson ATp. For in_ stance,yeastmutantsdefectivein Hsc60, a chaperoninin the mitochondrial matrix, can import matrix proteins and cleavetheir uptake-targerrngsequencenormally, but the imported polypeptidesfail to fold and assembleinto the native tertiary and quaternarystructures.

(a)

(b)

S t u d i e sw i t h C h i m e r i cP r o t e i n sD e m o n s t r a t e l m p o r t a n tF e a t u r e so f M i t o c h o n d r i a lm p o r t Dramatic evidencefor the ability of mitochondrial matrixtargettng sequencesto direct import was obtained with chimeric proteins produced by recombinant DNA techniques. For example, the matrix-targeting sequenceof alcohol dehydrogenasecan be fused to the N-terminus of dihydrofolate reductase(DHFR), which normally residesin the cytosol. In the presenceof chaperones,which prevent the Cterminal DHFR segmentfrom folding in the cytosol, cell-free translocation assaysshow that the chimeric protein is transported into the marrix (Figure 13-24a). The inhibitor methotrexate,which binds tightly ro the acive site of DHFR and greatly stabilizes its folded conformation, renders the chimeric protein resistant to unfolding by cytosolic chaperones. \il1'hentranslocation assaysare performed in the presence of methotrexate, the chimeric protein does not iompletely enter rhe matrix. This finding demonstratesthat a precursor must be unfolded in order to traverse the imoort poresin the mitochondrialmembranes.

Bound methotrexate inhibitor

(cl

Cytosol Outer

F o l d e dDHFR Cytosol Outer membrane Intermembrane space Intermembrane space

NHr* .d9" .s9' . ((.9. \$$o

Mitochondrial matrix

Cleaved targeting sequence

,

Spacerr"qr"n.i / EXPERIMENTAL FTGURE 13-24 Experimentswith chimeric proteins elucidate mitochondrial protein import. These experiments show that a matrix-targeting sequencealonedirects

energizem d i t o c h o n d r iaan d t h e m a t r i x _ t a r g e t isnigg n atl h e n i s removed.(b) When the C-terminusof the chimericproteinis locked in the foldedstateby bindingof methotrexate, translocalonrs blocked lf the spacersequenceis long enoughto extendacrossboth

560

CHAPTER 13

I

o'2 P'm

,

transport channels, a stabletranslocation intermediate, with the targeting sequence cleaved off, isgenerated in the presence of methotrexate, asshownhere.(c)TheC-terminus of thetranslocation intermediate in (b)canbe detected by incubating the mitochondria with antibodies thatbindto the DHFR segment, followedby gold particles coatedwith bacterial proteinA, whichbindsnonspecificallv to antibody (seeFigure9-21).An electron molecules micrograph of a sectioned sample goldparticles (redarrowhead) reveals boundto the translocation intermediate at a contactsrtebetween the innerand outermembranes Othercontactsites(blackarrows) alsoareevident (a)and(b)adapted IParts fromJ Rassow et al, 1990,FEBS Letters 2Tstigo Part(c) from M SchweigereI al , j987, I CellBiol. 105:235,courtesyof W N e u o e r tl

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Additional studies revealed that if a sufficiently long spacer sequenceseparatesthe N-terminal matrix-targeting sequenceand DHFR portion of the chimeric protein, then in the presenceof methotrexate a translocation intermediate that spansboth membranescan be trapped if enough of the polypeptideprotrudes into the matrix to preventthe polypeptide chain from sliding back into the cytosol, possibly by stably associatingwith matrix Hsc70 (Figure 13-24b).In order for such a stable translocation intermediateto form, the spacer sequencemust be long enough to span both membranes;a spacerof 50 amino acids extendedto its maximum possiblelength is adequateto do so. If the chimeracontains a shorter spacer-say, 35 amino acids-no stable translocation intermediateis obtained becausethe spacercannot span both membranes.These observationsprovide further evidence that translocated proteins can span both inner and outer mitochondrial membranes and traverse these membranesin an unfolded state. Microscopic studiesof stabletranslocation intermediates show that they accumulateat siteswhere the inner and outer mitochondrial membranes are close together, evidencethat precursor proteins enter only at such sites (Figure 1,3-24c). The distancefrom the cytosolic face of the outer membrane to the matrix face of the inner membrane at these contdct sitesis consistentwith the length of an unfolded spacer sequence required for formation of a stable translocation intermediate.Moreover, stabletranslocation intermediatescan be chemically cross-linkedto the protein subunits that comprise the translocation channelsof both the outer and inner membranes.This finding demonstratesthat imported proteins can simultaneouslyengagechannels in both the outer and inner mitochondrial membrane, as depicted in Figure 13-23. Since roughly 1000 stuck chimeric proteins can be observedin a typical yeast mitochondrion, it is thought that mitochondria have approximately 1000 general import pores for the uptake of mitochondrial proteins.

Three EnergyInputs Are Neededto lmport P r o t e i n si n t o M i t o c h o n d r i a As noted previously and indicated in Figure 13-23, ATP hydrolysis by Hsc70 chaperoneproteins in both the cytosol and the mitochondrial matrix is required for import of mitochondrial proteins. Cytosolic Hsc70 expends energy to maintain bound precursor proteins in an unfolded statethat is competent for translocationinto the matrix. The importance of ATP to this function was demonstratedin studiesin which a mitochondrial precursor protein was purified and then denatured (unfolded) by urea. \7hen tested in the cell-free mitochondrial translocation system,the denatured protein was incorporated into the matrix in the absenceof AIP. In contrast' import of the native, undenaturedprecursorrequired ATP for the normal unfolding function of cytosolic chaperones. The sequentialbinding and ATP-drivenreleaseof multiple matrix Hsc70 moleculesto a translocatingprotein may simply trap the unfolded protein in the matrix. Alternatively, the matrix Hsc70, anchoredto the membraneby the Tim44 protein, may act as a molecular motor to pull the

protein into the matrix (seeFigure 13-23).In this case'the lunctions of matrix Hsc70 andTim44 would be analogous to those of the chaperone BiP and Sec63 complex, respectivelS in post-translational translocation into the ER lumen ( s e eF i g u r e1 3 - 9 ) . The third energy input required for mitochondrial protein import is a H+ electrochemicalgradient, or protonmotive force, acrossthe inner membrane' Recall from Chapter 12 that protons are pumped from the matrix into the intermembrane space during electron transport, creating transmembranepotential across the inner membrane' In general,only mitochondria that are actively undergoing respiration, and thereforehave generateda proton-motive force acrossthe inner membrane' are able to translocateprecursor proteins from the cytosol into the mitochondrial matrix' of mitochondria with inhibitors or uncouplers of ir.u,-.n, oxidative phosphorylation' such as cyanide or dinitrophenol, dissipatesthis proton-motive force. Although precursor proteins still can bind tightly to receptorson such poisoned mitochondria, the proteins cannot be imported, either in intact cells or in cell-freesystems,even in the presenceof ATP and chaperone proteins. Scientistsdo not fully understand how the proton-motive force is used to facilitate entry of a p...rr.roi p.otein into the matrix. Once a protein is partially inserted into the inner membrane' it is subjectedto a trans-

M u l t i p l eS i g n a l sa n d P a t h w a y sT a r g e tP r o t e i n s t o S u b m i t o c h o n d r i aCl o m p a r t m e n t s Unlike targeting to the matrix, targeting of proteins to the intermembrane space,inner membrane, and outer membrane of mitochondria generally requires more than one targetlng sequenceand occurs via one of severalpathways' Figure 1325 summarizes the organization of targeting sequencesin proteins sorted to different mitochondrial locations. lnner-Membrane Proteins Three separatepathways are known to target proteins to the inner mitochondrial membrane. One pathway makes use of the same machinery that is used for targeting of matrix proteins (Figure 13-26, path A). A cytochrome oxidase subunit called CoxVa is a protein transported by this pathway' The precursor form of CoxVa' which contains an N-terminal matrix-targeting sequence recognizedby the Tom20l22 import receptor,is transferred through the generalimport pore of the outer membrane and the inier-membrane Tim23l17 translocation complex' In addition to the matrix-targeting sequence'which is cleaved during import, CoxVa contains a hydrophobic stop-transfer s.qr'r*ce. A. the protein passesthrough theTim23ll'7 chan.r.i th. stop-transfer sequenceblocks translocation of the

AND CHLOROPLASTS O SO M I T O C H O N D R I A S O R T I N GO F P R O T E I N T

561

Location of imported protein

lmported protein

Locations of targeting sequences in preprotein

Cleavageby matrix protease Alcohol d e h y d r o g e n a slel l

Matrix

*.*T**" Matrix-targeting sequence

Inner membrane PathA

Cytochrome o x i d a s es u b u n i t CoxVa

Cleavageby Hydrophobic matrix protease stop-transfersequence

Cleavageby matrix protease Path B

Mature protein

< FIGURE 13-25Targetingsequences in imported mitochondrialproteins.Most mitochondrial proteins havean N-terminal (pink)thatissimilar matrix-targeting sequence proteins but not identical in different proteins. destined for the innermembrane, the rntermembrane space, or the outermembrane haveoneor moreadditional targeting sequences thatfunctionto directthe proteins to theselocations by several different pathways. Theletteredpathways correspond to thoseillustrated in Figures 13-26and 13-27 [See W Neupert, 1997, Ann Rev. Biochem 66:863 l

Internalsequences recognizedby Oxal

ATP synthase s u b u n i t9 Internalsequencesrecognized by Tom70 receptor andTrm22 complex

Path c

ADP/ATP anttporter

Intermembrane space

PathA

Firstcleavageby matrix

Secondcleavageby protease

Cytochromeb2 Intermembrane-space-targeting sequence

Targetingsequencefor the generalimport pore path B

Cytochromec h e m el y a s e

Outer membrane

\--...P\-..^t

Stop-transferand outer-memorane localizationsequence Porin (P70)

C-terminus across the inner membrane. The membraneanchored intermediate is then transferred laterally into the bilayer of the inner membrane much as type I integral membrane proteins are incorporated into the ER membrane (see F i g u r e1 3 - 1 1 ) .

matrix via the Tom40 andTim23l17 channels.After cleav_ age of the matrix-targeting sequence,the protein is inserted into the inner membrane by a processthai requires interac_ 562

.

c H A p r E lR3

tion with Oxal and perhaps other inner-membraneproteins (Figure 13-26, parh B). Oxal is related to a bacterialprotein involved in inserting some inner-membraneproteins in bacteria. This relatednesssuggeststhat Oxal may have descendedfrom the translocation machinery in the endosymbiotic bacterium that eventually became the mitochondrion. However, the proteins forming the inner-membranechannels in mitochondria are not related to the proteins in bacterial translocons. Oxal also participates in ihe inner-membrane insertion of certain proteins (e.g., subunit II of cytochrome oxidase) that are encoded by mitochondrial DNA and synthesizedin the matrix by mitochondrial ribosomes. The final parhway for insertion in the inner mitochondrial membrane is followed by multipass proteins that

| M o v t N Gp R o r E t Nt sN T oM E M B R A NAENsD O R G A N E L L E S

Path B

Path A

Stop-transfer Matrix-targeting sequence sequence NHs NHs*

Tom40 Tom22

Cytosol

Tom20

Outer membrane

lntermembrane space Ttm23l17

; €

A Assembled protein

Mitochondrial matrix

Cleaved matrix-targeting SeQu€nCeS-_--_-

13-26Threepathwaysto the innermitochondrial A FIGURE with differenttargeting membranefrom the cytosol.Proteins pathways. viadifferent to the innermembrane aredrrected sequences via the proteins membrane pathways, the outer cross three In all A andB by pathways delivered importpore Proteins Tom40general thatis recognized sequence matrix-targeting containan N-terminal Although in theoutermembrane importreceptor bytheTom20/22 channel, 7 inner-membrane usetheTim23/1 boththesepathways proteinentersthe matrixand theydifferin thatthe entireprecursor in pathwayB MatrixHsc70 to the innermembrane thenisredirected

contain six membrane-spanning domains, such as the ADP/ATP antiporter. Theseproteins, which lack the usual N-terminal matrix-targeting sequence'contain multiple internal mitochondrial targeting sequences.After the internal sequencesare recognizedby a secondimport receptor composed of outer-membrane proteins Tom70 and Tom22, the imported protein passesthrough the outer membranevia the generalimport pore (Figure 13-26, path C). The protein then is transferredto a secondtranslocation complex in the inner membrane composedof the Tim22 and Tim54 proteins. Transfer to the Tim22l54

matrixproteins to itsrolein the importof soluble playsa rolesimilar by pathwayC containtnternal delivered (seeFigure13-23). Proteins importreceptor; by the Tom70/fom22 that arerecognized sequences (Tim22154) isused channel translocation inner-membrane a different (Tim9 Tim10) proteins and pathway. intermembrane Two in this betweenthe outerandinnerchannelsSeethe transfer facilitate andA Kuhn,2000,AnnRevCell [SeeR E Dalbey text for discussion

DevelBiol16:5l,andNPfannerandAGeissler,2OOl,NatureRevMolC Biol 2:339l

protein into the inner membrane.

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Intermembrane-Space Proteins Two pathways deliver cytosolicproteinsto the spacebetweenthe inner and outer mitochondrial membranes.The major pathway is followed by proteins, such as cytochrome b2, whose precursorscarry rwo different N-terminal targeting sequences,both of which ultimately are cleaved.The most N-terminal of the rwo seouences is a matrix-targeting sequence,which is removed by th. -a-

proteolytic cleavage,this pathway is similar to that of innermembraneproteins suchas CoxVa (seeFigure 13-26,parh A). Cytochrome c heme lyase, the enzyme responsiblefor the covalent attachment of heme to cytochrome c, illustrates a secondpathway for targetingto the intermembrane space.In this pathway, the imported protein does not con_ tain an N-terminal matrix-targetingsequenceand is deliv_ ered directly to the intermembranespace via the general

import pore without involvement of any inner-membrane translocationfactors (Figure 13-27, path B). Sincetranslocation through the Tom40 general import pore does not seemto be coupled to any energeticallyfavorable process such as hydrolysis of ATP or GTP, the mechanism rhat drives unidirectional translocation through the outer membrane is unclear. One possibility is that cytochrome c heme lyase passively diffuses through the outlr membrane and then is trapped within the intermembrane spaceby binding to another protein that is deliveredto that location by one of the translocationmechanismsdiscussedpreviously. Outer-Membrane Proteins Many of the proteinsthat reside in the mitochondrial outer membrane, including the Tom 40 pore itself and mitochondrial porin, have a B-barrel structure in which antiparallel strandsform the hydrophobic transmembranesegmentssurrounding a central channel. Such proteins are incorporated into the outer membrane by first interacting with the general import pore, Tom40, and then they are transferred to a complex known as the SAM (sorting and assembly machinery) complex, which is composedof at leastthree outer membraneproteins.presumably it is the very stable hydrophobic nature of B-barrel proteins

Path A

Path B

Intermembrane-space-targeti ng Matrix-targeting sequence /, 'NHs*

I n t e r m e mD r a n e-space-targeting sequence Protein -*..-

NHs

Tom22

Tom20

Tim23l17 Mitochondrial matrix

Cleaved

3:'J'J;'.T'"''"n FfGURE 13-27 Two pathwaysto the mitochondrial intermembrane space.pathway A, the majoronefor delivery of proteins fromthe cytosol to the intermembrane space, issimilar to pathway A for delivery to the innermembrane (seeFigure13_26) Themajordifference isthatthe internal targeting sequence in proteins suchascytochrome b2destined for the intermembrane spaceisrecognized by an inner-membrane protease. whichcleaves

564

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theproteinon the intermembrane-space sideof the membrane Thereleased proteinthenfoldsandbindsto itshemecofactor within the intermembrane spacePathway B involves directdelivery to the intermembrane spacethroughtheTom40general importporein the outermembrane. andA Kuhn,2OOO, [SeeR E Dalbey Ann Rev. Cett Devel Biol 16:51; N PfannerandA Geissler,2001, Nature Rev.Mol. CettBiot. 2 : 3 3 9 ;a n d K D i e k e r te t a l , 1 9 9 9 ,p r o c N a t , l A c a d S c i U S A 9 6 : 1 1 1 5 2 ] |

M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

that ultimately causesthem to be stably incorporated into the outer membrane, but precisely how the SAM complex facilitatesthis processis not known.

Targetingof ChloroplastStromalProteins l s S i m i l a rt o l m p o r t o f M i t o c h o n d r i a l Matrix Proteins Among the proteins found in the chloroplast stroma are the enzymesof the Calvin cycle,which function in fixing carbon dioxide into carbohydratesduring photosynthesis(Chapter 12). The large (L) subunit of ribulose 1,5-bisphosphate carboxylase(rubisco) is encodedby chloroplast DNA and synthesizedon chloroplastribosomesin the stromal space. The small (S) subunit of rubisco and all the other Calvin cycle enzymesare encodedby nuclear genesand transported to chloroplastsafter their synthesisin the cytosol.The precursor forms of these stromal proteins contain an N-terminal stromal-impolt sequence(seeTable 13-1). Experiments with isolated chloroplasts, similar to those with mitochondria illustrated in Figure 1'3-22, have shown that they can import the S-subunitprecursor after its synthesis. After the unfolded precursor entersthe stromal space,it binds transiently to a stromal Hsc70 chaperone and the N-terminal sequenceis cleaved. In reactions facilitated by Hsc60 chaperoninsthat reside within the stromal space, eight S subunits combine with the eight L subunits to yield the active rubisco enzyme. The generalprocessof stromal import appearsto be very similar to that for importing proteins into the mitochondrial matrix (seeFigure 1,3-23).At least three chloroplast outermembrane proteins, including a receptor that binds the stromal-import sequenceand a translocationchannelprotein' and five inner-membraneproteins are known to be essential for directing proteins to the stroma. Although theseproteins are functionally analogousto the receptor and channel proteins in the mitochondrial membrane,they are not structurally homologous. The lack of sequencesimilarity between these chloroplast and mitochondrial proteins suggeststhat they may have arisen independentlyduring evolutton. The available evidencesuggeststhat chloroplast stromal proteins, like mitochondrial matrix proteins, are imported in the unfolded state. Import into the stroma dependson ATP hydrolysis catalyzedby a stromal Hsc70 chaperone whose function is similar to that of Hsc70 in the mitochondrial matrix and BiP in the ER lumen. Unlike mitochondria, chloroplasts do not generatean electrochemicalgradient (protonmotive force) across their inner membrane. Thus protein import into the chloroplast stroma appears to be powered s o l e l yb y A T P h y d r o l y s i s .

ProteinsAre Targetedto Thylakoidsby MechanismsRelatedto TranslocationAcross t h e B a c t e r i aIln n e r M e m b r a n e In addition to the double membranethat surroundsthem, chloroplasts contain a series of internal interconnected

membranous sacs, the thylakoids (seeFigure 12-29). Proteins localized to the thylakoid membrane or lumen carry out photosynthesis.Many of theseproteins are synthesized in the cytosol as precursorscontaining multiple targeting sequences.For example, plastocyanin and other proteins destined for the thylakoid lumen require the successiveaction The first is an N-terminal of two uptake-targetingsequences. the protein to the directs that sequence stromal-import the rubisco S subimports pathway that same the stroma by protein from the the targets sequence second The unit. targeting these role of The lumen' thylakoid the to stroma the measuring in experiments shown has been sequences DNA recombinant by generated proteins mutant of uptake techniques into isolated chloroplasts. For instance' mutant plastocyanin that lacks the thylakoid-targeting sequence but contains an intact stromal-import sequenceaccumulates in the stroma and is not transported into the thylakoid Iumen. Four separatepathways for transporting proteins from the stroma into the thylakoid have been identified. All four pathways have been found to be closely related to analogolls t."ntport mechanismsin bacteria' illustrating the close evolutionary relationship between the stromal membrane and the bacterial inner membrane. Transport of plastocyanin and related proteins into the thylakoid lumen from the stroma occurs by a chloroplast SRP-dependentpathway that utilizes a translocon similar to SecY,the bacterial version of the Sec61complex (Figure 1'3-28,left). A second pathway for transporting proteins into the thylakoid lumen inuolues a protein related to bacterial protein SecA, which uses the energy from ATP hydrolysis to drive protein translocation through the SecY translocon. A third pathway, which targets proteins to the thylakoid membrane, depends on a protein related to the mitochondrial Oxal proiein and the homologous bacterial protein (see Figure 13-26,path B). Someproteinsencodedby chloroplastDNA and synthesizedin the stroma or transported into the stroma from the cytosol are inserted into the thylakoid membrane via this pathwaY. Finally, thylakoid proteins that bind metal-containing cofactors follow another pathway into the thylakoid lumen (Figure 13-28, rigbt). The unfolded precursorsof theseproteiis are first targeted to the stroma' where the N-terminal stromal-import sequenceis cleavedoff and the protein then folds and bittdt itt cofactor' A set of thylakoid-membrane proteins assistsin translocating the folded protein and tound cofactor into the thylakoid lumen, a processpowered by the pH gradient normally maintained across the thylakoid membrane.The thylakoid-targeting sequencethat directs a protein to this pH-dependent pathway includes two closely spacedarginine residuesthat are crucial for recognition. Baiterial cells also have a mechanismfor translocating folded proteins with a similar arginine-containing sequence across the inner membrane. The molecular mechanism whereby theselarge folded globular proteins are transported across the thylakoid membrane is currently under intense srudy.

SO M I T O C H O N D R I A N D C H L O R O P L A S T S S O R T I N GO F P R O T E I N T

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Thylakoid-targeting sequence

Plastocya nin Precursor

coo

COO

Stromal-import --..sequence NrLr Hat

Toc complex

Toc complex

Cytosol Outer membrane Intermembrane space I n n e rm e m b r a n e ,

,)

r,..r,

Tic comprex

Stroma

""\ "".*' g'"auudrnoon sequence Fl

E,[\

/ ,tChloroplast SRP

I SRP-dependent .' pathway

Metal-binding

:-.-.,** RR Cleavedimporl sequence

Bound metal tons

Chloroplast SRPreceptor RR.b.--

Mature plastocyani n

Mature metal-binding protein

Sorting of Proteins to Mitochondria and Chloroplasts r Most mitochondrial and chloroplast proteins are en_ coded by nuclear genes,synthesizedon cytosolic ribosomes, and imported post-translationallyinto the organelles.

r Cytosolic chaperonesmaintain the precursors of mito_ chondrial and chloroplast proteins in an unfolded state. Only unfolded proteins can be imported into the organelles. 556

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I

':,!.) 'j.:

< FIGURE 13-28Transporting proteins to chloroplastthylakoids.Twoof the four pathways for transporting proteins fromthecytosol to thethylakoid lumenare shownhere In thesepathways, unfolded precursors aredelivered to thestromavia thesameouter-membrane proteins that importstromal-localized proteins. Cleavage of the N-terminal stromal-import sequence by a stromalprotease thenreveals the thylakoid-targeting (steptr) sequence At thispointthetwo pathways diverge. ln the SRP-dependent (/efr), pathway plastocyanin proteins andsimilar arekept unfolded in the stromal spaceby a setof (notshown)and,directed chaperones by thethylakoid-targeting sequence, bindto proteins thatareclosely related to the bacterial SRP, SRPreceptor, andSecY translocon, whichmediate movement into the lumen(stepZ). Afterthe thylakoidtargeting sequence is removed in the thylakoid lumenbya separate endoprotease, theproteinfoldsintoitsmatureconformation (stepB) In the pH-dependent pathway (nght),metal-binding proteins fold in the stroma,andcomplexredoxcofactors are added(stepZ). Twoarginine (RR) residues at the N-terminus of thethylakoidtargeting sequence anda pHgradient across the innermembrane arereouired for transport of thefoldedproteinintothe thylakoid lumen(stepg). Thetranslocon in thethylakoid membrane iscomposed of at leastfourproteins related to proteins in thebacterial rnnermembrane. Thethylakoid targeting sequence containing thetwo arginine residues iscleaved in thethylakoid lumen(step4) [See R Datbey andC Robinson, 1999,TrendsBiochem Sci24:1j,R E Dalbey andA Kuhn,2000, Ann Rev. CellDevelBiol. 1 5 : 5 1 ;a n d C R o b i n s oann d A B o l h u i s2.0 0 1 . NatureRev.Mol CellBiol 2:350)

Translocation in mitochondria occurs at sites where the outer and inner membranesof the organellesare close together. r Proteins destined for the mitochondrial matrix bind to receptors on the outer mitochondrial membrane and then are transferred to the general import pore (Tom40) in the outer membrane.Translocationoccursconcurrentlythrough the outer and inner membranes,driven bv the Drotonmotive force acrossthe inner membraneand ATp hyirolysis by the Hsc70 ATPasein the matrix (seeFigure 13-23). r Proteins sorted to mitochondrial destinationsother than the matrix usually contain two or more targeting sequences, one of which may be an N-terminal matrix-targeting sequence (seeFigure 13-25).

M O V T N Gp R O T E t N |SN T O M E M B R A N E A SN D O R G A N E L L E S

r Somemitochondrial proteins destinedfor the intermembrane spaceor inner membrane are first imported into the matrix and then redirected; others never enter the matrix but go directly to their final location. r Protein import into the chloroplast stroma occurs through inner-membrane and outer-membrane translocation channelsthat are analogousin function to mitochondrial channels but composed of proteins unrelated in sequenceto the correspondingmitochondrial proteins. r Proteins destined for the thylakoid have secondarytargeting sequences.After entry of these proteins into the reveals stroma,cleavageof the stromal-targetingsequences the thylakoid-targetingsequences.

many different peroxisomal matrix proteins bear a sequence of this type, known as peroxisomal-targetingsequenceL' or simply PTS1. The pathway for import of catalaseand other PTSIbearing proteins into the peroxisomal matrix is depicted in Figure 13-29. The PTSl binds to a soluble carrier protein in the cytosol (Pex5), which in turn binds to a receptor in the peroxisomemembrane(Pex14).The solubleand membraneassociatedperoxisomal import receptors appear to have a function analogous to that of the SRP and SRP receptor in targeting proteins to the ER lumen, except that the soluble ptsf -bindlng protein functions post-translationally.The protein to be imported then moves through a multimeric

r The four known pathways for moving proteins from the chloropiast stroma to the thylakoid closely resemble translocation acrossthe bacterial inner membrane (seeFigure 13-28). One of these systemscan translocatefolded protelns.

Proteins Sortingof Peroxisomal Peroxisomesare small organellesbounded by a single membrane. Unlike mitochondria and chloroplasts, peroxisomes lack DNA and ribosomes.Thus all peroxisomalproteinsare encoded by nuclear genes,synthesizedon ribosomes free in the cytosol, and then incorporated into preexisting or newly generatedperoxisomes.As peroxisomesare enlargedby addition of protein (and lipid), they eventuallydivide, forming new ones,as is the casewith mitochondria and chloroplasts. The size and enzyme composition of peroxisomes vary considerablyin different kinds of cells. However, all peroxisomescontain enzymesthat use molecular oxygen to oxidize various substratessuch as amino acids and fatty acids, breaking them down into smaller components for use in biosyntheticpathways.The hydrogenperoxide (H2O2)generated by theseoxidation reactionsis extremely reactiveand potentially harmful to cellular componentsl however, the peroxisome also contains enzymes,such catalase,that efficiently convert H2O2 into H2O. In mammals,peroxisomes are most abundant in liver cells,where they constitute about 1 to 2 percent of the cell volume.

CytosolicReceptorTargetsProteinswith a n S K LS e q u e n c ea t t h e C - T e r m i n uisn t o l atrix t h e P e r o x i s o m aM The import of catalaseand other proteins into rat liver peroxisomes can be assayedin a cell-freesystemsimilar to that used for monitoring mitochondrial protein import (seeFigure 73-22). By testing various mutant catalaseproteins in this system, researchersdiscovered that the sequenceSerLys-Leu (SKL in one-lettercode) or a related sequenceat the C-terminus was necessaryfor peroxisomal targeting. Further, addition of the SKL sequenceto the C-terminus of a normally cytosolicprotein leadsto uptake of the alteredprotein by peroxisomes in cultured cells. All but a few of the

Pex14

E Peroxisomal matrix protein

13-29PTS1directedimportof peroxisomalmatrix A FIGURE matrix andmostotherperoxisomal proteins.Step[: Catalase (red) sequence PTSluptake-targeting proteins containa C-terminal with the Pex5 Step Pex5. receptor Z: thatbindsto the cytosolic on located receptor withthe Pex'14 boundmatrixproteininteracts StepB: Thematrixprotein-Pex5 membrane. the peroxisome (Pex'l 0, proteins to a setof membrane isthentransferred complex into the for translocation necessary thatare Pexl2, andPex2) Step4: At some matrixby an unknownmechanism. peroxisomal or in the lumen,Pex5dissociates point,eitherduringtranslocation that a process to the cytosol, fromthe matrixproteinandreturns and membrane andadditional complex IhePex2/10112 involves canbe proteins not shown.Notethatfoldedproteins cytosolic ls not sequence andthatthetargeting intoperoxisomes imported , Ann andP B Lazarow,2001 P E Purdue in the matrix[See removed et al, 2000,Ann RevBrcchem S Subramani Biot17:101, Rev. CellDevel S u b r a m a n i , ,2C0e0l1l 0 5 : 1 817 59:39a 9n ; d V D a m maanid S LROTEINS . S O R T I N GO F P E R O X I S O M AP

567

translocation channel while still bound to pex5, a feature that differs from protein import into the ER lumen. At some stage either during or after entry into the matrix, pex5 dissociatesfrom the peroxisomal matrix protein and is recycled back to the cytoplasm. In conrrasr to the N-terminal uptaketargetlng sequenceson proteins destined for the ER lumen, mitochondrial matrix, and chloroplast stroma, the pTSl sequence is not cleaved from proteins after their entry into a peroxisome. Protein import into peroxisomesrequires ATp hydrolysis, but it is not known how the energyreliased from ATP is used to power unidirectional translocation acrossthe peroxisomal membrane. The peroxisome import machinery, unlike mosr sysrems that mediate protein import into the ER, mitochondria, and chloroplasts, can translocate folded proteins across the membrane. For example, catalaseassumesa folded conformation and binds to heme in the cytoplasm before traversing the peroxisomal membrane. Cell-free studies have shown that the peroxisome import machinery can transport large macromolecular objects, including gold particles of about 9 nm in diameter,as long as they have a pTSl tag attached to them. However, peroxisomal membranes do nor appear to contain large stablepore structures,such as the nuclear Dore describedin the next section. The fundamental mechanism of peroxisomal matrix protein translocation is not well understood but is a topic under active investigation. Some of the mechanisms under consideration include the idea that peroxisomal membrane proteins pex10, pex12, and pex2 may assembleto form a relatively large transmembrane channel with a gated opening of about 10-15 nm (for refer-

peroxisomal matrix and Pex5 would be releasedback into the cytosol to complete another round of cargo import. A f e w p e r o x i s o m am l a r r i x p r o r e i n ss u c h - a st h i o l a s ea r e synthesized as precursors with an N-terminal uptaketargetingsequenceknown as PTS2.These proteins bind to a different cytosolic receptor prorein, but oiherwise import is thought to occur by the same mechanismas for piSlcontaining proteins.

P e r o x i s o m aM l e m b r a n ea n d M a t r i x p r o t e i n s Are Incorporatedby Different pathways Autosomal recessivemutations that cause defective peroxisome assemblyoccur naturally in the human population. Such defectscan lead to severedeveloomental defectsoften associatedwith craniofacialabnormaiities.In Zellweger syndrome and related disorders, for example. t h e t r a n s p o r to f m a n y o r a l l p r o t e i n si n t o t h e p e r o x i s o m a l matrix is impaired: newly synthesizedperoxisomal en_ zymes remain in the cytosol and are eventually degraded. Genetic analysesof cultured cells from different Zellweser patients and of yeast cells carrying similar mutations hive 568

CHAPTER 13

I

identified more than 20 genesthat are required for peroxisome biogenesis.I Studieswith peroxisome-assembly mutants have shown that different pathwaysare usedfor importing peroxisomalmatrix proteins versusinserting proteins into the peroxisomal membrane. For example, analysis of cells from some Zellweger patients led to identification of genesencoding the putative translocation channel proteins Pex10, Pex12, and pex2. Mutant cells defectivein any one of these proteins cannot rncorporate matrix proteins into peroxisomes;nonetheless, the cellscontain empty peroxisomesthat have a normal complement of peroxisomal membraneproteins (Figure 13-30b).

( a ) W i l d - t y p ec e l l s

Stainedfor PMPTO

Stainedfor catalase

Catalase PMP70

o, .o 9' O" , ' Peroxisome (b) Pex12mutants (deficient in matrix protein import)

(c) Pex3 mutants (deficient i n m e m b r a n eb i o g e n e s i s )

EXPERIMENTAL FTGURE 13-30Studiesrevealdifferent pathwaysfor incorporationof peroxisomalmembraneand matrixproteins.Cellswerestained with fluorescent antibodies to PMP70, protein, a peroxisomal membrane or with fluorescenr antibodies to catalase, a peroxisomal matrixprotein, thenviewed in a fluorescent (a)In wild-type microscope. cells,bothperoxisomal membrane andmatrixproteins arevisible asbrightfociin numerous peroxisomal bodies(b)In cellsfroma pex12-deficient patient, catalase isdistributed uniformly throughout the cytosol, whereas PMP70 is localized normally to peroxisomal bodies(c)In cellsfrom a Pex3-deficient patient,peroxisomal membranes cannotassemble, andasa consequence peroxisomal bodiesdo notform.Thusboth catalase andPMP70 aremis-localized to thecytosol[Courtesy of Stephen Gould, Johns Hopkins Universitvl

M O V I N GP R O T E I NISN T O M E M B R A N E A SN D O R G A N E L L E S

Precu rsor membrane

Peroxisomal memDrane proteins

Peroxisomal ghost

Mature peroxisome

PTSl-bearing matrix protein

PTS2-bearing matrix protein

Catalase

PMPTO and biogenesis 13-31 Modelof peroxisomal FIGURE rs of peroxisomes division.Thefirststagein the de novoformation proteins intoprecursor membrane of peroxisomal the incorporation for fromthe ER Pex19 actsasthe receptor derived membranes for the Pex3andPexl6arerequired sequences membrane-targeting membrane properinsertion intotheformingperoxisomal of proteins l n s e r t i oonf a l l o e r o x i s o mm ae l m b r a nper o t e i npsr o d u c eas Mutations in any one of threeother geneswere found to block insertion of peroxisomalmembraneproteins as well as import of matrix proteins (Figure 13-30c). These findings demonstrate that one set of proteins translocatessoluble proteins into the peroxisomalmatrix but a different set is required for insertion of proteins into the peroxisomalmembrane.This situation differs markedly from that of the ER, mitochondrion, and chloroplast, for which, as we have seen,membraneproteinsand solubleproteinssharemany of the samecomponents for their insertion into theseorganelles. Although most peroxisomes are generated by division of preexisting organelles,these organellescan arise de novo by the three-stageprocessdepicted in Figure 1'3-31'.In this case, peroxisome assembly begins in the ER' At least two peroxisomal membrane proteins, Pex3 and Pex16, are inserted into the ER membrane by the mechanismsdescribed in Section13 .2. Pex3 and Pex16 recruit additional peroxisomal proteins such as Pex19 forming a specializedregion of the ER membrane that can bud off of the ER to form a peroxisomal precursor membrane. Analysis of mutant cells revealed that Pex19 is the receptor protein responsiblefor targeting of peroxisomal membrane proteins' whereas Pex3 and Pex15 are necessaryfor their proper insertion into the membrane. These three proteins are thought to be responsible for peroxisomal membrane protein assemblyin mature peroxisomesas well as during the de novo formation of new peroxisomes.The insertion of peroxisomal membrane proteins generatesmembranesthat have all the componentsnecessaryfor import of matrix proteins, leading to the formation of mature, functional peroxisomes.Division of mature peroxisomes,which largely determinesthe number of peroxisomeswithin a cell, dependson still anotherprotein,Pex11. Overexpressionof the Pex11 protein causesalatge increase in the number of peroxisomes,suggestingthat this protein controls the extent of peroxisome division. The small peroxisomesgeneratedby division can be enlargedby incorporation of additional matrix and membrane proteins via the same pathways describedpreviously'

protetns targeted of importing ghost,whichiscapable peroxisomal PTS2-bearing and PTSlpathways for importing to the matrixThe receptor of thecytosolic differonlyin the identity matrixproteins (see sequence thatbindsthetargeting (Pex5 andPex7,respectively) yields a of matrixproteins incorporation Figure13-29)Complete division requires of peroxisomes Theproliferation matureperoxisome. on the Pexl1 protein' thatdepends a process of matureperoxisomes,

Sorting of PeroxisomalProteins r All peroxisomal proteins are synthesizedon cytosolic ribosomes and incorporated into the organelle posttranslationally. r Most peroxisomal matrix proteins contain a C-terminal PTS1 taigeting sequence;a few have an N-terminal PTS2 targeting sequence.Neither targeting sequenceis cleaved after import. r All proteins destinedfor the peroxisomal matrix bind to a cytosolic carrier protein, which differs for PTSI- and PTS2-bearingproteins' and then are directed to common import receptor and translocation machinery on the peroxisomal membrane (seeFigure 13-29). r Translocation of matrix proteins acrossthe peroxisomal membrane dependson ATP hydrolysis. Many peroxisomal matrix proteins fold in the cytosol and traverse the membrane in a folded conformation, which is different than for protein import into organellessuch as the ER' mitochondrion, and chloroplast. r Proteinsdestinedfor the peroxisomal membranecontain different targeting sequencesthan peroxisomal matrix proteins and are imported by a different pathway' r Unlike mitochondria and chloroplasts, peroxisomescan arise de novo from precursor membranesprobably derived from the ER as well as by division of preexistingorganelles ( s e eF i g u r e1 3 - 3 1 ) .

TransPortinto and out of the Nucleus The nucleus is separatedfrom the cytoplasm by two membranes,which form the nuclearenvelope(seeFigure 9-1)' The nuclear envelopeis continuous with the ER and forms a part T R A N S P O R ITN T O A N D O U T O F T H E N U C L E U S

.

569

of it. Transport of proteins from the cytoplasm into the nucleus and movement of macromolecules,including mRNps, tRNAs, and ribosomal subunits, out of the nucleus occur through nuclearpores,which span both membranesof the nuclear envelope. Import of proteins into the nucleus shares some fundamental features with protein import into other organelles. For example, imported nuclear proteins carry specific targeting sequencesknown as nuclear localization sequences, or NLSs. However, proteins are imported into the nucleusin a folded state,and thus nuclear import differs fundamentally from protein translocationacrossthe membranes of the ER, mitochondrion, and chloroplast,where proteins are unfolded during translocation.In this section we discussthe main mechanism by which proteins and some ribonuclear proteins such as ribosomesenter and exit the nucleus.\Wewill also discusshow mRNAs and other ribonuclear protein complexesare exported from the nucleusby a processthat differs mechanisticallyfrom nuclear protein import.

L a r g ea n d S m a l lM o l e c u l e sE n t e ra n d L e a v et h e Nucleusvia NuclearPoreComplexes Numerous pores perforate the nuclearenvelopein all eukaryotic cells. Each nuclear pore is formed from an elaborate

structure termed the nuclear pore complex (NPC), which is one of the largest protein assemblagesin the cell. The total massof the pore structureis 60-80 million Da in vertebrates, which is about 15 times larger than a ribosome. An NpC is made up of multiple copies of some 30 different proreins called nucleoporins. Electron micrographs of nuclear pore complexesreveal a roughly octagonal,membrane-embedded structure from which eight approximately 1O0-nm-longfilaments extend into the nucleoplasm(Figure 13-32). The distal ends of thesefilaments are joined by the terminal ring, forming a structure called the nuclear basket. The membraneembeddedportion of the NPC is also attacheddirectly to the nuclear lamina, a network of lamin intermediate filaments that form a meshwork extending over the inner surface of the nuclear envelope (seeFigure 20-1,6). Cytoplasmic filaments extend from the cytoplasmic side of the NPC into the cytosol. Ions, small metabolites, and globular proreins up to 2040 kDa can passivelydiffuse through the central aqueous region of the nuclear pore complex. However, large proteins and ribonucleoprotein complexescannot diffuse in and out of the nucleus. Ratheq these macromolecules are actively transported through the NPC with the assistanceof soluble transporter proteins that bind macromoleculesand also interact with nucleoporins.

(b)

Cytoplasm

Cytoplasmic filaments

O u t e rn u c l e a r memDrane

Nu c l e a r envelope

I n n e rn u c l e a r membrane Nucleoplasm N u c l e a rl a m i n a Nuclearbasket T e r m i n a rl i n g

A FIGURE 13-32Nuclearporecomplex.(a)Nuclear envelopes mrcrodissected fromthe largenucleiof Xenopus oocytes visualized by fieldemission in-lens scanning electron microscopy. Iop:Vrewof the cytoplasmic facereveals octagonal shapeof memorane_ embedded portionof nuclear porecomplexes Bottom: Viewof the 570

'

c H A p r E R1 3 |

nucleoplasmic faceshowsthe nuclear basket thatextends fromthe membrane portion.(b)Cutaway modelof the porecomplex. [part(a) fromV DoyeandE Hurt,1997, CurrOpinCellBiol9:401; courtesy of M W G o l d b e r ga n d T . D A l l e n P a r t( b ) a d a p t e df r o m M p R o u ta n d J D A t c h i s o n . 2001, J Biol. Chem. 276:165931

M o v r N Gp R o r E r N sr N T oM E M B R A N EAsN D o R G A N E L L E '

tLs

s n l l ) n N l H l _r o t n o c N V o t - N tl u o d s N V U l _

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The mechanismfor import of cytoplasmiccargo proteins mediated by an importin is shown in Figure 13-35 (the general mechanismis the samefor either a monomeric or dimeric importin). Freeimportin in the cytoplasm binds to its cognate NLS in a cargo protein, forming a bimolecwlar cargo complex. The cargo complex then translocatesthrough the NPC channel as the importin B subunit interacts with a class of nucleoporins called FG-nucleoporins. These nucleoporins, which line the channel of the nuclear pore complex and also are found in the nuclear basket and the cytoplasmic filaments, contain multiple repeats of short hydrophobic sequencesrich in phenylalanine (F) and glycine (G) residues (FG-repeats).The hydrophobic FG-repeatsare thought to occur in regionsof extended,otherwisehydrophilic polypeptide chains that fill the central transporter channel and in some way allow the relatively hydrophobic importin complexesto traverse the channel efficiently while excluding unchaperoned hydrophilic proteins larger than 20-40 kDa. 'Sfhen the cargo complex reachesthe nucleoplasm, the importin interacts with Ran.GTP, causing a conformational changein the importin that decreasesits affinity for the NLS, releasing the cargo protein into the nucleoplasm. The importin-Ran.GTP complex then diffuses back through the NPC. Once the importin-Ran.GTP complex reachesthe cytoplasmic side of the NPC, Ran inreracts with a specific GTPase-actiuatingprotein (Ran-GAP) that is a component of the NPC cytoplasmic filaments. This stimulates Ran to hydrolyze its bound GTP to GDP, causing it to convert to a

Ra n ' G D P R a n . G T P

conformation that has low affinity for the importin, so that the free importin is releasedinto the cytoplasm, where it can participate in another cycle of import. Ran.GDP travels back through the pore to the nucleoplasm,where it encountersa specificgwaninenucleotide-exchange factor (Ran-G EF) that causesRan to releaseits bound GDP in favor of GTP. The net result of this seriesof reactionsis the coupling of the hydrolysis of GTP to the transfer of an NlS-bearing protein from the cytoplasm to the nuclear interior, thus providing a driving force for nuclear transport. The import complex travels through the pore by diffusion, a random process.Yet transport is unidirectional.The direction of transport is a consequenceof the rapid dissociation of the import complex when it reachesthe nucleoplasm.As a result, there is a concentration gradient of the importin-cargo complex across the NPC: high in the cytoplasm, where the complex assemblesand low in the nucleoplasm,where it dissociates.This concentration gradient is responsiblefor the unidirectional nature of nuclear import. A similar concentration gradient is responsiblefor driving the importin in the nucleus back into the cytoplasm. The concentration of the importinRan.GTP complex is higher in the nucleoplasm,where it assembles,than on the cytoplasmicsideof the NPC, where it dissociates.Ultimately the direction of the transporr processesis dependenton the asymmetric distribution of the Ran-GEF and the Ran-GAP. Ran-GEF in the nucleoplasm maintains Ran in the Ran.GTP state,where it promotesdissociationof the cargo complex. Ran-GAP on the cytoplasmic side of the NPC

lmportin

ti

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M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

< FIGURE 13-35 Nuclearimport. M e c h a n i sf m o r n u c l e ai m r p o rot f " c a r g o " proteins. (boftorn), In the cytoplasm a free importinbindsto the NLSof a cargoprotein, forminga bimolecular cargocomplex. ln the caseof a basicNLS,theadapterprotein importino bridges the NLSandimportinp, (not forminga trimolecular cargocomplex shown). Thecargocomplex diffuses through the NPCby interacting with successive FGnucleoporins ln the nucleoplasm, interaction of Ran.GTP withthe imoortincauses a conformational change thatdecreases its affinityfor the NLS,releasing the cargoTo supportanothercycleof import,the importinRan'GTP complex istransported backto the cytoplasm. A GTPase-accelerating protein (GAP) associated with thecytoplasmic filaments of the NPCstimulates Ranto hydrolyze the boundGTPThisgenerates a conformational changecausing dissociation fromthe importin, whichcantheninitiate anotherroundof importRan.GDP isreturned t o t h en u c l e o p l a swmh,e r ea g u a n i n e nucleotide-exchange factor(GEF) causes release of GDPandrebindinq of GTP

converts Ran.GTP to Ran.GDP, dissociatingthe importinRan.GT? complex and releasingfree importin into the cytosol.

ExportinsTransportProteinsContaining N u c l e a r - E x p oS r ti g n a l so u t o f t h e N u c l e u s A very similar mechanism is used to export proteins, tRNAs, and ribosomal subunits from the nucleus to the cytoplasm. This mechanisminitiallv was elucidatedfrom studiesof certain ribonuclear protein complexes that "shuttle" between the nucleus and cytoplasm. Such "shuttling" proteins contain a nuclear-export signal /NES/ that stimulates their export from the nucleusto the cytoplasmthrough nuclearpores,in addition to an NLS that results in their uptake into the nucleus.Experiments with engineered hybrid genes encoding a nucleusrestricted protein fused to various segmentsof a protein that shuttles in and out of the nucleus have identified at least three different classesof NESs: a leucine-rich sequencefound in PKI (an inhibitor of protein kinaseA) and in the Rev protein of human immunodeficiency virus (HIV), as well as tvvo sequences identified in fwo different heterogeneousribonucleoprotein particles (hnRNPs). The functionally significant structural features that specifynuclearexport remain poorly understood. The mechanismwhereby shuttling proteins are exported from the nucleus is best understood for those containing a leucine-richNES. According to the current model, shown in Figure 13-36a, a specificexportin, or nuclear-exportreceptor, in the nucleus,called exportin 1, first forms a complex with Ran.GTP and then binds the NES in a cargo protein. Binding of exportin 1 to Ran.GTP causesa conformational changein exportin 1 that increasesits affinity for the NES so that a trimolecular cargo compler is formed. Like importins, exportin 1 interacts transiently with FG-repeatsin FG-nucleoporins and diffusesthrough the NPC. The cargo complex dissociates when it encountersthe Ran-GAP in the NPC cytoplasmicfilaments, which stimulates Ran to hydrolyze the bound GTI shifting it into a conformation that has low affinity for exportin 1. The releasedexportin 1 changesconformation to a structurethat has low affinity for the NES, releasingthe cargo into the cytosol. The direction of the export processis driven by this dissociationof the cargo from exportin 1 in the cytoplasm, which causesa concentration gradient of the cargo complex acrossthe NPC that is high in the nucleoplasmand low in the cytoplasm.Exportin 1 and the Ran'GDP are then transportedback into the nucleusthrough an NPC. By comparing this model for nuclear export with that in Figure 13-35 for nuclear import, we can seeone obvious difference:Ran.GTP is part of the cargo complex during export but not during import. Apart from this difference,the two transport processesare remarkably similar.In both processes, associationof a transport signal receptor with Ran'GTP in the nucleoplasmcausesa conformational changethat affects its affinity for the transport signal. During import, the interaction causesreleaseof the cargo, whereasduring export, the interaction promotes associationwith the cargo. In both export and import, stimulation of Ran'GTP hydrolysis in the cytoplasm by Ran-GAP producesa conformational changein Ran that releasesthe transport signal receptor. During nu-

clear export, the cargo is also released.Importins and exportins both are thought to diffuse through the NPC channel by successiveinteractions with FG-repeatsin FG-nucleoporins. Localization of the Ran-GAP and -GEF to the cytoplasm and nucleus,respectivelSis the basisfor the unidirectional transport of cargo proteins acrossthe NPC. In keeping with the similarity in function of importins and exportins, the two types of transport proteins are highly homologous in sequenceand structure. The entire family is called the importin B familS or karyopherins. There are'l'4 karyopherins in yeastand more than20 in mammalian cells. The NESs or NLSs to which they bind have been determined for only a fraction of them. Remarkably, some individual karyopherins function as both an importin and an exportin. A similar shuttling mechanismhas beenshown to export other cargoes from the nucleus. For example, exportin-t functions to export tRNAs. Exportin-t binds fully processed tRNAs in a complex with Ran'GTP that diffuses through NPCs and dissociateswhen it interacts with Ran-GAP in the NPC cytoplasmic filaments, releasingthe IRNA into the cytosol. A Ran-dependentprocess is also required for the nuclear export of ribosomal subunits through NPCs once the protein and RNA components have been properly assembledin the nucleolus. Likewise, certain specific mRNAs that associate with particular hnRNP proteins can be exported by a Ran-dependentmechanism.

Most mRNAsAre Exportedfrom the Nucleusby a R a n - l n d e p e n d e nMt e c h a n i s m Once the processingof an mRNA is completedin the nucleus, it remains associatedwith specifichnRNP proteins rn a messengerribonuclear protein complex, or mRNP. The principle transporter of mRNPs out of the nucleus is the nRNP exporter, a heterodimericprotein composedof a large subunit called nwclearexport factor 1 (NXFI) or TAP and a small subunit, nuclear export transporter L (Nxtl). The large subunit binds to nuclear mRNPs through cooperative interactions with the RNA and other mRNP adapter proteins that associatewith nascentpre-mRNAs during transcription elongation and pre-mRNA processing.It seemslikely that multiple TAPNxtl mRNP exportersbound along the length of an nRNP assistin its export. TAPAJxII acts like a karyopherin in the sensethat both subunits interact with the FG-domains of FG-nucleoporins, allowing them to diffuse through the central channel of the NPC. Tap also binds reversiblyto the protein Gle2, which in turn binds a nucleoporin in the nuclear basket, presumably positioning the mRNP for export through the nuclear pore. A nucleoporin in the cytoplasmic filaments of the NPC is also required for mRNP export. This nucleoporin binds an RNA helicasethat is proposed to function in the dissociationof the mRNP exporter and other hnRNP proteins from the mRNP as it reachesthe cytoplasm. The TAP/Nxt1 mRNP exporters do not appear to interact with Ran, and thus the unidirectional transport of mRNA out of the nucleusrequiresa sourceof energyother than GTP hydrolysis by Ran. As the mRNP complex is transported through an NPC, the proteins associatedwith INTOAND OUT OF THE NUCLEUS TRAN5PORT

.

573

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< FIGURE 13-36Ran-dependent and Ran-independent nuclearexport.(a)Randependent mechanism for nuclear exportof cargoproteins containing a leucine-rich nuclearexportsignal(NES)In the nucleoplasm, the proteinexportin1 bindscooperatively to the NESof thecargoproteinto betransported andto Ran.GTP Aftertheresulting cargo complexdiffuses throughan NPCviatransient interactions with FGrepeats in FG-nucleoporins, the Ran-GAP associated with the NPCcytoplasmic filaments stimulates conversion of Ran.GTP to Ran.GDP Theaccompanying conformational changein Ranleadsto dissociation of the complexTheNES-containing cargoproteinis released intothe cytosol, whereas exportin1 andRan.GDP are transported backintothe nucleus through N P C sR a n - G Ei nFt h en u c l e o p l a tshme n stimulates conversion of Ran.GDP to Ran.GTP. (b)Ran-independent nuclear exportof mRNAs Theheterodimeric TAP/Nxt1 complex bindsto (mRNPs) mRNA-protein complexes in the nucleusAssociation of TAP/Nxt1 with components of the NPCdirecttheassociated mRNP to the centralchannel of the pore.An (Dbp5)located RNAhelicase on thecytoplasmic sideof the NPCisthoughtto provide the drivingforceby hydrolysis for movingthe mRNP throughthe pore Thehelicase also freesthe mRNAfromTAPandNxtl proteins, whicharerecycled backintothe nucleus by the Ran-dependent importprocess depicted in F i g u r 1e 3 - 3 5

Trcnscription Re-import

it are exchanged for another set of proteins in the cytoplasm, a processcalled mRNP remodeling (Figure 13-36b). Severalnuclear mRNP proteins dissociatefrom the mRNp before it reachesthe cytoplasmic side of the NpC. These remain in the nucleus, where they bind to newly synthesized nascentpre-mRNA. Other nuclear mRNP proteins, including the TAPA{xt1 mRNP exporter, are exported through NPCs into the cytoplasm. Once they reach the cytoplasmic side of the NPC, they dissociate from the mRNp with the 574

.

c H A p r E R1 3 |

help of the RNA helicase,Dbp5, which associateswith cytoplasmic NPC filaments. Recall that RNA helicasesuse the energy derived from hydrolysis of ATP to move along RNA molecules,separatingdouble-strandedRNA chains and dissociatingRNA-protein complexes(Chapter4). This leads to the simple idea that Dpb5, which associateswith the cytoplasmic side of the nuclear pore complex, acts as an ATP-driven motor to move mRNP complexes through the nuclear pore.

M o v r N Gp R o r E r N sr N T o M E M B R A N EASN D o R G A N E L L E s

After remodelingis completed,the TAP and Nxtl proteins that have beenstrippedfrom the mRNA by Dbp5 helicaseare imported back into the nucleus by an importin, where they can function in the export of another mRNP. Consequently, thesenuclear mRNP proteins shuttle between the nucleus and cytoplasm,carrying mRNPs through NPCs (Figure 13-36b).

r Most mRNPs are exported from the nucleus by a heterodimeric mRNP exporter that interacts with FG-repeats of FG-nucleoporins.The direction of transport (nucleusto cytoplasm) may result from the action of an RNA helicase associatedwith the cytoplasmic filaments of the nuclear pore complexes.

Transport into and out of the Nucleus r The nuclear envelope contains numerous nuclear pore complexes(NPCs), large, complicated structurescomposed of multiple copies of 30 proteins called nucleoporins (see Figure 1,3-32).FG-nucleoporins,which contain multiple repeatsof a short hydrophobic sequence(FG-repeats),line the central transporter channel and play a role in transport of all macromoleculesthrough nuclear pores. r Transport of macromolecules larger than 20-40 kDa through nuclear pores requires the assistanceof proteins that interact with both the transported molecule and FGrepeatsof FG-nucleoporins. r Proteins imported to or exported from the nucleus contain a specificamino acid sequencethat functions as a nuclear-localization signal (NLS) or a nuclear-export signal (NES). Nucleus-restrictedproteins contain an NLS but not an NES, whereasproteins that shuttle betweenthe nucleus and cytoplasm contain both signals. r Severaldifferent types of NES and NLS have been identified. Each type of nuclear-transport signal is thought to interact with a specific receptor protein (exportin or importin) belonging to a family of homologous proteins termed karyopherins. r A "cargo" protein bearing an NES or NLS translocates through nuclear pores bound to its cognatereceptorprotein (karyopherin), which also interacts with FG-nucleoporins. Importins and exportins are thought to diffuse through the channel, filled with a hydrophobic matrix of FG-repeats. Both transport processesalso require participation of Ran, a monomeric G protein that exists in different conformations when bound to GTP or GDP. r After a cargo complex reachesits destination (the cytoplasm during export and the nucleusduring import), it dissociates,freeing the cargo protein and other components. The latter then are transportedthrough nuclearpores in the reversedirection to participate in transporting additional moleculesof cargo protein (seeFigures13-35 and 13-36). r The unidirectional nature of protein export and import through nuclear pores results from localization of the Ran guanine nucleotide-exchangefactor (GEF) in the nucleus and of Ran GTPase-activatingprotein (GAP) in the cytoplasm. The interaction of import cargo complexeswith the Ran-GTP in the nucleoplasmcausesdissociationof the complex, releasingthe cargo into the nucleoplasm(seeFigure 13-35). Export cargo complexesdissociatein the cytoplasm when they interact with Ran'GAP localized to the NPC cytoplasmic filaments (seeFigure 13-36).

As we have seen in this chapter, we now understand many aspectsof the basic processesresponsiblefor selectively transporting proteins into the endoplasmic reticulum (ER), mitochondrion, chloroplast, peroxisome, and nucleus. Biochemical and genetic studies, for instance, have identified cis-actingsignal sequencesresponsiblefor targeting proteins recepto the correct organellemembrane and the membrane 'We also have tors that recognize these signal sequences. learnedmuch about the underlying mechanismsthat translocate proteins across organelle membranes and have determined whether energyis usedto push or pull proteins across the membrane in one direction, the type of channel through which proteins pass, and whether proteins are translocated in a folded or an unfolded state. Nonetheless'many fundamental questions remain unanswered' including how fully folded proteins move acrossa membrane and how the topology of multipass membrane proteins is determined. The peroxisomal import machinery provides one example of the translocation of folded proteins. It not only is capable of translocating fully folded proteins with bound cofactors into the peroxisomal matrix but can even direct the import of a large gold particle decoratedwith a (PTS1)peroxisomal targetingpeptide.Someresearchershave speculated that the mechanismof peroxisomal import may be related to that of nuclear import, the best-understoodexample of posttranslational translocation of folded proteins. Both the peroxisomal and nuclear import machinery can transport folded moleculesof very divergentsizes,and both appear to involve a component that cycles between the cytosol and the organelle interior-the Pex5 PTS1 receptor in the caseof peroxisomal import and the Ran-importin complex in the case of nuclear import. However, there also appear to be crucial differencesbetweenthe two translocation processes.For example, nuclear pores representlarge, stable macromolecular assembliesreadily observedby electron microscopy,whereas analogousporelike structureshave not been observedin the peroxisomal membrane. Moreover' small molecules can readily pass through nuclear pores, whereas peroxisomal membranesmaintain a permanent barrier to the diffusion of small hydrophilic molecules.Taken together,these observations suggestthat peroxisomalimport may require an entirely new type of translocation mechanism. The evolutionarily conservedmechanismsfor translocating folded proteins acrossthe cytoplasmicmembraneof bacterial cells and across the thylakoid membrane of chloroplasts also are poorly understood.A better understandingof all of theseprocessesfor translocatingfolded proteins across P E R S P E C T I VFEO SRT H E F U T U R E

575

a membrane will likely hinge on future development of in vitro translocation systemsthat allow investigatorsto define the biochemical mechanisms driving translocation and to identify the structuresof trapped translocationintermediates. Compared with our understandingof how soluble proteins are translocatedinto the ER lumen and mitochondrial matrix, our understanding of how cis-acting sequencesspecify the topology of multipass membrane proteins is quite elementary. For instance,we do not know how the translocon channel accommodatespolypeptidesthat are oriented differently with respect to the membrane, nor do we understand how local polypeptide sequencesinteract with the translocon channel both to set the orientation of transmembranespansand to signal for lateral passageinto the membrane bilayer. A better understanding of how the amino acid sequencesof membrane proteins can specify membrane topology will be crucial for decoding the vast amount of structural information for membrane proteins contained within databasesof genomic sequences. A more detailed understanding of all translocation processesshould continue to emergefrom genetic and biochemical studies, both in yeasts and in mammals. These studies will undoubtedly reveal additional key proteins involved in the recognition of targeting sequencesand in the translocation of proteins across lipid bilayers. Finally, the structural studies of translocon channels will likely be extended in the future to reveal at resolutions on the atomic scalethe conformational statesthat are associatedwith each step of the translocation cycle.

KeyTerms biomolecular cargo complex 572 cotranslational translocation 537 dislocation 555 dolichol phosphate 550 exportin 573 FG-nucleoporins572 generalimport pore 559 hydropathy profile 548 importins 571 karyopherins 574

post-translational translocation 540 Ran protein 5Z1 signal-anchorsequence544 signal-recognitionparticle

(sRP) 537 signal (uptake-targeting) sequences535 stop-transferanchor sequence544 topogenic sequences543

molecular chaperones541 Nlinked oligosaccharides550 nuclear pore complex (NPC)570

topology of a membrane protein 543 translocon 539 trimolecular cargo complex J,/J unfolded-protein response555 1

O-linked oligosaccharides550

F - ^

Review the Concepts 7. Describethe sourceor sourcesof energyneededfor unidirectional translocation across the membrane in (a) co576

CHAPTER 13

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translational translocation into the endoplasmic reticulum (ER); (b) post-translationaltranslocation into the ER; (c) translocation across the bacterial cytoplasmic membranel and (d) translocation into the mitochondrial marnx. 2. Translocation into most organellesusually requires the activity of one or more cytosolic proteins. Describethe basic function of three different cytosolic factors required for translocation into the ER, mitochondria, and peroxisomes, respectively. 3. Describethe typical principles usedto identify topogenic sequenceswithin proteins and how thesecan be used to develop computer algorithms. How does the identification of topogenic sequenceslead to prediction of the membrane arrangementof a multipass protein? What is the importance of the arrangement of positive chargesrelative to the mem, brane orientation of a signal-anchorsequence? 4. The endoplasmicreticulum (ER) is an important site of "quality control" for newly synthesizedproteins. \7hat is meant by "quality control" in this context? I7hat accessory proteins are typically involved in the processingof newly synthesizedproteins within the ER? Cells generallydegradeER'Sfhere exit-incompetent proteins. within the cell does such degradation occur and what is the relationship of the Sec61 protein translocon and p97 to the degradationprocess? 5. Temperature-sensitive yeast mutants have been isolated that block each of the enzymatic stepsin the synthesisof the dolichol-oligosaccharideprecursor for N-linked glycosylation (seeFigure 1,3-1,7). Proposean explanation for why mutations that block synthesis of the intermediate with the structure dolichol-PP-(GlcNAc)2Man5 completely prevent addition of N-linked oligosaccharide chains to secretory proteins, whereas mutations that block conversion of this intermediate into the completed precursor-dolichol-PP(GlcNAc)2ManeGlca-allow the addition of N-linked oligosaccharidechains to secretoryglycoproteins. 6. Name four different proteins that facilitate the modification and/or folding of secretoryproteins within the lumen of the ER. Indicate which of theseproteins covalently modifies substrateproteins and which brings about only conformational changesin substrateproteins. 7. Becauseyou are interestedin studying how a particular secretoryprotein folds within the ER, you wish to determine whether BiP binds to the newly synthesizedprotein in ER extracts. You find that you can isolate some of the newly synthesizedsecretoryprotein bound to BiP when ADP is added to the cell extract but not when ATP is added to the extract. Explain this result basedon the mechanism for BiP binding to substrateproteins. 8. Describewhat would happen to the precursor of a mitochondrial matrix protein in the following types of mitochondrial mutants: (a) a mutation in the Tom22 signal receptor, (b) a mutation in the Tom70 signal receptor, (c) a mutation in the matrix Hsc70, and (d) a mutation in the matrix signal peptidase. 9. Describe the similarities and differences between the mechanismof import into the mitochondrial matrix and the chloroplast stroma.

M O V T N GP R O T E t NtSN T O M E M B R A N E A SN D O R G A N E L L E S

10. Design a set of experimentsusing chimeric proteins, composed of a mitochondrial precursor protein fused to dihydrofolate reductase(DHFR), that could be used to determine how much of the precursor protein must protrude into the mitochondrial matrix in order for the matrix-targeting sequenceto be cleaved by the matrix-processing protease (seeFigure 1,3-24). 11. Protein targeting to both mitochondria and chloroplasts involves the sorting of proteins to multiple sites within the respectiveorganelle.Briefly list thesesites.Taking the mitochondrion as an example and the proteins ADP/ATP anti-porter and cytochrome b2 as the specific cases,compareand contrast the extent to which a common mechanism is used for the site-specifictargeting of these two protelns. 12. Peroxisomescontain enzymesthat use molecular oxygen to oxidize various substrates,but in the processhydrogen peroxide forms and must be degraded.lfhat is the name of the enzyme responsiblefor the breakdown of hydrogen peroxide to water and what mechanismand associatedproteins allow for its import into the peroxisome? 13. Supposethat you have identified a new mutant cell line that lacks functional peroxisomes.Describe how you could determine experimentally whether the mutant is primarily defective for insertion/assemblyof peroxisomal membrane protelns or matrlx protelns. 14. Evidencethroughout Chapter 13 revealsthat specific motifs within polypeptidesare necessaryto direct or target these proteins acrossmembranesand into organelles. The nuclear import of proteins having a molecular mass more than approximately 40 kDa is no different, and they must be actively imported through nuclear pore complexes.What is the name given to the amino acid sequence that allows the selective transport of macromolecular cargo proteins into the nucleus?Name three proteins that are required for this import and briefly describehow they function. 'Sfhy 15. is localization of Ran-GAP in the nucleusand RanGEF in the cytoplasm necessaryfor unidirectional transport of cargo proteins containing an NES?

Analyze the Data Imagine that you are evaluating the early stepsin translocation and processingof the secretoryprotein prolactin. By using an experimental approach similar to that shown in Figure 1.3-7,you can usetruncated prolactin mRNAs to control the length of nascentprolactin polypeptidesthat are synthesized.\fhen prolactin mRNA that lacks a chain-termination (stop) codon is translated in vitro, the newly synthesized polypeptide ending with the last codon included on the mRNA will remain attached to the ribosome, thus allowing a polypeptide of defined length to extend from the ribosome. You have generateda set of mRNAs that encodesegmentsof the N-terminus of prolactin of increasing length, and each mRNA can be translated in vitro by a cytosolic translation extract containing ribosomes,tRNAs, aminoacyl-tRNA syn-

thetases,GTP, and translation initiation and elongation fac'When radiolabeled amino acids are included in the tors. translation mixture, only the polypeptide encoded by the addedmRNA will be labeled.After completion of translation, each reaction mixture was resolved by SDS poly-acrylamide gel electrophoresis,and the labeled polypeptides were identified by autoradiography. a. The autoradiogram depicted below shows the results of an experiment in which each translation reaction was carriedout eitherin the presence(+) or the absence(-) of microsomal membranes.Basedon the gel mobility of peptides synthesizedin the presenceor absenceof microsomes, deducehow long the prolactin nascentchain must be in order for the prolactin signal peptide to enter the ER lumen and to be cleaved by signal peptidase. (Note that microsomes carry significant quantities of SRP weakly bound to the membranes.)

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Given this length, what can you conclude about the b. conformational state(s)of the nascentprolactin polypeptide when it is cleavedby signal peptidase?The following lengths will be useful for your calculation: the prolactin signal sequenceis cleavedafter amino acid 31; the channel within the ,iboro-. occupied by a nascentpolypeptide is about 150 A long; a membrane bilayer is about 50 A thick; in polypeptides with an a-helical conformation, one residue extends 1.5 A, whereas in fully extended polypeptides, one residue extendsabout 3.5 A. c. The experimentdescribedin part (a) is carried out in an identical manner except that microsomal membranes are not present during translation but are added after translation is complete. In this case none of the samples shows a difference in mobility in the presenceor absence of microsomes. lfhat can you conclude about whether prolactin can be translocated into isolated microsomes posttranslationally? d. In another experiment, each translation reaction was carried out in the presenceof microsomes,and then the microsomal membranes and bound ribosomes were separated from free ribosomes and soluble proteins by centrifugation. For each translation reaction' both the total reaction (T) and the membrane fraction (M) were resolved in neighboring gel lanes. Basedon the amounts of labeled polypeptide in the membrane fractions in the autoradiogram depicted beloq deduce how long the prolactin nascent chain A N A L Y Z ET H E D A T A

577

must be in order for ribosomesengagedin translation to engage the SRP and thereby become bound to microsomal membranes.

Tsai, B., Y. Ye, and T. A. Rapoport.2002. Retro-translocation of proteins from the endoplasmicreticulum into the cytosol. Nature Reu.Mol. Cell Biol. 3:246-255 . Sorting of Proteins to Mitochondria and Chloroplasts

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References Translocation of Secretory Proteins Across the ER Membrane Egea,P. F., R. M. Srroud,and P. Walter.2005. Targetingproteins to membranes:structureof the signalrecognitionparticle. Curr. Opin. Struct. Biol. 75:213-220. Osborne,A. R., T. A. Rapoport, and B. van den Berg.2005. Protein translocationby the Sec51/SecY channel.Annu. Reu.Cell D e u .B i o l . 2 l : 5 2 9 - 5 5 0 . 'Wickner, S7.,and R. Schekman.2005. Protein rranslocation acrossbiological membranes.Science310:L452-1456. Insertion of Proteins into the ER Membrane Englund, P.T.1993. The structureand biosynthesisof glycosylphosphatidylinositolprotein anchors.Ann. Reu.Biochem.. 62:121-1.38. Goder,V., and M. Spiess.2001. Topogenesisof membraneproteins: determinantsand dynamics.FEBSLett. 504:87-93. Mothes, I7., et al. 1997. Molecular mechanismof membrane protein integrationinto the endoplasmicreticulum. Cel/ 89:523-53J. von Heijne, G. 1999. Recentadvancesin the understandingof membraneprotein assemblyand structure.Q. Reu.Biophys. 32:285-307. Protein Modifications, Folding, and euality Control in the ER . Helenius,A., and M. Aebi. 2004. Rolesof Nlinked glycansin the endoplasmicreticulum. Annu. Reu.Biochem. 73:1019-1049. Kornfeld, R., and S. Kornfeld. 1985. Assemblyof asparagine. linked oligosaccharides. Ann. Reu.Biochem. 45:631,-6G4. 'Walter. Patil, C., and P. 2001. Intracellularsignalingfrom the endoplasmicreticulum to the nucleus:the unfolded protein response in yeastand mammals. Curr. Opin. Cell Biol.73:349-355. Meusser,B., C. Hirsch, E. Jarosch,and T. Sommer.2005. ERAD: the long road ro destruction.Natwre Celt Biol.7:766-772. Sevier,C. S., and C. A. Kaiser.2002.Formation and transfer of disulphidebonds in living cells.Nature Reu.Mol. Cetl Biot. 3:836-847. . .Silberstein,S., and R. Gilmore. 1995. Biochemistry,molecular brology,and geneticsof the oligosaccharyltransferase. FASEB/. 10:849-85 8. Trombetta,E. S., and A. J. Parod. 2003. Quality control and protein folding in the secretorypathway.Annu. Reu.Cell Deu. Biol. 19:649-676.

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Koehler,C. M. 2004. New developmentsin mitochondrial assembly.Ann. Reu.Cell Deu. Biol.20:309-335. Dolezal,P.,V. Likic, J.Tachezy,and T. Lithgow 2006. Evolution of the molecular machinesfor protein import into mitochondria. Science313:31.4-31.8. Dalbey,R. E., and A. Kuhn. 2000. Evolutionarily relatedinsertion pathways of bacterial,mitochondrial, and thylakoid membrane proteins.Ann. Reu.Cell Deuel. Biol. 16:51-87. Matouschek,A., N. Pfanner,and S7.Voos. 2000. Protein unfolding by mitochondria:the Hsp70 import motor. EMBO Rept. l:404410. Neupert,'$7.,and M. Brunner.2002.The protein import motor of mitochondria.Nature Reu.Mol. Cell Biol.3:555-565. Rapaport, D. 2005. How doesthe TOM complex mediateinsertion of precursorproteins into the mitochondrial outer membrane? I. Cell Biol. 17l:479423. Robinson,C., and A. Bolhuis.2001. Protein targeringby the twin-argininetranslocationpathway.Nature Reu.Mol. Cell Biol. 2:350-356. Soll, J., and E. Schleiff.2003. Protein import into chloroplasts. Nature Reu.Mol. Cell Biol.5:198-208. Truscott, K. N., K. Brandner,and N. Pfanner.2003.Mechanismsof protein import into mitochondria. Curr. Biol. 13:R325-R337. Sorting of Peroxisomal Proteins Dammai, V., and S. Subramani.2001. The human peroxisomal targetingsignalreceptor,Pex5p,is translocatedinto the peroxisomal matrix and recycledto the cytosol. Cell 105:1,87-1,96. Gould, S. J., and C. S. Collins. 2002. Opinion: peroxisomal-protein import: is it really that complex?Nature Reu.Mol. Cell Biol. 3:382-389. Gould, S.J., and D. Valle. 2000. Peroxisomebiogenesisdisorders:geneticsand cell biology. TrendsGenet. 16:340-345. Hoepfner,D., D. Schildknegt,I. Braakman,P. Philippsen,and H. F. Tabak. 2005. Contribution of the endoplasmicreticulum to peroxisomeformation.Cell 122:85-9 5. Purdue,P. E., and P. B. Lazarow.2001. Peroxisomebiogenesis. Ann. Reu.Cell Deuel. Biol. 17:701-752. Subramani,S., A. Koller, and W. B. Snyder.2000. Import of peroxisomal matrix and membraneproteins.Ann. Reu.Biochem. 69:3994].8. Transport into and out of the Nucleus Chook, Y. M., and G. Blobel. 2001. Karyopherinsand nuclear import. Curr. Opin. Struct. Biol. ll:703-71,5. Cole, C. N., and J. J. Scarceili.2006. Transport of messenger RNA from the nucleusto the cytoplasm.Curr. Opin. Cell Biol. 18299-306. Johnson,A. \f., E. Lund, and J. Dahlberg.2002. Nuclear export of ribosomalsubunits.TrendsBiochem.Sci.27:580-585. Ribbeck, K., and D. Gorlich. 2001. Kinetic analysisof translocation through nuclearpore complexes.EMBO J. 20:1,320-1330. Rout, M. P., and J. D. Aitchison. 2001. The nuclearpore complex as a transport machine.J. Biol. Chem. 276:76593-1,6596. Schwartz,T. U. 2005. Modularity within the architectureof the nuclearpore complex. Curr. Opin. Struct. Biol. L5:221-226. Suntharalingam,M., and S. R. Wente.2003. Peeringthrough the pore: nuclearpore complex structure,assembly,and function. Deu. Cell.4:775-789.

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CHAPTER

TRAFFIC, VESICULAR AND SECRETION, ENDOCYTOSIS showingthe formationof clathrinelectronmicrograph Scanning on the cytosolic faceof the plasmamembrane coatedvesicles Washington School of Medicine University ] fJohnHeuser,

I n the previouschapter we explored how proteins are tarI g.ted to and translocatedacrossthe membranesof several I different intracellular organelles,including the endoplasmic reticulum, mitochondria and chloroplasts,peroxisomes,and the nucleus. In this chapter we turn our attention to the secretory pathway and the mechanismsthat allow soluble and membrane proteins to be deliveredto the plasma membrane and the lysosome. Ii7e will also discuss the related processesof endocytosisand autophagy,which deliver proteins and small moleculesfrom either outsidethe cell or from the cytoplasmto the interior of the lysosomefor degradation. Soluble and membrane proteins slated to function at the cell surfaceor in the lysosomeare transported to their final destination via the secretorypathway. Proteins delivered to the plasma membrane include cell-surfacereceptors, transporters for nutrient uptake, and ion channelsthat maintain the proper ionic and electrochemicalbalance across the plasma membrane.Solublesecretedproteins follow the same pathway to the cell surface as plasma membrane proteins, but instead of remaining embedded in the membrane, secreted proteins are releasedinto the aqueous extracellular environment in soluble form. Examples of secretedproteins are digestive enzymes, peptide hormones, serum proteins, and collagen. As describedin Chapter 9, the lysosomeis an organelle with an acidic interior that is generally used for degradation of unwanted proteins and the storage of small molecules such as amino acids. AccordinglS the types of proteins delivered to the lysosomal membrane are subunits of the V-classproton pump that pumps H* from the cytosol into the acidic lumen of the lysosomeas well as transporters to releasesmall molecules stored in the lvsosome into the

cytoplasm. Soluble proteins delivered by this pathway include lysosomal digestiveenzymessuch as proteases'glycosidases,phosphatases,and lipases. In contrast to the secretorypathway' which is generally used to deliver newly synthesizedmembrane proteins to their correct address,the endocyticpathway is usedto take up substancesfrom the cell surfaceinto the interior of the cell. The endocytic pathway is used to take up certain nutrients that are too large to be transported acrossthe plasma membrane by one of the transport mechanismsdiscussedin Chapter 1 1. For example, the endocytic pathway is utilized in the uptake of cholesterolcarried in LDL particlesand iron atoms carried by the iron-binding protein transferrin. In addition, the endocytic pathway can be usedremove receptorproteins from the cell surface as a way to down-regulate their activity.

OUTLINE 14.1 Techniquesfor Studyingthe Secretory Pathway

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14.2

MolecularMechanismsof VesicularTraffic

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14.3

EarlyStagesof the SecretoryPathway

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14.4

Later Stagesof the SecretoryPathway

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14.5

Endocytosis Receptor-Mediated

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14.6

DirectingMembraneProteinsand Cytosolic Materialsto the LYsosome

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A single unifying principle governs all protein trafficking in the secretoryand endocytic pathways: transport of membrane and soluble proteins from one membrane-bounded compartmentto another is mediatedby transport vesiclesthat collect " cargo" proteins in buds arisingfrom the membraneof one compartment and then deliver thesecargo proteins to the next compartment by fusing with the membraneof thar compartment. Importantly as transport vesiclesbud from one membrane and fuse with the next, the same face of the membrane remains oriented toward the cytosol. Therefore once a protein has been insertedinto the membraneor the lumen of the ER, the protein can be carried along the secretorypathway moving from one organelle ro the next without being translocatedacrossanother membraneor altering its orientation within the membrane. Similarly, the endocytic pathway usesvesicletraffic to transport proteinsfrom the plasmamembrane to the endosomeand lysosomeand thus preservestheir orientation in the membraneof theseorganelles.Figure 14-1 outlinesthe major routes for protein trafficking in the cell. Reducedto its simplest elements,the secretorypathway for delivery of newly synthesized proteins ro the plasma membrane or the lysosomeoperatesrn two stages.The first stage takes place in the rough endoplasmic reticulum (ER), as describedin Chapter 13. Newly synthesizedsoluble and membrane proteins are translocatedinto the ER, where they fold into their proper conformation and receivecovalent modifications such as N-linked and O-linked carbohydrates and disulfide bonds. Once newly synthesizedproteins are properly folded and have received their correct modifications in the ER lumen, they progressro the second stage of the secretory pathway, rransport through the Golgi. In the ER, secretory proteins are packaged into anterograde (forward-moving) transport vesicles.Thesevesiclesfusewith each other to form a flattened membrane-boundedcompartment known as the cis-Golgi cisterna. Certain proteins, mainly ER-localizedproteins, are retrieved from the cisGolgi to the ER via a different set of retrograde (backwardmoving) transport vesicles.A new cls-Golgi cisternawith its cargo of proteins physically moves from the cis position (nearestthe ER) to the trans position (farthest from the ER), successivelybecoming first a medial-Golgi cisterna and then a trans-GoIgi cisterna.This process,known as cisternalmaturation, does nor involve the budding off and fusion of anterograde transport vesicles.During cisternal maturation, enzymesand other Golgi-residentproteins are constantly being retrieved from later to earlier Golgi cisternae by retrograde transport vesicles,thereby remaining localized to the cis-, medial-, or trans-Golgi cisternae.As secreroryprorelns move through the Golgi, they can receivefurther modifications to linked carbohydratesby specific glycosyl transferasesthat are housed in the different Golgi comparrmenrs. Proteinsin the secretorypathway that are destinedfor the plasma membrane or lysosome eventually reach a complex network of membranes and vesiclestermed the trans-Golgi network (TGN). The TGN is a major branch point in the secretory pathway, and through a process known as protein sorttng, a protein can be loaded into one of at leastthree different kinds of vesiclesthat bud from the TGN. After buddine 580

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from the trans-Golgi network, the first type of vesicleimmediately moves to and fuses with the plasma membrane in a processknown as exocytosis,thus releasingits contentsto the exterior of the cell while the membraneproteinsfrom the vesicle becomeincorporatedinto the plasmamembrane.In all cell types,at leastsome proteins are loaded into such vesiclesand secretedcontinuouslyin this manner.The secondtype of vesicle to bud from the trans-Golgi network, known as secretory vesicles,are stored inside the cell until a signal for exocytosis causesreleaseof their contents at the plasma membrane. Among the proteins releasedby such regulatedsecretionare peptide hormones (e.g.,insulin, glucagon,ACTH) from various endocrinecells,precursorsof digestiveenzymesfrom pancreatic acinar cells, milk proteins from the mammary gland, and neurotransmittersfrom neurons.The third type of vesicle that buds from the trans-Golgi network is directed to the lysosome,an organelleresponsiblefor the intracellular degradation of macromolecules, and to lysosome-like storage organellesin certain cells.Secretoryproteinsdestinedfor lysosomes are first transported by vesiclesfrom the trans-Golgi network to a compartment usually called the late endosome; proteins then are transferred to the lysosome by direct fusion of the endosomewith the lysosomalmembrane. Endocytosis is related mechanistically to the secretory pathway. In the endocytic pathway, vesiclesbud from the plasma membrane, bringing membrane proteins and their bound ligands into the cell (see Figure 14-1). After being internalized by endocytosis,some proteins are transported to lysosomesvia the late endosome,whereasothers are recycled back to the cell surface. In this chapter we first discusshow our knowledge of the secretorypathway and endocytosishas expandedthrough experimental techniques.Then we focus on the generalmechanisms of membrane budding and fusion. We will seethat although different kinds of transport vesiclesutilize distinct sets of proteins for their formation and fusion, all vesiclesuse the same generalmechanismfor budding, selectionof particular setsof cargo molecules,and fusion with the appropriatetarget membrane.The following two sectionsshow how coordination betweenparticular vesicletrafficking stepscan maintain the identity (i.e.,a stableset of residentproteins) of the different compartments along the secretory pathway and how cargo selectionby vesiclesis usedto sort proteins to different intracellular locations.Next we will turn our attention to the endocytic pathway to examine how endocytosisis used to transport macromoleculesfrom the extracellularenvironment into the cell interior. Finally we will examine the variety of ways that membrane proteins and macromoleculesfrom the cell interior are transportedto the lysosomefor degradation.

Im

Techniques for Studying

the SecretoryPathway The key to understandinghow proteins are transporred through the organelles of the secretory pathway has been to develop a basic description of the function of transporr vesicles.Many components required for the formation and

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14-1 Overviewof < FIGURE the secretoryand endocytic pathwaysof protein sorting. pathway:Synthesis of Secretory proteins an ERsignal bearing on the iscompleted sequence roughERIl, andthe newlymade polypeptide areinserted chains or crossit intothe ERmembrane 13). intothe lumen(Chapter (e.g.,ERenzymes Someproteins proteins) remain or structural are withinthe ERTheremainder vesicles packaged intotransPort El that budfromthe ERandfuse cisternae to form newcr's-Golgi proteins ER-resident Missorted proteins membrane andvesicle that needto be reusedare to the ERbYvesicles retrieved E that budfromthe cts-Golgi andfusewith the ER.Eachctswith itsProtein Golgicisterna, movesfrom content,physically the crsto the fransfaceof the Golgicomplex @ by a process called nonvesicular Retrograde maturation. cisternal vesicles transport E moveGolgiproteins to the ProPer resident In allcells, compartment. Golgi proteins moveto certainsoluble in transPort the cellsurface vesicles El andaresecreted (constitutive continuously In certaincelltYPes, secretion). proteins arestored somesoluble vesicles in secretory Z andare onlyafterthe cell released neuralor an appropriate receives signal(regulated hormonal Lysosome-destined secretion). andsoluble membrane Proteins, in vesicles whicharetransported that budfromthe trans-Golgi E, firstmoveto the late andthento the endosome lysosomeEndocytic PathwaY: andsoluble Membrane proteins takenup in extracellular thatbudfromthe Plasma vesicles membrane I alsocanmoveto viathe endosome the lysosome

Proteinsvnthesison bound ribosomes; transportof proteins co-translational into or acrossER membrane

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fusion of transport vesicleshave been identified in the past decadeby a remarkable convergenceof the geneticand biochemical approachesdescribedin this section.All studies of intracellular protein trafficking employ some method for assaying the transport of a given protein from one compartment to another. \7e begin by describing how intracellular protein transport can be followed in living cells and then considergeneticand in vitro systemsthat have proved useful in elucidating the secretorypathway.

Transportof a ProteinThroughthe Secretory PathwayCan Be Assayedin Living Cells The classicstudiesof G. Paladeand his colleaguesin the 1960s first established the order in which proteins move from organelleto organelle in the secretoryp"ih*"y. Theseearly studies also showed that secretoryproteins are never releasedinto the cytosol, the first indication that transported proteins are always associatedwith some type of membrane,boundedintermediate. In theseexperiments,which combined pulse-chaselabeling (seeFigure 3-39) and autoradiography,radioactively labeledamino acidswere injectedinto the pancreasof a hamster.At different times after injection, the animal was sacrificed and the pancreatic cells were chemically fixed, sectioned,and subjectedto autoradiographyto visualizethe location of the radiolabeled proteins. Becausethe radioactive amino acids were administered in a short pulse, only those proteins synthesized immediately after injection were labeled, forming a distinct group, or cohort, of labeled proteins whose transport could be followed. In addition, becausepancrearicacinar cells are dedicatedsecretorycells, almost all of the labeled amino

acids in thesecells are incorporated into secretoryproteins, facilitating the observation of transported proteins. Although autoradiographyis rarely usedtoday to localize proteins within cells, these early experiments illustrate the two basic requirementsfor any assayof intercompartmental transport. First, it is necessaryto label a cohort ofproteins in an early compartment so that their subsequent transfer to Iater compartments can be followed with time. Second,it is necessaryto have a way to identify the compartment in which a labeled protein resides.Here we describetwo modern experimental procedures for observing the intracellular trafficking of a secretoryprotein in almost any type of cell. In both procedures, a gene encoding an abundant membrane glycoprotein (G protein) from vesicular stomaritis virus (VSV) is introduced into cultured mammalian cells either by transfection or simply by infecting the cells with the virus. The treated cells, even those that are not specializedfor secretion, rapidly synthesizethe VSV G protein on the ER like normal cellular secretoryproteins. Use of a mutant encoding a temperature-sensitiveVSV G protein allows researchersto turn subsequent transport of this protein on and off. At the restrictive temperature of 40 'C, newly made VSV G protein is misfolded and therefore retained within the ER by quality-control mechanismsdiscussedin Chapter 13, whereasat the permissivetemperatureof 32"C, the protein is correctly folded and is transported through the secretorypathway to the cell surface.This clever use of a temperature-sensitivemutation in effect defines a protein cohort whose subsequenttransport can be followed. In two variations of this basic procedure, transport of VSV G protein is monirored by different techniques.Studies using both of these modern trafficking assaysand Paladet

Video:Transportof VSVG-GFP Throughthe Secretorypathway 0 min

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A EXPERIMENTAL FIGURE 14-2 proteintransportthroughthe secretorypathway can be visualizedby fluorescence microscopy of cellsproducinga GFP-tagged membraneprotein.Culturedcells weretransfected witha hybridgeneencoding theviralmembrane glycoprotein VSVG proteinlinkedto the genefor greenfluorescent protein(GFP). A mutantversion of theviralgenewasusedsothat newlymadehybridprotein (VSVG-GFP) isretained in theERat 40 .C but isreleased for transport at 32 "C.(a)Fluorescence micrographs of cellsjustbeforeandat two timesaftertheywereshiftedto the lower temperature. Movement of VSVG-GFP fromthe ERto the Golqiand

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finallyto thecellsurface occurred within180minutes. Thescale baris 5 p.m (b)Plotof the levels of VSVG-GFp in the endoplasmic (ER), reticulum (pM)at different Golgi,andplasma membrane timesaftershiftto lowertemperature. Thekinetics of transport fromoneorganelle to anothercanbe reconstructed from computer analysis of thesedata.Thedecrease in totalfluorescence thatoccurs at latertimesprobably results fromslowinactivation of GFPfluorescence Jennifer Lippincott-Schwartz IFrom androret H i r s c h b e r gM, e t a b o l i s mB r a n c h N , a t i o n aIl n s t i t u t eo f C h i l dH e a l t ha n d H u m a nD e v e l o o m e nI t

VES|CULAR T R A F F t CS, E C R E T | O N A,N D E N D O C Y T O S t S

early experimentsall came to the same conclusron:In mammalian cellsvesicle-mediatedtransport of a protein molecule from its site of synthesison the rough ER to its arrival at the plasma membrane takes from 30 to 60 minutes. Microscopy of GFP-LabeledVSV G Protein One approach for observing transport of VSV G protein employs a hybrid genein which the viral geneis fused to qhegene encodinggreen fluorescent protein (GFP), a naturally fluorescent protein (Chapter 9). The hybrid geneis transfectpdinto cultured cells by techniques described in Chapter 5. \(hen cells expressing the temperature-sensitiveform of the hybrid protein (VSVGGFP) are grown at the restrictive temperature, VSVG-GFP accumulatesin the ER, which appearsas a lacy network of membranes when cells are observedin a fluorescentmicroscope. When the cells are subsequentlyshifted to a permissive temperature, the VSVG-GFP can be seento move first to the membranes of the Golgi apparatus,which are denselyconcentrated at the edge of the nucleus, and then to the cell surface (Figure 1.4-2a).By analyzing the distribution of VSVG-GFP at different times after shifting cells to the permissivetemperature' researchershave determined how long VSVG-GFP residesin each organelle of the secretorypathway (Figure 14-2b). Cis-Golgi

(a)

Detection of Compartment-Specific Oligosaccharide A second way to follow the transport of Modifications secretoryproteins takes advantageof modifications to their carbohydrate side chains that occur at different stagesof the secretorypathway. To understand this approach, recall that many secretoryproteins leaving the ER contain one or more copies of the N-linked oligosaccharide Mans(GlcNAc)2, which are synthesizedand attached to secretoryproteins in the ER (seeFigure 13-18). As a protein moves through the Golgi complex, different enzymeslocalized to the cis-, medial-, and trans-Golgi cisternae catalyze an ordered seriesof reactionsto thesecore Mans(GlcNAc)z chains, as discussed in a later section of this chapter. For instance,glycosidases that residespecificallyin the cis-Golgi compartment sequentially trim mannose residuesoff the core oligosaccharideto yield a "trimmed" form Man5(GlcNAc)2. Scientistscan use a specializedcarbohydrate-cleavingenzymeknown as endoglycosidase D to distinguish glycosylated proteins that remain in the ER from those that have entered the cisGolgi: trimmed cls-Golgi-specific oligosaccharides are cleaved from proteins by endoglycosidaseD, whereas the core (untrimmed) oligosaccharidechains on secretory proteins within the ER are resistant to cleavageby this enzyme (Figure 1'4-3a\.Becausea deglycosylatedprotein produced by endoglycosidaseD digestion moves faster on an SDS gel

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14-3 Transportof a membrane FIGURE A EXPERIMENTAL glycoproteinfrom the ERto the Golgicanbe assayedbasedon a D. Cellsexpressing sensitivityto cleavageby endoglycosidase with a pulse werelabeled VSVG protein(VSVG) temperature-sensitive sothat temperature aminoacidsat the nonpermissive of radioactive proteinwasretarned timesaftera return in the ERAt periodic labeled fromcells of 32"C,VSVGwasextracted temperature to the permissive moveto thec/sD.(a)Asproteins with endoglycosidase anddigested istrimmed Mans(GlcNAc)2 Golgifromthe ER,thecoreoligosaccharide compartment in the crs-Golgi that reside byenzymes to Mans(GlcNAc)2 fromproteins chains theoligosaccharide D cleaves Endoglycosidase

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0 T i m e( m i n ) in the ER.(b)SDSgel but notfromproteins processed in thecrs-Golgi the resistant, resolves mixtures of thedigestion electrophoresis (faster-migrating) cleaved (slower-migrating) and sensitive, uncleaved all shows,initially VSVGAsthiselectrophoretogram formsof labeled butwith timean increasing to digestion, of theVSVGwasresistant fromthe proteintransported reflecting to digestion, fractionissensitive 40'C, only at kept cells In control processed there. and ERto theGolgi after60 minutes VSVGwasdetected digestion+esistant slow-moving, to of VSVGthat issensitive (notshown).(c)Plotof the proportion of course time the reveals data, fromelectrophoretic derived digestion, 50t523 1987, Cell --; al, et ] Beckers C J [From ER Golgitransport. T E C H N I Q U EF SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y

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ClassA

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Normal secretion

Defective function

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Accumulation i n r o u g hE R

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Accumulation in Golgi

Accumulation In secretory vesicles

Transport i n t ot h e E R

B u d d i n go f vesiclesfrom t h e r o u g hE R

Fusionof transportvesicles with Golgi

Transportfrom Golgi to secretory vesicles

Transportfrom secretoryvesicles to cell surface

EXPERIMENTAL FTGURE l4-4 phenotypes of yeastsec mutantsidentifiedstagesin the secretorypathway.These temperature-sensitive mutants canbe grouped intofiveclasses based on thesitewherenewlymadesecreted (reddots)accumulate proteins whencellsareshiftedfromthe permissive temperature to the

ClassD

ClassE

highern , o n p e r m i s s i voen e . A n a l y s i o s f d o u b l em u t a n t sp e r m i t t e d the sequentialorder of the stepsto be determined.[Seep Novick e t a | , 1 9 8 1C , e l l 2 5 : 4 6a1n, d C A K a i s e r a nRdS c h e k m al g ng . O.Cett 61:723l

than the corresponding glycosylatedprotein, these proteins A Iarge number of yeast mutants initially were identified can be readily distinguished(Figure14-3b). basedon their ability to secreteproteins at one temperature This type of assaycan be usedto track movement of VSV and inability to do so at a higher, nonpermissivetemperaG protein in virus-infectedcellspulse-labeledwith radioacture. Sfhen these temperature-sensitivesecretion (sec) mwtive amino acids. Immediately after labeling, all the extants are transferred from the lower to the higher temperatractedlabeledVSV G protein is still in the ER and is resistture, they accumulate secretedproteins at the point in the ant to digestion by endoglycosidaseD, but with time an pathway blocked by the mutation. Analysis of such mutants increasingfraction of the glycoprotein becomessensitivero identified five classes(A-E) characterizedby protern accudigestion. This conversion of VSV G protein from an endomulation in the cytosol, rough ER, small vesiclestaking proglycosidase D-resistant form ro an endoglycosidase teins from the ER to the Golgi complex, Golgi cisternae,or D-sensitive form corresponds to vesicular transport of the constitutive secretory vesicles (Figure 14-4). Subsequent protein from the ER to the cis-Golgi. Note that transport of characterization of sed mutants in the various classeshas VSV G protein from the ER to the Golgi takes about 30 minhelped elucidate the fundamental components and molecuutes as measuredby either the assaybasedon oligosaccha- lar mechanismsof vesicletrafficking that we discussin later ride processing or fluorescencemicroscopy of VSVG-GFp sectlons. (Figure 14-3c). A variety of assaysbased on specific carboTo determine the order of the steps in the pathw ay, rehydrate modifications thar occur in later Golgi compartsearchersanalyzeddouble sec mutants. For instance,when ments have been developedto measureprogression of VSV yeast cells contain mutarions in both class B and class D G protein through each stageof the Golgi apparatus. functions, proteins accumulate in the rough ER, not in the Golgi cisternae.Since proteins accumulate at the earliest blocked step; this finding shows that classB mutatrons must YeastMutants Define Major Stagesand Many act at an earlier point in the secretoryparhway than classD Componentsin VesicularTransport mutations do. These studies confirmed that as a secreted The generalorganization of the secretorypathway and many protein is synthesizedand processed,it moves sequentially of the molecular componentsrequired for vesicletrafficking from the cytosol -+ rough ER --> ER-to-Golgi transport are similar in all eukaryotic cells. Becauseof this .o.r.ruul vesicles-+_Golgi cisternae-+ secreroryvesiclesand finally is exocytoseo. The three methods outlined in this sectionhave delineated the major steps of the secretory pathway and have contributed to the identification of many of the proteins responsible for vesiclebudding and fusion. Currently eachof the individual steps in the secretorypathway is being studied in mechanisticdetail, and increasinglSbiochemical assaysand moleculargeneticstudiesare usedto study eachof thesesteps in terms of the function of individual protein molecules. 584

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Addition of N-acetylglucosamine to G protein

FIGURE 14-5A cell-freeassaydemonstrates EXPERIMENTAL protein transpoftfrom one Golgi cisternato another.(a)A in thistypeof assayIn fibroblasts isessential mutantlineof cultured N-acetylglucosamine thecellslacktheenzyme thisexample, is thisenzyme cells, | (stepE in Figure14-14)ln wild-type transferase N-linked oligosaccharides andmodifies to themedr,afGolgi localized wild-type InVSV-infected of oneN-acetylglucosamine, bytheaddition to a typical on theviralG proteinismodified cells, theoligosaccharide panel.In asshownin the trans-Golgi oligosaccharide, complex the cellsurface the G proteinreaches infectedmutantcells,however,

onlytwo containing oligosaccharide high-mannose with a simpler (b)WhenGolgi residues. andfivemannose N-acetylglucosamine with Golgi mutantcellsareincubated frominfected isolated cisternae produced protein VSV G the cells, unrnfected fromnormal, cisternae Thismodification N-acetylglucosamine theadditional in vitrocontains enzymethat is movedby transport iscarriedout by transferase to the mutantctscisternae fromthewild-typemedial-Golgi vesrcles Balch et al, 1984'Cell W E in the reactionmixture[See Golgicisternae and J E Rothman 1; and 39:51 1984, Cell et al A Braell , and525;W 39:405 275:1212 1997,Science l I S6llner,

Cell-FreeTransportAssaysAllow Dissection o f I n d i v i d u a lS t e p si n V e s i c u l aTr r a n s p o r t

purified away from the donor wild-type Golgi membranes ty centrifugation. By examining the proteins that are enriched in thise vesicles,scientistshave been able to identify

In vitro assaysfor intercompartmental transport are powerful complementary approachesto studieswith yeast sec mu' tants for identifying and analyzingthe cellular components responsible for vesicular trafficking. In one application of this approach, cultured mutant cells lacking one of the enzymes that modify N-linked oligosaccharidechains in the Golgi are infected with vesicular stomatitis virus (VSV). For example, if infected cells lack N-acetylglucosamine transferaseI, they produce abundant amounts of VSV G protein but cannot add N-acetylglucosamine residues to the oligosaccharidechains in the medial-Golgi as wild-type cells do (Figure 14-5a). When Golgi membranes isolated from such mutant cells are mixed with Golgi membranes from wild-type, uninfected cells, the addition of N-acetylglucosamineto VSV G protein is restored(Figure14-5b). This modification is the consequenceof vesiculartransport of NacetylglucosaminetransferaseI from the wild-type medialGolgi to the cls-Golgi compartment from virally infected mutant cells.Successfulintercompartmentaltransport in this cell-freesystem dependson requirementsthat are typical of a normal physiologicalprocess,including a cytosolic extract, a sourceof chemicalenergyin the form of ATP and GTP, and incubation at physiological temperatures' In addition, under appropriate conditions a uniform population of the transport vesiclesthat move N-acetylglucosaminetransferaseI from the medial- to cls-Golgi can be

targeting and fusion of vesicleswith appropriate acceptor In vitro assayssimilar in general design to the -.-br".t.t. one shown in Figure 1,4-5 have been used to study various transport stepsin the secretorypathway.

Techniquesfor Studyingthe SecretoryPathway r AII assays for following t he trafficking of proteins rn living cells require a way through the secretorypathway ln and a way to identity proteins secretory to label a cohort of subsequentlyare proteins labeled where the compartments located. r Pulse labeling with radioactive amino acids can specifically label a cohort of newly made proteins in the ER' AIternatively, a temperature-sensitivemutant protein that is retained in the ER at the nonpermissivetemperaturewill be releasedas a cohort for transport when cells are shifted to the permissivetemperature. T E C H N I Q U EF SO R S T U D Y I N GT H E S E C R E T O RPYA T H W A Y

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r Transport of a fluorescentlylabeled protein along the secretory pathway can be observed by microscopy (seeFigure 14-2). Transport of a radiolabeledprotein commonly is tracked by following comparrment-specificcovalent m o d i f i c a t i o n sr o r h e p r o t e i n . Many of the componentsrequired for intracellularprotein afficking have been identified in yeast by analysisof temperature-sensitive sec mutants defectivefor the secretionof proteins at the nonpermissivetemperarure(seeFigure 14-4). r Cell-free assaysfor intercompartmentalprorein transport have allowed the biochemicaldissectionof individual stepsof the secretorypathway. Such in vitro reactronscan be used to produce pure transport vesiclesand to rest the biochemicalfunction of individual transportproteins.

Molecular Mechanisms

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Small membrane-boundedvesiclesthat transporr prorelns from one organelle to another are common elemenisin the

type of vesicle, studies employing genetic and biochemical techniqueshave revealedthat each of the different vesicular transport stepsis simply a variation on a common theme. In this sectionwe explore the basicmechanismsunderlying vesicle budding and fusion, that all vesicletypeshave in common. The budding of vesiclesfrom their parent membrane is driven by the polymerization of soluble protein complexes onto the membrane to form a proteinaceousvesiclecoat (Figure 14-6a). Interactionsbetweenthe cytosolic portions of integral membraneproteins and the vesiclecoat gather the appropriate cargo proteins into the forming vesicle.Thus the coat not only gives curvature to the membrane to form a vesiclebut also acts as the filter to determine which oroteins are admitted into the vesicle. The integral membrane proteins in a budding vesicleinclude v-SNAREs, which are crucial to eventual fusion of the

protein

complex

FIGURE 14-6 Overviewof vesiclebuddingand fusionwith a target membrane.(a)Budding isinitiated by recruitment of a small proteinto a patchof donormembraneComplexes GTP-binding of coatproteins in thecytosol thenbindto thecytosolic domainof membrane cargoproteins, someof whichalsoactasreceptors that proteins bindsoluble in the lumen,therebyrecruiting luminal cargo proteins intothe buddingvesicle(b)Afterbeingreleased and shedding itscoat,a vesicle fuseswith itstargetmemorane In a process thatinvolves proteins. interaction of coqnate SNARE

reversiblepolymerization of a distinct set of protein subunits (Table 14-1). Each rype of vesicle,named for its primary coat proterns,transports cargo proteins from particular parent organellesto particular destination organelles: r COPI vesiclestransport proteins from the rough ER to the Golgi. COPI vesiclesmainly transport proteins in the retrograde irection betweenGolgi cisternaeand from the cls-Golei ack to the rough ER.

Assemblyof a ProteinCoat DrivesVesicle F o r m a t i o na n d S e l e c t i o no f C a r g oM o l e c u l e s Three types of coated vesicleshave been characterized,each with a different type of protein coat and each formed by 586

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c H A p r E R1 4 I

r Clathrin vesiclestransport proteins from the plasma membrane (cell surface)and the trans-GoIgi network to late endosomes. Every vesicle-mediatedtrafficking step is thought to utilize some kind of vesicle coat; however, a specific coat

vEstcuLAR T R A F F t cs,E c R E T t o N A,N D E N D o c y r o s t s

TYPI VESICIE

MEDIATEI) STEP IRANSPORT

PROTEINS C()AT

GTPase ASS0CIATED

COPII

ER to cls-Golgi

1 Sec23lSec24and Secl3/Sec3 Sec16 complexes,

Sarl

COPI

cls-Golgi to ER Later to earlier Golgi cisternae

Coatomerscontalnrngseven differentCOP subunits

ARF

Clathrinand adapterprotelns

tr ans-G olgi to endosome

Clathrin* AP1 comPlexes

ARF

tr ans -Golgi to endosome

Clathrin + GGA

ARF

Plasmamembrane to endosome

Clathrin * AP2 comPlexes

ARF

Golgi to lysosome,melanosome, or plateletvesicles

AP3 complexes

ARF

.Each

vesiclescontains clathrin t1,peof Ap complex consists of four different subunits. It is not known whether the coat of AP3

protein complex has not been identified for every type ol have not yet identifiedthe vesicle.For example,researchers that move protelns the vesicles coat proteins surrounding during either membrane plasma the from the trans-Golgito secretion. constitutive or regulated The general scheme of vesicle budding shown in Figure 14-6a appliesto all three known types of coated vesicles. Experimentswith isolated or artificial membranesand purified coat proteins have shown that polymerizationof the coat proteins onto the cytosolic face of the parent membrane is necessaryto producethe high curvatureof the membranethat is typical of a transport vesicleabout 50 nm in diameter.Electron micrographs of in vitro budding reactions often reveal structufes that exhibit discrete regions of the parent membrane bearing a dense coat accompanied by the curvature characteristicof a completedvesicle(Figure14-7). Suchstructures, usually calleduesiclebwds, appearto be intermediates that are visible after the coat has begunto polymerizebut before the completedvesiclepinchesoff from the parent membrane. The polymerized coat proteins are thought to form sometype of curved lattice that drivesthe formation of a vesicle bud by adheringto the cytosolicface of the membrane'

and clathrin vesicles,this GTP-binding protein is known as ARF protein A different but related GTP-binding protein known as Sarl protein is present in the coat of COPII vesicles. Both ARF and Sarl are monomeric proteins with an overail structuresimilar to that of Ras' a key intracellular signal-transducingprotein (seeFigure 16-24)' ARF and Sarl pr"ot.inr, like Ras, belong to the GTPase superfamily of switch proteins that cycle between inactive GDP-bound and activeGTP-boundforms (seeFigure 3-32)' The cycle of GTP binding and hydrolysis by ARF and Sarl are tho,tght to control the initiation of coat assembly, as schematicafy depicted for the assemblyof COPII vesicles

A ConservedSet of GTPaseSwitch Proteins ControlsAssemblyof Different VesicleCoats Based on in vitro vesicle-buddingreactions with isolated membranesand purified coat proteins,scientistshave determined the minimum set of coat components required to form each of the three maior types of vesicles.Although most of the coat proteinsdiffer considerablyfrom one type of vesicleto another,the coats of all three vesiclescontain a small GTP-bindingprotein that acts as a regulatorysubunit to control coat assembly(seeFigure 1,4-6a).For both COPI

14-7 Vesiclebudscan be visualized FIGURE r, EXPERIMENTAL coat WhenpurifiedCOPII during in vitro buddingreactions. or artificial ERvesicles with isolated areincubated components of the coatproteins polymerization (liposomes), vesicles phospholipid curvedbuds ln highly of emergence induces surface on thevesicle notethe reaction, budding vitro in of an micrograph th electron present on the asa darkproteinlayer, coat,visible di inctmembrane 93(2):263 Ceil ] etal, 1988, K Matsuoka buds lFrom vesicle M E C H A N I S MO S F V E S I C U L ATRR A F F I C MOLECULAR

.

587

E

Sarl membranebinding, GTP exchange GTP

Cytosol

Sart

/

GDP

Hydrophobic N-terminus

\ Jec tz -

ER lumen

Sec23/Sec24

E :3:'liT'

p

.

in Figure 14-8. First, an ER membrane protein known as Sec1.2catalyzesrelease of GDp from cyrosolic Sarl.GDp and binding of GTP. The Sec12 guanine nucleotideexchangefactor apparently receivesand integratesmultiple as yet unknown signals,probably including the presenceof cargo proteins in the ER membrane that are ready to be transported. Binding of GTp causes a conformational change in Sarl that exposes its hydrophobic N-terminus, which then becomesembeddedin the phospholipid bilayer and tethers Sarl.GTP to the ER membrane. The membrane_ attached Sarl.GTP drives polymerization of cytosolic complexes of COPII subunits on the membrane.eventually leading to formation of vesicle buds. Once COPII vesiciesare releasedfrom the donor membrane, the Sarl GTpase activ_ ity hydrolyzes Sarl.GTP in the vesicle membrane to Sarl.GDP with the assistanceof one of the coat subunits. This hydrolysis triggers disassemblyof the COpII coat. Thus Sarl couples a cycle of GTp binding and hydrolysis to the formation and then dissociationof the COpII coat. ARF protein undergoesa similar cycle of nucleotide ex_ change and hydrolysis coupled to the assembly of vesicle coats composedeither of COpI or of clathrin and other coat proteins (AP complexes),discussedlater. A covalent protein modification known as a myristate anchor on the N-termi_

cre hydrotysis

P;

I

ARF'GTP with the membrane servesas the foundation for further coat assembly. Drawing on the structural similarities of Sarl and ARF to other small GTPaseswitch proteins, researchershave con_ structed genesencoding mutant versionsof the two proteins that have predictable effectson vesiculartraffic when trans-

Coatdisassembly

Uncoated vesicle FIGURE 14-8 Modelfor the role of Sarl in the assemblyand disassembly of COPIIcoats.Step[: Interaction of soluble GDp_ boundSarlwith theexchange factorSecl2, an ERinregrar membrane protein, catalyzes exchange of GTpfor GDpon Sarl In the GTP-bound formof Sar1,itshydrophobic N-terminus extends outwardfromthe protein's surface andanchors Sarlto the ER membrane. Stepf,l: Sarlattached to the membrane serves asa bindingsitefor the Sec23/Sec24 coatproteincomplex. Membrane cargoproteins arerecruited to theformingvesicle budby bindingof specific shortsequences (sorting signals) in theircytosolic regions to siteson the Sec23/Sec24 complex, Somemembrane cargoproteins alsoactasreceptors that bindsoluble proteins in the lumen.The coatiscompleted by assembly of a second typeof coatcomplex composed of Sec13 (notshown)StepB: Afterthe andSec31 vesicle coatiscomplete, the Sec23coatsubunitpromotes GTp h y d r o l y sbiysS a r l S t e p@ : R e l e a soef S a r l . G Dfpr o mt h ev e s i c l e membrane causes disassembly of thecoat.[See S Sprrngeret at, 1999, Cell97:145I

588

.

c H A p r E R1 4 |

with target membranes.Addition of a nonhydrolyzable GTp analog to in vitro vesicle-buddingreactions causesa similar blocking of coat disassembly.The vesiclesthat form in such reactions have coats that never dissociate,allowing their composition and structure to be more readily analyzed.The purified COPI vesiclesshown in Figure 14-9 were produced in such a budding reacrion.

TargetingSequenceson CargoproteinsMake SpecificMolecularContactswith Coat proteins In order for transport vesiclesto move specificproteins from one compartment to the next, vesicle buds must be able to discriminate among potential membrane and soluble cargo proteins, acceptingonly those cargo proteins that should J_ vance to the next compartment and excluding those that

vEstcuLAR T R A F F I cs,E c R E T t o N A.N D E ND O C Y T O S I S

14-9 Coatedvesiclesaccumulate FIGURE A EXPERIMENTAL of a during in vitro buddingreactionsin the presence Golgimembranes Whenisolated analogof GTP. nonhydrolyzable COPIcoatproteins, extractcontaining with a cytosolic areincubated of a Inclusion formandbudoff fromthe membranes vesicles prevents analogof GTPin the buddingreaction nonhydrolyzable shows Thismicrograph release of the coataftervesicle disassembly from generated andseparated in sucha reaction COPIvesicles prepared in thisway Coatedvesicles by centrifugation membranes andproperties theircomponents to determine canbe analyzed of L Orci] [Courtesy

should remain as residentsin the donor compartment' In addition to sculpting the curvature of a donor membrane' the vesiclecoat functions in selectingspecificproteins as cargo' The primary mechanism by which the vesicle coat selects cargo moleculesis by directly binding to specific sequences, or sorting signals, in the cytosolic portion of membrane cargoproteins(seeFigure 14-6a).The polymerizedcoat thus acts as an affinity matrix to cluster selectedmembrane cargo proteins into forming vesicle buds. Since soluble proteins within the lumen of parent organelles cannot contact the coat directly, they require a different kind of sorting signal' Soluble luminal proteins often contain what can be thought of as luminal sorting signals,which bind to the luminal domains of certain membrane cargo proteins that act as receptors for luminal cargo proteins. The properties of several known sorting signalsin membrane and soluble proteins are summarizedin Table 14-2.We describethe role of thesesignals in more detail in later sectlons.

Control Dockingof Vesicles Rab GTPases on TargetMembranes A secondset of small GTP-binding proteins, known as Rab proteins, participate in the targeting of vesiclesto the appropriate target membrane' Like Sarl and ARF, Rab proteins

LUMENAL SORTING SIGNALS (KDEL) Lys-Asp-Glu-Leu

ER-resident soluble protelns

KDEL receptorin cls-Golgi membrane

(M6P) Mannose6-phosphate

Soluble lysosomal enzymes after processingin cls-Golgi

M6P receptor in trans-Golgr membrane

Secretedlysosomal enzymes

M6P recepror in plasma membrane

Clathrin/AP2

CYTOPLASMICSORTING SIGNALS Lys-Lys-X-X(KKXX)

ER-resident membrane protelns

COPI ct and P subunits

COPI

Di-acidic(e.g.,Asp-X-Glu)

Cargo membrane proteins in ER

COPIISec24subunit

COPII

Asn-Pro-X-Tyr(NPXY)

LDL receptor in plasma membrane

AP2 complex

ClathridAP2

Tyr-X-X-
Membrane proteins in trans-Go|gi

AP1 (p1 subunit)

Clathrin/AP1

Plasma membrane proteins

AP2(p.2subunit)

Clathrin/AP2

Plasma membrane proteins

AP2 complexes

Clathrin/AP2

Leu-Leu(LL)

in parentheses' X : any amino acid; O - hydrophobic amino acid. Single-letteramino acid abbreviations are S F V E S I C U L ATRR A F F I C MOLECULAM R E C H A N I S MO

589

(a)

Transport vesicle

V e s i c l e d o cki n sl E

Rab.GTP

Target memDrane

< FIGURE 14-10Modelfor dockingand fusionof transport vesicleswith their target membranes. (a)Theproteins shownin thisexample participate in f usionof secretory vesicles with the plasmamembrane, but similarproteins mediate allvesicle-fusion events. Step[:A Rabproteintethered viaa lipidanchorto a secretory vesicle bindsto an effectorproteincomplex on the plasma membrane, therebydockingthetransport vesicle on the appropriate targetmembrane. StepE: A v-SNARE protein(inthiscase,VAMp) interacts with the cytosolic domains of the cognatet-SNAREs (inthis case,syntaxin andSNAP-25) Theverystablecoiled-coil SNARE complexes thatareformedholdthe vesicle closeto the target membraneStepB: Fusion of the two membranes immediately followsformationof SNARE complexes, but precisely how this occursrsnot known Step@: Following membrane fusion,NSFin conjunction with ct-SNAP proteinbindsto the SNARE complexes. TheNSF-catalyzed hydrolysis of ATpthendrivesdissociation of the SNARE complexes, freeingthe SNARE proteins for anotherroundof vesicle fusionAlsoat thistime,Rab.GTp ishydrolyzed to Rab.GDp anddissociates fromthe Rabeffector(notshown)(b)TheSNARE complexNumerous noncovalent interactions betweenfour longo helices, two fromSNAP-25 andoneeachfromsyntaxin andVAMp, stabilize the coiled-coil structure[See J E Rothman andT Sollner, 1997, Science2T6:1212, part andW Weis andR Scheller, 1gg1,Nature 395:328 (b)fromY A ChenandR H Scheller, 2001,Nat Rev. Mot Cett Biot2(2):98 I

SNARE complex\

tu.'on JE

Membrane

o-SNAP cis-SNARE comptex

D i s a s s e m b l yo f SNAREcomplexes

( b ) S N A R Ec o m o l e x

Syntaxin

belong to the GTPasesuperfamily of switch proteins. Con_ version of cytosolic Rab.GDp to Rab.GTp, iatalyzed by a specific guanine nucleotide-exchangefactor, induces a conformational changein Rab that enablesit to interact with a surface protein on a particular transport vesicle and insert its isoprenoid anchor into the vesicle membrane. Once 590

.

c H A p r E R1 4 |

Rab'GTP is tetheredto the vesiclesurface,it is thought to interact with one of a number of different large proteins, known as Rab effectors, attached to the target membrane. Binding of Rab.GTP to a Rab effector docks the vesicleon an appropriate target membrane (Figure 14-10, step [). After vesiclefusion occurs, the GTp bound to the Rab protein is hydrolyzed to GDP, triggering the releaseof Rab.GDp, which rhen can undergo another cycle of GDp-GTp exchange,binding, and hydrolysis. Severallines of evidencesupport the involvement of spe_ cific Rab proteins in vesicle-fusionevents.For instance,ihe yeast SEC4 gene encodesa Rab protein, and yeast cells expressingmutant Sec4proteins accumulate secretoryvesicles that are unable to fuse with the plasma membrane (classE mutants in Figure 14-4).In mammalian cells,Rab5 protein is localized to endocytic vesicles,also known as early endosomes. These uncoated vesiclesform from clathrin-coated vesiclesjust after they bud from the plasma membrane during endocytosis(seeFigure 14-1, step pt). The fusion of early endosomeswith eachother in cell-freesysremsrequrres the presenceof Rab5, and addition of Rab5 and GTp to cellfree extracts acceleratesthe rate at which thesevesiclesfuse with each other.A long coiled protein known as EEA1 (early endosome antigen 1), which resideson the membrane of the early endosome,functions as the effector for Rab5. In this case, RabS.GTP on one endocytic vesicle is thought to specifically bind to EEA1 on the membrane of another endocytic vesicle, setting the stage for fusion of the two vesicles. A different type of Rab effector appearsto function for each vesicletype and at each step of the secretorypathway. Many questions remain about how Rab proteins are rar_ geted to the correct membrane and how specific complexes

v E S t c u L ATRR A F F I sc E , c R E T t oA NN . DE N D O C Y T O S I S

form between the different Rab proteins and their corresponding effector protetns.

PairedSetsof SNAREProteinsMediate Fusion of Vesicleswith TargetMembranes As noted previously,shortly after a vesiclebuds off from the to uncover a donor membrane, the vesiclecoat disassembles (seeFigure protein, a v-SNARE membrane vesicle-specific 14-6b1.Likewise, eachtype of target membranein a cell contains I-SNARE membrane proteins. After Rab-mediated docking of a vesicleon its target (destination)membrane,the interaction of cognate SNAREs brings the two membranes close enough together that they can fuse. One of the best-understood examples of SNAREmediatedfusion occursduring exocytosisof secretedproteins (Figure 14-10, steps f,l and B). In this case,the v-SNARE, membrane protein), is known as VAMP (zesicle-associated incorporated into secretory vesiclesas they bud from the trans-Golgi network. The I-SNAREs aresyntaxin, anintegtal membrane protein in the plasma membrane, and SNAP-25, which is attachedto the plasma membraneby a hydrophobic lipid anchor in the middle of the protein. The cytosolic region in each of thesethree SNARE proteins contains a repeating heptad sequencethat allows four a helices-one from VAMP, one from syntaxin, and two from SNAP-2S-Io coil around one another to form a four-helix bundle. The unusual stability of this bundled SNARE complex is conferred by the arrangementof hydrophobic and charged amino residuesin the heptad repeats.The hydrophobic amino acids are buried in the central core of the bundle, and amino acidsof opposite chargeare alignedto form favorableelectrostaticinteractions between helices.As the four-helix bundles form, the vesicle and target membranesare drawn into closeapposition by the embeddedtransmembranedomains of VAMP and syntaxin. In vitro experiments have shown that when liposomes containingpurified VAMP are incubatedwith other liposomes containing syntaxin and SNAP-25, the two classesof membranesfuse,albeit slowly. This finding is strong evidencethat the close apposition of membranesresulting from formation of SNARE complexesis sufficient to bring about membrane fusion. Fusion of a vesicleand target membraneoccurs more rapidly and efficiently in the cell than it does in liposome experiments in which fusion is catalyzed only by SNARE proteins. The likely explanation for this differenceis that in the cell, other proteinssuch as Rab proteinsand their effectorsare involved in targetingvesiclesto the correct membrane. Yeastcells,like all eukaryotic cells,expressmore than 20 different related v-SNARE and I-SNARE proteins. Analysesof yeast mutants defectivein each of the SNARE geneshave identified specific membrane-fusion events in which each SNARE protein participates.For all fusion eventsthat have been examined, the SNAREs form four-helix bundled complexes,similar to the VAMP/syntaxin/SNAP-25 complexes that mediate fusion of secretoryvesicleswith the plasma membrane.However, in other fusion events(e.g.,fusion of COPII vesicleswith the crs-Golgi network), each participating SNARE protein contributesonly one ct helix to the bundle (unlike SNAP-25'which

contributes two helices);in thesecasesthe SNARE complexes comprise one v-SNARE and three I-SNARE molecules. Using the in vitro liposome fusion assay'researchershave tested the ability of various combinations of individual v-SNARE and I-SNARE proteins to mediate fusion of donor and target membranes.Of the very large number of different combinations tested,only a small number could efficiently mediate membrane fusion. To a remarkable degree,the functional combinations of v-SNAREs and I-SNAREs revealedin thesein vitro experimentscorrespond to the actual SNARE protein interactions that mediate known membrane-fusioneventsin the yeastcell. Thus the specificityof the interaction betweenSNARE oroteins can account for much of the specificity of fusion between a particular vesicletype and its target membrane'

Dissociationof SNAREComplexesAfter MembraneFusionls Driven by ATPHydrolysis After a vesicle and its target membrane have fused, the SNARE complexes must dissociateto make the individual SNARE proteins available for additional fusion events' Becauseof the stability of SNARE complexes,which are held together by numerous noncovalent intermolecular interactiJrs, their dissociation dependson additional proteins and the input of energY. The first clue that dissociation of SNARE complexesrequired the assistanceof other proteins came from in vitro tiansport reactions depleted of certain cytosolic proteins' The observedaccumulation of vesiclesin these reactions indicated that vesiclescould form but were unable to fuse with a targetmembrane.Eventually two proteins, designatedNSI o-SNaR were found to be required for ongoing vesicle "nd fusion in the in vitro transport reaction. The function of NSF in vivo can be blocked selectively by N-ethylmaleimide -SH group (NEM), a chemical that reactswith an essential on NSF (hencethe name, NEM-sensitive /actor)' Among the classC yeastsecmutants are strains that lack functionaf Sec18or Secl7, the yeast counterpartsof mammalian NSF and o-SNAP, respectively.\fhen these class C mutants are placed at the nonpermissivetemperature, they accumulate nR-to-Golgi transport vesicles;when the cells are shifted to the lower, permissivetemperature'the accumulated vesiclesare able to fuse with the cis-Golgi' Subsequentto the initial biochemicaland geneticstudies identifying NSF and ct-SNAP,more sophisticated in vitro transport assayswere developed.Using thesenewer assays'researchershave shown that NSF and a-SNAP proteins are not

sion assaysand in the yeast mutants after a loss of Sec17or of free SNARE proteinsrapidly beSec18were a consequence SNARE complexesand in undissociated coming sequestered fusion' membrane mediate to unavailable thus b-ing TRAFFIC M O L E C U L A RM E C H A N I S M 5O F V E 5 I C U L A R

.

591

Molecular Mechanismsof VesicularTraffic

!,''ir*,

r The three well-characterizedtransport vesicles-COpl, COPII, and clathrin vesicles-aredistinguishedby the proteins that form their coats and the transport routes they mediate(seeTable 14-1). r All types of coated vesiclesare formed by polymerization of cytosoliccoat proteinsonto a donor (parent)membrane to form vesicle buds that eventually pinch off from the membrane to releasea complete vesicle.Shortly after vesicle release,the coat is shed,exposingproteinsrequiredfor fusion with the target membrane(seeFigure 14,6). Small GTP-bindingproteins (ARF or Sarl) belongingto e GTPase superfamily control polymerization of coat proteins, the initial step in vesicle budding (see Fig_ ure 14-8).After vesiclesare releasedfrom the donor mem_ brane, hydrolysis of GTP bound to ARF or Sarl triggers disassemblyof the vesiclecoats.

$cotgi

j' network

iF,,f-q'"',

pecific sorting signalsin membrane and luminal pros_ofdonor organellesinteracrwith coat proteinsd.riing c l e b u d d i n g .r h e r e b yr e c r u i t i n gc a r g o p r o t e i n sr o v e s i (seeTable 14-2). r A secondset of GTP-bindingproteins,the Rab proteins, regulate docking of vesicleswith the correct rarget membrane. Each Rab appearsto bind to a specificRab effector associatedwith the target membrane. r Each v-SNARE in a vesicular membrane specifically binds to a complex of cognateI-SNARE proteins in the target membrane, inducing fusion of the two membranes. After fusion is completed, the SNARE complex is disassembledin an MP-dependent reacrionmediatedby other cytosolic proteins (seeFigure 14-10).

EarlyStagesof the Secretory M Pathway In this section we take a closer look at vesicular traffic

tain newly synthesizedproteins destined for the Golgi, cell surface,or lysosomesas well as vesiclecomponents such as v-SNAREs that arerequired ro targer vesiclesto the cls-Golei membrane. Proper sorring of proteins between the ER anld Golgi also requires reverse retrograde transport from the cls-Golgito the ER and is mediatedby COpI vesicles(Fig_ ure 14-11). This retrogradevesicletransport servesro rerrreve v-SNARE proteins and rhe membraneitielf back to the ER to provide the necessarymaterial for additional rounds of vesi_ cle budding from the ER. COpl-mediated retrograde trans, port also retrieves missorted ER-resident proteins from the cis-Golgi to correct sorting mistakes.proteins that have been 592

.

c H A p r E R1 4 I

Rough ER

A FIGURE 14-11 Vesicle-mediated protein trafficking between the ERand crs-Golgi. (anterograde) Stepsfi-f,t: Forward transport is mediated by COPII vesrcles, whichareformedby polymerization of soluble COPII (green) coatproteincomplexes on the ERmembrane (orange) v-SNAREs (blue)in the ER andothercargoproteins membrane areincorporated intothe vesicle by interacting with coat proteinsSoluble (magenta) cargoproteins arerecruited by binding to appropriate receptors in the membrane of buddingvesicles Dissociation of the coatrecycles freecoatcomplexes andexposes v-SNARE proteins on the vesicle surfaceAfterthe uncoated vesicle becomes tetheredto the crs-Golgi membrane in a Rab-mediated process, pairingbetweenthe exposed v-SNAREs andcognate I-SNARE i nst h e G o l g m i e m b r a nael l o w sv e s i c lfeu s i o nr.e l e a s i n o the contentsintothe crs-Golgi (seeFigure14-101. compartment (retrograde) Steps@-@: Reverse transport, mediated by vesicles coatedwith COPIproteins (purple), recycles the membrane bilayer andcertainproteins, suchasv-SNAREs and missorted ER-resident proteins (notshown),fromthe crs-Golgi to the ER All SNARE p r o t e i nasr es h o w ni n o r a n g ea l t h o u g vh- S N A R aEnsdI - S N A R E a rse d i s t i n cpt r o t e i n s correctly delivered to the Golgi advance through successive compartmentsof the Golgi by cisternalmaturarion.

COPIIVesiclesMediate Transportfrom the ER to the Golgi COPII vesicleswere first recognizedwhen cell-free extracts of yeast rough ER membraneswere incubated with cytosol and a nonhydrolyzable analog of GTp. The vesicles that

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Video:KDELReceptorTrafficking < FIGURE 14-13Roleof the KDEtreceptorin retrievalof ER-resident luminalproteinsfrom the Golgi.ERluminal proteins, especially thosepresent at highlevels, canbe passively incorporated intoCOPII vesicles andtransported to the Golgi (stepsE and Z). Manysuchproteins beara C-terminal KDEL (Lys-Asp-Glu-Leu) (red)thatallowsthemto be retrieved sequence TheKDELreceptor, locatedmainlyin the crs-Golgi networkandin bothCOPII andCOPIvesicles, bindsproteins bearing the KDEL sortingsignalandreturnsthemto the ER(stepsEl and 4). This retrieval prevents system depletion of ERluminalproteins suchas thoseneededfor properfoldingof newlymadesecretory proteinsThebindingaffinityof the KDELreceptor isvery sensitrve to pH Thesmalldifference in the pHof the ERand Golgifavorsbindingof KDEl-bearing proteins to the receptor in Golgi-derived vesicles andtheirrelease in the ER [Adapted fromJ Semenza etal, 1990, Cell61:13491 COPIIcoat

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COPIVesiclesMediate RetrogradeTransport w i t h i n t h e G o l g i a n d f r o m t h e G o l g it o t h e E R COPI vesicleswere first discoveredwhen isolated Golgi fractions were incubated in a solution containing .ytorJl and a nonhydrolyzable analog of GTp (seeFiguie 14-9). Subsequentanalysisof thesevesiclesshowed that the coat is formed from large cytosolic complexes, called coatomers,composedof sevenpolypeptide subunits.yeast cells containing temperature-sensitive murations in COpI proteins accumulateproteins in the rough ER at the non_ permissive temperature and thus are categorizedas class B secmutants (seeFigure 14-4). Although discoveryof these mutants initially suggestedthat COpI vesiclesmediateER_ to-Golgi transport, subsequentexperiments showed that their main function is retrograde transport, both between Golgi cisternaeand from the cis-Golgi to the rough ER (seeFigure 14-11, right). BecauseCOpI mutanrs cannor recyclekey membrane proteins back to the rough ER, the ER gradually becomesdepleted of ER proteins such as vSNAREs necessaryfor COpII vesiclefunction. Eventually. vesicleformation from the rough ER grinds to a halt; se_ cretory proteins continue to be synthesizedbut accumu_ late in the ER, the defining characteristicof classB secmutants. The generalability of secmurants involved in either COPI or COPII vesiclefunction to eventually block both anterograde and retrograde transport illustrates the 594

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fundamental interdependence of these two rransporr processes. As discussedin Chapter 13, the ER contains severalsoluble proteins dedicated to the folding and modification of newly synthesizedsecretory proteins. These include the chaperone BiP and the enzymeprotein disulfide isomerase,which are necessaryfor the ER to carry out its functions. Although such ER-residentluminal proteins are not specificallyselected by COPII vesicles,their sheer abundancecausesthem to be continuouslyloadedpassivelyinto vesiclesdestinedfor the clsGolgi. The transport of thesesolubleproteins back to the ER, mediated by COPI vesicles,preventstheir eventualdepletion. Most soluble ER-residentproteins carry a Lys-Asp-GIuLeu (KDEL in the one-lettercode) sequenceat their C-terminus (seeTable 14-2). Severalexperimentsdemonstratedthat this KDEL sorting signal is both necessaryand sufficient to causea protein bearingthis sequenceto be located in the ER. For instance,when a mutant protein disulfide isomerase lacking thesefour residuesis synthesizedin cultured fibroblasts, the protein is secreted.Moreover, if a protein that normally is secretedis alteredso that it containsthe KDEL signal at its C-terminus,rhe protein is locatedin the ER. The KDEL sorting signalis recognizedand bound by the KDEL receptor, a transmembraneprotein found primarily on small transport vesiclesshuttling between the ER and the cls-Golgi and on the cis-Golgi reticulum. In addition, soluble ER-residentproteins that carry the KDEL signal have oligosaccharidechains

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Video: 3-D Model of a Golgi Complex K.€l < EXPERIMENTAL FIGURE 14-15 Electron micrographof the Golgicomplexin an Formingsecretory exocrinepancreatic cellrevealsboth vesicle anterogradeand retrogradetransport vesicles. A Iargesecretory trans-Golginetwork vesicle canbe seenformingfromthe trans-Golgi network -l Elements of the rough ER are on the bottom trans andleftin thismicrograph Adjacent to the ,,,uaiut lG.olsi roughERaretransitional elements from Crsrernae . I whichsmoothprotrusions appearto be cts ,) buddingThesebudsformthe smallvesicles cis-Golginetwork thattransport proteins secretory fromthe roughERto the Golgicomplexlnterspersed ER-to-Golgi a m o n gt h eG o l gci i s t e r n aaer eo t h e rs m a l l t r a n s p o r vt e s i c l e s vesicles now knownto functionin Smooth protrusion retrograde, not anterograde, transport G Palade [Courtesy ] T r a n s i t i o n aell e m e n t s

the Golgi appearsto have a highly dynamic organization. To see the effect this retrograde transport has on the organization of the Golgi, consider the net effect on the medial-Golgi compartment as enzymesfrom the trans-Golgi move to the medial-Golgi while enzymesfrom the medialG o l g i a r e r r a n s p o r t e dr o t h e c i s - G o l g i . A s t h i s p r o c e s s continues, the medial-Golgi acquires enzymes from the trans-Golgi while losing medial-Golgi enzymesand thus g r a d u a l l y b e c o m e sa n e w t r a n s - G o I g i c o m p a r t m e n t . I n this wa5 secretory cargo proteins can acquire carbohydrate modification in the proper sequentialorder without being moved from one cisternato another vra antero, grade vesicletransport. The first evidencethat the forward transporr of cargo proteins from the cis- to the trans-Goigi occurs by a nonvesicular mechanism, called cisternal maturation, came from careful microscopic analysisof the synthesisof algal scales.These cell-wall glycoproteins are assembledin the cls-Golgi into large complexes visible in the electron mi-

gen precursor often form in the lumen of the cls-Golgi (see Figure 19-24). The procollagen aggregaresare roo l a r g et o b e i n c o r p o r a t e di n t o s m a l l t r a n s p o r tv e s i c l e sa, n d

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CHAPTER 14

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investigators could never find such aggregaresin transport vesicles.These observationssuggestthat the forward m o v e m e n t o f t h e s e a n d p e r h a p s a l l s e c r e t o r yp r o t e i n s from one Golgi compartment to another does not occur via smallvesicles. A particularly elegant demonstration of cisrernal maturation in yeast takes advantageof different-coloredversions of GFP to image two different Golgi proteins simultaneously.Figure 14-16 shows how a cis-Golgiresidentprotein labeled with a green fluorescent protein and a trans-Golgt protein labeled with a red fluorescentprorein behavein the same yeast cell. At any given moment individual Golgi cisterna appear to have a distinct compartmental identity, in the sensethat they contain either the cls-Golgi protein or the trans-Golgi protein but only rarely contain both proteins. However, over time an individual cisterna labeled with the cis-Golgi protein can be seento progressivelylose this protein and acquire the trans-Golgi protein. This behavior is exactly that predicted for the cisternal maturation model, in which the composition of an individual cisterna changesas Golgi residentproteins move from later to earlier Golgi compartments. Numerous controversial questions concerning membrane flow within the Golgi stack remain unresolved. For example, although most protein traffic appears to move through the Golgi complex by a cisternal maturation mechanism,thereis evidencethat at leastsomeof the COpI trans-

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< FIGURE 14-17Vesicle-mediated protein trafficking from the trans-golginetwork. (ll) mediate COPI(purple) vesicles retrograde transport withintheGolgi.Proteins that functionin the lumenor in the membrane of the lysosome aretransported fromthe transGolginetworkviaclathrin coated(red)vesicles (B). Afteruncoating, thesevesicles fusewith lateendosomes, whichdeliver theircontents to the lysosome Thecoaton mostclathrin vesicles (AP contains proteins additional complexes) not indicated hereSomevesicles fromthe trans-Golgi carrying cargodestined for the lysosome fusewith the lysosome (E), bypassing directly theendosome These vesicles arecoatedwith a typeof APcomplex (blue); it is unknownwhetherthesevesicles alsocontainclathrin. Thecoatproteins (4) andregulated surrounding constitutive ($) secretory vesicles arenot yetcharacterized; thesevesicles proteins carrysecreted and plasma-membrane proteins fromthe transGolginetworkto thecellsurface

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FIGURE14-18 Structureof clathrin coats. (a)A clathrin molecule,calleda triskelion,is composedof threeheavyand three lightchains lt hasan intrinsiccurvaturedue to the bend in the heavychains(b) Clathrincoatswere formed in vrtroby mlxrng purifiedclathrinheavyand Iightchainswith Ap2 complexes in the absenceof membranesCryoelectron mrcrographs of morethan 1000assembled hexagonalclathrrnbarrelparticles were analyzedby digitalimageprocessing to generatean averagestructural representation. The processed imageshowsonlythe clathrinheavy chainsin a structurecomposedof 36 triskelions. Threerepresenralve triskelions are highlightedin red,yellow,and green partof the Ap2 complexes packedinto the interrorof the clathrincagearealso visiblein this the processed representation andG [SeeB pishvaee Payne,1998, Cell95:443 part(b)from Fotinet al , 2004,Nature432:573 I

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The best-characterizedvesicles that bud from the transGolgi network (TGN) have a two-layered coat: an outer layer composed of the fibrous protein clathrin and an inner Iayer composedof adapter protein (AP) complexes.Purified clathrin molecules,which have a three-limbed shape, are calledtriskelions, from the Greek for "three-legged" (Figure 14-18a). Each limb contains one clathrin heavy chain (180,000MV) and one clathrinlight chain (=35,000-40,000 M'W). Triskelions polymerize to form a polygonal lattice with an intrinsic curvature (Figure 14-18b). V/hen clathrin polymerizeson a donor membrane, it does so in association with AP complexes,which assemblebetweenthe clathrin lattice and the membrane. Each AP complex (340,000 M\f) contains one copy eachof four different adapter subunit proteins. A specificassociationbetween the globular domain at the end of each clathrin heavy chain in a triskelion and one subunit of the AP complex both promotes the co-assemblyof clathrin triskelions with AP complexesand adds to the stability of the completed vesiclecoat. By binding to the cytosolic face of membraneprorelns, adapter proteins determine which cargo proreins are specificallyincluded in (or excludedfrom) a budding transport vesicle. Three different AP complexes are known (4P1, AP2, AP3), each with four subunits of different, though related,proteins.Recently,a secondgeneraltype of adapterprotein known as GGA has beenshown to contain

V E S T C U L ATRR A F F | CS , E C R E T T OA NN , D ENDOCYTOStS

in a single 70,000 M\7 polypeptide both clathrin- and cargo-bindingelementssimilar to those found in the much larger hetero-tetramericAP complexes.Vesiclescontaining each type of adapter complex (AP or GGA) have been found to mediate specifictransport steps(seeTable 14-1). All vesicleswhose coats contain one of these complexes utilize ARF to initiate coat assemblyonto the donor membrane. As discussedpreviously,ARF also initiatesassembly of COPI coats.The additional featuresof the membraneor protein factors that determine which type of coat will assemble after ARF attachment are not well understood at this time. Vesicles that bud from the trans-GoIgi network en route to the lysosome by way of the late endosomehave clathrin coats associatedwith either AP1 or GGA. Both AP1 and GGA bind to the cytosolic domain of cargo proteins in the donor membrane. Recent studies have shown that membrane proteins containing a Tyr-X-X-O sequence,where X is any amino acid and O is a bulky hydrophobic amino acid, are recruited into clathrin/AP1 vesicles budding from the trans-Golgi network. This YXXQ sorting signal interacts with one of the AP1 subunits in the vesiclecoat. As we discussin the next section, vesicles with clathrin/AP2 coats, which bud from the plasma membrane during endocytosis,also can recognize the YXXO sorting signal. Vesiclescoated with GGA proteins and clathrin bind cargo molecules with a different kind of sorting sequence.Cytosolic sorting signals that specificallybind to GGA adapter proteins include Asp-XLeu-Leu and Asp-Phe-Gly-X-Osequences(where X and O are defined as above). Some vesiclesthat bud from the trans-Golgi network have coats composed of the AP3 complex. Although the AP3 complex does contain a binding site for clathrin similar to the AP1 and AP2 complexes,it is not clear whether clathrin is necessaryfor function of AP3-containing vesicles since mutants of AP3 that lack the clathrin binding site appear to be fully functional. AP3-coatedvesiclesmediate trafficking to the lysosome, but they appear to bypass the late endosomeand fuse directly with the lysosomal membrane.In certain types of cells, such AP3 vesicles mediate protein transport to specializedstoragecompartments related to the lysosome. For example, AP3 is required for delivery of proteins to melanosomes,which contain the black pigment melanin in skin cells, and to platelet storage vesiclesin megakaryocytes,a large cell that fragments into dozens of platelets.Mice with mutations in either of two different subunits of AP3 not only have abnormal skin pigmentation but also exhibit bleeding disorders.The latter occur becausetears in blood vesselscannot be repaired without plateletsthat contain normal storagevesicles.

D y n a m i nl s R e q u i r e df o r P i n c h i n gO f f of ClathrinVesicles A fundamental step in the formation of a transport vesicle that we have not yet considered is how a vesicle bud is

Exoplasmicface

Integral

vesicle Clathrin-coated pinchingoff of 14-19Modelfor dynamin-mediated a FIGURE forms, dynamin bud vesicle a After vesicles. clathrin/AP-coated thatisnotwellunderstood, polymerizes overthe neck.Bya mechanism of thevesicle of GTPleadsto release hydrolysis dynamin-catalyzed proteins in the fromthe donormembraneNotethatmembrane with by interacting vesicles into Incorporated are donormembrane Nature et al, 1995, fromK Takel in the coat lAdapted APcomplexes 374:186]l

oinched off from the donor membrane. In the case of clathrin/AP-coated vesicles' a cytosolic protein called dynamin is essentialfor releaseof complete vesicles.At the later stages of bud formation, dynamin polymerizes around the neck portion and then hydrolyzes GTP. The energy derived from GTP hydrolysis is thought to drive a conformational changein dynamin that stretchesthe vesicle neck until the vesiclepinchesoff (Figure 14-1,9).Intetestingly, COPI and COPII vesicles appear to pinch off from-donor membraneswithout the aid of a GTPasesuch as dynamin. At presentthis fundamental differencein the processof pinching off among the different types of vesicles is not understood. Incubation of cell extracts with a nonhydrolyzable detivative of GTP provides dramatic evidencefor the importance of dynamin in pinching off of clathrin/AP vesiclesduring endocytosis.Such treatment leadsto accumulation of clathrincoated vesicle buds with excessively long necks that are surrounded by polymeric dynamin but do not pinch off (Figure 14-20). Likewise, cells expressingmutant forms of dynamin that cannot bind GTP do not form clathrin-coated vesiclesand instead accumulate similar long-necked vesicle buds encasedwith polymerized dynamin. PATHWAY L A T E RS T A G E sO F T H E S E C R E T O R Y

.

599

< EXPERIMENTAL FIGURE 14-20 cTp hydrolysisby dynaminis requiredfor pinchingoff of clathrin-coated vesiclesin cell-free extracts.A preparation of nerveterminals, whichundergoextensive e n d o c y t o sw l sa, sl y s e db y t r e a t m e nwt i t h d i s t i l l ew d a t e ra n d incubated with GTP-1-S, a nonhydrolyzable derivative of GTpAfter sectioning t h, e p r e p a r a t i owna st r e a t e dw i t h g o l d - t a g g eadn t i d y n a m ia n n t i b o dayn dv i e w e di n t h e e l e c t r om n i c r o s c o pTeh i s image,whichshowsa long-necked clathrin/AP-coated budwith polymerizd e yd n a m i lni n i n gt h e n e c k r, e v e a tl sh a tb u d sc a nf o r mi n the absence of GTPhydrolysis, but vesicles cannotpinchoff. The polymerization extensive of dynaminthat occursin the presence of G T P - 1 -p5r o b a b ldy o e sn o t o c c u rd u r i n gt h e n o r m abl u d d i n g process[From K Takel et al , 1995,Nature 374:186; courtesy of pietro D eC a m i l l i

As with COPI and COPII vesicles,clathrin/Ap vesicles normally lose their coat soon after their formation. Cytosolic Hsc70, a constitutive chaperoneprotein found in all eukaryotic cells, is thought to use energy derived from the hydrolysis of ATP to drive depolymerization of the clathrin coat into triskelions.Uncoatingnot only releasestriskelions for reusein the formation of additional vesiclesbut also exposes v-SNAREs for use in fusion with targer membranes. Conformational changes thar occur when ARF switches from the GTP-bound to GDP-bound state are thousht to regulatethe timing of clathrin coat depolymerization.How the action of Hsc70 might be coupled to ARF switching is not well understood.

M a n n o s e6 - P h o s p h a t R e e s i d u eT s a r g e tS o l u b l e Proteinsto Lysosomes Most of the sorting signals that function in vesicular trafficking are short amino acid sequencesin the targeted protein. In contrast, the sorting signal that directs soluble lysosomal enzymes from the trans-Golgi network to the late

endosomeis a carbohydrateresidue,mlnnose 5-phospbate (M6P), which is formed in the cis-Golgi. The addition and initial processing of one or more preformed N-linked oligosaccharideprecursors in the rough ER is the same for lysosomal enzymesas for membrane and secretedproteins, yielding core Man6(GlcNAc)2chains (seeFigure 13-18). In the cls-Golgi, the N-linked oligosaccharidespresenton most lysosomal enzymesundergo a two-step reaction sequence that generatesM6P residues(Figure 14-21.).The addition of M6P residuesto the oligosaccharide chains of solublelysosomal enzymesprevents theseproteins from undergoing the further processing reactions characteristic of secretedand membrane proteins (seeFigure 14-14). As shown in Figure 14-22, the segregationof M6pbearing lysosomal enzymesfrom secretedand membrane proteins occurs inthe trans-Golgi network. Here, transmembrane mannose 6-phosphate receptors bind the M6p residues on lysosome-destinedproreins very tightly and specifically.Clathrin/AP1 vesiclescontaining the M6p receptor and bound lysosomal enzymesthen bud from the traisGolgi network, lose their coats, and subsequentlyfuse with

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FIGURE 14-21Formationof mannose6-phosphate (M6p) andboundby thisenzyme, phosphorylated groupsare GlcNAc residuesthat target solubleenzymesto lysosomes. TheM6p addedspecifically to lysosomal enzymesStepA: Afterrelease of a residues thatdirectproteins to lysosomes aregenerated in the crsmodifiedproteinfromthe phosphotransferase, a phosphodiesterase Golgibytwo Golgi-resident enzymes Step[: An N-acetylglucosamine removes the GlcNAc a phosphorylated mannose Aroup,leaving (GlcNAc) phosphotransferase transfers a phosphorylated r e s i d u e otnh el y s o s o meanl z y m e[ S GlcNAc , e e AB c a n t o r eatl ,j 9 9 2 , JB i o l groupto carbonatom6 of oneor moremannose residues Because Chem 267:23349,and S Kornfeld, 1987, FASEBI 1i4621 onlylysosomal enzymes containsequences (red)thatarerecoqnized

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CHAPTER 14

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V E S T C U L ATR R A F F t CS, E C R E T T OANN, D E N D O C Y T O S t S

M6P receptor

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'14-22frallicking of solublelysosomalenzymes FfGURE from the trans-Golginetwork and cell surfaceto lysosomes. produced in the ER,acquire Newlysynthesized lysosomal enzymes, (M6P)residues (seeFigure 6-phosphate in thecrs-Golgi mannose oligosaccharide chain 14-21)Forsimplicity, onlyonephosphorylated althoughlysosomal enzymes typically havemanysuch isdepicted, c h a i n sI n t h e t r a n s - G o lngei t w o r kp, r o t e i ntsh a tb e a rt h e M 6 P and with M6Preceptors in the membrane sortingsignalinteract (steptr) Thecoat aredirected intoclathrin/AP1 vesicles thereby (stepE), and released vesicles is rapidly depolymerized surrounding (stepB) vesicles fuse with late endosomes the uncoated transport fromthe M6Preceptors Afterthe phosphorylated enzymes dissociate

fusewith a subsequently lateendosomes andaredephosphorylated, are (step4) Notethatcoatproteins andM6Preceptors lysosome to aredelivered (steps recycled EEI andEE), andsomereceptors (stepE). Phosphorylated lysosomal enzymes thecellsurface and to the cellsurface aresortedfromthe trans-Golgi occasionally by receptorcanbe retrieved enzymes secretedThesesecreted parallels (steps6-ts), a process thatclosely endocytosis mediated networkto from the trans-Golgi enzymes traffickingof lysosomal 1992,Ann et al, 1988,Cell52:329,5Kornfeld, G Griffiths lysosomes [See Trends Cell 1991, andJ,Gruenberg, andG Griffiths 61:307; Rev. Biochem B i o1 l :51

the late endosome by mechanismsdescribed previously. BecauseM6P receptorscan bind M6P at the slightly acidic pH (=6.5) of the trans-Golgi network but not at a pH less than 6, the bound lysosomal enzymes are releasedwithin Iate endosomes,which have an internal pH of 5.0-5.5.

Furthermore, a phosphatasewithin late endosomesusually removesthe phosphatefrom M6P residueson lysosomal enzymes, preventing any rebinding to the M6P receptor that might occur in spite of the low pH in endosomes.Vesicles budding from late endosomesrecyclethe M6P receptor back L A T E RS T A G E SO F T H E S E C R E T O RPYA T H W A Y

601

to the trans-Golgi network or, on occasion, to the cell surface. Eventually, mature late endosomes fuse with lysosomes,delivering the lysosomal enzymes to their final destination. The sorting of soluble lysosomal enzymesin the transGolgi network (Figure L4-22, steps[-4) sharesmany of the features of trafficking between the ER and cls-Golgi compartments mediated by COPII and COPI vesicles.First, mannose 6-phosphateacrs as a sorring signal by interacting with the luminal domain of a receptor protein in the donor membrane.Second,the membrane-embedded receptorswith their bound ligands are incorporated into the appropriate vesicles-in this case, either GGA or AP1-containing clathrin vesicles-by interacting with the vesiclecoat. Third, thesetransport vesiclesfuse only with one specificorganelle, here the late endosome,as the result of interactions between specificv-SNAREs and I-SNAREs. And finally, intracellular transport receptorsare recycledafter dissociatingfrom their bound ligand.

Study of LysosomalStorageDiseases RevealedKey Componentsof the Lysosomal Sorting Pathway termedlysosomal storFE A groupof geneticdisorders

age diseasesare causedby the absenceof one or more 3l lysosomalenzymes.As a result, undigestedglycolipids and extracellularcomponentsthat would normally be degraded by lysosomalenzymesaccumulatein lysosomesas large inclusions.I-cell diseaseis a particularly severetype of lysosomal storagediseasein which multiple enzymesare missing from the lysosomes.Cells from affected individuals Iack the N-acetylglucosaminephosphotransferasethat is required for formation of M6P residueson lysosomal enzymes in the cls-Golgi (see Figure 1,4-21). Biochemical comparisonof lysosomalenzymesfrom normal individuals with those from patientswith I-cell diseaseled to the initial discoveryof mannose6-phosphateas rhe lysosomalsorting signal. Lacking the M6P sorting signal, the lysosomalenzymes in I-cell patients are secreted rather than being sorted to and sequestered in lysosomes. lVhen fibroblasts from patients with I-cell disease are grown in a medium containing lysosomalenzymesbearing M6P residues,the diseasedcellsacquirea nearly normal intracellular content of lysosomal enzymes.This finding indicatesthat the plasmamembraneof thesecellscontainsM6p receptors,which can internalize extracellular phosphorylated lysosomalenzymesby receptor-mediated endocytosis. This process,used by many cell-surfacereceptorsto bring bound proteins or particlesinto the cell, is discussedin detail in the next section. It is now known that even in normal cells, some M6P receptorsare transported to the plasma membrane and some phosphorylated lysosomal enzymes are secreted(seeFigure 14-22). The secretedenzymescan be retrieved by receptor-mediatedendocytosisand directed to lysosomes.This pathway thus scavenges any lysosomalenzymes that escapethe usual M6P sorting parhway.

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Hepatocytes from patients with l-cell diseasecontain a normal complementof lysosomalenzymesand no inclusions, even though these cells are defective in mannose phosphorylation.This finding implies that hepatocytes(the most abundant type of liver cell) employ an M6P-independent pathway for sorting lysosomal enzymes.The nature of this pathway, which also may operate in other cells types,is unknown. I

ProteinAggregationin the trans-GolgiMay Functionin Sorting Proteinsto Regulated SecretoryVesicles As noted in the chapter introduction, all eukaryotic cells continuously secretecertain proteins, a process commonly called constitwtiue secretion. Specialized secretory cells also store other proteins in vesiclesand secretethem only when triggered by a specific stimulus. One example of such regulated secretioz occurs in pancreatic B cells, which store newly made insulin in special secretoryvesiclesand secrete insulin in responseto an elevation in blood glucose(seeFigure 15-33). These and other secretorycells simultaneously utilize two different types of vesiclesto move proteins from the trans-Golgi network to the cell surface:regulated transport vesicles,often simply called secretoryvesicles,and unregulated transport vesicles,also called constitutive secretory vesicles. A common mechanismappearsto sort regulatedproteins as diverseas ACTH (adrenocorticotropichormone), insulin, and trypsinogen into regulated secretory vesicles.Evidence for a common mechanismcomesfrom experimentsin which recombinant DNA techniquesare used to induce the synthesis of insulin and trypsinogenin pituitary tumor cells already synthesizingACTH. In these cells, which do not normally express insulin or trypsinogen, all three proteins segregate into the same regulated secretory vesiclesand are secreted together when a hormone binds to a receptor on the pituitary cellsand causesa rise in cytosolic Ca2*. Although these three proteins share no identical amino acid sequencesrhat might serveas a sorting sequence,they obviously have some common feature that signals their incorporation into regulated secretoryvesicles. Morphologic evidencesuggeststhat sorting into the regulated pathway is controlled by selectiveprorein aggregadon. For instance,immature vesiclesin this pathway-those that have just budded from the trans-Golginerwork-contain diffuse aggregatesof secretedprotein that are visible in the electron microscope.These aggregatesalso are found in vesicles that are in the processof budding, indicating that proteins destinedfor regulated secretoryvesiclesselectivelyaggregate together before their incorporation into the vesicles. Other studies have shown that regulated secretoryvesicles from mammalian secretorycells contain three proteins, chromogranin A, chromogranin B, and secretogranin II, that together form aggregateswhen incubated at the ionic conditions (pH = 6.5 and 1 mM Ca2*; thought to occur in the trans-Golgi network; such aggregatesdo not form at the

VEStCULAR T R A F F t CS, E C R E T T OA NN , D ENDOCYTOStS

14-23Proteolyticcleavageof < EXPERIMENTAL FIGURE proinsulinoccursin secretoryvesiclesafter they have budded sections of the Golgiregion network.Serial from the trans-Golgi with (a)a monoclonai cellwerestained of an insulin-secreting proinsulin or (b)a different but not insulin that recognizes antibody Theantibodies, insulinbut not proinsulin antibody that recognizes goldparticles, appearasdark whichwereboundto electron-opaque (seeFigure 9-21).lmmature micrographs dotsin theseelectron (closed buddingfromthe arrowheads) andvesicles vesicles secretory (arrows) antibody but notwith stainwiththe proinsulin trans-Golgi vesicles containdiffuseproteinaggregates insulin antibodyThese proteinsMature proinsulin secreted andotherregulated that include (openarrowheads) but notwith antibody stainwith insulin vesicles proinsulin antibody andhavea densecoreof almostcrystalline vesicles contain secretory insulinSincebuddingandimmature (notinsulin), to of proinsulin conversion proinsulin the proteolytic aftertheybudfromthe insulinmusttakeplacernthesevesicles secretory networkTheinsetin (a)showsa proinsulin-rich trans-Golgi ltne)lrromL orcietal, by a proteincoat(dashed vesicle surrounded

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neutral pH of the ER. The selectiveaggregationof regulated secretedproteins together with chromogranin A, chromogranin B, or secretograninII could be the basisfor sorting of theseproteins into regulatedsecretoryvesicles.Secretedproteins that do not associatewith theseproteins,and thus do not form aggregates,would be sorted into unregulatedtransp o r t v e s i c l e bs y d e f a u l t .

For somesecretoryproteins(e.g.,growth hormone) and certain viral membraneproteins (e.g.,the VSV glycoprotein), removal of the N-terminal ER signal sequencefrom the nascent chain is the only known proteolytic cleavagerequired to convert the polypeptide to the mature, active species(see Figure 13-6). However, some membraneand many soluble secretoryproteins initially are synthesizedas relatively longlived, inactive precursors, termed proproteins, that require further proteolytic processingto generatethe mature, active proteins. Examples of proteins that undergo such processing are soluble lysosomal enzymes)many membrane proteins such as influenza hemagglutinin (HA), and secretedproteins such as serum albumin, insulin, glucagon, and the yeast c{. mating factor. In general, the proteolytic conversion of a proprotein to the correspondingmature protein occurs after the proprotein has been sorted in the trans-Golgi network to appropriate vesicles. In the caseof soluble lysosomalenzymes,the proproteins are called proenzymes,which are sorted by the M5P receptor as catalytically inactive enzymes.In the late endosomeor lysosomea proenzymeundergoesa proteolytic cleavagethat generatesa smaller but enzymaticallyactive polypeptide. Delaying the activation of lysosomal proenzymesuntil they reach the lysosomepreventsthem from digestingmacromoleculesin earlier compartments of the secretorypathway. Normall5 mature vesiclescarrying secretedproteins to the cell surface are formed by fusion of several immature ones containing proprotein. Proteolytic cleavageof proproteins, such as proinsulin, occurs in vesiclesafter they move away from the trans-Golgi network (Figure 14-23). The proproteins of most constitutively secreted proteins (e.g., albumin) are cleaved only once at a site C-terminal to a L A T E RS T A G E SO F T H E S E C R E T O RPYA T H W A Y

603

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and thus were unable to mate with cells of the opposite mating type (seeFigure 21,-19).The wild-type KEX2 gene encodesan endoproteasethat cleavesthe cr-factorprecursor at a site C-terminal to Arg-Arg and Lys-Arg residues.Using the KEX2 gene as a DNA probe, researcherswere able to clone a famtly of mammalian endoproteases,all of which cleavea protein chain on the C-terminal side of an Arg-Arg or LysArg sequence.One, calledfurin, is found in all mammalian cells; it processesproteins such as albumin that are secreted by the continuous pathway. In contrast, the PC2 and PC3 endoproteasesare found only in cells that exhibit regulated secretion;theseenzymesare localized to regulated secretory vesiclesand proteolytically cleave the precursors of many hormones at specificsites.

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SeveralPathwaysSort MembraneProteins to the Apical or BasolateralRegion o f P o l a r i z e dC e l l s

The plasma membrane of polarized epithelial cells is divided into two domains, apical and basolateral; tight juncIIS-S tions located between the two domains prevent the movecarbox'Pe'tidase ment of plasma-membrane proteins between the domains (seeFigure 19-9). Severalsorting mechanismsdirect newly [ [^E-] t-^Er synthesizedmembrane proteins to either the apical or basolateral domain of epithelial cells, and any one protein may be sorted by more than one mechanism.Although these s__l__s I .[' -l---lsorting mechanisms are understood in general terms, the Insutin|ll IBI molecular signals underlying the vesicle-mediatedtransport S-S of membrane proteins in polarized cells are not yet known. As a result of this sorting and the restriction on protein A FIGURE 14-24 Proteolyticprocessingof proproteinsin the movement within the plasma membrane due to tight juncconstitutiveand regulatedsecretionpathways.Theprocessing of proalbumin andproinsulin istypical tions, distinct setsof proteins are found in the apical or baof theconstitutive andregulated pathways, respectively. Theendoproteases that functionin such solateral domain. This preferential localization of certain processing cleave at the C-terminal sideof two consecutive amino transport proteins is critical to a variety of important physacids(a)Theendoprotease furinactson the precursors of constitutive iological functions, such as absorption of nutrients from the proteins(b)Twoendoproteases, secreted PC2andPC3,acton the intestinal lumen and acidification of the stomach lumen (see precursors of regulated proteinsThefinalprocessing secreted of Figures11-29 and 11-30). manysuchproteins iscatalyzed by a carboxypeptidase that Microscopic and cell-fractionation studies indicate that sequentially removes two basicaminoacidresidues at theC-terminus proteins destined for either the apical or the basolateral of a polypeptide[See D Steiner etal,1992,] Biol.Chem261:234351 membranes are initially located together within the membranes of the trans-Golgi network. In some cases,proteins destined for the apical membrane are sorted into their own transport vesiclesthat bud from the trans-Golgi network dibasic recognition sequencesuch as Arg-Arg or Lys-Arg and then move to the apical region, whereas proteins des(Figure 14-24a). Proteolytic processingof proteins whose tined for the basolateral membrane are sorted into other secretionis regulated generally entails additional cleavages. vesiclesthat move to the basolateral region. The different In the case of proinsulin, multiple cleavagesof the single vesicle types can be distinguished by their protein conpolypeptide chain yields the N-terminal B chain and the stituents, including distinct Rab and v-SNARE proteins, C-terminal A chain of mature insulin, which are linked by which apparently target them to the appropriate plasmadisulfide bonds, and the central C peptide, which is lost and membrane domain. In this mechanism, segregationof prosubsequentlydegraded(Figure14-24b). teins destined for either the apical or basolateral memThe breakthrough in identifying the proteasesresponsrbranes occurs as cargo proteins are incorporated into ble for such processingof secretedproteins came from analyparticular types of vesicles budding from the trans-Golgi sis of yeastwith a mutation inthe KEX2 gene.Thesemutant network. cells synthesizedthe precursor of the cr mating factor but Such direct basolateral-apicalsorting has been investicould not proteolytically process it to the functional form gated in cultured Madin-Darby canine kidney (MDCK)

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< FIGURE 14-25 Sortingof proteins destinedfor the apicaland basolateral plasmamembranesof polarizedcells.When culturedMDCKcellsareinfectedsimultaneously w i t h V S Va n d i n fl u e n z av i r u st,h e V S VG g l y c o p r o t e (i np u r p l ei)s f o u n do n l yo n t h e HA whereas the influenza membrane, basolateral (green)isfoundonlyon the apical glycoprotein S.o m ec e l l u l aprr o t e i n(so r a n g cei r c l e ) , membrane especially thosewith a GPIanchor,arelikewise and to the apicalmembrane sorteddirectly (notshown) membrane othersto the basolateral vesicles that bud fromthe transport viaspecific cells, network.In certainpolarized trans-Golgi proteins are someapicaland basolateral surface; togetherto the basolateral transported (yellowoval)then move the apicalproteins to andtranscytosis, by endocytosis selectively, andA the apicalmembrane. [AfterK Simons 1990,Cell62:201 Wandinger-Ness, , andK Mostov et al , 1992, I CellBiol 116:577 l

cells, a line of cultured polarized epithelial cells (seeFigwe 9-34).In MDCK cellsinfectedwith the influenzavirus, progeny viruses bud only from the apical membrane, whereas in cells infected with vesicular stomaritis virus (VSV), progeny viruses bud only from the basolateralmembrane. This difference occurs becausethe HA glycoprotein of influenza virus is transported from the Golgi complex exclusively to the apical membrane and the VSV G protein is transported only to the basolateral membrane (Figure 14-25). Furthermore, when the geneencoding HA protein is introduced into uninfected cells by recombinant DNA techniques, all the expressedHA accumulatesin the apical membrane, indicating that the sorting signal resides in the HA glycoprotein itself and not in other viral proteins produced during viral infection. Among the cellular proteins that undergo similar apicalbasolateral sorting in the Golgi are those with a glycosylp h osph atidy lino sito I (G PI ) m embrane an chor. In MD CK cells and most other types of epithelial cells, GPl-anchored proteins are targeted to the apical membrane.In membranes GPl-anchored proteins are clustered into lipid rafts, which are rich in sphingolipids (seeChapter 10). This finding suggests that lipid rafts are localized to the apical membrane along with proteins that preferentially partition them in many cells.However, the GPI anchor is not an apical sorting signal in all polarized cells; in thyroid cells, for example, GPl-anchored proteins are targeted to the basolateralmembrane. Other than GPI anchors, no unique sequenceshave been identified that are both necessaryand sufficient to target proteins to either the apical or basolateral domain. Instead, each membrane protein may contain multiple sorting

signals, any one of which can target it to the appropriate plasma-membranedomain. The identification of such complex signals and of the vesicle coat proteins that recognize them is currently being pursued for a number of different proteins that arc sorted to specific plasma-membranedomains of polarized epithelial cells. Another mechanism for sorting apical and basolateral proteins, also illustratedin Figure 14-25, operatesin hepatocytes. The basolateral membranesof hepatocytesface the blood (as in intestinalepithelialcells),and the apical membranes line the small intercellular channels into which bile is secreted.In hepatocytes,newly made apical and basolateral proteins are first transported in vesiclesfromthe transGolgi network to the basolateralregion and incorporated into the plasma membraneby exocytosis(i.e.,fusion of the vesiclemembranewith the plasma membrane)'From there, both basolateral and apical proteins are endocytosedin the same vesicles, but then their paths diverge. The endocytosed basolateralproteins are sorted into transport vesicles that recycle them to the basolateral membrane. In contrast, the apically destined endocytosed proteins are sorted into transport vesiclesthat move across the cell and fuse with the apical membrane, a process called transcytosis. This process also is used to move extracellular materials from one side of an epithelium to another. Even in epithelial procells,such as MDCK cells,in which apical-basolateral tein sorting occurs in the Golgi, transcytosismay provide a "fail-safe" sorting mechanism.That is, an apical protein sorted incorrectly to the basolateral membrane would be subjectedto endocytosisand then correctly delivered to the apical membrane. PATHWAY L A T E RS T A G E SO F T H E S E C R E T O R Y

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Later Stages of the Secretory Pathway t The trans-Golgi network (TGN) is a major branch point in the secretory pathway where soluble secretedproteins, Iysosomal proteins, and in some cells membrane proteins destinedfor the basolateralor apical plasma membrane are segregatedinto different transport vesicles. r Many vesiclesthat bud from the trans-Golgi network as well as endocytic vesicles bear a coat composed of AP (adapterprotein) complexesand clathrin (seeFigure 1.4-1.8). r Pinching off of clathrin-coatedvesiclesrequiresdynamin, which forms a collar around the neck of the vesiclebud and hydrolyzes GTP (seeFigure 14-191. r Soluble enzymesdestined for lysosomesare modified in the cls-Golgi, yielding multiple mannose 5-phosphate (M6P) residueson their oligosaccharide chains. r M6P receptors in the membrane of the trans-Golgi network bind proteins bearing M6P residuesand direct their transfer to late endosomes,where receptors and their ligand proteins dissociate.The receptorsthen are recycledto the Golgi or plasma membrane, and the lysosomalenzymes are deliveredto lysosomes(seeFigure 14-22). r Regulated secretedproteins are concentratedand stored in secretory vesiclesto await a neural or hormonal signal for exocytosis.Protein aggregationwithin the trans-GoIgi network may play a role in sorting secretedproteins to the regulatedpathway. r Many proteins transported through the secretory pathway undergo post-Golgi proteolytic cleavagesthat yield the mature, active proteins. Generally,proteolytic maturation can occur in vesiclescarrying proteins from the trans-Golgi network to the cell surface,in the late endosome,or in the lysosome. r In polarized epithelial cells, membrane proteins destined for the apical or basolateral domains of the plasma membrane are sorted in the trans-Golgi network into different transport vesicles(seeFigure 14-25). The GPI anchor is the only apical-basolateralsorting signal identified so far. r In hepatocytes and some other polarized cells, all plasma-membraneproteins are directed first to the basolateral membrane. Apically destined proteins then are endocytosed and moved acrossthe cell to the apical membrane (transcytosis).

flp

Receptor-Mediated Endocytosis

In previous sectionswe have explored the main pathways whereby secretoryand membraneproteins synthesizedon the rough ER are delivered to the cell surface or other destinations. Cells also can internalize materials from their surroundings and sort theseto particular destinations.A few cell types (e.g., macrophages)can take up whole bacteria and other large particles by phagocytosis,a nonselectiveacrin605

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mediated processin which extensionsof the plasma membrane envelop the ingested material, forming large vesicles called phagosomes(seeFigure 9-2).ln contrast, all eukaryotic cells continually engage in endocytosis, a process in which a small region of the plasma membraneinvaginatesto form a membranelimited vesicleabout 0.05-0.1 pm in diameter. In one form of endocytosis, called pinocyrosls, small droplets of extracellular fluid and any material dissolvedin it are nonspecificallytaken up. Our focus in this section,however, is on receptor-mediatedendocytosis,in which a specific receptor on the cell surface binds tightly to an extracellular macromolecular ligand that it recognizes;the plasma-membrane region containing the receptorJigand complex then buds inward and pinches off, becoming a transport vesicle. Among the common macromolecules that vertebrate cellsinternalizeby receptor-mediatedendocytosisare cholesterol-containing particles called low-density lipoprotein (LDL), the iron-binding protein transferrin, many protein hormones (e.g., insulin), and certain glycoproteins. Receptor-mediated endocytosis of such ligands generally occurs via clathrin/AP2-coatedpits and vesiclesin a processsimilar to the packaging of lysosomal enzymesby mannose 6-phosphate (M6P) in the trans-Golgi network (seeFigure 1.4-22). As noted earlier, some M6P receptors are found on the cell surface,and theseparticipate in the receptor-mediatedendocytosis of lysosomalenzymesthat are mistakenly secreted.In general, the transmembranereceptor proteins that function in the uptake of extracellular ligands are internalized from the cell surface during endocytosisand are then sorted and recycled back to the cell surface,much like the recycling of M5P receptors to the plasma membrane and trans-Golgi. The rate at which a ligand is internalized is limited by the amount of its correspondingreceptor on the cell surface. Clathrin/AP2 pits make up about 2 percent of the surface of cells such as hepatocytesand fibroblasts. Many internalized ligands have been observed in these pits and vesicles, which are thought to function as intermediatesin the endocytosis of most (though not all) ligands bound to cell-surface receptors (Figure 1,4-26).Some receptors are clustered over clathrin-coated pits even in the absenceof ligand. Other receptors diffuse freely in the plane of the plasma membrane but undergo a conformational change when binding to ligand, so that when the receptor-ligandcomplex diffuses into a clathrin-coatedpit, it is retained there. Two or more types of receptor-boundligands, such as LDL and transferrin, can be seenin the samecoated pit or vesicle.

CellsTakeUp Lipidsfrom the Blood in the Form of Large,Well-DefinedLipoproteinComplexes Lipids absorbed from the diet in the intestinesor stored in adipose tissue can be distributed to cells throughout the body. To facilitate the mass transfer of lipids between cells, animals have evolved an efficient way to packagefrom hundreds to thousands of lipid molecules into water-soluble, macromolecular carriers, called lipoproteins, that cells can take up from the circulation as an ensemble.A lipoprotein particle has a shell composed of proteins (apolipoproteins)

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EXPERIMENTAL FIGURE 14-26The initialstagesof receptormediatedendocytosisof low-densitylipoprotein(LDL) particlesare revealedby electronmicroscopy. human Cultured fibroblasts wereincubated in a mediumcontarning LDLparticles protein linkedto theelectron-dense, covalently iron-containing ferritin;eachsmallironparticle in ferritinisvisible asa smalldot microscope. underthe electron Cellsinitially wereincubated at 4'C; at thistemperature LDLcanbindto itsreceptor, but internalization doesnot occur. Afterexcess LDLnot boundto the cellswaswashed for away,thecellswerewarmedto 37 'C andthenprepared

(a)A coatedpit,showingthe intervals at periodic microscopy of the pit,soonafterthe surface coaton the inner(cytosolic) clathrin (b) pit closing LDLapparently containing raised. A was temperature containing on itselfto forma coatedvesicle(c)A coatedvesicle (d)Ferritin-tagged in a LDLparticles LDLparticles ferritin-tagged afterinternalization 6 minutes earlyendosome smooth-surfaced from bypermission Reprinted of R Anderson courtesy began.[Photographs Journals l979,Macmillan Copyright 279:619 etal, Nature J Goldstern 232:341 1986,Scrence andJ Goldstein, Lrmrted SeealsoM 5 Brown

and a cholesterol-containingphospholipid monolayer. The shell is amphipathic becauseits outer surfaceis hydrophilic, making theseparticles water soluble, and its inner surfaceis hydrophobic. Ad;acent to the hydrophobic inner surface of the shell is a core of neutral lipids containing mostly cholesteryl esters,triglycerides, or both. Mammalian lipoproteins fall into different classes,defined by their differing buoyant densities.The class we will consider here is low-density lipoprotein (LDL). A typical LDL particle, depicted in Figve 1.4-27,is a sphere20-25 nm in diameter.The amphipathic outer shell is composed of a phospholipid monolayer and a single molecule of a large protein known as apoB-100; the core of a particle is packed with cholesterol in the form of cholesterylesters. Two generalexperimental approacheshave been used to study how LDL particles enter cells.The first method makes use of LDL that has been labeledby the covalent attachment 12sIto the side chains of tyrosineresiduesin of radioactive apoB-100 on the surfacesof the LDL particles.After cul-

tured cells are incubated for severalhours with the labeled LDL, it is possibleto determine how much LDL is bound to the surfacesof cells. how much is internalized,and how much of the apoB-100 component of the LDL is degradedby enzymatic hydrolysis to individual amino acids. The drp:l125Id"tion of apoB-100 can be detectedby the releaseof tyrosine into the culture medium. Figure 14-28 shows the time course of eventsin receptor-mediatedcellular LDL processingdetermined by pulse-chaseexperimentswith a fixed 12sI-labeledLDL. These experiments concentration of clearly demonstrate the order of events:surface binding of LDL -+ internalization -+ degradation. The second approach involves tagging LDL particles with an electrondense label that can be detected by electron microscopy. Such studiescan revealthe details of how LDL particles first bind to the surface of cells at clathrin-coated endocytic pits and then remain associatedwith the coated pits as they invaginate and bud off to form coated vesiclesand finally are transportedto endosomes(seeFigure 1'4-26). R E C E P T O R - M E D I A TE EN DD O C Y T O S I S

607

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FIGURE 14-27Modelof low-densitylipoprotein(LDL).This class andthe otherclasses of lipoproteins havethesamegeneral structure: an amphipathic shell,composed of a phospholipid (notbilayer), monolayer cholesterol, andprotein, anda hydrophobic core,composed mostlyof cholesteryl esters or triglycerides or both b u tw i t hm i n o a r m o u n tosf o t h e rn e u t r al il p i d (seg , ,s o m ev i t a m i n s ) Thismodelof LDLis basedon electron microscopy andotherlowr e s o l u t i obni o p h y s i cmael t h o d sL D Li su n i q u ei n t h a ti t c o n t a i nosn l y a single molecuo l ef o n et y p eo f a p o l i p o p r o t (eai np o Bw ) ,h i c h appears to wraparoundtheoutside of the particle asa bandof proteinTheotherlipoproteins containmultiple apolipoprotein molecules, oftenof different types[Adapted fromM Krieger, 1995, in E Haber, ed , Molecular Cardiovascular Medrcine. Scientific American pp 31-47I Medicrne,

Receptorsfor Low-DensityLipoprotein a n d O t h e r L i g a n d sC o n t a i nS o r t i n gS i g n a l s That TargetThem for Endocytosis The key to understandinghow LDL particlesbind to the cell surface and are then taken up into endocytic vesiclescame from discovery of the LDL receptor (LDLR). The LDL receptor is an 839-residueglycoprotein with a single transmembrane segment; it has a short C-terminal cytosolic segmentand a long N-terminal exoplasmic segmentthat containsa B-propellerdomain and a ligand-bindingdomain. Seven cysteine-rich imperfect repeats form the ligandbinding domain, which interacts with the apoB-100 moleculein a LDL particle. Figure 14-29 shows how LDL receptor proteins facilitate internalization of LDL particles by receptor-mediatedendocytosis. After internalized LDL particles reach lysosomes,lysosomal proteaseshydrolyze their surface apolipoproteins and lysosomal cholesteryl esteraseshydrolyze their core cholesterylesters.The unesterified cholesterolis then free to leavethe lysosomeand be used as necessaryby the cell in the synthesisof membranesor various cholesterolderivatrves. The discoveryof the LDL receptor and an understand-

diseasethat is marked by elevated plasma LDL cholesterol 508

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A EXPERIMENTAL FIGURE 14-28Pulse-chase experiment demonstratesprecursor-product relationsin cellularuptakeof LDL.Cultured normalhumanskinfibroblasts wereincubated in a t"I-LDLfor 2 hoursat 4 "C(thepulse)After mediumcontaining 12sl-LDL excess notboundto thecellswaswashed away,thecellswere incubated a|37"Cfor theindicated amounts of timein theabsence of external LDL(thechase)Theamounts of surface-bound, internalized. 12sl-LDL (hydrolyzed) anddegraded weremeasured. Binding but not internalization or hydrolysis of LDLapoB-100 occurs duringthe4'C pulse, Thedatashowtheveryrapiddisappearance of bound1251-LDL fromthesurface it internalized as is afterthecellshavebeenwarmed to allowmembrane movements Aftera lagperiodof 15-20minutes, 12sl-LDL lysosomal degradation of theinternalized commences tV] [See S BrownandJ L Goldstein, 1976, Ce//9:663 1 and is now known to be causedby mutations in the LDLR gene.In patientswho have one normal and one defectivecopy of the LDLR gene (heterozygotes),LDL cholesterol in the blood is increasedabout twofold. Those with rwo defective LDLR genes(homozygotes)have LDL cholesterollevelsthat are from fourfold to sixfold as high as normal. FH heterozygotes commonly develop cardiovasculardiseaseabout 10 years earlier than normal people do, and FH homozygotes usually die of heart attacks beforereachingtheir late 20s. A variety of mutations in the geneencodingthe LDL receptor can causefamilial hypercholesterolemia. Some mutations prevent the synthesisof the LDLR protein; others prevent proper folding of the receptor protein in the ER, l e a d i n g t o i t s p r e m a t u r e d e g r a d a t i o n( C h a p t e r 1 3 ) ; s t i l l other mutations reduce the ability of the LDL recepror to bind LDL tightly. A particularly informative group of mutant receptorsare expressedon the cell surface and bind LDL normally but cannot mediate the internalization of bound LDL. In individualswith this type of defect,plasmamembranereceptorsfor other ligands are internalizednormally, but the mutant LDL receptor is not recruited into coated pits. Analysis of this mutant receptor and other mutant LDL receptors generatedexperimentally and expressedin fibroblastsidentified a four-residuemotif in the cytosolic segmentof the receptor that is crucial for its internalization: Asn-Pro-X-Tyr,where X can be any amino acid. This NPXY sorting signalbinds to the AP2 complex,

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FIGURE 14-29Endocyticpathwayfor internalizinglowdensitylipoprotein(tDL).Step[: Cell-surface LDLreceptors bind to an apoBproteinembedded in the phospholipid outerlayerof LDL particles Interaction between the NPXYsortingsignalin thecytosolic tailof the LDLreceotor andthe AP2comolexincoroorates the intoformingendocytic receptor-ligand complex vesicles StepE: pits(orbuds)containing receptor-LDL are Clathrin-coated complexes pinched off bythe samedynamin-mediated mechanism usedto form vesicles on the trans-Golgr network(seeFigure14-19) clathrin/AP1 coatisshed,the uncoated endocytic vesicle StepB:After thevesicle

TheacidicpH in this (earlyendosome) fuseswith the lateendosome. changein the LDLreceptor causes a conformational compartment of the boundLDLparticleStepZl: Thelate that leadsto release andlipidsof the andthe proteins fuseswith the lysosome, endosome partsby arebrokendownto theirconstituent freeLDLparticle recycles to the Step[: TheLDLreceptor in the lysosome. enzymes mediumthe whereat the neutralpHof the exterior cellsurface, changesothatit canbind a conformational receptor undergoes 1986,science andJ L Goldstein, M S Brown anotherLDLparticle. [See 298:2353 etal,2002,Sclence andG Rudenko l 232:34,

linking the clathrin/AP2 coat to the cytosolic segmentof the LDL receptor in coated pits. A mutation in any of the conservedresiduesof the NPXY signalwill abolishthe ability of the LDL receptor to be incorporated into coated pits. A small number of individuals who exhibit the usual symptoms associatedwith familial hypercholesterolemia produce normal LDL receptors.In these individuals, the geneencodingthe AP2 subunit protein that binds the NPXY sorting signal is defective.As a result, LDL receptorsare not incorporated into clathrin/AP2 vesiclesand endocytosisof LDL particles is compromised.Analysis of patients with this genetic disorder highlights the importance of adapter proteins in protein trafficking mediated by clathrin vesicles.I

Mutational studieshave shown that other cell-surfacereceptors can be directed into forming clathrin/AP2 pits by a different sorting signal: Tyr-X-X-O, where X can be any amino acid and Q is a bulky hydrophobic amino acid. This Tyr-X-X-O sorting signal in the cytosolic segment of a teceptor protein binds to a specific cleft in one of the protein subunits of the AP2 complex. Becausethe tyrosine and O residues mediate this binding' a mutation in either one reducesor abolishesthe ability of the receptor to be incorporated into clathrin lAP2-coatedpits.Moreover, if influenza HA protein, which is not normally endocytosed,is genetically engineeredto contain this four-residue sequencein its cytosolic domain, the mutant HA is internalized' Recall D I A T E DE N D O C Y T O S I S RECEPTORE -M

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from our earlier discussionthat this same sorting signal recruits membraneproteins into clathrin/AP1 vesiclesthat bud from the trans-Golgi network by binding to a subunit of AP1 (seeTable 14-2). All these observationsindicare rhat Tyr-XX-O is a widely used signal for sorting membraneproteins to clathrin-coatedvesicles. In some cell-surfaceproteins, however, other sequences (e.g.,Leu-Leu)or covalentlylinked ubiquitin moleculessignal endocytosis.Among the proteins associatedwith clathrin/AP2 vesicles,several contain domains that specifically bind to ubiquitin, and it has been hypothesizedthat thesevesicle-associated proteins mediate the selectiveincorporation of ubiquitinated membrane proteins into endocytic vesicles.As described later, the ubiquitin tag on endocytosedmembrane proteins is also recognized at a later stage in the endocytic pathway and plays a role in delivering these proteins into the interior of the lysosome,where they are degraded.

The Acidic pH of Late EndosomesCausesMost Receptor-Ligand Complexesto Dissociate The overall rate of endocytic internalization of the plasma membrane is quite high: cultured fibroblasts regularly internalize 50 percent of their cell-surfaceproteins and phospholipids each hour. Most cell-surfacereceptorsthat undergo endocytosis will repeatedlydeposit their ligands within the cell and then recycle to the plasma membrane, once again to mediate internalization of ligand molecules.For instance,the LDL receptor makes one round trip into and out of the cell every 10-20 minutes,for a total of severalhundred trips in its 20-hour life span.

Internalizedreceptor-ligandcomplexescommonly follow the pathway depicted for the M5P receptor inFigure 14-22 and the LDL receptor in Figure L4-29. Endocytosedcell-surface receptorstypically dissociatefrom their ligands within late endosomes,which appearas sphericalvesicleswith tubuIar branching membraneslocateda few micrometersfrom the cell surface.The original experimentsthat definedthe late endosome sorting vesicleutilized the asialoglycoproreinreceptor. This liver-specific protein mediates the binding and internalization of abnormal glycoproteinswhose oligosaccharides terminate in galactose rather than the normal sialic acid; hence the name asialoglycoprotein. Electron microscopy of liver cells perfusedwith asialoglycoproteinreveal that 5-10 minutes after internalization, ligand molecules are found in the lumen of late endosomes,while the tubular membraneextensionsare rich in receptor and rarely contain ligand. These findings indicate that the late endosomeis the organelle in which receptorsand ligands are uncoupled. The dissociationof receptor-ligandcomplexesin late endosomesoccurs not only in the endocytic pathway but also in the delivery of soluble lysosomal enzymesvia the secretory pathway (seeFigure 1.4-22).As discussedin Chapter 11, the membranesof late endosomesand lysosomescontain Vclassproton pumps that act in concert with Cl channelsto acidify the vesiclelumen (seeFigure 11-13). Most receptors, including the M5P receptor and cell-surfacereceptors for LDL particles and asialoglycoprotein, bind their ligands tightly at neutral pH but releasetheir ligands if the pH is Iowered to 6.0 or below. The late endosomeis the first vesicle encounteredby receptor-ligandcomplexeswhose luminal

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FIGURE 14-30Modelfor pH-dependent bindingof LDt particlesby the LDLreceptor.Schematic depictionof LDLreceptor at neutralpHfoundat thecellsurface (a)andat theacidicpHfound in the interiorof the lateendosome (b) (a)nt the cellsurface, apoB100on thesurface of a LDLparticle bindstightlyto the receptor. Of (R1-R7) thesevenrepeats in the ligand-binding arm,R4andR5 appearto be mostcritical (b,top)Withinthe for LDLbinding. endosome, histidine residues in the B-propeller domainof the LDL receptorbecomeprotonatedThepositively chargedpropeller can bindwith highaffinityto the ligand-binding arm,whichcontains negatively charged residues, causing release of the LDLparticle(b, bottom)Experimental electron density andCotracemodelof the extracellular regionof the LDLreceptor at pH 5 3 basedon x-ray crystallographic analysis. Inthisconformation, extensive hydrophobic andionicinteractions occurbetween the B propeller andthe R4and R5repeats. et al, 2002,Science2gS:2353] lPart(b)fromG Rudenko 610

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ceptor-ligandcomplex doesnot dissociatein late endosomes. Nonetheless,changesin pH also mediate the sorting of receptors and ligands in the transferrin pathway, which functions to deliver iron to cells. A major glycoprotein in the blood' transferrin transports iron to all tissuecellsfrom the liver (the main site of iron storage in the body) and from the intestine (the site of iron absJrption). The iron-free form, apotransferrin, binds two Fe3* ions very tightly to form ferrotransferrin. Arllmammalian cells contain cell-surfacetransferrin receptors that avidly bind ferrotransferrin at neutral pH, after which the receptor-bound ferrotransferrinis subjectedto endocytosis.Like the componentsof an LDL particle,the two bound Fe'* atoms remain in The EndocyticPathway Deliverslron to Cells the cell, but the apotransferrinpart of the ligand doesnot diswithout Dissociationof the Receptor-Transferrin sociate from the receptor, and within minutes after being enC o m p l e xi n E n d o s o m e s docytosed,apotransferrinis secretedfrom the cell. As depicted in Figure 14-3'J',the explanation for the beThe endocytic pathway involving the transferrin receptor havior of the transferrin receptor-ligand complex lies in the and its ligand differs from the LDL pathway in that the re-

pH is sufficiently acidic to promote dissociationof most endocytosedreceptorsfrom their tightly bound ligands. The mechanism by which the LDL receptor releases bound LDL particles is now understood in detail (Figure 1,4-30).At the endosomalpH of 5.0-5.5, histidineresidues in the B-propeller domain of the receptor become protonated, forming a site that can bind with high affinity to the negatively charged repeats in the ligand-binding domain. This intramolecular interaction sequestersthe repeats in a conformation that cannot simultaneouslybind to apoB-100, thus causingreleaseof the bound LDL particle.

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14-31Thetransferrincycle,which operatesin all A FIGURE dimercarrying growing mammaliancells.StepE: Thetransferrin bindsto the two boundatomsof Fe*3,calledferrotransferrin, between receptor at thecellsurfaceStep[: Interaction transferrin complex receptor andtheAP2adapter thetailof thetransferrin clathrinintoendocytic the receptor-ligand complex incorporates coatisshedandthe StepsB and4:The vesicle coatedvesicles

of the endosomeFe*3is fusewith the membrane vesicles endocytic in the acidiclate complex fromthe receptor-ferrotransferrin released proteinremains StepE: Theapotransferrin compartment endosome to thecellsurface at thispH,andtheyrecycle boundto itsreceptor mediumcauses Step@: TheneutralpHof the exterior together. etal, 1983, A Ciechanover apotransferrin iron-free [See of the release J BiolChem258:9681 l R E C E P T O R - M E D I A T E D E N D O C Y T O .S I S 6 1 1

unique ability of apotransferrin to remain bound to the transferrin receptor at the low pH (5.0-5.5) of late endosomes.At a pH of lessthan 5.0, the two bound Fe3* atoms dissociatefrom ferrotransferrin, are reduced to Fe2* by an unknown mechanism,and then are exported into the cytosol by an endosomal transporter specific for divalent metal ions. The receptor-apotransferrincomplex remaining after dissociationof the iron atoms is recycledback to the cell surface. Although apotransferrin binds tightly to its receptor at a pH of 5.0 or 6.0, it doesnot bind at neutral pH. Hence the bound apotransferrin dissociatesfrom the transferrin receptor when the recycling vesiclesfuse with the plasma membrane and the receptor-ligandcomplex encountersthe neutral pH of the extracellular interstitial fluid or growth medium. The recycled receptor is then free to bind another molecule of ferrotransferrin, and the releasedapotransferrin is carried in the bloodstream to the liver or intestineto be reloaded with iron.

Receptor-Mediated Endocytosis r Some extracellular ligands that bind to specific cellsurface receptors are internalized, along with their receptors, in clathrin-coated vesicleswhose coats also contain AP2 complexes. r Sorting signalsin the cytosolic domain of cell-surfacereceptors target them into clathrin/AP2-coatedpits for internalization. Known signals include the Asn-Pro-X-Tyr, Tyr-X-X-O, and Leu-Leu sequences(seeTable 14-2). r The endocytic pathway delivers some ligands (e.g., LDL particles) to lysosomes,where they are degraded. Transport vesiclesfrom the cell surfacefirst fuse with late endosomes,which subsequentlyfuse with the lysosome. r Most receptor-ligandcomplexes dissociatein the acidic milieu of the late endosomel the receptors are recycled to the plasma membrane,while the ligands are sorted to lysosomes (seeFigurel4-29). Iron is -imported into cells by an endocytic pathway in hich Fe3+ ions are releasedfrom ferrotransferrin in the late endosome.The receptor-apotransferrincomplex is recycled to the cell surface, where the complex dissociates. releasingboth rhe receptorand apotransfeirinfor reuse.

DirectingMembraneproteins M and CytosolicMaterialsto the Lysosome The major function of lysosomesis to degradeextracellular materials taken up by the cell and intracellular components under certain conditions. Materials to be degraded must be delivered ro the lumen of the lysosome, where the various degradativeenzymesreside.As just discussed,endocytosedligands (e.g.,LDL particles) that dissociate from their receptors in the late endosome subsequently enter the lysosomal lumen when the membrane of 612

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the late endosome fuses with the membrane of the lysosome (seeFigure 1,4-29).Likewise, phagosomescarrying bacteria or other particulate matter can fuse with lysosomes,releasingtheir contents into the lumen for degradation. It is apparent how the general vesicular trafficking mechanism discussedin this chapter can be used to deliver the luminal contents of an endosomal organelle to the lumen of the lysosome for degradation. However, vesicular trafficking cannot allow for delivery of membrane proteins or cytosolic materials to the lysosomal lumen. As we will seein this section.the cell has two different specializedpathways for delivery of these molecules to the lysosome interior for degradation. The first pathway is used to degrade endocytosedmembrane proteins and utilizes an unusual type of vesiclethat buds into the lumen of the endosometo produce a multivesicular endosome.The secondpathway, known as autophagS involves the de novo formation of a double membrane organelle known as an autophagosomethat envelopscytosolic material, such as soluble cytosolic proteins or sometimesorganellessuch as peroxisomesor mitochondria. Both pathways lead to fusion of either the multivesicular endosome or autophagosomewith the lysosome,depositingthe contents of these organelles into the lysosomal lumen for degradation.

MultivesicularEndosomesSegregate MembraneProteinsDestinedfor the LysosomalMembranefrom Proteins Destinedfor LysosomalDegradation Residentlysosomalproteins, such as V-classproton pumps and amino acid transporters, can carry out their functions and remain in the lysosomal membrane, where they are protected from degradation by the soluble hydrolytic enzymes in the lumen. Such proteins are delivered to the lysosomal membrane by transport vesiclesthat bud from the trans-Golgi network by the same basic mechanisms described in earlier sections.In contrast, endocytosedmembrane proteins such as receptor proteins that are to be degraded are transferred in their entirety to the interior of the lysosome by a specializeddelivery mechanism. Lysosomal degradation of cell-surface receptors for extracellular signaling molecules is a common mechanism for controlling the sensitivityof cells to such signals(Chapter 15). Receptors that become damaged also are targeted for lysosomal degradation. Early evidencethat membranescan be delivered to the lumen of compartments came from electron micrographs showing membrane vesiclesand fragments of membranes within endosomesand lysosomes(seeFigure 9-2c).parallel experimentsin yeastrevealedthat endocytosedreceptor proteins targeted to the vacuole (the yeast organelle equivalent to the lysosome)were primarily associatedwith membrane fragments and small vesicleswithin the interior of the vacuole rather than with the vacuole surfacemembrane.

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proteinsto the 14-32Deliveryof plasma-membrane FIGURE carrying endosomes lysosomalinteriorfor degradation.Early (blue)andvesicles proteins plasma-membrane carrying endocytosed (green) proteins network membrane fromthe trans-Golgi lysosomal proteins transferring theirmembrane fusewith the lateendosome, s be a l e m b r a n(es t e p tsr a n d Z ) P r o t e i nt o t o t h ee n d o s o m m areincorporated degraded, suchasthosefromtheearlyendosome, intovesicles that bud into the interiorof the lateendosome, containing manysuch forminga multivesicular endosome eventually

(stepB) Fusion endosome of a multivesrcular vesicles internal intothe lumen vesicles the internal releases with a lysosome directly (step Because be degraded they can Zl) where of the lysosome, proteins normally are membrane protonpumpsandotherlysosomal theyaredelivered vesicles, endosomal intointernal not incorporated fromdegradation. [see andareprotected membrane to the lysosomal 1:11, andD J Katzmann Cell Eukaryot andD J Klionsky,20Q2, F.Reggiori Mol CellBiol3:893l Rev. et al. 2002.Nature

These observations suggest that endocytosed membrane proteins can be incorporatedinto specializedvesicles that form at the endoso-ai -e-brane (Figure 1,4-32).Although thesevesiclesare similar in size and appearancero ,r"nrlo., vesicles,they differ topologically.Transport vesicles bud outward from the ,.rifu.. of a donor trganelle into the cytosol, whereas vesicleswithin the endosomebud inward from the surface into the lumen (away from the cytosol). Mature endosomescontaining numerous vesiclesin their interior are usually calledmultiuesicularendosomes (or bodies). Eventually,the surfacemembrane of a multivesicular endosome fuses with the membrane of a lysosome,thereby deliveringits internal vesiclesand the membrane proteins they contain into the lysosomeinterior for degradation.Thus the sorting of proteins in the endosomal membrane determineswhich onei will remain on the lysosome surface (e.g., pumps and transporters) and *hi.h ones will be incolpor"t.d i.rto internal vesicles and ultimately degradedin lysosomes. Vany of the proteins required for inward budding of the endosomal membrane were first identified by mutations in yeast that blocked delivery of membraneproteins to the interior of the vacuole. More than 10 ,rr.h "bodding" proteins have beenidentified in yeast,most with significanl similaritiesto mammalian proteins that evidentiy perform the same function in mammalian cells. The current model of endosomalbuddine to form multivesicularendo-

somesin mammalian cells is basedprimarily on studiesin yeast (Figure 1'4-33).Most cargo proteins that enter the multivesicularendosomearetaggedwith ubiquitin. Cargo proteins destined to enter the multivesicular endosome usually receivetheir ubiquitintagat the plasma membrane' the TGN, or the endosomal membrane. We have already seenhow ubiquitin tagging can serve as a signal for degradation of cytosolic or misfolded ER proteins by the proteasome (seeChapters 3 and 13). When used as a signal for proteasomaldegradation,the ubiquitin tag usualjy consists of a chain of covalently linked ubiquitin molecules(polyubiquitin), whereasubiquitin used to tag proteins for entry into the multivesicularendosomeusually takes the form of a single(monoubiquitin) molecule.In the membraneof the endosome a ubiquitin-tagged peripheral membrane protein, known as Hrs, facilitates loading of specific monoubiquitinatedmembranecargo proteins into vesiclebuds directed into the interior of the endosome. The ubiquitinated Hrs protein then recruits a set of three different protein complexesto the membrane.TheseESCRT (endosomal sorting complexes required for /ransport) proteins include the ubiquitin-binding protein Tsg101..The membrane-associatedESCRT proteins act to complete vesicle budding, Ieading to releaseof a vesicle carrying specific membranecargo into the interior of the endosome'Finally, an ATPase, known as Vps4, usesthe energy from ATP hydrolysis to disassemblethe ESCRT' releasingthem into the

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< FIGURE 14-33 Model of the mechanismfor formation of m u l t i v e s i c u l aern d o s o m e sI .n e n d o s o m a l b u d d i n gu, b i q u i t i n a t eHdr so n t h e endosomm a l e m b r a ndei r e c t lso a d i n g of s p e c i f im c e m b r a ncea r g op r o t e i n(sb l u ei)n t o vesicle budsandthenrecruits cytosolic E S C Rt T o t h e m e m b r a n(es t e pE ) N o t et h a t both Hrsandthe recruited cargoproteins are taggedwith ubiquitinAfterthe setof b o u n dE S C RcTo m p l e x emse d i a t m e embrane f u s i o na n dp i n c h i nogf f o f t h ec o m p l e t e d (stepZ), theyaredisasssembled vesicle by the ATPase Vps4and returnedto the cytosol (stepB) Seetextfor discussion [Adapted fromO Pornillos et al , 2002,Trends CellBiol. 12:569,1

Cytosol

Ubiquitin Hrs protein

Cargo proTer ns

Endosomar vesicle Lumen of endosome

cytosol for another round of budding. In the fusion event that pinchesoff a completedendosomalvesicle,the ESCRT proteins and Vps4 may function like SNAREs and NSF, respectively, in the typical membrane-fusion process discussedpreviously (seeFigure 14-10).

RetrovirusesBud from the plasmaMembrane by a ProcessSimilarto Formationof M u l t i v e s i c u l aE r ndosomes The vesiclesthat bud into the interior of endosomeshave a topology similar to that of envelopedvirus particles that bud from the plasma membrane of virus-infected cells. Moreover, recent experimentsdemonstratethat a common set of proteins is required for both types of membrane-budding events. In fact, the two processesso closely parallel each other in mechanistic detail as to suggestthat enveloped viruses have evolved mechanismsto recruit the cellular proteins used in inward endosomal budding for their own purposes. The human immunodeficiency virus (HIV) is an enveloped retrovirus that buds from the plasma membrane of infected cells in a process driven by viral Gag protein, the major structural component of completed virus particles. Gag protein binds to the plasma membrane of an infected cell and =4000 Gag molecules polymerize into a spherical shell, producing a structure that looks like a vesicle bud protruding outward from the plasma membrane. Mutational studies with HIV have revealed that the N-terminal segmentof Gag protein is required for associationwith the plasma membrane, whereas the C-terminal segmentrs required for pinching off of complete HIV particles. For instance, if the portion of the viral genome encoding the Cterminus of Gag is removed, HIV buds will form in infected

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cells, but pinching off does not occur, and thus no free virus particles are released. The first indication that HIV budding employs the same molecular machinery as vesicle budding into endosomes came from the observation that Tsg101, an ESCRT protein, binds to the C-terminus of Gag protein. Subsequent findings have clearly established the mechanistic parallels between the two processes.For example, Gag is ubiquitinated as part of the processof virus budding, and in cells with mutations in Tsg101 or Vps4, HIV virus buds accumulatebut cannot pinch off from the membrane (Figure 14-34). Moreover, when a segment from the cellular Hrs protein is added to a truncated Gag protein, proper budding and releaseof virus particles is restored. Taken together, these results indicate that Gag protein mimics the function of Hrs, redirecting ESCRT to the plasma membrane, where they can function in the budding of virus particles. Other enveloped retroviruses such as murine leukemia virus and Rous sarcomavirus also have beenshown to require ESCRT complexesfor their budding, although eachvirus appearsto have evolveda somewhatdifferent mechanismto recruit ESCRT complexesto the site of virus budding. The Autophagic Pathway Delivers Cytosolic proteinr or Entire Organelles to Lysosomes When cells are placed under stresssuch as conditions of starvation, they have the capacity to recycle macromoleculesfor use as nutrients in a process of lysosomal degradation known as autophagy ("eating oneself"). The autophagicpathway involves the formation of a flattened double-membrane cup-shapedstructure that envelops a region of the cytosol or an entrre organelle (e.g., mitochondrion), forming an autophagosome, or autophagic uesicle (Figure 1,4-35).The outer membrane

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Core particle

HIV envelope

Extracellular space Plasmamembrane Cytosol

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as

(c)

of an autophagic vesiclecan fuse with the lysosome,delivering a large vesicle,bounded by a singlemembrane bilayer,to the interior of the lysosome.Similar to the situation that occurs when multivesicular endosomes are delivered to the lysosome,lipasesand proteaseswithin the lysosomewill degrade the autophagic vesicleand its contents into their molecular components.Amino acid permeasesin the lysosomal membrane then allow for transport of free amino acids back into the cytosol for use in synthesisof new proteins. The formation and fusion of autophagic vesicles are thought to take place in three basic steps.Although the underlying mechanismsfor each of these steps remain poorly understood,they are thought to be related to the basicmechanisms for vesiculartrafficking discussedin this chapter. Autophagic Vesicle Nucleation The autophagicvesicleis thought to originate from a fragment of a membrane-

14-34 Mechanismfor < FIGURE budding of HIVfrom the Plasma for formation required membrane.Proteins exploited are endosomes multivesicular of by HIVfor virusbuddingfromthe plasma (a)Budding of HIVparticles membrane. cellsoccursby a similar fromH|V-infected asin Figure14-33,usingthe mechanism Gagproteinandcellular encoded virally andVps4(stepsn-B). ESCRT Gagneara buddingparticle Ubiquitinated likeHrs Seetextfor discussion. functions (b)Inwild-type with HIVvirus cellsinfected membrane particles budfromthe plasma intothe andarerapidlyreleased (c)In cellsthatlackthe space. extracellular protein theviral Tsg101, ESCRT functional Gagproteinformsdenseviruslike but buddingof thesestructures structures, cannotbe membrane fromthe plasma viral of incomplete andchains completed plasma membrane the to budsstillattached of University accumulate. [WesSundquist, UtahI

bounded organelle.Although the origin of this membrane is not known, most studiessuggestthat the autophagic vesicle

autophagic vesicle. Autophagic Vesicle Growth and Completion New membrane must be delivered to the autophagosomemembrane in order for this cup-shapedorganelle to grow' This

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Autophagic pathways

A FIGURE 14-35 The autophagicpathway.Theautophagic pathwayallowscytosolic proteinsandorganelles to be delivered to t h e l y s o s o mianlt e r i ofro r d e g r a d a t i oInn,t h e a u t o p h a g pi ca t h w a y , a cup-shaped structure formsaroundportionsof the cytosolor a n o r g a n e l lseu c ha sa p e r o x i s o maes,s h o w nh e r eC o n t i n u e d addition o f m e m b r a neev e n t u a llleya d st o t h ef o r m a t i o o nf a n autophagosome vesicle that envelops itscontentsby two complete m e m b r a n e( s t e pE ) F u s i o on f t h e o u t e rm e m b r a nwei t h t h e

membrane of a lysosome releases a single-layer vesicle andits contents (stepfl) Afterdegradation intothe lysosome interior of the protern andlipidby hydrolases in the lysosome interior, the released aminoacidsaretransported across the lysosomal membrane intothecytosolProteins knownto participate in theautophagic pathwayinclude Atg8,whichformsa coatstructure aroundthe autophaqosome

growth is likely to occur by the fusion of some type of transport vesiclewith the membrane of the autophagosome.Although the origin of such vesiclesis not known, the endosome is a likely candidate. Some of the proteins that participate in the formation of autophagosomeshave been identified in geneticscreensin yeast for mutants that are defective in autophagy. A subset of these proteins appear to form a coat structure on the surfaceof the autophagoso-e. One of theseproteins is Atg8, shown in Figure 14-35, which is covalently linked to the lipid phosphatidylethanolamine and thus becomesattached to the cytoplasmic leaflet of the autophagic vesicle.This coat may give the autophagosome its cup-shapedstructure.

to mask fusion proteins and to prevent premature fusion of the autophagosomewith the lysosome.

Autophagic Vesicle Targeting and Fusion The outer membrane of the completed autophagosomeis thought to contain a set of proteins that target fusion with the mem_ brane of the lysosome. Two vesicle-tetheringproteins have been found to be required for autophagosomefusion with the lysosome, but the corresponding SNARE proteins have not been identified. Fusion of the autophagosomewith the lysosomeoccurs after Atg8 has beenreleasedfrom the membrane by proteolytic cleavage,and this proteolysis step only occurs once the autophagic vesiclehas completely formed a sealeddouble,membranesystem.Thus AtgS protein appears 616

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Directing Membrane Proteins and Cytosolic Materials to the Lysosome r Endocytosed membrane proteins destined for degradation in the lysosomeare incorporated into vesiclesthat bud into the interior of the endosome. Multivesicular endosomes,which contain many of these internal vesicles,can fuse with the lysosometo deliver the vesiclesto the interior of the lysosome(seeFigure 14-32). r Some of the cellular components (e.g.,ESCRT) that mediate inward budding of endosomalmembranesare usedin the budding and pinching off of envelopedviruses such as HIV from the plasma membrane of virus-infectedcells (see Figures 14-33 and 14-34). r A portion of the cytoplasm or an entire organelle (e.g., peroxisome)can be envelopedin a flattened membrane and eventually incorporated into a double-membrane autophagic vesicle.Fusion of the outer vesiclemembranewith the lysosomedeliversthe envelopedcontentsto the interior of the lysosomefor degradation (seeFigure 14-35).

vEstcuLAR T R A F F t cs,E c R E T t o N A,N D E N D o c y r o s t s

The biochemical, genetic, and structural information presented in this chapter shows that we now have a basic understanding of how protein traffic flows from one membrane-boundedcompartment to another.Our understanding of theseprocesseshas come largely from experimentson the function of various types of transport vesicles.Thesestudies have led to the identification of many vesicle components and the discovery of how these components work together to drive vesicle budding, to incorporate the correct set of cargo moleculesfrom the donor organelle' and then to mediate fusion of a completed vesiclewith the membrane of a target organelle. Despite these advances,important stagesof the secretory and endocytic pathways remain about which we know relatively little. For example,we do not yet know what types of proteins form the coats of either the regulatedor the constitutive secretoryvesiclesthat bud from the trans-Golgi network. Moreover, the types of signals on cargo proteins that might target them for packaging into secretoryvesicleshave not yet been defined. Another baffling processis the formation of vesiclesthat bud away from the cytosol, such as the vesiclesthat enter multivesicular endosomes.Although some of the proteins that participate in formation of these "internal" endosome vesiclesare known, we do not know what determinestheir shapeor what type of processcausesthem to pinch off from the donor membrane. Similarly, the origin and growth of the membrane of the autophagic vesicle is also poorly understood. In the future, it should be possible for these and other poorly understood vesicle-trafficking steps to be dissectedthrough the use of the same powerful combination of biochemical and genetic methods that have delineated the working parts of COPI, COPII, and clathrin/AP vesicles. Questions still remain about vesicletrafficking between the ER and cls-Golgi, betweenGolgi stacks,and betweenthe tr ans- G olgi and endosome, the best-char acterized transport steps. In particular, our understanding of how proteins are actually sorted between these organelles is incomplete largely becauseof the highly dynamic nature of all the organelles along the secretory pathway. Although we know many of the details of how particular vesicle components function, we cannot account for why their functions are restricted to specific stagesin the overall flow of anterograde and retrograde transport steps.For example' we cannot explain why COPII vesiclesfuse with one another to form a new cls-Golgi stack, whereas COPI vesiclesfuse with the membrane of the ER, since both vesicletypes appear to contain similar sets of v-SNARE proteins. In the same vein, we do not know what feature of the Golgi membrane actually distinguishesa COPl-coated vesiclebud from a clathrin/APcoated bud. In both cases,binding of ARF protein to the Golgi membrane appearsto initiate vesiclebudding. The solution to these problems will require a more integrated understandingof the flow of vesicular traffic in the context of the entire secretory pathway. Recent improvements in our

ability to image vesicular transport of cargo proteins in live cells gives hope that some of these more subtle aspectsof vesiclefunction may be clarified in the near future.

KeyTerms AP (adapter protein) complexes598 anterogradetransPott 592 ARF protein 587 autophagy 51.4 cisternal maturation 596 clathrin 585 constitutive sectetiol 602 COPI586 COPII585 dynamin 599 endocytic pathway 579 ESCRT proteins613 late endosome580 low-density lipoprotein (LDL) 607

endosomes multivesicular 613 Rab proteins589 receptor-mediated 605 endocytosis regulatedsecretion602 retrogradetransPort592 secmutants584 secretorypathwaY580 sortingsignals589 505 transcytosis tr ans G olgi network (TGN) 580 580 transportvesicles I-SNAREs586 v-SNAREs585

mannose6-phosphate (M6P) 600

Reviewthe ConcePts 1.. The studies of Palade and colleaguesused pulse-chase labeling with radioactively labeled amino acids and autoradiography to visualizethe location of newly synthesizedproteins in pancreaticacinar cells.These early experimentsprovided invaluable information on protein synthesis and intercompartmental transport. New methods have evolved, but two basic requirementsare still necessaryfor any assay to study this type of protein transport. What two requirements must be met? Briefly describe the experimental approachesneededto meet the criteria. NSF. It is a class C 2. Sec18is a yeast gene that encodes'$fhat is the mechapathway. mutant in the yeast secretory indicated by As trafficking? nistic role of NSF in membrane produce mutation NSF an does why its class C phenotype, one only be to appears what at accumulation of vesicles stageof the secretorYPathway? IUfhatis 3. Vesiclebudding is associatedwith coat proteins' coat How are the role of coat proteins in vesicle budding? molecules of proteins recruited to membranes?!7hat kinds are likely to be included or excluded from newly formed vesicles?What is the best-known example of a protein likely to be involved in vesiclepinching off? 4. Treatment of cells with the drug brefeldin A (BFA) has the effect of decoating Golgi apparatus membranes, resulting in a cell in which the vast majority of Golgi proteins are found in the ER.'What inferencescan be made from this R E V I E WT H E C O N C E P T S

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observation regarding roles of coat proteins other than promoting vesicle formation? Predict what type of mutation in Arfl might have the same effect as treating cells with BFA. 5. An antibody to an exposed '.hinge" region of BCOpI known as EAGE blocks the function of BCOpI when microinjected into HeLa cells. predict what the consequencesof this functional block might be for anterograde transport from the ER to the plasma membrane. propose an experiment to test whether the effect of EAGE microinjection is initially on anterograde or retrograde transport. 6. Specificity in fusion between vesiclesinvolves two discrete and sequentialprocesses.Describe the first of the two processesand its regulation by GTpase switch proteins. What effect on the sizeof early endosomesmight reiult from overexpressionof a mutant form of Rab5 that is stuck in the GTP-bound state? 7. Sorting signalsthat causeretrograde transport of a protein in the secretory pathway are sometimesknown as retrieval sequences.List the two known examplesof retrieval sequencesfor soluble and membrane prot;ins of the ER. How does the presenceof a retrieval sequenceon a soluble ER protein result in its retrieval from the cls-Golgi complex? Describehow the concept of a retrieval sequenceis essential to the cisternal-progressionmodel. 8. _Clathrinadapter protein (Ap) complexesbind directly to the cytosolic face of membrane proteins and also interact with clathrin. Sfhat are the four known adapter pro_ tein complexes?rVhy may clathrin be consideredto be an accessoryprotein to a core coat composedof adapter pro_ teins?

hibitor/competitor of HIV budding and decideto mimic in a synthetic peptide a portion of the HIV Gag protein. Which portion of the HIV Gag protein would be a logical choice? 'Sfhat normal cellular processmight this inhibitor block? 13. The phagocytic and autophagicpathways servetwo fundamental roles, but both deliver their vesiclesto the lysosome. !7hat are the fundamental differences between the two pathways? Describe the three basic steps in the formation and fusion of autophagic vesicles.

Analyze the Data In order to examine the specificity of membrane fusion conferred by specificv-SNAREs and I-SNAREs (seeMacNew et aI., 2000, Nature 407:15 3-1 59), liposomes (artificial lipid membranes)were reconstitutedwith specificI-SNARE complexes or with v-SNAREs. To measurefusion, the v-SNARE liposomes also contained a fluorescent lipid at a relatively high concentration such that its fluorescenceis quenched. (Quenchingis reducedfluorescencerelative to that exoected. In this case,quenching occurs becausethe fluoresceni lipid, are too concentrated and interfere with each other's ability to becomeexcited.) On fusion of theseliposomeswith those lacking the fluorescent lipid, the fluorescent lipids are diluted, and quenching is alleviated. Three sets of liposomes were prepared using yeast t-SNARE complexes:those containing plasma membrane I-SNAREs, Golgi I-SNAREs, or vacuolar I-SNAREs. Each of these was mixed with fluorescent liposomes containing one of three different yeast vSNAREs. The following data were obtained.

9, I-cell diseaseis a classicexample of an inherited human defect in protein targeting that affects an enrire classof proteins, soluble enzymesof the lysosome. !7hat is the molecular defectin I-cell disease?Why doesit affectthe targeting of an entire class of proteins? lfhat other types of mutati,ons might produce the samephenotype? 10. The TGN, trans-Golgi network, is the site of multiple

v-SNARE1

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12. \What mechanisticfeaturesare shared by (a) the forma_ tion of multivesicular endosomesby budding into the inte_ rior of the endosome and (b) the outward Uuaaing of HIV virus at the cell surface?You wish to design a peptide in-

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a. $fhat can be deduced from these data about the specificity of membrane fusion events? b. Ifhere might you expect to find v-SNAREs 1. 2. and 3 in yeast? c. \What kind of experiment could be designedto determine where in the secretorypathway a given v-SNARE is requiredin vivo?

VES|CULAR T R A F F | CS, E C R E T | O N A,N D E N D O C Y T O S | S

d. The cytoplasmic domain of v-SNARE 2 has been expressedand purified from E. coli. Yarious amounts of this domain are incubated either with the Golgi I-SNARE liposomes or with v-SNARE 2 liposomes' The liposomes are then washed free of unbound protein. The various liposomes are then mixed, as indicated below, and the fluorescenceof each sample is measured t hour after mixing' How can the data be explained? What would you predict the outcome to be if yeastwere to overexpressthe cytoplasmic domain of vSNARE 2?

= v - S N A R E2 l i p o s o m e si n c u b a t e dw i t h t h e c y t o p l a s m i cd o m a i n o f v - S N A R E2 a n d t h e n m i x e d w i t h G o l g i I - S N A R El i p o s o m e s

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'Wickner, \7., and A. Haas. 2000. Yeasthomotypic vacuole on organelletrafficking mechanisms'Ann' Reu' window fusion: a Biochem. 69:247-275. Zerial,M., and H. McBride.2001. Rab proteins as membrane organizers.Nature Reu.MoI. Cell Biol. 22L07-tI7 ' Early Stages of the Secretory Pathway Barlowe, C. 2003. Signalsfor COPll-dependentexport from the ER: what's tire ticket out? Trends CeII Biol' t3:295-300'

Lee, M. C., et al. 2004. Bi-directionalprotein-transportbetween the ER and Golgr. Ann. Reu. Cell Deu. Biol' 20:87-123' Letourneut,F, et al. 1994. Coatomeris essentialfor retrieval of to the endoplasmicreticulum' Cell dilysine-tagged.proteins 79:1.L99-1207. Losev, E., et al. 2006. Golgi maturation visualized in living y east.N atur e 441:10 02-t00 6. Pelham,H. R. 1995' Sorting and retrieval befiveenthe endoplasmic reticulum and Golgi apparat.ts'Curr' Opin' Cell Biol' /:)JU-)J).

References Techniques for Studying the Secretory Pathway Beckers,C. J., et al. 1.987.Semi-intactcellspermeableto macromolecules:use in reconstitutionof protein transport from the endoplasmic reticulum to the Golgi complex' Cell 5O:523-534. Kaiser,C. A., and R. Schekman.1990. Distinct setsof SEC genesgovern transport vesicleformation and fusion early in the secretorypathway. Cell 6l:723-7 33. Novick, P.,et al. 1981. Order of eventsin the yeastsecretory pathway. Cell 25:461'469. Lippincott-Schwartz,J.,et al. 2001. Studyingprotein dynamics in living cells.Nature Reu.Mol. Cell Biol.2:444456. Orci, L., et al. 1989. Dissectionof a singleround of vesicular transport: sequentialintermediatesfor intercisternalmovementin the Golgi stack.Cell 56:357-368. Palade,G.1975. Intracellularaspectsof the processof protein synthesis.Science789:347-3 5 8. Molecular Mechanisms of Vesicular Traffic Bonifacino,J. S., and B. S. Glick. 2004.The mechanismsof vesicle budding and fusion. Cell 116:153-66. Grosshans,B. L., D. Ortiz, and P. Novick. 2006. Rabs and their effectors: achieving specificity in membrane traffic' Proc. Natl. Acad. Sci.USA 103:11821-11'827. Jahn,R., et al. 2003. Membranefusion.Cel/ 112:51'9-533' Kirchhausen,T. 2000. Three ways to make a vesicle.Nature Reu.Mol. Cell Biol.7:187-1'98. McNew, J. A., et al. 2000. Compartmentalspecificityof cellular membranefusion encodedin SNARE proteins.Nature 407 1.53-159. Ostermann,J., et al. 1993. Stepwiseassemblyof functionally activetransport vesicles.Cell 7 5 :1'015-1'025' Schimmoller,F., I. Simon, and S. Pfeffer.1998. Rab GTPases, directorsof vesicledocking'J. Biol' Cbem.273:22161-221'64' 'Weber, T., er.al. 1998. SNAREpins:minimal machineryfor membranefusion. Cell 92:759-772.

Later Stages of the Secretory Pathway Bonifacino,J. S. 2004. The GGA proteins:adaptorson the move. Na/. Reu.MoI. Cell Biol. 5:23-32' Bonifacino,J. S., and E. C. Dell'Angelica' 1'999'Molecular basesfor the recbgnition of tyrosine-baied sorting signals'J' Cell Biol. 745:923-926. Edeling,M. A., C. Smith, and D. Owen' 20-0-6'I'ife of a clathrin from clathrin and AP structures' Nat' Reu' MoL Cell ..";i;tGi; Biol.7:3244. Fotin, A., et aI.2004. Molecular model for a complete-clathrin lattice from .i..tro.t cryomicroscopy'Nature 432:573-579' new Ghosh,P.,et al. 2003' Mannose 6-phosphate-receptors: twists in the tale. Nature Reu.Mol. CeIl Bio' 4:202-213' Mostov, K. E., M. Verges,and Y. Altschuler'2.000'Membrane traffic in polarizeiepithelial ceIls.Curr' Opin' Cell Biol' 122483490. Schmid,S. t997. Clathrin-coatedvesicleformation and protein sorting: an integratedprocess'Ann. Reu' Biochem' 66:511-548' Simons.K.. and E. Ikonen. 1997. Functional rafts in cell membranes.Nature 387:569-572. Song,B. D., and S. L. Schmid.2003' A molecular motor or a ,.gulatoii Dynamin's in a classof its own' Biochemistry 42:1369-1376. Steiner,D. F.' et al. 1'996.The role of prohormone convertases in insulin biosynihesis:evidencefor inherited defectsin their action i., -"n ,nd experimentalanimals.DiabetesMetab' 22294-104' Tooze.S. A.. et al. 2001. Secretorygranule biogenesis:rafting to Trends CeIl Biol' ll:11'6-122' SNARE. the Receptor-Mediated EndocYtosis Brown, M. S., and J' L. Goldstein' 1986' Receptor-mediated pathway for cholesterolhomeostasis'Nobel Prize Lecture' Science 232:34-47. Kaksonen,M., C. P.Toret' and D' G' Drubin' 2006' Harnessing Jy""-i.s for clathrin-mediatedendocytosis'Nat' Reu'Mol' ".ri" Biol.7A044l4. Cell Rudenko, G., et al. 2002. Structureof the LDL receptorextracellular domain at endosomalpH. Science298:2353-2358'

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Lemmon, s. K.,andL. M. Traub.2000.sortingin theendosomalrystemin yeasiandanimalcells.c"* oil". iri-ai.t. t2:457466'

.Katzmann,D._J.,et al. 2002.Receptordownregulationand multivesicular-body sorting.Nature Riu.Mot. CatTiii.i,SiigOs. Khalfan,W. A., and D..J.Klionsky.2002.Molecularmachinery requiredfor autophagyan_d the cytopias- to vacuoletargeting - (Cvi) pathwayin S. cereuisiae. Curr. Opin. Cell Biol. t+469475.

Shintani,J..,an{ D..J. Klionsky.2004.A_u_toplragy in healthand ,. disease: a double-edged sword.Science30699C-l995'.

Ain.ii,;rii.;ti:;;If:lgi:,:;#;):;-rrarricking ""iiiu'

RNAvirus b"dl,:;"#;;3,';:;t;1ii.'?;,H;1;;"-'orenveroped

I 620

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cHAprER 14 | vEsrcuLAR TRAFFT., sEcRETroN, ANDENDocyrosrs

cLASSIC

EXPERIMENT

14

OUT OF THECELL A PROTEIN FOLLOWING J. Jamieson and G. Palade, 1965, Proc. Natl. Acad. Sci.IJSA 55(2):424-431

The advent of electron microscopy allowed researchersto seethe cell and its structuresat an unprecedentedlevel of detail. George Palade utilized this tool not only to look at the fine details of the cell but also to analyze the process of secretion. By combining electron microscopy with pulse-chaseexperiments, Paladeuncoveredthe path proteins follow to leave the cell.

Background In addition to synthesizingproteinsto carry out cellular functions, many cells must also produce and secreteadditional proteins that perform their duties outside the cell. Cell biologists, including Palade, wondered how secreted proteins make their passage from the inside to the outside of the cell. Early experimentssuggestingthat proteins destinedfor secretionare synthesizedin a particular intracellular location and then follow a pathway to the cell surface employed methods to disrupt cells synthesizing a particular secreted protein and to separate their various organelles by centrifugation. Thesecell-fractionationstudiesshowed that secretedproteins can be found in membrane-bounded vesicles derived from the endoplasmic reticulum (ER), where they are synthesized,and with zymogen granules, from which they are eventually released from the cell. Unfortunately, results from thesestudies were hard to interpret due to difficulties in obtaining clean separationof all of the different organellesthat contain secretoryproteins. To further clarify the pathway, Palade turned to a newly developedtechnique, high-resolution autoradiography, that allowed him to detect the position of radioactively labeled proteins in thin cell sections that had been prepared for elec-

tron microscopy of intracellular organelles.His work led to the seminal finding that secreted proteins travel within vesicles from the ER to the Golgi complex and then to the plasma membrane.

TheExperiment Palade wanted to identify which cell structuresand organellesparticipate in protein secretion. To studY such a complex process,he carefully chosean appropriate model systemfor his studies, the pancreaticexocrine cell, which is responsible for producing and secreting large amounts of digestive enzymes. Becausethese cells have the unusual property of expressing only secretory proteins, a general label for newly synthesized protein' such as radioactively labeled leucine,will only be incorporated into protein molecules that are following the secretory pathway. Palade first examined the Protein secretionpathway in vivo by injecting live guinea pigs with ['H]-leucine, which was incorporated into newlY made proteins, thereby radioactively labeling them. At time points from 4 minutes to 15 hours. the animals were sacrificed,and the pancreatictissue was fixed. By subjectingthe speci mens to autoradiography and viewing them in an electronmicroscope,Palade could trace where the labeled proteins were in cells at various times. As expected, the radioactivity localized in vesiclesat the ER at tim^epoints immediately following the [rH]-leucine iniection and at the plasma membrane at the later time points. The surprise came in the middle time points. Rather than traveling straight from the ER to the plasma membrane, the radioact i v e l y l a b e l e d p r o t e i n s a p p e a r e dt o

stop off at the Golgi comPlex in the middle of their iourney. In addition, there never was a time point where the radioactively labeledproteins were not confined to vesicles. The observation that the Golgi complex was involved in protein secretion was both surprising and intriguing. To thoroughly addressthe role of this organelle in protein secretion, Palade turned to in vitro pulse-chase experiments, which Permitted more precise monitoring of the fate of labeled proteins. In this labeling technique,cells are exposedto radiolabeled precursor' in this case ['H]leucine, for a short Period known as the pulse. The radioactive precursor is then replacedwith its nonlabeled form for a subsequentchaseperiod. Proteins synthesized during the pulse period will be labeled and detectedby autoradiography, whereas those synthesized during the chase period, which are nonlabeled, will not be detected. Palade began by cutting guinea Pig pancreasinto thick slices,which were then incubated for 3 minutes in media containing [3H]-leucine.At the end of the pulse, he added excess unlabeled leucine. The tissue sliceswere then either fixed for autoradiography or used for cell fractionation. To ensure that his results were an accurate reflection of protein secretion in vivo' Palade meticulously charucterized the system. Once convinced that his in vitro system accurately mimicked protein secretion in vivo, he Proceeded to the critical experiment. He pulse-labeled tissue slicis with [3Hl-leucine for 3 minutes, then chased the label for 7, 1,7, 37 , 57 , and 1 17 minutes with unlabeled leucine. Radioactivity, again confined in vesicles'began at the ER, then traveled in vesiclesto the Golgi complex and remained in the vesicles

F O L L O W I N GA P R O T E I NO U T O F T H E C E L L

621

< FIGURE 1 The synthesisand movementof guineapig pancreaticse€retoryproteinsas revealedby electronmicroscope autoradiography. Aftera periodof labeling with [3H]-leucine, the tissueisfixed,sectioned for electron microscopy, andsubjected to autoradiography. Theradioactive decayof [3tt]in newlysynthesized proteins produces autoradiographic in an emulsion placedover Arains (whichappear thecellsection inthemicrograph asdense, wormlike granules) thatmarkthe position of newlysynthesized proteins(a)At the endof a 3-minute periodautoradiographic labeling areoverthe Arains roughER(b)Following a 7-minute period chase withunlabeled leucine, mostof the labeled proteins havemovedto the Golgivesicles (c)Aftera 37-minute chase, mostof the proteins areoverimmature secrerory (d)Aftera 117-minute vesicles chase, the majority of theproteins are overmaturezymogen granulesfCourtesyof J Jamieson andG palade ] (c)

(d)

as they passedthrough the Golgi and onto the plasma membrane (seeFigure 1). As the vesiclestraveledfarther along the pathway, they becamemore densely packed with radioactive prot e i n . F r o m h i s r e m a r k a b l es e r i e so f a u toradiograms at different chasetimes, Palade concluded that secretedproteins rravel in vesiclesfrom rhe Ei{ ro the Golgi and onto the plasma membrane and that throughout this process,they remain in vesiclesand do not mix with the rest of the cell.

Discussion Palade'sexperimenrs gave biologists the first clear look at the stagesof the

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secretorypathway. His studieson pancreatic exocrine cells yielded two fundamental observations.First, that secreted proteins pass through the Golgi complex on their way out of the cell. This was the first function assignedto the Golgi complex. Second, secrered proteins never mix with cellular proteins in the cytosol; they are segregated into vesiclesthroughout the pathway. These findings were predicated from two rmportant aspects of the experimental design. Palade'scareful use of electronmicroscopy and autoradiography allowed him to look at the fine details of the pathway. Of equal importance was the choice of a cell tyoe devoted to secretion, the pancreatic

v E s t c u L A RT R A F F | CS, E C R E T T O A NN , D ENDOCYTOSTS

exocrine cell, as a model system. In a different cell type, significant amounts of nonsecretedproteins would have also been produced during the labeling, obscuring the fate of secretory proteins in particular. Palade's work set the stage for more detailed studies. Once the secretory pathway was clearly described, entire fields of research were opened u p t o i n v e s t i g a t i o inn t h e s y n t h e s ias n d movement of both secretedand membrane proteins. For this groundbreaking work, Palade was awarded the Nobel Prize for Physiology and Medicine in 1974.

CHA PTER

is regulatedby many Thestorageand metabolismof triglycerides isstainedfor three This3T3-11adipocyte importanthormones. proteinsthat Iinetriglyceride dropletsat differentstagesof their in blue,adipophilin in green,andTlP47in maturation-perilipin red.Courtesv PerrvBickeland NathanWolins

o cell lives in isolation; cellular communication is a fundamental property of all cellsand shapesthe function and abilities of every living organism. Even single-celledorganismshave the ability to communicatewith each other or other organisms.Eukaryotic microorganisms, such as yeasts, slime molds, and protozoans, use secreted moleculescalled pheromones to coordinate the aggregation of free-living cells for sexual mating or differentiation under certain environmental conditions. Yeastmating-type factors are a well-understood example of pheromone-mediatedcellto-cell signaling (Chapter 21). More important in plants and animals are extracellular signaling molecules that function within an organism to control metabolism of sugars, fats' and amino acids, the growth and differentiation of tissues, the synthesisand secretionof proteins, and the composition of intracellular and extracellular fluids. Animals also respond to many signals from their environment, including light, oxygen, odorants, and tastantsin food. Many extracellular signaling molecules are synthesized and releasedby signaling cells within the organism. In all cases signaling molecules produce a specific response only in target cells that have receptors for the signaling molecules' Many types of chemicalsare used as signals:small molecules(e.g., amino acid or lipid derivatives,acetylcholine),peptides(e.g', ACTH and vasopressin),soluble proteins (e.g., insulin and growth hormone) and many proteinstetheredto the surfaceof a cell or bound to the extracellular matrix. Most receptors bind a singlemoleculeor a group of closelyrelatedmolecules. Some signaling molecules,especiallyhydrophobic moleculessuchas steroids,retinoids,and thyroxine' spontaneously diffuse through the plasma membrane and bind to intracellular

I: CELLSIGNALING SIGNAL TRANSDUCTION AND SHORT-TERM RESPONSES CELLULAR

receptors; signaling from such intracellular receptors is discussedin detail Chapter 7. Somesmall signaling moleculesare hydrophilic and are transported by membrane proteins into the cell cytoplasm in order to influence cell behavior' Most signalingmolecules,however,are too large and too hydrophilic to penetrate through the plasma membrane' These bind to cell-surfacereceptors that are integral proteins

OUTLINE 15.1

F r o mE x t r a c e l l u l aSri g n a tl o C e l l u l a r ResPonse

62s

15.2

Receptors StudyingCell-Surface

627

15.3

Highly ConservedComponentsof Pathways IntracellularSignal-Transduction

632

15.4

GeneralElementsof G Protein-Coupled ReceptorSystems

15.5

G Protein-CoupledReceptorsThat R e g u l a t el o n C h a n n e l s

15.6

G Protein-CoupledReceptorsThat Activate or Inhibit AdenYlYlCYclase

646

15.7

G Protein-Coupled ReceptorsThat Activate C Phospholipase

6s3

15.8

of Cellsto IntegratingResponses E n v i r o n m e n t aI ln fl u e n c e s

623

OverviewAnimation:Extracellular tlltl Signaling

o o

Cell surface receptor

,a

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uo @/\

Modificationof Modificationof cellularmetabolism, geneexpression, function,movement develooment

on the plasma membranes.Cell-surfacereceptors generally consist of three discretesegments:a segmenton the extracelIular surface, a segment that spans the plasma membrane, and a segmentfacing the cytosol. The signalingmoleculeacts as a ligand, which binds to a structurally complementarysite on the extracellular or membrane-spanningdomains of the receptor. Binding of the ligand induces a conformational change in the receptor that is transmitted through the membrane-spanning domain to the cytosolicdomain, resulting in binding to and subsequentactivation(or inhibition) of othei proteins in the cytosol or attached to the plasma membrane. The overall process of converring extracellular signals into intracellular responses,as well as the individual stepsin this process,is termed signal transduction (Figure 15-1). In all eukaryotesthere are only about a dozen classesof

bined with biochemical analyseshave enabledresearchersto trace many entire signalingpathways from binding of ligand to receptorsto the final cellular resDonses.

624

'

C H A P T Et R 5

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< FIGURE 15-1 Generalprinciplesof signalingby cell-surface receptors. Communication by extracellular signals usually involves t h ef o l l o w i n g s t e p sS: y n t h e soi sf t h e s i g n a l i nm g o l e c u lbey t h e signaling cellanditsrncorporation intosmallintracellular vesicles fil), itsrelease intothe extracellular (Z), andtransport spaceby exocytosis o f t h e s i g n atlo t h et a r g e ct e l l( B ) w h e r et h e s i g n a l i nm g olecule bindsto a specific cell-surface receptor proteinleading to activation (4). Theactivated of the receptor receptor theninitiates one or m o r ei n t r a c e l l u lsai g r n a l - t r a n s d u cpt iaotnh w a y(sE t )l e a d i n tgo specif ic changes, usually short-term, in cellular function,metabolism. (EE)or to long-term or movement changes in geneexpresston or ((p). Termination development of the cellular response iscaused by intracellular signaling molecules thatinhibitreceptor function(Z) andby removal of the extracellular signal(S) the samepathway may be referred to by different names. FortunatelS as researchershave discoveredthe molecular details of more and more receptorsand pathways, some overarching principles and mechanismsare beginning to emerge.These shared featurescan help us make senseof the wealth of new information concerningcell-to-cellsignaling. Perhapsthe most numerous class of receptors-found in organismsfrom yeast to human-are commonly called G protein-coupled receptors (GPCRs). The human genome encodes about 900 G protein-coupled receptors including receptorsin the visual, olfactory (smell),and gustatory (taste) systems,many neurotransmitter receptors, and most of the receptorsfor hormones that control carbohydrate, amino acid, and fat metabolism. Essentiallyrhe same signaling pathway is usedin yeastfor signalingby mating factors(Chapter21). This chapter focusesmainly on these receptors, which usually induce short-term changesin cell function. Activation of many cell-surfacereceptors alters the pattern of gene expression by the cell, leading to cell differentiation and other long-term consequences. Thesereceptorsand the intracellular signaling pathways they activate are explored in Chapter 16. Certain other cell-surfacereceptorsare normally closed ion channels that open in responseto ligand binding, allowing a particular type of ion to cross the plasma membrane.These receptors, called ligand-gatedion channels,are especiallyimportant in nerve cells;they are discussedin Chapter 23. In this chapter we first review the generalprinciples of cell signaling and describehow cell-surfacereceptorsare identified and characterized.Next we discussseveral features of many signal-transduction pathways and their regulation that have been conservedthroughout evolution and are found in a wide variety of organisms.We then describethe common elementsin G protein-
c E L Ls T G N A L TrN: sGr c N A LT R A N S D U c T oANN Ds H o R T - T E RCME L L U L ARRE s p o N s E s

FromExtracellular Signal to CellularResponse Communicationby extracellularsignalswithin an organism usually involves the following steps (seeFigure 15-1): tr synthesis and Z release of the signaling molecule by the signalingcell; E transport of the signalto the targetcell; 4 binding of the signal by a specific receptor protein Ieading to a conformationalchange;E initiation of one or more lntracellular signal-transductionpathways by the activated receptor; 6 specific changes in cellular function, metabolism, or development;and feedbackregulation usually involving Z deactivation of the receptor and B removal of the signaling molecule, which together terminate the cellular response.

S i g n a l i n gM o l e c u l e sC a nA c t L o c a l l y or at a Distance Releasedsignaling moleculestravel to their target cells (see Figure 15-1, step B). Some are transportedlong distances by the blood; others have more local effects.In animals, signaling by extracellular moleculescan be classifiedinto three types-endocrine, paracrine,or autocrine-based on the distance over which the signalacts (Figure 15-2a-c).In addition, certain membrane-bound proteins on one cell can directly signalan adiacentcell.

( a ) E n d o c r i nsei g n a l i n g ooo o^ o

.

S i g n a l i n gC e l l sP r o d u c ea n d R e l e a s e S i g n a l i n gM o l e c u l e s In humans rapid responsesto changesin the environment are primarily mediated by the nervous system and by hormonesincluding small peptides(e.g.,insulin and adrenocorticotropic hormone,ACTH) and small (nonpeptide)molecules such as the catecbolamines(e.g., epinephrine, norepinephrine, and dopamine).The cellsthat make thesesignalingmoleculesare found in the pancreas(insulin), the pituitary gland (ACTH), the adrenal glands (epinephrine and norepinephrine), neurons (norepinephrine)and the part of the brain termed the hypothalamus (dopamine).Small signalingmolecules,including catecholamineneurotransmitters,are synthesizedin the cytosol and then transported into secretoryvesicles (Chapter 23), whereaspeptide and protein hormones are synthesizedand processedby the secretorypathway detailed in Chapter 14. In both casesvesiclescontaining thesesignaling moleculesaccumulatejust under the plasma membrane where they are held, awaiting a releasesignal (seeFigure 15-1, s t e p[ ) . Stimulation of signalingcells is almost always coupled to a rise in the local concentration of Ca"- near the vesicles, causing immediate fusion of the membranes of the v e s i c l e sa n d t h e p l a s m a m e m b r a n e ,l e a d i n gt o e x o c y t o s i s of the storedpeptideor protein hormone or small molecule i n t o t h e s u r r o u n d i n gm e d i u m o r b l o o d ( s e eF i g u r e 1 5 - 1 , s t e pZ ) . Releasedpeptidehormonespersistin the blood for only secondsto minutes before being degraded by blood and tissue proteases.Releasedsmall molecules such as catecholaminesare rapidly inactivatedby enzymesor taken up via transporters into specific cells. The initial actions of these signalingmoleculeson target cells (the activation or inhibition of specificenzymes)usually only lastssecondsor minutes. Thus the catecholaminesand some peptide hormones can mediate short-term responsethat are terminated by their own degradation. For sustainedor longer-term responses,such as cell division or cell differentiation,the cells must be exposedto signalingmoleculesfor extendedperiods. usually hours.

,.1

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( b ) P a r a c r i n se i g n a l i n g

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( c ) A u t o c r i n es i g n a l i n g Key: E x t r a c e l l u l asri g n a l

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( d ) S i g n a l i n gb y p l a s m am e m b r a n e - a t t a c h epdr o t e i n s

\"'_-.'**--,-,,-,.."*'-.'"_S i g n a l i n cg e l l

signaling' 15-2 Generalschemesof intracellular a FIGURE over occurs chemicals by extracellular (a-c)Cell-to-cell signaling paracrine and in autocrine micrometers few a from distances (d)Proteins signaling. metersin endocrine to several signaling directly of onecellcaninteract membrane to the plasma attached cells on adjacent receptors with cell-surface RESPONSE F R O M E X T R A C E L L U L ASRI G N A LT O C E L L U L A R

625

In endocrine signaling, the signaling molecules are synthesized and secrered by signaling cells (endocrine cells), transported through the circulatory systemof the organism, and finally act on target cells distant from their site of synthesis.The term hormone generally refers to signaling moleculesthat mediate endocrine signaling. In paracrine signaling, the signaling molecules releasedby a cell affect only those target cells in close proximity. The conduction by a neurotransmitter of a signal from one nerve cell to another or from a nerve cell to a muscle cell (inducing or inhibiting muscle contraction) occurs via paracrine signaling. Many growth factors regulating development in multicellular organisms also act at short range. Some of these molecules bind tightly to the extracellular matrix, unable to signal, but subsequentlycan be releasedin an active form. Many developmentally important signals diffuse away from the signaling cell, forming a concentration gradient and inducing differeni cellular responsesdepending on the distance of a particular target cell from the site of signal release(Chapter 22). In autocrine signaling cells respond to substancesthat they themselvesrelease.Somegrowth factors act in this fashion, and cultured cellsoften secretegrowth factors that stimulate their own growth and proliferation. This type of signaling is particularly characteristicof tumor cells, many of which overproduceand releasegrowth factors that stimulate inappropriate, unregulatedself-proliferation as well as influ-

(a)

Residuesessentialto tight binding with receptor

(b)

encing adjacentnontumor cells;this processmay lead to formation of a tumor mass. Signalingmoleculesthat are integral membrane proteins located on the cell surfacealso play an important role in development (Figure 15-2d).In some cases,such membranebound signalson one cell bind receptorson the surfaceof an adjacent target cell to trigger its differentiation. In other cases,proteolytic cleavageof a membrane-bound signaling protein releasesthe extracellular segment,which functions as a soluble signaling molecule. Some signaling molecules can act at both short and long ranges. Epinephrine, for example, functions as a neurotransmitter (paracrine signaling) and as a systemichormone (endocrine signaling). Another example is epidermal growrh factor (EGF), which is synthesizedas an integral plasma membrane protein. Membrane-bound EGF can bind to and signal an adjacentcell by direct contact. Cleavageby an extracellularmatrix proteasereleasesa soluble form of EGF, which can signalin either an autocrineor a paracrinemanner.

Bindingof SignalingMoleculesActivates Receptorson TargetCells When a signaling molecule arrives at atargetcell, it binds to a receptor(seeFigure 15-1, step 4). The vast majority of receptors are activated by binding of secretedor membrane-bound

(c)

Growth hormone

Residuesessential to tight binding with hormone

H3' Growth normone receptor

-ooc EXPERIMENTAL FTGURE 15-3 Smallpatchesof aminoacids are important for specificbinding between growth hormone and its receptor.Theoutersurfaceof the plasmamembrane is towardthe bottomof the figure,andeachreceptormolecule is anchored to the membrane by a hydrophobic membrane-spanning cthelix(notshown)thatisa continuation of the carboxyl terminus depicted in thefigureAs determined fromthethree-dimensional structure of the groMh hormone-growth hormonerecepror comptex, 28 aminoacidsin the hormone areat the bindinginterface with one receptorEachof theseaminoacidswasmutated, oneat a ttme.to alanine, andthe effecton receptor (a)From bindingwasdetermined thisstudyit wasfoundthatonlyeightaminoacidson growthhormone (pink)contribute 85 percent of the bindingenergy; theseaminoacids 626

C H A P T E R1 5

I

aredistantfromeachotherin the primarysequence but adjacent in the foldedproteinSimilar studies showedthat two tryptophan (blue)in the receptor residues contributemostof the energyfor bindinggrowthhormone, although otheraminoacidsat the interface (yellow) with the hormone (b)Binding arealsoimportant. of growth hormone to onereceptor molecule isfollowedby (c)bindingof a (purple) second receptor to theopposing sideof the hormone; this involves thesamesetof yellowandblueaminoacidson the receptor but differentresidues on the hormone.As we seein the nextchapter, suchhormone-induced receptor dimerization isa commonmechanism for activation of receptors for proteinhormones[AfterB Cunningham andJ,Wells, 1993, L Mol Biol234:554, andT.Clackson andJ Wells. 1995. Sclence 267:383 l

C E L LS I G N A L I N GI : S I G N A LT R A N S D U C T I OA NN D S H o R T . T E R Mc E L L U L A RR E s P o N s E s

molecules(e.g.,hormones,growth factors,neurotransmitters, and pheromones).Somereceptors,however,are activatedby changesin the concentrationof a metabolite (e.g.,oxygen or nutrients) or by physicalstimuli (e.g.,light, touch, heat). As noted earlier,the specificreceptor protein for any hydrophilic extracellular signaling molecule is almost always located on the surfaceof the target cell. The signaling molecule, or ligand, binds to a site on the extracellular domain of the receptor as a consequenceof their molecular complementarity (Figure15-3; seealso Chapter2). Binding of a ligand causes a conformational change in the membranebound receptor that initiates a sequenceof reactionsleading to a specific cellular responseinside the cell. Different cell types may have different sets of receptors for the same ligand, each of which can induce a different response.Or the samereceptor may be found on various cell types, and binding of a particular ligand to a particular receptor may trigger a different responsein each type of cell. In theseways, one ligand can induce different cells to respond in a variety of ways. Ligand binding to G protein-coupled receptors triggers an intracellular protein (a trimeric G protein) to exchange one bound GDP nucleotide for a GTP. The conformational change induced by GTP binding, in turn, affects interaction of the G protein with downstream signal-transductionproteins. The human receptors for the hormones epinephrine, serotonin, and glucagon are examplesof G protein-coupled receptors. As we discuss in other chapters, the conformational change resulting from ligand binding to some other classesof receptors activates (or occasionally inhibits) the phosphorylating, or kinase, activity of the receptor's intracellular domain, or of an associatedcytosolic kinase enzyme. Subsequentphosphorylation of substrate proteins in the cytosol modifies their activities. Activation of still other receptors involves proteolysis or disassemblyon intracellular multiprotein complexes, leading to releaseof signaltransductionproteins.

From ExtracellularSignal to Cellular Response r Extracellular signalingmoleculesregulate interactions between unicellular organisms and are critical regulators of physiology and development in multicellular organlsms. r Extracellular signaling molecules bind to receptors, inducing a conformational change in the receptor (activation) and consequent alteration in intracellular activities and functions. r Signalsfrom one cell act on nearby cells in paracrine signaling, on distant cells in endocrine signaling, or on the signaling cell itself in autocrine signaling (see Figure 15-2). r External signals include membrane-anchoredand secreted proteins and peptides (both dissolved in the extra-

cellular fluid or embedded in extracellular matrix), small lipophilic molecules (e.g., steroid hormones, thyroxine), small hydrophilic molecules(e.g.,epinephrine),gases(e.g., nitric oxide), and physicalstimuli (e.g.,light). r Binding of extracellularsignalingmoleculesto cell-surface receptorstriggers a conformational changein the receptor, which in turn leads to activation of intracellular signaltransduction pathways that ultimately modulate cellular metabolism, function, or geneexpression(seeFigure 15-1).

Receptors StudyingCell-Surface The responseof a cell or tissueto specificexternal signalsis dictated (a) by the cell's complement of receptors that can recognize the signals, (b) the signal-transductionpathways activated by those receptors, and (c) the intracellular processesaffected by those pathways. Recall that the interaction betweenligand and receptor causesa conformational changein the receptor protein that enablesit to interact with other proteins, thereby initiating a signalingcascade(seeFigure 15-1, steps4 and E). In this sectionwe explorethe biochemical basisfor the specificity of receptor-ligand binding, as well as the ability of different concentrationsof ligand to activate a pathway. $(/e also examine experimental techniques used to purify and characterize receptor proteins. Many of thesemethods are applicable to receptorsthat mediate endocytosis(Chapter 1'4\or cell adhesion(Chapter 19) as well as those receptorsthat mediate signaling. Typical cell-surface receptors are present in 1000 to 50,000 copies per cell. This may seem like a-large number, but a "typical" mammalian cell contains -10tu total protein moleculesand:106 proteins on the plasma membrane.Thus the receptor for a particular signaling molecule commonly constitutes only 0.1 to 5 percent of plasma membrane proteins. This low abundance complicates the isolation and purification of cell-surface receptors. The purification of receptors is also difficult becausethese integral membrane proteins first must be solubilized from the membrane with a nonionic detergent (see Figure 1'0-23) and then separated from other cellularproteins.

ReceptorProteinsBind LigandsSpecifically Each receptor generallybinds only a singlesignalingmolecule (ligand) or a group of very closely structurally related molecules. The binding specificity of a receptor refers to its ability to distinguishclosely related substances.Ligand binding depends on weak, multiple noncovalent forces (i.e., ionic, van der \faals, and hydrophobic interactions) and molecular complementarity between the interacting surfacesof a receptor and ligand (seeFigure 2-1,2).The insulin receptor, for example, binds insulin and related hormones called insulin-like growth factors 1' and2 (IGF-1 and IGF-2), but no other hormones.SimilarlS the receptor that binds growth hormone does not bind other hormones of similar structure, and acetylcholinereceptorsbind only this small molecule and not others that differ only slightly S T U D Y I N GC E L L - S U R F A CREE C E P T O R S

627

in chemical structure. As these examplesillustrate, all receptors are highly specificfor their ligands. Not only is each receptor protein characterized by its binding specificityfor a particular ligand, the resulting receptor-ligand complex exhibits effector specificity becausethe complex mediatesa specificcellular response.Many signaling molecules bind to multiple types of receprors,each of which can activate different intracellular signalingpathways and thus induce different cellular responses.For instance,the surfacesof skeletal muscle cells, heart muscle cells, and the pancreatic acinar cells that produce hydrolytic digestiveenzymes each have different types of receptors for acetylcholine. In a skeletalmusclecell, releaseof acetylcholinefrom an adjacent neuron triggers contraction by activating an acetylcholine-gatedion channel.In the autoimmune paralytic diseasemyasthenia gravis, the body makes antibodies that block the activity of its own acetylcholinereceptors.In heart muscle,the releaseof acetylcholineby different neurons activatesa G protein-coupled receptorand slows the rate ofcontraction, and thus the heart rate. Releaseof acetylcholine near pancreaticacinar cells triggers a rise in cytosolic [C"t*] that induces exocytosis of the digestive enzymes stored in secretorygranules to facilitate digestion of a meal. Thus formation of different acetylcholine-receptorcomplexes in different cell types leadsto different cellular responses. On the other hand, different receptorsof the same class that bind different ligands often induce the same cellular responsesin a cell; thus different extracellularsignalscan cause a common changein the behaviorof a cell. In liver cells,for instance,the hormonesepinephrine,glucagon,and ACTH bind to different membersof the G protein{oupled receptor familS but all these receptorsactivate the same signal-transduction pathway, one that promotes synthesisof a small intracellular signalingmolecule(cyclicAMP) that in turn regulatesvarious metabolic functions, including glycogenbreakdown. As a result, all three hormones have the same effect on liver-cell metabolism, as further detailedin Sections15.6 and 15.8.

The DissociationConstantls a Measureof the Affinity of a Receptorfor tts Ligand Ligand binding to a receptor usually can be viewed as a simple reversiblereaction, where the receptor is representedas R, the ligand as L, and the receptor-ligand complex as RL: koff

R+L

=

RL

(1s-1)

kot

Ao66 is the rate constant for dissociation of a ligand from its receptor,and Ao, is the rate constant for formation of a receptor-ligandcomplex from free ligand and receptor. At equilibrium the rate of formation of the receptor-ligand complex is equal to the rate of its dissociation,and can be describedby the simple equilibrium-binding equation

ro : 628

'

c H A P T E R1 5

|

f R l fL l

lnl_l

( 1s- 21

where [R] and [L] are the concentrations of free receptor (that is, receptorwithout bound ligand) and ligand, respectively, at equilibrium, and [RL] is the concentration of the receptor-ligand complex. Ka, the dissociation constant, is a measure of the affinity of the receptor for its ligand. For a simple binding reaction, Ka : ko,;.lko,.The lower ftos is relative to &o., the more stable the RL complex-the tighter the binding-and thus the lotuer the value of K6. Another way of seeingthis key point is that K6 equals the concentration of ligand at which half of the receptorshave a ligand bound; at this ligand concentration [R] : [RL] and thus, from Equation 1,5-2,Kd: [L]. The lower the K6, the lower the ligand concentration required to bind 50 percent of the cell-surfacereceptors. The K6 for a binding reaction here is essentiallyequivalentto the Michaelis constant K-, which reflects the affinity of an enzyme for its substrate ( C h a p t e r3 ) . Like all equilibrium constants,however, the value of K6 does not depend on the absolute values of &o6and Ao,, only on their ratio. In the next sectionwe learn how K6 valuesare experimentally determined.

BindingAssaysAre Usedto DetectReceptors a n d D e t e r m i n eT h e i rA f f i n i t i e sf o r L i g a n d s Usually receptorsare detectedand measuredby their ability to bind radioactive ligands to intact cells or to cell fragments. Figure 15-4 illustrates such a binding assayfor interaction of insulin with insulin receptors on liver cells. The amounts of radioactive insulin bound to its receotor on cells growing in Petri dishes(vertical axis) were miasured as a function of increasinginsulin added to the extracellular fluid (horizontal axis). Both the number of ligand-binding sitesper cell and the K6 value are easilydetermined from the specificbinding curve (curve B), usually by applying readily available computer curve-fitting programs to the experimentalvalues.Assuming each receptor generally binds just one ligand molecule, the number of ligand-binding sitesequals the number of active receptors per cell. In the example shown in Figure 1.5-4,the value of Ka is 1.4 x 10-10 M. In other words. an insulin concentration of 1.4 x 10-10 M in the extracellular fluid is required for 50 percent of a cell'sinsulin receptorsto have a bound insulin. A receptor usually has a different affinity for each of the ligands that it can bind. For instance,similar binding assaysshowed that the Ka for binding of insulin-like growth factor 1(IGF-1) to the insulin receptor is 3 x 10-o M. Thus an -200-foId higher concentration of IGF-1 than insulin is required to bind 50 percent of the insulin receptors. Direct binding assayslike the one in Figure 1.5-4 are feasible with receptors that have a strong affinity for their ligands, such as the erythropoietin receptor (K6 : 1 x 10-10 M) and the insulin receptoron liveriells (Ka : 1.4 x 10-10 M). However,many ligands such as epinephrineand other catecholaminesbind to their receptors with much lower affinity. If the K6 for binding is greater than -1 X 10-7 M, a casewhen the rate .orrrt".rt &"s is relativelylarge

C E L LS T G N A L T NrG : s T G N A LT R A N S D U C T o NA N D s H o R T - T E R MC E L L U L A R REspoNSEs

manipulations required for the measurement(relatively low kor). c)

6 40,000 Specificbinding

th 0)

o

30,000

E c

= 20,000 o o

Nonspecificbinding

10,000

N

= 0

0.2

0.4 0.6 0.8 [ 1 2 5i1n]s u l i n( n M )

1.0

FIGURE 15-4 Fot high-affinityligands, A EXPERIMENTAL bindingassayscandeterminethe K6 and the numberof receptors receptorsper cell.Shownherearedatafor insulin-specific o n t h e s u r f a c oe f l i v e rc e l l sA. s u s p e n s i o nf c e l l si s i n c u b a t efdo r insulin; concentrations of 12sl-labeled t hourat 4'C with increasing is usedto preventendocytosis of the cell-surface the low temperature Thecellsareseparated f rom unboundinsulin,usually by receptors. i toy u n dt o t h e mi s c e n t r i f u g a t i oann,dt h e a m o u not f r a d i o a c t i v b m e a s u r eT d h et o t a lb i n d i n gc u r v eA r e p r e s e ni tnss u l i sn p e c i f i c a l l y r ssw e l la si n s u l i n o n s p e c i f i c a l l y b o u n dt o h i g h - a f f i n irtey c e p t o a on the cellsurfaceThe boundwith low affinityto othermolecules bindingto totalbindingis determined contribution of nonspecific in the presence of a 10O-fold excess the bindingassay by repeating high-affinity sites insulin, whichsaturates allthespecific of unlabeled insulinbindsto nonspecific sites,yielding In thiscase,allthe labeled bindingcurveB iscalculated asthedifference curveC Thespecific by the maximumof the betweencurvesA and C As determined sites ic insulin-binding ic bindingcurveB,the numberof specif specif (surface percellis33,000TheK6istheconcentration of receptors) insulinreceptors to bindto 50 percent of the surface insulinrequired (inthiscaseabout16,500receptors/cell) Thus,the K6isabout1 4 x 1 0 - 1 0M , o r O 1 4 n M [ A d a p t ef rdo mA C i e c h a n oevtea rl, 1 9 8 3 , Cell32:267l

compared to fro,, then it is likely that during the seconds to minutes required to measurethe amount of bound ligand, some of the receptor-boundligand will dissociateand thus the observedvalueswill be systematicallytoo low. One way to detect weak binding of a ligand to its receptor is in a competition assaywith another ligand that binds to the samereceptor with high affinity (low K6 value). In this type of assay,increasingamounts of an unlabeled,low-affinity ligand (the competitor) are added to a cell sample with a constant amount of the radiolabeled,high-affinity ligand (Figure15-5). Binding of unlabeledcompetitor blocks binding of the radioactive ligand to the receptor; the concentration of competitor required to inhibit binding of half the radioactive ligand approximates the K6 value for binding of the competitor to the receptor. It is possible to accurately measurethe amount of the high-affinity ligand bound in this assay because little dissociatesduring the experimental

Competitive binding is often used to study synthetic analogsof natural hormones that activate or inhibit receptors.Theseanalogs,which are widely usedin research on cell-surfacereceptorsand as drugs, fall into two classes: agonists, which mimic the function of a natural hormone by binding to its receptor and inducing the normal response,and antagonists,which bind to the receptor but induce no response.By occupying ligand-binding sites on a receptor, an antagonist can block binding of the natural hormone (or agonist)and thus reducethe usual physiological activity of the hormone. In other words' antagonists inhibit receptorsignaling. Consider for instance the drug isoproterenol used to treat asthma.Isoproterenolis made by the addition of two methyl groups to epinephrine(seeFigure 1'5-5,right).lsoproterenol, an agonist of the epinephrine-responsiveG protein-coupled receptors on bronchial smooth muscle cells, binds about tenfold more strongly (lO-fold lower K6) than does epinephrine(seeFigure 15-5, left). Because activation of these receptors promotes relaxation of bronchial smooth muscle and thus opening of the air passagesin the lungs, isoproterenol is used in treating bronchial asthma, chronic bronchitis, and emphysema.In contrast' activation of a different type of epinephrine-responsive G protein-coupled receptors on cardiac muscle cells increasesthe heart contraction rate. Antagonists of this receptor, such as alprenolol and related compounds' are referred to as beta-blockers; such antagonists are used to slow heart contractionsin the treatment of cardiac arrhythmias and angina. I

M a x i m a lC e l l u l a rR e s p o n s teo a S i g n a l i n g MoleculeUsuallyDoesNot RequireActivation of AII Receptors All signalingsystemsevolvedsuch that a rise in the level of extracellularsignalingmoleculesinducesa proportional responsein the respondingcell. For this to happen the binding affinity (Ka value) of a cell-surfacereceptor for a circulating hormone must be greater than the normal (unstimulated) Ievel of that hormone in the extracellular 'We can see this principle in practice by fluids or blood. comparing the levelsof insulin presentin the body and the K6 for binding of insulin to its receptoron liver cells' 1.4 x 10-10 M. Suppose,for instance,that the normal concentration of insulin in the blood is 5 x 10-12 M. By substituting this value and the insulin K6 into Equation 1'5-2,we can calculatethe fraction of insulin receptorswith bound insulin-[Rl-]/(lRLl +[R])-at equilibrium as 0.0344; that is, about 3 percent of the total insulin receptors will be bound with insulin. If the insulin concentration rises fivefold to 2.5 x L0-11 M, the number of receptor-hormone complexes will rise proportionately, almost fivefold, so that about 15 percent of the total receptors will have bound insulin. If the extent of the inducedcellular response S T U D Y I N GC E L L - S U R F A CREE C E P T O R S

629

OH

100

l*

cH -cH2 -

I

NH2- CH3

(EP) Epinephrine

P80

?'

EOU c o

. ?'.

cH-cH2-NH2-CH

E40

(lPl lsoproterenol

CHs

oH

gH.

c

CH,

il-

E20

l*l cH2-cH -cH2 -NH2-CH

I

0-

CHs

10-8 10-6 1O-4 (M) Competitor concentration

Alprenolol(AP)

A EXPERIMENTAL FTGURE 15-5 Forlow-affinityligands, bindingcan be detectedin competitionassays.In thisexample, the synthetic ligandalprenolol, whichbindswith highaffinityto the e p i n e p h r i nr e c e p t oorn l i v e rc e l l s( K a= 3 x 1 O - eM ) ,i s u s e dt o detectthe bindingof two low-affinity ligands, the naturalhormone (EP) epinephrine anda synthetic (lp)Assays ligandcalledisoproterenol areperformed asdescribed in Figure 15-4butwith a constant amount of [3H]alprenolol to whichincreasing amounts of unlabeled epinephrine or isoproterenol areadded.At eachcompetitor concentration, the amountof boundlabeled alprenolol isdetermined In a plotof the

inhibition of ['H]alprenolol binding versus epinephrine or isoproterenol concentration, suchasshownhere,theconcentration of the competitor thatinhibits alprenolol bindingby 50 percent approximates theK6 valuefor competitor binding.Notethat the concentrations of competitors areplottedon a logarithmic scaleTheK6for bindingof epinephrine to itsreceptor on livercellsisonly-5 x 1O-sM and wouldnot be measurable by a directbindingassay with TheK6for bindingof isoproterenol, [3H]epinephrine. whichinduces thenormalcellular response, ismorethantenfoldlower

parallels the number of hormone-receptor complexes, [RL], as is often the case,then the cellular responsesalso will increaseby about fivefold. On the other hand, supposethat the normal concentration of insulin in the blood is the same as the K6 value of 1.4 x 10-10 M; in this case,50 percent of the toial receptors would have a bound insulin. A fivefold increasein the insulin concentrationto 7 x 10-10 M would result only in a 66 percent increase in the fraction of receptors with bound insulin (to 83 percent bound). Thus, in order for a rise in hormone concentrationto causea proportional increase in the fraction of receptors with bound ligand, the normal concentration of the hormone must be well below the Ka value. In generalthe maximal cellular responseto a particular ligand is induced when much less than 100 percent of its receptorsare bound to the ligand. This phenomenoncan be revealed by determining the extent of the responseand of receptor-ligand binding at different concenrrations of ligand (Figure 15-6). For example, a typical red blood (erythroid) progenitor cell has -1000 surface receptors for erythropoietin, the protein hormone that induces these cells to proliferate and differentiate into red blood cells. Becauseonly 100 of these receptors need to bind erythropoietin to induce division of a progenitor cell, the ligand concentration neededto induce 50 percent of the maximal cellular responseis proportionally lower than the K6 value for binding. In such cases,a plot of the percentageof max-

imal binding versus ligand concentration differs from a plot of the percentageof maximal cellular responseversus ligand concentration.

630

C H A P T E R1 5

|

1.0

P h y s i o l o g i c ar el s p o n s e

!L"X,

-=

0.8

G6

xL OL

Fractionof surfacereceptors with boundligand

0.6

bf h=

0.4 LL

Ligandconcentration for 50Yophysiologicalresponse

0.2 K6for ligand binding ll

01234 Relative concentration of ligand EXPERIMENTAL FIGURE 15-6 The maximalphysiological responseto an externalsignaloccurswhen only a fraction of the receptorsare occupiedby ligand.Forsignaling pathways t h a te x h i b itth i sb e h a v i opr ,l o t so f t h e e x t e not f l i g a n db i n d i n gt o the receptor and of physiological response at differentligand concentrations differ.In the exampleshownhere,50 percentof the maximal physiological response isinduced at a ligandconcentration at whichonly 18 percentof the receptors areoccupiedLikewise, 8 0 p e r c e not f t h e m a x i m ar le s p o n si sei n d u c e w d h e nt h e l i g a n d concentration equalsthe K6value,at which50 percentof the receptors areoccupied

C E L LS I G N A L I N Gl : S I G N A LT R A N S D U C T I O A NN D S H O R T - T E R M CELLULAR RESpONSEs

Sensitivityof a Cellto ExternalSignals ls Determinedby the Numberof Surface Receptorsand Their Affinity for Ligand Becausethe cellular responseto a particular signaling molecule dependson the number of receptor-ligand complexes, the fewer receptors present on the surface of a cell, the less sensitiuethe cell is to that ligand. As a consequence,a higher ligand concentration is necessaryto induce the physiological response than would be the case if more receptorswere present To illustrate the important relationship betweenreceptor number and hormone sensitivity,let's extend our example of a typical erythroid progenitor cell. The K6 for binding of ery10 M. As we thropoietin (Epo) to its receptor is about 10 :1000 noted above, only 10 percent of the erythropoietin receptorson the surfaceof a cell must be bound to ligand to induce the maximal cellular response.'Wecan determine the ligand concentration, [L], needed to induce the maximal responseby rewriting Equation 15-2 as follows:

(15-3)

where R1 : [R] + [RL], the total number of receptorsper cell. If the total number of Epo receptorsper cell, R1, is 1000, K6 is 10-10 M, and tRLl is 100 (the number of Epo-occupied receptorsneededto induce the maximal response),then an 11M will elicit the maxEpo concentration([L]) of 1.1 x 10 imal response.If the total number of Epo receptors (R1) is reducedto 200/cell,then a ninefold higher Epo concentration (10-to M) is required to occupy 100 receptorsand induce the maximal response.Clearly, therefore, a cell's sensitivity to a hormone is heavily influencedby the number of receptorsfor that hormone that are presentas well as the Kd. Epithelial growth factor (EGF), as its name implies, stimulates the proliferation of many types of epithelial cells, including those that line the ducts of the mammary gland. In about 25 percent of breast cancers the tumor cells produce elevated levels of one particular EGF receptor called HERZ. The overproduction of HER2 makes the cells hypersensitiveto ambient levels of EGF that normally are too low to stimulate cell proliferation; as a consequencegrowth of these tumor cells is inappropriately stimulated by EGF. Understanding of the role of HER2 in certain breast cancersled to development of monoclonal antibodies that bind HER2, and thereby block binding of EGF; these antibodies have proven useful in treatment of thesebreastcancerpatients.I The HER2-breast cancer connection vividly demonstratesthat regulation of the number of receptorsfor a given signaling molecule expressedby a cell plays a key role in directing physiological and developmentalevents.Suchregulation can occur at the levelsof transcription, translation, and

post-translational processing,or by controlling the rate of receptor degradation.Alternatively, endocytosisof receptors on the cell surfacecan sufficiently reducethe number present to terminate the usual cellular response at the prevailing signal concentration. As we discussin later sections,other mechanismscan reduce a receptor's affinity for ligand, and thus reduce the cell's responseto a given concentration of ligand. Thus reduction in a cell's sensitivity to a particular ligand, calleddesensitization,can result from various mechanisms and is critical to the ability of cells to respond appropriately to external signals.

ReceptorsCan Be Purifiedby Affinity Techniques Becauseof their low abundancespecialtechniquesare necessary to isolate and purify receptors. Cell-surfacereceptors can be identified and followed through isolation procedures by affinity labeling.In this technique,cellsare mixed with an excessof a radiolabeled ligand for the receptor of interest. After unbound ligand is washed away the cells are treated with a chemical agent that covalently crosslinks the bound labeled ligand molecules to receptors on the cell surface. Once a radiolabeled ligand is covalently cross-linked to its receptor,it remains bound even in the presenceof detergents and other denaturing agents that are used to solubilize receptor proteins from the cell membrane. The labeled ligand provides a means for detectingthe receptor during purification procedures. Another technique often used in purifying cell-surface receptorsthat retain their ligand-binding ability when solubilized by detergentsis similar to affinity chromatography using antibodies (seeFigure 3-37c). To purify a receptor by this technique, a ligand for the receptor of interest, rather than an antibody, is chemically linked to the beads used to form a column. A crude, detergent-solubilizedpreparation of membrane proteins is passedthrough the column; only the receptor binds, while other proteins are washed away. Passageof an excessof the soluble ligand through the column causesthe bound receptor to be displaced from the beadsand eluted from the column. In some cases'a receptor can be purified as much as 100,000-fold in a single affinity chromatographic step.

ReceptorsAre Frequently Expressed from ClonedGenes Once the amino acid sequenceof a purified receptor has been determined its gene can be cloned. A functional expression assay of the cloned cDNA in a mammalian cell that normally lacks the encoded receptor can provide definitive proof that the proper protein indeed has been obtained (Figure 15-7). Suchexpressionassaysalso permit investigators to study the effects of mutating specific amino acids on ligand binding or on "downstream" signal transduction, thereby pinpointing the receptor amino acids responsiblefor interacting with the ligand or with c r i t i c a l s i g n a l - t r a n s d u c t i opnr o t e l n s . S T U D Y I N GC E L L - S U R F A CREE C E P T O R S

631

$

Receotorfor

|( :

ilgano orner than X

Tf_

..

ii

,l

.t,

iI

{

! L i g a n dX

No binding of X; no cellular response

clone encoding the receptor is identified, the sequenceof the cDNA can be determinedand that of the receptorprotein deduced from the cDNA sequence.Cells overexpressingthe receptor protein can be usedto purify large amounts of the protein, which can be used to determine its three-dimensional structure.This structural information can provide additional insightsinto the mechanismsby which the receptorfunctions, as well as suggestinghow new types of drugs might interact with the receptor to treat diseases. Genomic studies coupled with functional expressionassaysare now being used to identify genesfor previously unknown receptors. In this approach, stored DNA sequences are analyzed for similarities with sequencesknown to encode receptor proteins (Chapter 6). Any putative receptor genesthat are identified in such a searchthen can be tested for their ability to bind a signaling molecule or induce a responsein cultured cells by a functional expressionassay.

CharacterizingCell-SurfaceReceptors r Receptorsbind ligandswith considerablespecificity,which is determinedby noncovalent interactions betweena ligand and specificamino acids in the receptorprotein (seeFigure 15-3). r The concentration of ligand at which half its receprors are occupied, the K4, can be determined experimentally and is a measureof the affinity of the receptor for the ligand (seeFigure 15-4). Binding of X; normal cellular response

A EXPERIMENTAL FIGURE 15-7 Functional expression assay can identify a cDNAencodinga cell-surfacereceptor.Targetcells lackingreceptors for a particular ligand(X)arestablytransfected with a cDNAexpression vectorencoding the receptor Thedesignof the expression vectorpermits selection of transformed cellsfromthose thatdo not incorporate thevectorintotheirgenome(seeFigure 5-32b)Providing thatthesecellsalready express allthe relevant signal-transduction proteins, thetransfected cellsexhibitthe normal cellular response to X if the cDNAin factencodes thef unctional receptor. Cell-surfacereceptors for many signaling molecules are presentin such small amounts that they cannot be purified by affinity chromatographyand other conventional biochemical techniques.Theselow-abundancereceptor proreins can now be identified and cloned by various recombinant DNA techniques, eliminating the need to isolate and purify them from cell extracts.In one technique,a library of cloned cDNAs prepared from the entire nRNA extracted from cells that produce the receptor is inserted into expression vectors by techniques describedin Chapter 5. The recombinanr vectors then are transfected into cells that normally do not synthesizethe receptorof interest(seeFigure 15-7). Only the very few transfected cells that contain the cDNA encoding the desiredreceptor synthesizeit; other transfectedcellsproduce irrelevant proteins. The rare cellsexpressingthe desiredreceptorcan be detectedand purified by various techniquessuch as fluorescence-activatedcell sorting using a fluorescent-labeledligand for the receptor of interest (seeFigure 9-28). Once a cDNA

632

CHAPTER 15

I

r The maximal responseof a cell to a particular ligand generally occurs at ligand concentrationsat which most of its receptorsare still not occupied(seeFigure 15-6). r Becausethe amount of a particular receptor expressedis generallyquite low (rangingfrom -1000 to 50,000 molecules per cell), biochemical purification may not be feasible. Genes encoding low-abundance receptors for specific ligands often can be isolated from cDNA libraries rransfected into cultured cells. r Functional expression assayscan determine if a cDNA encodesa particular receptor and are useful in studying the effects on receptor function of specific mutarions in irs sequence(seeFigure 15-7).

HighlyConserved Components of Intracellular Signal-Transduction Pathways External signals induce two major types of cellular responses:( 1 ) changesin the activity or function of specificenzymes and other proteins that pre-exist in the cell, and (2) changesin the amounts of specific proteins produced by a cell, most commonly by modification of transcription factors that stimulate or repressgene expression (seeFigure 15-1, step Et). In general, the first type of responseoccurs more rapidly than the second. Signaling from G protein-coupled receptors, described in detail later in this chapter. often

C E L LS I G N A L I N G I : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RcME L L U L A RR E s P o N s E s

results in changesin the activity of pre-existingproteins, although activation of these receptors on some cells also induceschangesin geneexpression.Other classesof receptors operate primarily but not exclusively to modulate gene expression. Transcription factors activated in the cytosol by thesepathways move into the nucleus,where they stimulate (or occasionally repress) transcription of specific target genes.'Weconsider thesesignalingpathways, which regulate transcription of many genesessentialfor cell division and for many cell differentiation processes,in the following chapter. Several intracellular proteins or small molecules are employed in a variety of signal-transductionpathways. These include cytosolic enzymesthat add or remove phosphate groups from specifictarget proteins. Ligand binding to a receptor activates or inhibits these enzymes,whose action in turn activates or inhibits the function of their target proteins. G proteins, another component of many signal-transductionpathways, shuttle betweena statewith a bound GTP that is capableof activating other proteins and a statewith a bound GDP that is inactive. A number of small molecules (e.g., Ca2* and cyclic AMP) are also frequently used in intracellular signal-transduction pathways: A rise in the concentration of one of thesemoleculesresults in its binding to an intracellular target protein, causing a conformational changein the protein that modulates

its function. Here we review the basicproperties of theseintracellular signal-transducingmolecules.The rules and exceptions that govern how they are usedin particular signaling pathways are developedfurther in subsequentsectionsof this chapter.

GTP-BindingProteinsAre FrequentlyUsed As On/Off Switches We introduced the large group of intracellular switch proteins that form the GTPase superfamily in Chapter 3' These guanine nucleotide-binding proteins are turned "on" when they bind GTP and turned "off" when the GTP is hydrolyzed to GDP (seeFigure 3-32). Signal-inducedconversion of the inactive to active state is mediated by a guanine nucleotide-exchangefactor (GEF), which causesreleaseof GDP from the switch protein. Subsequentbinding of GTP' favored by its high intracellular concentration relative to its binding affinity, induces a conformational change in at least two highly conserved segmentsof the protein' termed switch I and switch II, allowing the protein to bind to and activate other downstream signaling proteins (Figure 15-8). The intrinsic GTPase activity of the protein then hydrolyzes the bound GTP to GDP and P1,thus changingthe conformation of switch I and switch II from the active form back to the

(b) GDP-bound"off" state

(a) GTP-bound"on" state

Gly-60 Thr-35 Switch ll

Gly-60

Switch I

GDP

for G proteins.Theability 15-8 Switchingmechanism A FIGURE a with otherproteins andthustransduce of a G proteinto interact "off" "on" stateandGDP-bound signaldiffersin the GTP-bound termedswitchI state(a)Inthe active"on" state.two domains, (green) andswitchll (blue), areboundto theterminal 1 phosphate groupsof a interactions with backbone amide through the of GTP of the1 residue(b)Release threonine andglycine conserved

switchI andswitch causes hydrolysis phosphate by GTPase-catalyzed "off" stateThe the inactive conformation, ll to relaxintoa different of Ras,a conformations both shownhererepresent ribbonmodels switches mechanism protein spring-loaded A similar G monomeric the activeandinactive between G proteins thectsubunitin trimeric from of threeswitchsegmentsfAdapted by movement conformations 29411299 2001,Scrence | andA Wittinghofel I Vetter

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inactiveform. The rate of GTP hydrolysisregulatesthe length of time the switch protein remainsin the activeconformation and able to signal downstream: The slower the rate of GTP hydrolysis, the longer the protein remains in the active state. The rate of GTP hydrolysis is often modulated by other proteins. For instance,both GTPase-actiuatingproteins (GAP) and regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis.Many regulatorsof G protein activity are themselvescontrolled by extracellular signals. Two large classesof GTPaseswitch proteins are used in signaling. Trimeric (large) G proteins, ai noted already, directly bind to and are activated by certain cell-surfacereceptors. The activated receptor functions as a GEF and triggers releaseof GDP and binding of GTP. Monomeric (small) G proteins, such as Ras and various Ras-likeproteins,play crucial roles in many parhwaysthat regulatecell division and cell motility; theseG proteinsfrequentlyundergo activaringmutations in cancers. Ras is linked indirectly to receptors via adapterproteins and GEF proteins discussedin the next chapter. All G switch proteins contain regions like switch I and switch II that modulate the activity of specificeffector proteins by direct protein-protein interactions when the G protein is bound to GTP. Despite these similarities, the nvo classesof GTP-binding proteins are regulatedin very different ways.

ProteinKinasesand Phosphatases are Employed i n V i r t u a l l yA l l S i g n a l i n gP a t h w a y s Activation of virtually all cell-surfacereceptorsleads directly or indirectly to changesin protein phosphorylation through the activation of protein kinases,which add phosphategroups to specific residues,or protein phosphatases,which remove phosphategroups. Animal cells contain two types of protein kinases:those that add phospharero rhe hydroxyl grorrp ort tyrosine residues and those that add phosphate to the hydroxyl group on serineor threonine (or both) residues.phosphatasescan act in concertwith kinasesto switch the function of various proteins on or off (seeFigure 3-33). At last count the human genome encodesabout 500 protein kinases and 100 different phosphatases.In some signalingpathways, rhe receptor itself possesses intrinsic kinase or phosphataseactivity; in other pathways, the receptor interacts with cytosolic or membrane-associated kinases.Importantly the activity of all kinasesis highly regulated.Commonly the catalytic activity of a protein kinase itself is modulated by phosphorylation by other kinases, by direct binding to other proteins or by changesin the levels of various small intracellular signaling molecules.The resultingcascadesof kinaseactiviry .o-"r. " mon feature of many signalingpathways. In general, each protein kinase phosphorylates specific residuesin a set of target proteins whose patterns of expression generally differ in different cell types.Many proteins are substratesfor multiple kinaseseachof which phosphorylates differentamino acids.Each phosphorylationevenican modify the activity of a particular target protein in different ways, some activating its function, others inhibiting it. An example we encounter later is glycogen phosphorylase kinase, a key regulatory enzymein glucosemetabolism. 634

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The activity of all protein kinasesis opposed by the activity of protein phosphatases,some of which are themselves regulated by extracellular signals.Thus the activity of a pro, tein in a cell can be a complex function of the activitiesof the usually multiple kinasesand phosphatasesthat act on it. Several examplesof this phenomenon occur in regulation of the cell cycle and are describedin Chapter 20.

S e c o n dM e s s e n g e rC s a r r ya n d A m p l i f y S i g n a l s from Many Receptors The binding of ligands ("first messengers")to many cellsurfacereceptorsleads to a short-lived increase(or decrease) in the concentration of certain low-molecular-weight intracellular signaling molecules termed second messengers. These,in turn, bind to other proteins modifying their activity. One secondmessengerusedin virtually all metazoancells is Ca2* ions. I(e noted in Chapter 11 that the concentration of Ca2* free in the cytosol is kept very low (-10-7 M) by ATP-poweredpumps that continually transport Ca2* out of the cell or into the endoplasmicreticulum (ER). The cytosolic Ca2* level can increasi from l0- ro 100-fold by a signalinduced releaseof Ca2* from ER srores or by its import through calcium channelsfrom the extracellularenvironment. In muscle,a signal-inducedrise in cytosolicCa2* triggerscontraction (see Figure 17-33). In endocrine cells, a similar increasein Ca"* inducesexocytosisof secretoryvesiclescontaining hormones.In nerve cells,a Ca2* increaseleadsto the exocytosis of neurotransmitter-containing vesicles (see Chapt^er23).ln all cells this rise in cytosolic Ca2* is sensed by Ca'*-binding proteins, particularly those of the EF hand family, such as calmodulin, all of which contain the helixloop-helix motif (seeFigure 3-9a). The binding of Caz* to calmodulin and other EF hand proteins causesa conformational changethat permits the protein to bind various target proteins, thereby switching their activities on or off (seeFigu r e3 - 3 1 ) . Another nearly universally used second messengeris cyclic AMP (cAMP). In many eukaryoric cells, a rise in cAMP triggers activation of a particular protein kinase that in turn inducesvarious changesin cell metabolism in different types of cells. In other cells, cAMP regulatesthe activity of certain ion channels.The structures of cAMP and three other common secondmessengersare shown in Figure 15-9. In later sectionsof this chapter,we examine the specificroles of secondmessengers in signalingpathways activated by various G protein-coupled receptors. Becausesecondmessengerssuch as Ca2* and cAMP diffuse through the cytosol much faster than do proteins, they are employed in pathways where the downstream target is located in an intracellular particle or organelle (such as a secretory vesicle)distant from the plasma membrane receptor. Another advantageof secondmessengersis that they facilitate amplification of an extracellular signal. Activation of a single cell-surfacereceptor moleculecan result in an increase in perhapsthousandsof cAMP moleculesor Ca2* ions in the cytosol. Each ofthese, in turn, by activating its target protein affects the activity of multiple downstream proteins.

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A c t i v a t e s p r o t e i n k i n a s eA ( P K A )

Activates protein kinaseG (PKG) and ooens cation channelsin rod cells

A FIGURE 15-9 Fourcommonintracellular secondmessengers. Themajordirecteffector effectsof eachcompoundareindicated belowitsstructural formula.Calcium ion(Ca2*)andseveral

Highly Conserved Components of Intracellular Signal-TransductionPathways r Protein kinasesand phosphatasesare employed in virtually all signaling pathways; their activities are highly regulated by receptors. r Other conserved proteins that act in many signaltransduction pathways include monomeric and trimeric G switch proteins(seeFigure 15-8). r The cytosolic concentrationsof secondmessengers, such as Ca2* and cAMP, increase or occasionally decreasein responseto binding of ligand to cell-surfacereceptors(see Figure 15-9).Thesenonprotein, low-molecular-weightintracellular signalingmolecules,in turn, regulatethe activitiesof enzymesand nonenzymaticproteins in signalingpathways.

GeneralElementsof G Protein-CoupledReceptorSystems As noted above, perhaps the most numerous class of receptors-found in organisms from yeast to man-are the G proteircoupled receptors (GPCRs). Receptor activation by ligand binding triggers activation of the coupled trimeric G protein, which interacts with downstream signal-transduction proteins. All GPCR signaling pathways share the following common elements: (1) a receptor that contains seven membrane-spanningdomains; (2) a coupled trimeric G protein, which functions as a switch by cycling between actrve and inactive forms; (3) a membrane-boundeffector protein; and (4) feedbackregulation and desensitizationof the signaling pathway. A secondmessengeralso occursin many GPCR pathways.Thesecomponentsare modular and can be "mixed and matched" to achievean astonishinqnumber of different

proteinkinase C Activates (PKC)

lnositol 1,4,5-trisphosphate (tP3)

in channels OpensCa2+ reticulum theendoPlasmic

phosphatidylinositol derivatives alsoact membrane-bound messenqers. assecond

pathways. GPCR pathways usually have short-term effects in the cell by quickly modifying existing proteins' either enzymes or ion channels.Thus thesepathways allow cells to respond rapidly to a variety of signals,whether they be environmental stimuli such as light or hormonal stimuli such as epinephrine. In this section, we first consider the general features of GPCR signaltransductionand then discusseach of the membrane-bound components in turn: the receptor,the trimeric G protein, and the effectorproteins.In Section15.5-15'7 we describe GPCR pathways that involve several different effector proteins. The short-term signalingdescribedin thesesections of this chapter often can be turned into long-term signaling involving changesin transcription and, as a consequence,cell differentiation,as describedin Chapter 16.

G Protein-CoupledReceptorsAre a Large and DiverseFamilywith a CommonStructure and Function All G protein-
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Exterior

Cytosol

cooG protein i nteraction

FIGURE 15-10Generalstructureof G protein-coupled receptors. All receptors of thistypehavethe sameorientation in the membrane andcontainseventransmembrane o-helical regions ( H 1 - H 7 f)o, u re x t r a c e l l usl a , df o u rc y t o s o l i c e rg m e n(t E s 1 - E 4a) n (C1-C4)Thecarboxyl-terminal segments (C4),the C3 loop, segment and,in somereceptors, alsothe C2 loopareinvolved in interactions with a coupled trimeric G protein

Iight-absorbing visual pigment 11-cls-retinal.In rhodopsin, the sevenmembrane-embedded o helicesof opsin completely surround a central segment to which retinal is covalently bound. In this case, binding of the ligand, retinal, does not trigger a conformational change in the receptor; rather, absorption of a quantum of light by the bound retinal inducesa changein opsin conformation that activatesit, as we discuss in Section15.5. The amino acids that form the interior of different G protein-coupledreceptorsare diverse,allowing differenrreceprors to bind very different small molecules,be they hydrophilic like

epinephrineor hydrophobic like many odorants and retinal. Figure 15-11 depictsa model of the complex formed betvveen the B2-adrenergicreceptor and the hormone epinephrine. As with retinal, epinephrineis thought to bind (in this case,noncovalently)in the middle of the plane of the membrane,interactingwith amino acidsin the interior-facingside of severalof the membrane-spanning a helices. As an exampleof the diversity and functionality of GPCR proteins, we will considerthe different G protein-coupled receptors for epinephrine that are found in different types of mammalian cells. The hormone epinephrine is particularly important in mediating the body's responseto stress(fightor-flight response),such as fear or heavy exercise,when tissuesmay have an increasedneed to catabolizeglucose and fatty acids to produce ATP. These principal metabolic fuels can be supplied to the blood in secondsby the rapid breakdown of glycogen to glucosein the liver and of triacylglycerols to fatty acids in adipose(fat) cells. In mammals, the liberation of glucoseand fatty acids can be triggered by binding of epinephrine (or its derivative norepinephrine) to B-adrenergicreceptors on the surface of hepatic (liver) and adiposecells. Epinephrine bound to Badrenergicreceptorson heart musclecellsincreasesthe contraction rate, which increasesthe blood supply to the tissues.In contrast, epinephrinestimulation of B-adrenergic receptors on smooth muscle cells of the intestrne causes them to relax. Another type of epinephrine receptor,the cvadrenergic receptor, is found on smooth muscle cells lining the blood vesselsin the intestinal tract, skin, and kidneys. Binding of epinephrineto thesereceptorscausesthe arteries to constrict,cutting off circulation to theseperipheral organs.

View from external surfaceface

Exterior

Membrane

Gytosol

H e l i x5

A FIGURE 15-11 Structuralmodelof complexformed between epinephrineand the B2-adrenergic receptor.(left)sideview.The approximate location of the membrane phospholipid bilayer is indicated Thethreect-heltces thatparticipate in epinephrine binding arecolored red(Helix5),green(Helix3),andpurple(Helix6) (right) viewfromexternal face.Epinephrine grey(C), atomsarecolored red(O)andpurple(N) Epinephrine interacts with several residues in the receptor thatarewithinthe planeof the membraneltsamino 636

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groupforms an ionicbond with the carboxylate sidechainof '1 ) H 3 ; t h e c a t e c h orli n ge n g a g e si n h y d r o p h o b i c a s p a r t a t e 1 3 ( D 1 1 3i n interactions with phenylalanine 290 (F2e0) in H6; and two hydroxyl groupson the catecholring hydrogen-bond to the hydroxylgroupsin (5203, threeserineresidues 5204and S2o7) in H5 iseep L Freddolino et al ,2004,Proc Nat'lAcad SciUSA101:2736, adaptedfrom modelprovided byW A Goddardl

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Effect on adenylyl cyclase I n h i b i t s( b i n d sG " i )

o,2-Adrenergicreceptor (wild typel

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Although all ct- and B-adrenergicreceptorsbind epinephrine, different receptors are coupled to different G proteins that induce different downstream signaling pathways, Ieading to different cellular responses.Studies with chimeric adrenergicreceptors,like those outlined in Figure 15-12' suggest that the long C3 loop between ct helices5 and 5 is important for interactionsbetweena receptorand its coupled G protein. PresumablS ligand binding causesthese helicesto move relative to each other in a way that allows the loop to bind and activatethe transducing G protein. Other evidence indicatesthat the C2 loop, joining helices3 and4, also contributes to the interaction of somereceptorswith a G protein.

G Protein-CoupledReceptorsActivate Exchange of GTPfor GDPon the q.Subunit of a Trimeric G Protein

B2-Adrenergicreceptor (wild type)

Trimeric G proteins contain three subunits designatedct, B, and 1. Both the G. and G, subunits are linked to the membrane by covalently attached lipids. During intracellular signaling, the B and 1 subunits remain bound together and are usually referred Chimeric receptor 1 to as the Gs, subunit. In the resting state' when no ligand is bound to the receptor,the Go subunit has a bound GDP and is complexed with Gpr. Binding of a normal hormonal ligand (e.g.,epinephrine)or an agonist (e.g.,isoproterenol)to a G I n h i b i t s( b i n d sG " i ) protein{oupled receptor changesthe conformation of its cytosol-facing loops and enablesthe receptor to bind to the G. subunit (Figure15-13, stepsI and Z). This binding releases Chimeric receptor 2 the bound GDP; thus the activatedligand-bound receptor functions as a guanine nucleotide-exchangefactor (GEF) for the G* subunit (stepB). Next GTP rapidly binds to the "empty" guanine nucleotide site in the Go subunit, causing a change in CONCLUSION the conformation of its switch segments(seeFigure 15-8). These changesweaken the binding of G* with both the receptor and the Gs, subunit (step4). R e g i o nd e t e r m i n i n gs p e c i f i c i t o y f G p r o t e i nb i n d i n g In most cases,Go'GTl which remains anchored in the ( c o m p a r ec h i m e r a s1 a n d 2 ) membrane, then interacts with and activates an associated effector protein, as depictedin Figure 15-13 (step E). In FIGURE 15-12Studieswith chimeric A EXPERIMENTAL some cases,G.'GTP inhibits the effector.Moreover, dependadrenergicreceptorsidentifythe long C3loop as criticalto ing on the type of cell and G protein, the G9" subunit, freed interactionwith G proteins.Xenopus oocytes weremicroinjected from its a subunit, will sometimestransduce a signal by ina wild-type o2-adrenergic, or withmRNAencoding B2-adrenergic, teractingwith an effectorprotein. o-B receptors AlthoughXenopus oocytes do not normally chimeric In any case,the active G.'GTP state is short-lived beadrenergic receptors, theydo express G proteins thatcancouple express the bound GTP is hydrolyzed to GDP in minutes, cause expressed on thesurface of microinjected to theforeignreceptors by the intrinsic GTPase activity of the G' subcatalyzed cellsin the presence oocytesTheadenylyl cyclase activity of the injected (see 15-13, step El). The conformation of the Figure unit whether the agonists wasdetermined andindicated of epinephrine (G*,)or Go then switches back to the inactive G*'GDP state, receptor boundto thestimulatory expressed adrenergic (G")typeof oocyteG proteinComparison of chimeric inhibitory blocking any further activation of effector proteins' The receptor 2, which receptor 1,whichinteracts withG..,andchimeric rate of GTP hydrolysis is further enhancedby binding of isdetermined with G", showsthatthe G proteinspecificity interacts the G*'GTP complex to the effector; the effector thus primarily between bythesourceof thecytosol-facing C3 loop(yellow) functions as a GTPase-activating protein (GAP). This B Kobilka cthelices 5 and6. [See etal,1988, Sclence240:1310] mechanism significantly reducesthe duration of effector activation and avoids a cellular overreaction. In many These diverse effects of epinephrine help orchestrate intecases,a noneffector RGS protein also acceleratesGTP hygrated responsesthroughout the body all directed to a drolysis by the Go subunit, further reducing the time during which the effector remains activated' The resulting common end: supplying energy that can be used for the rapid movementof major locomotor musclesin responseto G..GDP quickly reassociateswith Gp" and the complex becomesready to interact with an activated receptor and bodily stress. Activates(bindsG"r)

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start the process all over again. Thus the GPCR signaltransduction system contains a built-in feedback mechanism that ensures the effector protein becomes activated only for a few seconds or minutes following receptor activation; continual activation of receptors via Iigand binding is essentialfor prolonged activation of the effector. Early evidencesupporting the model shown in Figure 15-13 came from studieswith compounds that can bind to Go subunits as well as GTP does, but cannot be hydrolyzed by the intrinsic GTPase. In some of these compounds, the P-O-P phosphodiesterlinrcageconnecr-

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bindsto andactivates an effectorprotein(stepS) Hydrolysis of GTP terminates signaling andleadsto reassembly of thetrimeric G protein, returning thesystem to the resting state(step6) Binding of another ligandmolecule causes repetition of thecycleInsomepathways, the proteinisactivated effector bythefreeGs,subunit[After W Oldham andH Hamm, 2006,(QuaftRev. Biophys 40:(tnpress)l

ing the B and 1 phosphatesof GTP is replaced by a nonhydrolyzable P-CH2-P or P-NH-P linkage. Addition of such a GTP analog to a plasma membrane prepararion in the presence of the natural ligand or an agonist for a particular receptor results in a much longer-lived activation of the associatedeffector protein than occurs with GTP. In this experiment, once the nonhydrolyzable GTP analog is exchangedfor GDP bound to Go, it remainspermanently bound to Go. Becausethe Go.analogcomplex is as functional as the normal G*.GTP complex in activating the effector protein, the effector remains permanently actrve.

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T i m e( s ) 15-14Activationof G proteinsoccurs A EXPERIMENTAL FIGURE of ligandbindingin amoebacells.Intheamoeba within seconds signaling Dictyostelium discoideum cell,cAMPactsasan extracellular it isnota second andbindsto a G protein-coupled receptor; molecule Amoebacellsweretransfected with genesencoding two messenger (CFP), protein a mutant fusionproteins: a G* fusedto cyanfluorescent protein(GFp), formof greenfluorescent anda Gsfusedto another (YFP)CFPnormally yellowfluorescent protein fluoresces GFpvariant, light (a)WhenCFpandYFParenearby, as 490-nmlight;yFB521-nm fluorescence energy transfer canoccur in theresting G" Gp"complex, restingcellswith Asaresult,irradiationof betweenCFPandYFP(eft) excites CFPbutnotYFP) causes emission 440-nmlight(whichdirectly

if ligand of YFPHowever, li9ht,characteristic oI 527-nm(yellow) then of theG" andGu"subunits, to dissociation bindingleads of energytransfercannotoccur.In thiscase,irradiation fluorescence characteristic of 490-nmlight(cyan) emission cellsat 440nmcauses of yellowlight(527nm)from oI CFP(right)(b)Plotof the emission of before andafteraddition cell amoeba transfected a single protein-
GPCR-mediated dissociation of trimeric G proteins recently has been detectedin living cells. These studieshave exploited the phenomenon of fluorescenceenergy transfer, which changesthe wavelength of emitted fluorescencewhen two fluorescent proteins interact. Figure 15-14 shows how this experimental approach has demonstrated the dissociation of the Go'Gs" complex within a few secondsof ligand addition, providing further evidencefor the model of G protein cycling.This generalexperimentalprotocol can be usedto follow the formation and dissociationof other protein-protein complexesin living cells.

Table 15-1 summarizesthe functions of the maior classes of G proteins with different Go subunits. For example, the different typesof epinephrinereceptorsmentionedpreviously are coupled to different G proteins that influence effectors differently,and thus have distinct effectson cell behavior in a target cell. Both subtypesof B-adrenergicreceptors,termed B1 and 82, arecoupled to a stimulatory G protein (G,) whose alpha subunit (G.,) activatesa membrane-boundeffector enzyme called adenylyl cyclase. Once activated' this enzyme catalyzessynthesisof the second messengercAMP. In contrast, the cr2-adrenergicreceptor is coupled to a Go; protein that inhibits adenylyl cyclase,the sameeffector enzymeassociated with B-adrenergicreceptors.The Gooprotein, which is coupled to the ct1-adrenergicreceptor' activates a different effector enzyme)phospholipase C, that generatestwo other secondmessengers(DAG and IP3)' Examples of signaling pathways that use each of the G. proteins listed in Table 151 are describedin the following three sections.

Different G ProteinsAre Activated by Different GPCRs and ln Turn Regulate Different Effector Proteins All effector proteins in GPCR pathways arc either membrane-boundion channelsor enzymesthat catalyzeformation of the secondmessengersshown in Figure 15-9. The variations on the theme of GPCR signaling that we examine in Sections1,5.5-1.5.7 arise becausemultiple G proteins are in encoded eukaryotic genomes.At last count humans have 2L different Go subunits encoded by 16 genes, several of which undergo alternative splicing; 6 Gp subunits; and 12 G" subunits. So far as is known, the different GB, subunits function similarly.

Some bacterial toxins contain a subunit that penetrates the plasma membrane of target mammalian cellsand in the cytosol catalyzesa chemicalmodification of Go proteins that prevents hydrolysis of bound GTP to GDP. For example, toxins produced by the bacterium Vibrio cholera, which causes cholera' or certain strains of E. coli modify the Go, protein in intestinal epithelialcells.

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cAMP (decreased) Change in membrane potential

ct2-Adrenergic receptor Muscarinic acetylcholine receptor

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Odorant receptors in nose

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cGMP (decreased)

Rhodopsin (light receptor) in rod cells

"A given G. subclassmay be associatedwith more than one effector protein. To date, only one major G.. has been identified, but multiple G.o and G"; proteins have been described.Effector proteins commonly are regulated by G* but in some casesby Gu" or the combined action of G* a.td ip". IP3 : lnor',ot 1,4,5-trisphosphate;DAG : 1,2-diacylglycerol. souRcEs: SeeL. Birnbaum eg 1992, Cell 77:1069; Z. Farfel et al., 1999, New Eng. Med. 340:1,012;and K. Pierce er al., 2002, Nature Reu. Mol. Cell J. Biol. 32639.

As a result, Go, remains in the active state, continuously activating the effector adenylyl cyclase in the absence of hormonal stimulation. The resultingexcessiverise in intracellular CAMP leads to the loss of electrolytesand water into the intestinal lumen, producing the watery diarrhea characteristicof infection by thesebacteria.The toxin produced by Bordetella pertwssis,a bacterium that commonly infects the respiratory tract and causeswhooping cough, catalyzesa modification of Goi that prevents releaseof bound GDP. As a result, G.; is locked in the inactive state, reducing the inhibition of adenylyl cyclase.The resulting increase in cAMP in epithelial cells of the arrways promotes loss of fluids and electrolytesand mucus secretionI

General Elements of G Protein-Coupled Receptor Systems r G protein-coupled receptors are a large and diverse family with a common structure of sevenmembrane-spanning cr helices. r Trimeric G proteins transducesignalsfrom coupled cellsurface receptors to associatedmembrane-bound effector proteins, which are either enzymesthat form second messengers(e.g.,adenylylcyclase)or ion channelproteins (see T a b l e1 5 - 1 ) . r Signalsmost commonly are transducedby G., a GTpase switch protein that alternatesbetween an active (,,on") state with bound GTP and inacive ("off',) state with GDp. The B and 1 subunits, which remain bound togethe! occasionally transducesignals. 640

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r Hormone-occupied receptors act as guanine nucleotide-exchangefactors (GEFs) for Go proreins, caralyzingdissociationof GDP and enabling GTP to bind. The resulting change in conformation of switch regions in Go causesit to dissociatefrom the GB" subunit and the receptor and interactwith an effectorprotein (seeFigure 15-13). r Fluorescenceenergy transfer experiments demonstrate receptor-mediateddissociationof coupled Go and Gs, subunits in living cells(seeFigure 15-14).

G Protein-Coupled Receptors ThatRegulatelon Channels One of the simplest cellular responsesto a signal is the opening of ion channels essentialfor transmission of nerve impulses.Nerve impulsesare essentialto the sensoryperception of environmental stimuli such as light and odors, to transmission of information to and from the brain, and to the stimulation of muscle movement. During transmission of nerve impulses, the rapid opening and closing of ion channelscauseschangesin the membranepotential. Many neurotransmitter receptors are simply ligand-gated ion channels,which open in responseto binding of a ligand. Such receptorsinclude some types of glutamate,seroronin, and acetylcholine receptors, including the acetylcholine receptor found at nerve-muscle synapses.Ligand-gated ion channels that function as neurotransmitter receDrorsare c o v e r e di n C h a p r e r2 3 . Some neurotransmitter receptors, however, are G protein-coupled receptorswhose effector proteins are a Na* or K* channel. Neurotransmitter bindins to these receDrors

C E L LS I G N A L I N G I : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RcME L L U L A RR E s P o N S E S

causesthe associatedion channel to open or close,leading to changesin the membrane potential. Still other neurotransmitter receptors,as well as odorant receptorsin the noseand photoreceptors in the eye, are G protein-coupled receptors that indirectly modulate the activity of ion channelsvia the In this section,we considertwo action of secondmessengers. G protein-coupled receptorsthat illustrate the direct and indirect mechanismsfor regulating ion channels:the muscarinic acetylcholinereceptor of the heart and the lightactivatedrhodopsin of the eye.

AcetylcholineReceptorsin the Heart Muscle Activate a G ProteinThat OpensK* Channels Activation of muscarinic acetylcholine receptors in cardiac muscle slows the rate of heart muscle contraction. (Because muscarine,an acetylcholineanalog,also activatesthesereceptors, they are termed "muscarinic.") This type of acetylcholine receptor is coupled to a Go; protein, and ligand binding leads to opening of associatedK* channels(the effectorprotein) in the plasmamembrane(seeTable 15-1). The subsequentefflux of K* ions from the cytosol causesan increasein the magnitude of the usual inside-negativepotential acrossthe plasma membrane that lasts for several seconds.This state of the membrane, called hyperpolarization, reducesthe frequency of muscle contraction. This effect can be determinedexperimentally by direct addition of acetylcholineto isolatedheart musclecells and measurementof the potential using a microelectrodeinsertedinto the cell (seeFigure 11-18).

As depicted in Figure 15-15, the signal from activated muscarinic acetylcholine receptors is transduced to the effectorprotein by the releasedGp" subunit rather than by G..GTP. That GB" directly activates the K* channel was demonstratedby patch-clampingexperiments,which can measureion flow through a single ion channel in a small \fhen purified GB" patch of membrane (seeFigure 1'1-21'1. protein was added to the cytosolic face of a patch of heart muscle plasma membrane, K* channels opened immediately, even in the absenceof acetylcholine or other neurotransmitters-clearly indicating that it is the Gg" protein that is responsiblefor opening the effector K- channels and not G.'GTP.

Rhodopsins Light ActivatesGo.-Coupled The human retina contains two types of photoreceptor cells, rods and cones, which are the primary recipients of visual stimulation. Cones are involved in color vision, while rods are stimulated by weak light like moonlight over a range of wavelengths.The photoreceptorssynapseon layer upon Iayer of interneuronsthat are innervated by different combinations of photoreceptor cells. All these signals are processedand interpreted by the part of the brain called the

uisual cortex. As noted aheady,rhodopsin consistsof the protein opsin' which has the usual GPCR structure,covalently linked to the light-absorbing pigment 11-cis-retinal.Rhodopsin is localized to the thousand or so flattened membrane disks that make up the outer segmentof rod cglls (Figure 15-16). A huAcetylcholine man rod cell contains about 4 x 10/ moleculesof rhodopsin. Kt channel The trimeric G protein coupled to rhodopsin, called transExterior ++ ducin (Gr), contains the Go, subunit (seeTable 15-1); like rhodopsin, G*. is found only in rod cells. Upon absorption of a photon, the retinal moiety of Cytosol rhodopsin is immediatelyconvertedfrom the cis to the alltrans isomeq causinga conformational changein the opsin Active muscarinic portion that activatesit (Figure 1'5-1'7).This is equivalent acetylcholinereceptor to the conformational change that occurs upon Iigand binding by other G protein-coupled receptors.Analogous K* to other G protein-coupled receptors' the light-activated form of rhodopsin interacts with and activatesa Go protein. in this caseGo,. Activated opsin is unstableand spontaneously dissociatesinto its component parts, releasing oosin and all-trans-retinal,thereby terminating visual sign"ling. In the dark, free all-trans-retinal is converted back to LI-cis-retinal,which can then rebind to opsin' re-forming rhodopsin. A FIGURE15-15 Activation of the muscarinicacetylcholine In the dark, the membrane potential of a rod cell is receptor and its effector K* channel in heart muscle.Bindingof about -30 mV, considerablylessthan the resting potential triggersactivationof the G. subunitand its dissociation (-60 acetylcholine to -90 mV) typical of neurons and other electrically l a y ( s e eF i g u r e1 5 - 1 3 )I.n t h i s f r o m t h e G u , s u b u n i ti n t h e u s u a w active cells.This state of the membrane,called depolarizacase,the releasedGu" subunit(ratherthan G"i GTP)bindsto and tion, causesrod cells in the dark to constantly secreteneueffectorprotein,a K* channel The increasein opensthe associated rotransmitters, and thus the neurons with which they hyperpolarizes the membrane,which reducesthe K* permeability synapse are continually being stimulated. The depolarized frequencyof heart musclecontractionThoughnot shown here, stateof the plasmamembraneof restingrod cellsis due to the to activatronis terminatedwhen the GTPbound to G*| is hydrolyzed presenceof a large number of open nonselectiueion chanwith Gsn [SeeK Hoet al , 1993,Nafure GDPand G"i GDPrecombines nels that admit Na+ and Ca2*, as well as K*. Absorption 362:127 362:31. andY Kuboet al . 1993,Nature | ION CHANNELS G P R O T E I N - C O U P L ERDE C E P T O RTSH A T R E G U L A T E

641

(a)

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'

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F I G U R1E5 - 1 6H u m a nr o d c e l l .( a )S c h e m a tdi ica g r a m of an entirerodcell.At thesynaptic body,the rodcellformssynapses with oneor morebipolarinterneurons Rhodopsin, a light-sensitive G protein-coupled receptor, is located in theflattened membrane disks of the outersegment. (b)Electron micrograph of the regionof the

rodcellindicated by the bracket in (a),Thisregionincludes the j u n c t i o on f t h ei n n e r a n o d u t e r s e g m e nltpsa r t ( b ) f r oRmG K e s s e l

of light by rhodopsin leads to closing of these channels, causing the membrane potential to become more jnsjde negatrve. The more photons absorbed by rhodopsin, rhe more c h a n n e l sa r e c l o s e d ,t h e f e w e r N a * a n d C a 2 * i o n s c r o s s the membrane from the outside, the more negative the membrane potential becomes,and the lessneurotransmitter is released.This change is transmitted to the brain where it is perceivedas light. Remarkably,a singlephoton absorbed by a resting rod cell produces a measurableresponse,a more inside negative change in the membrane potential of about 1 mV which in amphibians lasrsa second or two. Humans are able to derecia flash of as few as five photons.

A c t i v a t i o no f R h o d o p s i nI n d u c e sC l o s i n g o f c G M P - G a t eC d a t i o nC h a n n e l s

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CHAPTER 1s

I

and R H Kardon,1979, Tissuesand Organs.A Text-Atlas of ScanninqElectron Microscopy,W H Freemanand Company.p 91 l

Opening of GPCR-stimulatedK* channelsin the heart requiresonly an acrivatedG protein (seeFigure 15-15).In contrast, the closing of cation channelsin the rod-cell plasma membranerequireschangesin the concentrationof the second messengercyclic GMP, or cGMP (seeFigure 15-9). Rod outer segmentscontain an unusually high concentration (:0.07 mM) of cGMP, which is continuouslyformed from GTP in a reaction catalyzedby guanylyl cyclase,which appears to be unaffectedby light. However, light absorption by rhodopsin inducesactivation of a cGMP phosphodiesterase, which hydrolyzescGMP to 5'-GMP. As a result,the cGMp

C E L LS I G N A L I N G l : S I G N A LT R A N S D U C T T O A N D S H O R T - T E RcME L L U L A R RESpONSEs

< F I G U R 1E5 - 1 7T h e l i g h t - t r i g g e r esdt e p i n v i s i o n .T h el i g h t '1 scl o v a l e n tbl yo u n dt o t h e a m i n o a b s o r b i npgi g m e n1t - c i s - r e t i ni a r e s i d uien o p s i nt,h e p r o t e i np o r t i o no f g r o u po f a l y s i n e of rapidphotoisomerrzation of lightcauses rhodopsinAbsorption formingthe unstable isomer, to the all-trans the boundcrs-retinal opsin,whichactivates ll, or activated meta-rhodopsin intermediate fromopsin dissociates all-trans-retinal seconds, G, proteinsWithin whichthen by an enzymebackto the ctsisomer, and is converted 1992, Biochemistry J Nathans, to anotheropsinmolecule. [See rebinds 31:4923 I

11-cis-Retinal moietv

Lysineside chain + :N-(CH?)d-

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moietv all-trans-Retinal tra N S '11

c:N-(cHz)o-E@ H

ll Mefa-rhodopsin (activatedopsin) concentration decreasesupon illumination. The high level of cGMP present in the dark acts to keep cGMP- gated cation channelsopen; the light-induced decreasein cGMP leads to channel closing, membrane hyperpolarization, and reduced neurotransmitterrelease.

is As depictedin Figure 15-18, cGMP phosphodiesterase the effector protein for Go.. The free G*,'GTP complex that is generatedafter light absorption by rhodopsin binds to the two inhibitory 1 subunits of cGMP phosphodiesterase'releasingthe active catalytic ct and B subunits,which then convert cGMP to GMP. This is a cleat example of how signalinduced removal of an inhibitor can quickly activate an enzyme,a common mechanismin signalingpathways. A single moleculeof activatedopsin in the disk membranecan activate 500 Go, molecules,each of which in turn activatesone cGMP phosphodiesterase,thereby amplifying the original light signal early in this pathway. Direct support for the role of cGMP in rod-cell activity has been obtained in patch-clamping studies using isolated patches of rod outer-segmentplasma membrane, which contains abundant cGMP-gated cation channels. When cGMP is added to the cytosolic surface of these patches, there is a rapid increasein the number of open ion channels; cGMP binds directly to a site on the channel protein to keep channels. them open,indicatingthat theseare nucleotide-gated

Lig Cytosol Disk membrane Disk lumen

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q TSI"

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l /p\ GG TD PP

cGMP GMP

\tr \

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rhodopsinpathwayand the closingof cation 15-18 Light-activated A FIGURE nucleotide-gated rodcells, a highlevelof cGMPkeeps in rod cells. In dark-adapted channels generates opsin,O* (steptr), activated open Lightabsorption cationchannels nonselective of GDPwithGTP(step replacement andmediates G*,protein GDP-bound whichbindsinactive (PDE) by bindingto its cGMPphosphodiesterase thenactivates Z) ThefreeG*,GTPgenerated (step41 (step subunits cr and from the catalytic them inhibitory B 1 subunits E) anddissociating cGMPto GMP(stepE) The of PDEconvert thectandB subunits Relieved of theirinhibition, of cGMPfromthe nucleotide-gated in cytosolic cGMPleadsto dissociation decrease resulting (step51 Themembrane then of thechannels membrane andclosing intheplasma channels Neuron20|11 1998, and 1 fromV Arshavsky E Pugh, hyperpolarized transiently becomes lAdapted

L-------J

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Closed cGMP-gated ion channel

High Dark,.li&pted cytosolic ti€{f. : .'::"' cGMP (a)

Na* Ca2'

Open cGMP-gated ion channel

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state for only a fraction of a second.Thus cGMP phosphodiesteraserapidly becomesinactivated, and the cGMP level gradually risesto its original level when the light stimulus is removed. This allows rapid responsesof the eye roward moving or changing objects. Recentx-ray crystallographicstudiesrevealhow the subunits of G, protein interact with each other and with lightactivatedrhodopsin and provide clues about how binding of GTP leadsto dissociationof Go from GB". As revealedin the structuralmodel in Figure 15-19,two surfacesof Go. interact with GB: an N-terminal region near the membrane surface and the two adjacent switch I and switch II regions, which are found in all G, proteins. G" directly contacts Gp but not Gro. These models also suggestthat the nucleotidebinding domain of Go., together with the lipid anchors at the C-terminus of G" and the N-terminus of Got, form a surface that binds to light-activatedrhodopsin(O', in Figure15-18), promoting the releaseof GDP from Go. and the subsequent binding of GTP. The subsequentconformational changesin G.o, particularly those within switches I and II, disrupt the molecular interactions between Go. and Gpr, leading to their dissociation.The structural studieswith rhodopsin and Go, are consistentwith data concerningother G protein-coupled receptors and are thought to be generally applicable to all receptorsof this type.

Rhodopsin

Exterior ligand

Membrane

Cytosol

GDP N_

Gq

GIl,

A FIGURE 15-19 Structuralmodelof rhodopsinand its associated trimericG protein,transducin(GJ. Thestructures of r h o d o p s iann dt h e G o .a n dG B "s u b u n i tws e r eo b t a i n ebdyx - r a y crystallography TheC-terminal segment of rhodopsin that follows t r a n s m e m b r ahneel i xH 7a n de x t e n disn t ot h ec y t o s oi sl n o ts h o w n in thismodelTheorientation of G*,withrespect to rhodopsin and t h e m e m b r a niesh y p o t h e t i c iat li;sb a s e d o n t h ec h a r g e ano hydrophobicity of the proteinsurfaces andthe knownrhodopsinbindingsiteson G.,.As in othertrimericG proteins, the Go,andG-, s u b u n i tcso n t a i n c o v a l e n tal yt t a c h eldi p i d s( r e da n db l u ej a g g e d l i n e st)h a ta r et h o u g h to b e i n s e r t eidn t ot h e m e m b r a n Ien t h e G D P - b o u nf odr ms h o w nh e r et,h eo s u b u n i(tb l u ea) n dt h e B ) t e r a cwt i t he a c ho t h e ra, sd o t h e s u b u n iat n d s u b u n i(tg r e e ni n B 1 s u b u n r( tr e d )b, u tt h es m a l^l ys u b u n i tw, h i c hc o n t a i nl u s s tt w o c t helices, doesnot contactthe ctsubunitSeveral segments of the a subunitarethoughtto interact with an activated rhodopsin, c a u s i nag c o n f o r m a t i o ncahla n g teh a tp r o m o t erse l e a soef G D pa n d binding o f G T PB i n d i nogf G T pi,n t u r n ,i n d u c elsa r g e conformational changes in the switchregions of G.. leadingto its dissociation from GBr.[Adapted fromH Hamm, 2OO1 , procNat'lAcad SciUSA98:48'19, andW Oldham andH Hamm2006euart Rev. Biophvs 40:(lnpress) l Like the K+ channelsdiscussedin Chapter 11, the cGMpgated channel protein contains four subunits [see Figure 7I-191. In this case each of the subunits is able to bind a CGMP molecule. Three or four cGMp moleculesmust bind per channel in order to open it; this allosteric interaction makes channel opening very sensitive to small changes in cGMP levels. Conversion of active G.t.GTp back to inactive G...GDP is acceleratedby a specificGTPase-activatingprorein (GAp). In mammals Go, normally remains in the active GTp-bound 644

t

c H A P T E R1 s

I

Rod CellsAdapt to Varying Levelsof Ambient LightBecause o f O p s i nP h o s p h o r y l a t i o n a n d B i n d i n go f A r r e s t i n Cone cellsare insensitiveto low levelsof illumination, and the activity of rod cells is inhibited at high light levels. Thus when we move from bright daylight into a dimly lighted room, we are initially blinded. As the rod cells slowly becomesensitiveto the dim light, we gradually are able to seeand distinguish objects.This processof uisual adaptation permits a rod cell to perceivecontrast over a 100,000-fold range of ambient light levels.This wide range of sensitivity is possible becausedifferencesin light levels in the visual field, rather than the absolute amount of absorbed light, are used to form visual images. Lightdependentregulation of the rhodopsin signaling pathway (seeFigure 15-18) is responsiblefor this extraordinarily wide sensitivityrange. One process contributing to visual adaptation involves phosphorylation of opsin in its active conformarion (O',) but not in its inactive, or dark form (O) by rhodopsin kinase (Figure 15-20), a member of a classof GPCR kinases.Each opsin molecule has three principal serine phosphorylation sites on its cytosol-facing surface; the more sites that are phosphorylated,the less able O', is to activate Go. and thus induce closing of cGMP-gated cation channels.Becausethe extent of opsin phosphorylation by rhodopsin kinase is proportional to the amount of time each opsin molecule spends in the light-activatedform, it is a measureof the background (ambient) level of light. Under bright-light conditions, opsin phosphorylation is increased,and consequentlyits ability to activate Go, is reduced.In other words, rhodopsinis desensitized

c E L L s T G N A L T Nr G : sTGNAL T R A N S D U c I o NA N D s H o R T - T E R M c E L L U L A RR E S p o N s E s

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Activated opsrn Very high l i gh t

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adaptationto ambientlight level 15-20Rod-cell A FIGURE Light-activated opsin(O*), changesand opsinphosphorylation. kinase. for rhodopsin isa substrate rhodopsin, but notdark-adapted proportional to the isdirectly Theextentof opsinphosphorylation in the light-activated spends amountof timeeachopsinmolecule ambientlightleveloverthe previous formandthusto theaverage proportional G*,isinversely few minutesTheabilityof O* to activate

by bright light, and thus a greater increasein light intensity is necessaryto generatea change in cGMP levels and a visual signal. When the level of ambient light is reduced' the opsins become dephosphorylatedand the ability to activate Go, increases;in this case,relatively fewer additional photons are necessaryto generatea visual signal. The importance of opsin phosphorylation in visual adaptation is supported by studies with rod cells from mice with mutant rhodopsins bearing zero or only one of the target serineresidues.These rod cells show a much slower than normal rate of deactivation in bright light. The light-dependentdesensitizationof rod cellsis further increasedby binding of the cytosolic protein B-arrestin. At high ambient light (such as noontime outdoors), B-arrestin binds to the phosphorylatedserineresidueson the C-terminal opsin segment.Bound B-arrestincompletelypreventsinteraction of Go. with phosphorylatedO*, totally blocking formation of the active G*.'GTP complex and causinga shutdown of all rod-cell activity. The negative-feedbackregulation of rod-cell activity by rhodopsinkinaseand arrestinis similarto adaptation(or desensitization)of other G protein-coupled receptors to high ligand levels.Another mechanism,which appearsunique to rod cells, also contributes to visual adaptation. In darkadapted cellsvirtually all the G., and GB" subunits are in the outer segments.But exposure for 10 minutes to moderate daytime intensitiesof light causesover 80 percent of the Go, and Gp., subunits to move out of the outer segmentsinto other cellular compartments. Although the mechanism by which theseproteins move is not yet known, the result is that Go, proteins are physically unable to bind activated opsin. As occurs in other signalingpathways, multiple mechanisms are thus used to inactivate signaling during visual adaptation, presumably to allow strict control of activation of the signaling pathway over broad rangesof illumination'

Greatlyreduced Gd activation

No Go, activation

on O* Thusthe higher residues to the numberof phosphorylated of opsin greater extent the level, the light the ambient in lightlevelneededto phosphorylation andthe largerthe increase At veryhighlightlevels, the samenumberof G*,molecules activate phosphorylated opsin,forminga anestinbindsto the completely andD Baylor, Lagnado L all. at G*. activate [See thatcannot complex 28:153 Neuron etal,2OOO, l andA Mendez Neuron8:995, 1992,

G Protein-Coupled Receptors That Regulate lon Channels r The cardiac muscarinic acetylcholine receptor is a GPCR whose effector protein is a K* channel. Receptor activation causesreleaseof the GB" subunit, which opens K* channels(seeFigure 15-15). The resultinghyperpolarization of the cell membrane slows the tate of heart muscle contraction. dopsin, the photosensitiveGPCR in rod cells' comthi protein opsin linked to 11-cis-tetinal. Lightd isomerization of the ll-cis-retinal moiety produces activated opsin, which then activates the coupled trimeric G protein transducin (Gr) by catalyzingexchange of free GTP for bound GDP on the Go. subunit. r The effector protein in the rhodopsin pathway is cGMP phosphodiesterase'which is activated by the G..'GTPmediated release of inhibitory subunits. Reduction in the cGMP level by this enzyme leads to closing of cGMP-gated Na+/Ca2+ channels, hyperpolarization of the membrane, and decreasedreleaseof neurotransmitter ( s e eF i g u r e1 5 - 1 8 ) . r As with other Go proteins, binding of GTP to Go. causes conformational changesin the protein that disrupt its molecular interactions with GB" and enable G.I'GTP to bind to its downstream effector. r Phosphorylation of light-activated opsin by rhodopsin kinase interfereswith its ability to activate Gorl subsequent binding of arrestin to phosphorylatedopsin further inhibits its ability to activate Go, (seeFigure 15-20)' This general mechaniim of adaptation' or desensitization,is utilized by other GPCRs at high ligand levels.

G P R O T E I N - C O U P L ERDE C E P T O RTSH A T R E G U L A T EI O N C H A N N E L S

645

G Protein-Coupled Receptors That Activateor InhibitAdenylylCyclase GPCR pathways that utilize adenylyl cyclaseas an effector protein and cAMP as the secondmessengerare found in most mammalian cells. These pathways follow the general GPCR mechanismoutlined in Figure 15-13: Ligand binding to the receptor activatesa coupled trimeric G protein that activates adenylyl cyclase,which synthesizesthe diffusible secondmessengercAMP. cAMP, in turn, activates a cAMp-dependent protein kinase that phosphorylatesspecifictarger proteins. To explore this GPCR/cAMP pathway we focus on the first such pathway discovered-the hormone-stimulated generation of glucose from glycogen, a srorage polymer of glucose.The breakdown of glycogen (glycogenolysls/occurs

Positive (activation) and negative (inhibition) regulation of adenylyl cyclaseactivity occurs in many cell types, providing fine-tuned conrrol of the cAMP level (Figure 75-21). For example,the breakdown of triacylglycerolsto fatty acids in adipose cells (lipolysls) is stimulated by binding of epinephrine, glucagon, or ACTH to receptors that activate adenylyl cyclase.On the other hand, binding of two other hormones, prostaglandin PGEI or adenosine,to their respective G protein-coupled receptors inhibits adenylyl cyclase.The prostaglandin and adenosinereceptorsactivarean inhibitory G1protein that contains rhe sameB and ^ysubunits as the stimulatory G. protein but a different ct subunit (G.1). After the active G*;.GTP complex dissociatesfrom Gs,, it binds to but inhibits (rather than stimulates) adenylyl cyclase,resulting in lower cAMP levels.

StructuralStudiesEstablishedHow G.r.GTp Bindsto and ActivatesAdenylyl Cyclase

A d e n y l y lC y c l a s et s S t i m u l a t e da n d I n h i b i t e d by Different Receptor-LigandComplexes

G protein-coupled receptors,but both receptorsinteract with and activate the same Go, that activates adenylyl cyclase. Hence, both hormones induce the samemetabolic responses. Activation of adenylyl cyclase,and thus the cAMp level, is proportional ro the total concentrationof Go, .GTp resulting from binding of both hormones to their respectivereceptorsl

X-ray crystallographic analysis has pinpointed the regions in G.,.GTP that interact with adenylyl cyclase.This enzyme is a multipass transmembraneprotein with two large cltosolic segmentscontaining the catalytic domains (Figure l5-22a). Because suchtransmembraneproteins are notoriously difficult to crystallize, scientistspreparedtlvo protein fragmentsencompassingthe fwo catalytic domains of adenylyl cyclasethat tightly associate with one another in a heterodimer.When these catalytic fragments are allowed to associatein the presenceof G*..GTp and forskolin, they are stabilizedin their activeconformations. The resulting water-soluble complex (two adenylyl cyclasedomain fragments/G.,.GTP/forskolin) was catalytically active and showedpharmacologicaland biochemicalproperties similar to those of intact full-length adenylyl cyclase.In this complex, two regionsof G*,.GTP,the switch II helix and the o3-B5 loop, contact the adenylyl cyclasefragments (Figure 15-22b). Thesecontactsare thought to be responsiblefor the activation of the enzymeby G*,.GTP.Recallthat switch II

S t i m u l a t o r yI E p i n e p h r i n e hormone , { Glucagon

| [ AcrH

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Adenytyl ; cycrase l /tr\ :

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P \___-_____\aJ

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A FIGURE 15-21Hormone-induced activationand inhibitionof adenylylcyclasein adiposecells.Ligandbindingto G*,-coupled receptors causes activation of adenylyl cyclase, whereas ligandbindingto G" -coupled receptors causes inhibition of theenzyme. TheGp,,subunit in bothstimulatory andinhibitory G proteins isidenticar; theG^subunits 646

o

CHAPTER 15

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andtheircorresponding receptors differ.Ligand-stimulated formation of activeGoGTPcomplexes occursbythesamemechanism in bothG*, (seeFrgure andG* proteins 15-13) However; G*,GTpandG.,.GTp interact differently with adenylyl cyclase, sothatonestimulates andthe otherinhibits itscatalytic activity. A G Gilman, 1984, [See Cett36:57j ]

C E L Ls t c N A L I N Gr : s T G N A L TRANSDUCToN AND SHoRT-TERM c E L L U L A RR E S p o N s E s

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15-22Structureof mammalianadenylylcyclases A FIGURE (a)Schematic diagram of and their interactionwith G"'.GTP. enzyme Themembrane-bound adenylyl cyclases mammalian faceof the on thecytosolic catalytic domains two simrlar contains e a c ho f w h i c hi s m e m b r a n d e o m a i n s , i n t e g r a l m e m b r a naen dt w o o helices(b)Modelof the thoughtto containsixtransmembrane with two structure of G., GTPcomplexed three-dimensional cyclase domainof adenylyl thecatalytic encompassing fragments Thect3-B5loopandthe helixin byx-raycrystallography determined wttha simultaneously theswitchll region(blue)of G"sGTPinteract portionof G., Thedarker-colored cyclase regionof adenylyl specific to Ras(seeFigure in structure whichissimilar domain, isthe GTPase 1 5 - 8 )t;h el i g h t epr o r t i o nr sa h e l i c adlo m a i nT h et w o a d e n y l y l (green) areshownin orangeandyellowForskolin fragments cyclase (a)see in theiractiveconformations fragments [Part locksthecyclase 9e 2 l, l T O : 8P6a9r t ( b ) a d a p t e d f Jr oGm J A G i l m a n , 1 9C W - 1T a n g a n dG 278:1907 et al, 1997,Science Tesmer ) is one of the segmentsof a Go protein whose conformation is different in the GTP-bound and GDP-bound states (see Figure 15-8). The GTP-inducedconformation of Go. that favors its dissociationfrom Gs, is preciselythe conformation essentialfor binding of Go, to adenylyl cyclase.Other studies indicate that Go1binds to a different region of adenylyl cyclase,accountingfor its different effect.

cAMPActivatesProteinKinaseA by Releasing C a t a l y t i cS u b u n i t s In multicellular animals, virtually all the diverse effects of cAMP are mediated through protein kinase A (PKA), also called cAMP-dependent protein kinase. Inactive PKA is a tetramerconsistingof two regulatory(R) subunitsand two catalytic (C) subunits(Figure t5-23a). Each R subunit contains a

cNB_A-

d-,

CNB-B

o

(c) Conformational c h a n g e sf r o m c A M Pb i n d i n g cAMP bound Catalytic s ub u n i t bound

15-23Structureof the regulatory(R)subunitsof A FIGURE A kinase proteinkinaseA and its activationby cAMP'(a)Protein (green) (R)subunits andtwo catalytic (PKA) of two regulatory consists subunit, bindsto theregulatory (C)subunits WhencAMP(redtriangle) (b) The two PKA thusactivating subunitisreleased, thecatalytic linkeranda dimerization/ bya flexible arejorned subunits regulatory (AKABFigure 15-28) protein activating A-kinase where domain Oocting and CNB-A domains, hastwo cAMP-binding canbind.EachRsubunit (arrow)(c)Binding of subunit sitefor a catalytic anda binding CNB-8, cAMPtotheCNB-Adomaindisp|acesthecata|yticsubunit|eading domain WithoutboundcAMBoneloopof theCNB-A activation (C)subunitA thatcanbindthecatalytic (purple) istna conformation of participate in binding (R209) residue (E200) andarginine glutamate (green) loop in the change a conformational whichcauses cAMP(red), (b)after5 S bindingof the loopto the C subunitlPart thatprevents (c) C Kim,N-H after Part 1754i25 Acta Biophys Biochim 2005, al et Taylor , 307:690 science 2005, l andS 5 Taylor, Xuong,

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Glycogen (n + 1 residues)

P'--rl n nn

en rorylasr:

J

HOCH2

OH

?+o

o-',

( oHo

Glucose 1-phosphate

Glycogen (n residuesl

FIGURE 15-24Synthesis and degradationof glycogen. catalyzed by glycogen phosphorylase Because two different enzymes Incorporation of glucose fromUDp-glucose intoglycogen iscatalyzed catalyze theformation anddegradation of glycogen, thetwo by glycogen synthase. Removal of glucose unitsfromgrycogen reactions ts canbe independently regulated pseudosubstratesequencethat binds to the active site in a catalytic domain. By blocking substratebinding, the R subunits inhibit the activity of the catalytic subunits.Inactive PKA is turned on by binding of cAMp. Each R subunit has two distinct cAMP-binding sites,called CNB-A and CNB_B (Figure I5-23b). Binding of cAMp ro an R subunit causesa conformational change in the pseudosubstratedomain that leadsto releaseof the associatedC subunit, unmaskingits catalytic site and activatingits kinase activity (Figure 15-23c). Binding of cAMP by an R subunit of protein kinase A

activity. Rapid activation of enzymesby hormone-triggered dissociation of an inhibitor is a common feature of-manv signaling pathways.

G l y c o g e nM e t a b o l i s ml s R e g u l a t e d by Hormone-lnduced Activation of protein K i n a s eA Glycogen, alarge glucosepolymer, is the major storageform of glucosein animals. Like all biopolymers,glycogenls syn648

'

C H A P T E1Rs I

thesizedby one set of enzymesand degradedby another (Fig, ure 15-24). Degradation of glycogen, or glycogenolysis,involvesthe stepwiseremoval of glucoseresiduesfrom one end of the polymer by a phosphorolysisreaction, catalyzedby glycogen p h osph oryla.se,yielding glucose1-phosphate. In both muscle and liver cells, glucose 1-phosphateproduced from glycogenis convertedto glucose6-phosphate.In musclecells,this metaboliteentersthe glycolyticpathway and is metabolizedto generateATP for usein powering musclecontraction (Chapter 12). Unlike musclecells,liver cellscontain a phosphatasethat hydrolyzesglucose6-phosphateto glucose, which is exported from these cells in parr by a glucose transporter (GLUT2) in the plasma membrane(Chapter 11). Thus glycogenstoresin the liver are primarily broken down to glucose,which is immediatelyreleasedinto the blood and transported to other tissues,particularly the musclesand brain. The epinephrine-stimulated activation of adenvlvl cyclase,resulting increasein cAMp, and subsequentactivation of protein kinase A (PKA), enhancesthe conversion of glycogen to glucose 1-phosphatein two ways: by inhibiting glycogen synthesisand by stimulating glycogen degradation (Figure 15-25a).PKA phosphorylatesand in so doing inactivates glycogensynthase,the enzymethat synthesizesglycogen. PKA promotes glycogen degradation indirectly by phosphorylating and thus activating an intermediate kinase,

c E L Ls t c N A L t N rG: s T G N ATLR A N S D U c T oANN D s H o R T - T E RCM E L L U L ARRE s p o N s E s

(a) Increased cAMP

GSO

G l u c o s e1 - p h o s p h a t e (b) DecreasedcAMP :;;;;;",t,*, GPK

________-_> G P

UDP-glucose----------> Glycogen + UDP

i

; e K A e r o t " i n k i n a s eA n hosphatase I P P P h o s p h o P r o t e iP I G P K G l y c o g e np h o s p h o r y l a s ek i n a s e I G P G l y c o g e np h o s P h o r Y l a s e G S G l y c o g e ns y n t h a s e I n h i b i t o ro f P h o s p h o p r o t e l n i lP phosphatase .

1 i I l

15-25Regulationof glycogenmetabolismby cAMP A FIGURE in darker arehighlighted in liver and musclecells.Activeenzymes in cytosolic forms,in lightershades(a)An increase inactive shades; glycogen whichinhibits proteinkinase A (PKA), cAMPactivates glycogen viaa protein promotes degradation and directly synthesis an inhibitor At highcAMBPKAalsophosphorylates kinase cascade

(PP). of the phosphorylated Binding phosphatase of phosphoprotein the dephosphorylating from phosphatase prevents this PP to inhibitor glycogen or the inactive cascade in the kinase enzymes activated to release PKA,leading (b)A decrease in cAMPinactivates synthase promotes this enzyme of of the activeformof PPTheaction glycogen degradation andinhibits glycogen synthesis

glycogen phosphorylase kinase (GPK), that in turn phosphorylates and activatesglycogen phosphorylase,the enzyme that degradesglycogen. The entire processis reversedwhen epinephrineis removed and the level of cAMP drops, inactivating proteln kinaseA (PKA). This reversalis mediatedby phosphoprotein phosphatase,which removes the phosphateresiduesfrom the inactiveform of glycogensynthase,therebyactivatingit' and from the activeforms of glycogenphosphorylasekinase and glycogen phosphorylase, thereby inactivating them (Figure15-25b). Phosphoproteinphosphataseitself is regulated by PKA. An inhibitor of phosphoproteinphosphatase is normally inactive. When activatedPKA phosphorylates this inhibitory protein, it can bind to phosphoproteinphosphatase,inhibiting its activity (seeFigure 1'5-25a).At low cAMP levels, when PKA is inactive, the inhibitor is not phosphorylatedand phosphoproteinphosphataseis active. As a result, in the absence of cAMP the synthesis of glycogen by glycogen synthase is enhanced and the degradation of glycogenby glycogenphosphorylaseis inhibited' Epinephrine-inducedglycogenolysisthus exhibits dual regulation: activation of the enzymescatalyzing glycogen degradation and inhibition of enzymespromoting glycogen synthesis.Such coordinate regulation of syntheticand degradativepathways provides an efficient mechanismfor

achieving a particular cellular responseand is a common phenomenonin regulatory biology.

cAMP-MediatedActivation of Protein KinaseA ProducesDiverseResponses in Different CellTYPes

array of hormone-induced cellular responsesin multiple b o d y c e l l s ( T a b l e1 5 - 2 ) ' Although protein kinase A acts on different substratesin different typei of cells, it always phosphorylatesa serine or threonine residuethat occurs within the same sequencemotif: X-Arg-(Arg/Lys)-X-(Ser/Thr)-O' where X denotes any O denotesa hydrophobic amino acid' Other amino ".id "ttd kinases phosphorylate target residues serine/threonine within other sequencemotifs.

CYCLASE G P R O T E I N - C O U P L ERDE C E P T O RTSH A T A C T I V A T EO R I N H I B I TA D E N Y L Y L

.

649

TlsslJE

H0BM0NE tNDuctNG RtsE tNcAMp

cELtutAR RESP0NSE

Adipose

Epinephrine; ACTH; glucagon

Increasein hydrolysis of triglyceride; decreasein amino acid uptake

Liver

grucagon lH,?Tr'1":1ffi:'J::;:,1'f:ff:trf:T:,T'"'J,:,li:',::,." "3,iil,i.oiii,'e; rn gluconeogenesis(synthesisof glucose from amino acids)

ovarian follicle

FSH; LH

Increasein synthesisof estrogen,progesterone

Adrenal cortex

ACTH

Increasein synthesisof aldosterone, cortisol

Cardiac muscle

Epinephrine

Increasein contractron rate

Thyroid gland

TSH

Secretionof thyroxine

Bone

Parathyroid hormone

Increasein resorption of calcium from bone

Skeletal muscle

Epinephrine

Conversion of glycogen to glucose

Intestine

Epinephrine

Fluid secretion

Kidney

Vasopressin

Resorption of water

Blood platelets

Prostaglandin I

Inhibition of aggregation and secretion

''Nearly all the effects of cAMP are mediated through protein kinase A (PKA), which is activated by binding of cAMp. s o u R C E :E . W . S u t h e r l a n d ,1 , 9 7 2 ,S c i e n c e1 7 7 : 4 0 1 .

S i g n a lA m p l i f i c a t i o nC o m m o n l yO c c u r si n M a n y S i g n a l i n gP a t h w a y s Receptors are low-abundance proteins, typically present in only a few thousand copies per cell. yet the'..llul", .._ sponsesinduced by binding of a relatively small number of hormone molecules to the available receptors mav require production of tens of thousandsor even millions of second messenger or activatedenzymemoleculesper cell. Thus sub_ stantial signal amplification often -urt oi.u, in order for a hormone signal to induce a significant cellular response. In the caseof G protein--coupledreceptors,signal amplifi_ cation is possiblein part becauseboth recepto.sand G pro_ teins can diffuse rapidly in the plasma membrane. A sinele epinephrine-GPcR complex causesconversionof up to 1-00 inactive Go, moleculesto the active form before epinephrine dissociatesfrom the receptor.Each active G.,.GTq in turn, activatesa single adenylyl cyclasemolecule. which then cat_ alyzes synthesisof many cAMp molecules during the time G.,.GTP is bound to it. The amplification that occurs in such an amplification cas_ cade dependson the number of steps in it and the relative concentrationsof the various components.In the epinephrine_ induced cascadeshown ^in Figure 15-26, blood lwels of epi_ nephrineas low as 10-10 M can stimulateliver glycogenolysis and releaseof glucose.An epinephrine stimulus of thii maeni_ tude generatesan intracellularcAMp concentrationof 10-5"M. 650

.

c H A p r E R1 s I

_.- Epinephrine -------.-"

AmPlificatiorr

AAA

r

Amprification . r-l.l.\.

ttttt

(10-10 M)

Adenylyl cyctase

c A M P ( 1 0 - 6M ) Protein k i n a s eA

Am plification @

Activated enzyme

A m p l i fi c a t i o n lz a

Product

FIGURE 15-26Amplificationof an externalsignaldownstream from a cell-surface receptor.Inthisexample, bindlngof a single epinephrine molecule to oneGosprotein-coupled receptor molecule induces synthesis of a largenumber of cAMpmolecules, thefirstlevelof amplification. Fourmolecules of cAMpactivate two molecules of proteinkinase pKAphosphorvlates A (PKA), buteachactivated and activates multiple product molecules. Thissecond levelof amplification mayinvolve several sequential reactions in whichthe productof one reaction activates the enzyme catalyzing the nextreactionThemore stepsin sucha cascade, thegreater thesignal amplification posslble

c E L LS I G N A L I N G l : S t G N A LT R A N S D U c I o NA N D s H o R T - T E R M c E L L U L A RR E s p o N s E s

an amplificationof 104fold. Becausethreemore catalyticsteps precedethe releaseof glucose,another 104 amplification can occur,resultingin a 106amplificationof the epinephrinesignal. In striated muscle the amplification is less dramatic, because the concentrations of the three successiveenzymes in the glycogenolyticcascade-protein kinase A, glycogen phosphorylase kinase, and glycogen phosphorylase-are tn a 1:10:240 ratio (a potential 240-foldmaximal amplification). The epinephrine-inducedGPCR pathway leadingto glycogenolysis,whether in liver or striatedmusclecells,dramaticallyillustrates how the effectsof an external signal can be amplified.

of the B-adrenergicreceptor, not those phosphorylated by PKA, can be phosphorylatedby the enzymeB-adrenergicreceptor kinase (BARK/, but only when epinephrineor an agonist is bound to the receptor and the receptor is in its active conformation. This processis called homologous desensitization,becauseonly those receptorsthat are in their activeconformations are subject to deactivation by phosphorylation' Another exampleof this regulatory mechanismis the desensitization of rhodopsin by rhodopsin kinase. Recall from our discussionof the rhodopsin pathway that

s o w n ' R e g u l a t eS i g n a l i n g S e v e r aM l e c h a n i s mD from G Protein-CoupledRecePtors For cellsto respond effectivelyto changesin their environment, mechanismsmust exist to terminate the activation of signaling pathways. Severalmechanismscontribute to termination of cellular responsesto hormonesmediatedby B-adrenergicreceptors and other G protein-coupled receptorscoupled to Go,. First, the affinity of the receptor for its ligand decreaseswhen the GDP bound to Go, is replacedwith GTP.This increasein the K6 of the receptor-hormone complex enhancesdissociationof the ligand sis. These interactions promote the formation of coated pits from the receptor and thereby limits the number of Go, proteins and endocytosisof the associatedreceptors'thereby decreasthat are activated. Second,the intrinsic GTPaseactivity of G.s ing the number of receptorsexposedon the cell surface(Figconvertsthe bound GTP to GDP, resulting in inactivation of the the ure 15-27).Eventually some of the internalizedreceptorsare protein and decreasedadenylyl cyclaseactivity' Importantly' Go, when rate of hydrolysis of GTP bound to Go, is enhanced binds to adenylyl cyclase,lesseningthe duration of cAMP production; thus adenylyl cyclasefunctions as a GAP for Go,. More generally,binding of most if not ail G*'GTP complexesto their Exterior respectiveeffector proteins acceleratesthe rate of GTP hydrolyactsto hydrolyze cAMP to sis.Finally, cAMP phosphodiesterase Thus the continuous response. 5'-AMB terminatingthe cellular is required concentration high enough presenceof hormone at a of maintenance and cyclase of adenylyl for continuous activation CYtosol falls concentration hormone the level. Once an elevatedcAMP sufficiently,the cellular responsequickly terminates' Receptors can also be down-regulated by feedback reActivationof l*rnoo.u,or,. pression, in which the end product of a pathway blocks an c - J u nk i n a s e proGo, a when instance, pathway. For in the step early cascade tein-coupled receptor is exposedto hormonal stimulation for severalhours, severalserineand threonineresiduesin the cytosolicdomain of the receptorbecomephosphorylatedby of MAP Activation protein kinaseA (PKA), the end product of the Go, pathway. kinasecascade The phosphorylatedreceptorcan bind its ligand but cannot and desensitization 15-27Roleof p-arrestinin GPCR FIGURE efficiently activate Go,; thus ligand binding to the phosphoand serine phosphorylated to binds signaltransduction' P-Arrestin rylated receptorleadsto reducedactivation of adenylyl cyof G protein-coupled segment in theC-terminal residues threonine clasecomparedwith ligand binding to a nonphosphorylated receptors boundby (GPCRt. andAP2,two otherproteins Clathrin receptor.Becausethe activity of PKA is enhancedby the high also of the receptor' promoteendocytosis B-Arrestin B-arrestin, to binding by cAMP levelinducedby any hormone that activatesGo,,proreceptors activated from signals in transducing functions MAP the activates c-src protein kinases. longed exposure to one such hormone, say, epinephrine, cytosolic several andactivating factors of keytranscrrption not only B-adrenergicreceptorsbut also other to phosphorylation desensitizes pathway, leading kinase protelns, other withthree of B-arrestin (Chapter 16).Interaction Go, protein-coupled receptorsthat bind different ligands in phosphorylation results (aJunN-terminal kinase), JNK-3 (e.g., glucagon receptor in liver). This cross-regulationis including fromW c-Jun factor, fAdapted transcription of another andactivation calledh eterologows desensitization. Pierce K and 13:139, Biol Cell Opin Curr' 2OO1 Lefkowitz, R J , and forms Miller Exposure of cells to epinephrinealso leadsto other 3:6391 Biol Cell Mol Rev. Nature 2002, al et domain , Particularresiduesin the cytosolic of desensitization.

J

CYCLASE G P R o T E I N _ C O U P L ERDE C E P T O RTSH A T A C T I V A T EO R I N H I B I TA D E N Y L Y L

.

651

degradedintracellularlS and some are dephosphorylatedin endosomes.Following dissociationof B-arrestin,the resensitized (dephosphorylated)receptorsrecycleto the cell surface, similar to recyclingof the LDL receptor (Chapter 14). Desensitizationof many GpCRs and other classesof r e c e p t o r so c c u r s b y r e c e p t o r p h o s p h o r y l a t i o n , a r r e s t i n b i n d i n g , a n d e n d o c y t o s i so f l i g a n d - o c c u p i e dr e c e p t o r s , leading to their sequestrationinside the cell. In addition to its role in regulating receptor activity, B-arrestin also functions as an adapter protein in transducingsignals frgm Gprotein-coupled receptorsto the nucleurlCh"pt., 16). The multiple functions of p-arrestinillustrate the importance of adapter proteins in both regulating signalingand transducingsignalsfrom cell-surfacereceptors.

One such anchoring protein (AKAP15) is tethered to the cytosolic face of the plasma membranenear a particular type of gated Ca2* channel in certain heart muscle cells. In the heart, activarion of B-adrenergicreceptorsby epinephrine(as part of the fight-or-flight response)leads to pKA-catalyzed phosphorylation of these Ca2+ channels, causing them to open; the resulting influx of Ca2* increasesthe rate of heart

A different AKAP in heart muscle anchors both protein kinase A and cAMP phosphodiesterase(pDE) to the outer nuclear membrane.Becauseof the closeproximity of pDE to protein kinase A, negativefeedbackprovides tight local control of the cAMP concentration and hencelocal pKA activity (Figure15-28).The localizationof protein kinaseA near the nuclear membrane also facilitates entry of its catalytic subunits into the nucleus, where they phosphorylate and activatecertain transcriptionfactors (seeChapter 16).

A n c h o r i n gP r o t e i n sL o c a l i z eE f f e c t so f c A M p t o S p e c i f i cR e g i o n so f t h e C e l l In many cell types,a rise in the cAMp level may produce a responsethat is required in one part of the cell but is un_

G Protein-Coupled Receptors That Activateor Inhibit AdenylylCyclase r Ligand activationof G protein-coupledreceptorsthat activateGo, resultsin the activationof the membrane-

a

E

B a s a lP D Ea c t i v i t y= resting state

E

I n c r e a s e dc A M P : PKA activation

P D Ep h o s p h o r y l a t i o n a n d a c t i v a t i o nr ;e d u c t i o n i n c A M Pl e v e l

cAMP >a t

mAKAP

I

Cytosol Outer nuclear memDrane

!f FIGURE 15-28 Localization of proteinkinaseA (pKA)to the nuclearmembranein heartmuscleby an A kinase-associated protein.Thismemberof theAKAPfamily,designated mAKAB anchors bothcAMPphosphodiesterase (pDE) andthe regulatory s u b u no i tf P K At o t h en u c l e amr e m b r a nm e ,a i n t a i n i n t hge mi n a negative feedback loopthatprovides closelocalcontrolof the cAMp levelandPKAactivity. Step[:The basallevelof pDEactivity in the absence (resting of hormone state)keepscAMplevels belowthose necessary for PKAactivation. StepsA and E: Activation of B_ adrenergic receptors causes an increase in cAMplevelin excess of

652

CHAPTER 15

I

Returnto restingstate

t h a tw h i c hc a nb e d e g r a d ebdy p D ET h er e s u l t i nbgi n d i n o gf cAMp (R)subunits to the regulatory of pKAreleases (C) theactivecatalytic subunits intothecytosolSomeC subunits enterjntothe nucleus, wheretheyphosphorylate andthusactivate certaintranscrrptron factors(Chapter 16),Concomitant phosphorylation of pDEby active PKAcatalytic subunits stimulates itscatalytic activity, therebyhydrolyzing cAMPanddriving cAMPlevels backto basal andcausing reformation of theinactive PKAStep@: Subsequent dephosphorylation of pDE returns the complex to the resting state,[Adapted fromK L Dodqe et al , 200 1, EMBOT 20:1921 )

C E L LS I G N A L I N G I : S I G N A LT R A N S D U C T I OANN D S H o R T - T E R M cELLULAR REsPoNsEs

bound enzymeadenylyl cyclase,which converts ATP to the cyclic AMP (cAMP). secondmessenger r Ligand activation of G protein-coupled receptors that activate Goi results in the inhibition of adenylyl cyclaseand lower levelsof cAMP. r The switch regions in the activated forms of G.,'GTP and Go1'GTP bind to the heterodimericactive site domains in adenylyl cyclaseto activate or inhibit the enzyme, respectively.

the receptors.The consequentreduction in the number of cell-surfacereceptorsrendersthe cell lesssensitiveto additional hormone. r Localization of PKA to specificregions of the cell by anchoring proteins restricts the effectsof cAMP to particular subcellularlocations.

r cAMP binds cooperativelyto a regulatory subunit of protein kinase A (PKA) releasingthe active kinase catalytic subunit (seeFigure 15-23).

That RecePtors G Protein-CouPled C ActivatePhosPholiPase

r PKA mediatesthe diverse effects of cAMP in most cells (seeTable 15-2). The substratesfor PKA and thus the cellular responseto hormone-inducedactivation of PKA vary among cell types.

Calcium ions play an essentialrole in regulating cellular responsesto external signals and internal metabolic changes' A, *. r"* in Chapter 11 the level of Ca2* in the cytosol is maintainedat a submicromolarlevel (<0.2 ptM) by the continuous action of AlP-powere d Ca2* pumps' which transport Ca2+ ions acrossthe plasma membrane to the cell exteiio, o, into the lumens of the endoplasmic reticulum and other vesicles.Much intracellular Ca2* is also sequesteredin the mitochondria. A small rise in cytosolicCa2* inducesa variety of cellular responsesincluding hormone secretion by endocrine cells, secietionof digestiveenzymesby pancreaticexocrinecells,and contractionof muscle(Table15-3).For example,acetylcholine stimulationof G protein--coupledreceptorsin secretorycellsof

r In liver and muscle cells, activation of PKA induced by epinephrine and other hormones exerts a dual effect, inhibiting glycogen synthesis and stimulating glycogen breakdownvia a kinasecascade(seeFigure 15-25)' and kir Signalingpathwaysinvolving secondmessengers amplify an externalsignaltremendously(see nasecascades Figure 75-26). r BARK phosphorylatesligand-boundB-adrenergicreceptors, leadingto the binding of B-arrestinand endocytosisof

Pancreas(acinar cells)

RISIINCAz+ INDUCI]I|G ll()RMt)NE

RESPONSE CETLULAR

Acetylcholine

Secretion of digestive enzymes' such as amylase and trypsinogen Secretion of amylase

Parotid (salivary) gland Vascular or stomach smooth muscle

Acetylcholine

Contractton

Liver

Vasopressin

Conversion of glycogen to glucose

Blood platelets

Thrombin

Aggregation, shape change, secretion of hormones

Mast cells

Antigen

Histamine secretion

Fibroblasts

Peptide growth factors (e.g., bombesin and PDGF)

DNA synthesis,cell divisron

',Hormone stimulation leads to production of inositol 1,4,5-trisphosphate (lP:), a second messengerthat promotes releaseof Ca2* stored in the endop l a s m i cr e t i c u l u m . 31,2231.5. iou*.o, M. J. Berridge, 7987, Ann. Reu.Biochem. 5621.59;M. J. Berridge and R. F. hvine, 1984, Nature

RD E C E P T O RTSH A T A C T I V A T EP H O S P H O L I P A SCE G PROTEIN_COUPLE

653

I

I

o

.)I

o

A v tUt 2

QHH- a; to ID _

-LH-

I .H

I

I

ATP ->

o aH-a! I'

o I

OH

o

_, 'e6l<

\n

ADP

lr

4

9l =L

OH Phosphatidylinositol

Pl 4-phosphate (PIP}

Pl 4,5-bisphosphate (ptpzl

,lIl3iil3lh.;l; (lP3) A FIGURE 15-29 Synthesis of secondmessengers DAGand lp3 from phosphatidylinositol (pl).Eachmembrane-bound pl kinase places (yellowcircles) a phosphate on a specific groupon hydroxyl the inositol ring,producing plpand the phosphorylated derivatives

PlP2Cleavageof PlP2by phospholipase C yieldsthe two important secondmessengers DAG and lP3 [SeeA Toker andL C Cantley, 1997, N a t u r e 3 8 7 i 6 7a3n,d C L C a r p e n t e r a n dCLC a n t l e y , 1 9 9C6u,r r . O p i C n ell Biol8:153I

the pancreasand parotid (salivary)gland inducesa risein Ca2* that triggers the fusion of secretoryvesicleswith the plasma membraneand releaseof their protein contentsinto the extracellular space.In blood platelers,the rise in Ca2* induced by thrombin stimulation triggers a conformational change in thesecell fragmentsleadingto their aggregation,an lmporranr step in blood clotting to preventleakageout of blood vessels. In this secrion,we discussan important GpCR-triggered signal-transducdonpathway that resultsin an elevarionof cy_ tosolic Ca'* ions. Binding of many hormonesto their G protein-
Chapter 16. One derivarive of PI, the lipid phosphatidyl inositol 4,5-bisphosphate (PIP2), is cleaved by activated phospholipase C into two important second messengers: 1,2diacylglycerol (DAG), a lipophilic molecule that remains associated with the membrane, and inositol 1,4,5-trisphosphate (IP3), which can freely diffuse in the cytosol (Figure 15-29). 'We refer to downstream events involving these two second messengers collectively as the IP j/DAG pathtuay.

PhosphorylatedDerivativesof InositolAre l m p o r t a n tS e c o n dM e s s e n g e r s A number of important second messengers,used in several signal-transduction pathways, are derived from tire mem_ brane lipid_phosphatidylinositol (pI). The inositol group in this phospholipid, which always faces the cytosoll .u., b. reversiblyphosphorylatedat one or more positionsby the com_ bined actionsof variouskinasesand phosphatases discussedin 654

'

CHAPTER ts

I

C a l c i u ml o n R e l e a s e from the Endoplasmic R e t i c u l u ml s T r i g g e r e db y l p 3 G protein-coupled receptors that activare phospholipaseC inducean elevationin cytosolicCa2* evenwhen Ca2* ions are absentfrom the surrounding extracellularfluid. In this situation, Ca2+ is releasedinto the cytosol from the ER lumen through operation of the lPj-gated Ca2+ channel in the ER membrane,as depicted in Figure 15-30 (step @). This large channelprotein is composedof four identicalsubunits,eachof which containsan IP3-bindingsite in the N-terminal cytosolic domain. IPj binding inducesopening of the channel,allowing Ca'* to flow down its concentrationgradientfrom the ER into the cytosol. rX/henvarious phosphorylatedinositols found in cellsare added to preparationsof ER vesicles,only Ip3 causes releaseof Ca2* ions from the vesicles.This simpleexperiment demonstratesthe specificityof the IP3effect. The IP3-mediatedrise in the cytosolic Ca2* level is transient becauseCa2* pumps locatedin the plasmamembrane and ER membrane actively transport Ca2l from the cytosol to the cell exterior and ER lumen, respectively.Furthermore, within a secondof its generation,the phosphatelinked to the

c E L L s t c N A L ' N Gr : s T G N A L T R A N s D U c t o N A N D ' H o R T - T E R MC E L L U L A R E s p o N S E S

llll+ FocusAnimation:SecondMessengers in SignalingPathwa

G coupledproteinreceptor(GPCR)

Store-operated o 6 Ca2* @ - 9o^oo o a o "Oov o oao o c^ hha nan na ln e l .

PhospholipaC se

Exterior Plasma membrane

Cytosol

\o

ooo

PlP2 lPa

pathwayand the 15-30|P3/DAG > FIGURE canbe Thispathway cytosolic Ca2*. of elevation thatactivate by ligandbindingto GPCRs triggered to eitherthe Gooor G*oalphasubunitleading of C (stepIl). Cleavage of phospholipase activation C yieldslP3andDAG(stepZ). PlP2 by phospholipase with lP3interacts throughthecytosol, Afterdiffusing a n do p e n sC a 2 *c h a n n eilnst h em e m b r a noef t h e (stepB), causing release of reticulum endoplasmic (stepZl) Oneof storedCa2*ionsintothecytosol by a risein cytosolic responses induced several cellular C (PKC) to the of proteinkinase Ca2*is recruitment (stepE), whereit isactivated by plasma membrane membrane-associated DAG(stepEl) Theactivated k i n a scea np h o s p h o r y l vaat er i o ucse l l u l aern z y m easn d (stepZ) As theiractivity therebyaltering receptors, a aredepleted, Ca2-stores reticulum endoplasmic Ca2*channels proteinassociated with the lP3-gated in Ca'* channels bindsto andopensstore-operated membrane, allowinginfluxof extracellular the plasma 1999,Proc fromJ W Putney, Ca2*(stepEl) [Adapted Nat'lAcadSciUSA96z14669 l

(;)

,/

r"'Vil '^

kinaseC

"o oo

lP3-gated Ca2* channel

ooo OO

oO

o

A - oo o o-

i^)+

reticulum Endoplasmic

carbon-S of IP3 (seeFigure 15-29\ is hydrolyzed,yielding This compoundcannot bind to the inositol 1,4-bisphosphate. protein and thus does not stimulate IP3-gatedCa2* channel ER. Ca2* releasefrom the \ilithout some means for replenishingdepletedstores of intracellular Ca2*, a cell would soon be unableto increasethe cytosolicCa2* levelin responseto hormone-inducedIP3.Patchclampingstudies(seeFigure 11-21)haverevealedthat a plasma membrane Ca2* channel, called the store-operatedchannel, opensin responseto depletionof ER Ca2* stores.In a way that is not fully understood,depletionof Ca2* in the ER lumen leads with the IP3to a conformationalchangein a protein associated gatedCa2* channelthat allows it to bind to the store-operated Ca2* channel in the plasma membrane,causingthe latter to open (seeFigure15-30,step E). Continuous activation of certain G protein-coupled re-

tion of the cell-surface G protein-coupled receptor' How-

cytosolicCa2*, is not understood.

i no m p l e xM e d i a t e sM a n y T h e C a 2 + / C a l m o d u lC l ignals C e l l u l a rR e s p o n s etso E x t e r n a S potentiatesopening of thesechannelsby IP3, thus facilitating ihe rapid rise in cytosolicCa2* following hormone stimula-

The small ubiquitous cytosolic protein calmodulin functions as a multipurptse switch protein that mediatesmany cellular

RD E C E P T O RTSH A T A C T I V A T EP H O S P H O L I P A SCE G PROTEIN_COUPLE

65s

effects of Ca2* ions. Binding of Ca2* to four sites on calmodulin yields a complex that interacts with and modulates the activity of many enzymesand other protelns (see Figure 3-31). Becausefour Ca2* bind to calmoJulin in a cooperative fashion, a small change in the level of cytosolic Ca2* leadsto a large changein the level of activecalmodulin. One well-studied enzyme activated by the Ca2+/calmodulin complex is myosin light-chain kinase,which regulatesthe activity of myosin in muscle cells (Chapter 17). Another is cAMP phosphodiesrerase,the enzyme that degradescAMp to 5'-AMP and terminates its effects.This reaction thus links Ca2+ and cAMP, one of many examples in which two second messenger-mediated pathways interact to fine-tune certain aspectsof cell regulation. In many cells, the rise in cytosolic Ca2* following recep_ tor signaling via phospholipaseC-generated Ip3 leads io

phosphategroups from a transcription factor. An important example of this mechanism involves T cells of the immune systemin which Ca2* ions enhancethe activity of an essential transcription factor called NFAT (nuclear factor of acti-

sequencethat allows NFAT to move into the nucleus and stimulate expressionof genesessentialfor the function of T cells.

Diacylglycerol(DAG)Activatesprotein KinaseC, W h i c h R e g u l a t e sM a n y O t h e r p r o t e i n s After rts formation by phospholipaseC-catalyzed hydrolysis of PIP2, the secondary messengerDAG remains associated

phosphorylatesvarious transcription factors; depending on the cell type, these induce synthesisof mRNAs that trigger cell division.

S i g n a l - l n d u c eR d e l a x a t i o no f V a s c u l a S r mooth Musclels Mediated by cGMp-Activatedprotein K i n a s eG ffi Nitroglycerinhasbeenusedfor overa centuryas a

treatment lor the intense chest pain of angina. It was Ill known to slowly decompose in the body to nitric oxide (NO/, which causesrelaxation of the smooth muscle cells surrounding the blood vesselsthat "feed" the heart muscle itself, thereby increasingthe diameter of the blood vessels and increasing the flow of oxygen-bearingblood to the heart muscle. One of the mosr intriguing discoveriesin modern medicine is that NO, a toxic gas found in car exhaust, is in fact a natural signalingmolecule.I Definitive evidencefor the role of NO in inducins relaxation of smooth muscle came from a set of experirnentsin which acetylcholinewas added to experimentalpreparations of the smoorh muscle cells that surround blood vessels. Direct application of acetylcholineto thesecellscausedthem to contract, the expected effect of acetylcholine on these muscle cells. But addition of acetylcholine to the lumen of small isolated blood vesselscaused the underlying smooth muscles to relax, not contract. Subsequentstudies showed that in responseto acetylcholinethe endothelialcellsthat line the lumen of blood vesselswere releasingsome substancethat in turn triggeredmusclecell relaxation.That substanceturned out to be NO. \7e now know that endothelial cells contain a Go protein-coupled receptor that binds acetylcholine and activates phospholipaseC, leading to an increasein the level of cytosolic Ca2*. After Ca2* binds to calmodulin, the resulting complex stimulates the activity of NO synthase,an enzyme that catalyzes formation of NO from 02 and the amino acid arginine. BecauseNO has a short half-life (2-30 seconds),it can diffuse only locally in tissuesfrom its site of synthesis.In particular NO diffusesfrom the endothelial cell into neighboring smooth muscle cells,where ir tnggers muscle relaxation (Figure15-31). The effect of NO on smooth muscle is mediated by the secondmessengercGMP, which is formed by an intracellular NO receptor expressedby smooth muscle cells. Binding of NO to the heme group in this receptor leads to a conformational change that increasesits intrinsic guanylyl cyclase activity, leading to a rise in the cytosolic cGMp level. Most

IP3/DAG pathway. The activation of protein kinase C in different cells re_ blood vessel.In this case,cGMP acts indirectly via protein kinase G, whereasin rod cellscGMp acts directiy by bindlng to and thus opening cation channels in the plasma membrane (seeFigure 15-18). 556

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15-31The nitricoxide < FIGURE (NO)/cGMP pathwayand the relaxationof arterialsmoothmuscle.Nitricoxideis to cellsin response in endothelial synthesized in elevation andthesubsequent acetylcholine locally ca'* (n-E). No diffuses cytosolic an intracellular andactivates throughtissues in activity cyclase with guanylyl NOreceptor cells(E) Theresulting smoothmuscle nearby proteinkinase G (6 and risein cGMPactivates and muscle the of to relaxation Z), leading (ts) Thecell-surface receptor thusvasodilation alsohas factor(ANF) for atrialnatriuretic (notshown); guanylyl activity cyclase intrinsic on smooth of thisreceptor stimulation cGMP increased to leads also muscle cells PP;: relaxation muscle andsubsequent etal, C S Lowenstein pyrophosphate [See 1994,Ann lnternMed 120:227 )

Smooth musclecells

Relaxation of vascular smooth muscle also is triggered by binding of atrial natriureticfactor (ANF) and someother peptidehormonesto their receptorson smooth musclecells. The cytosolicdomain of thesecell-surfacereceptors,like the intrinsic guanylyl cyintracellular NO receptor,possesses '$fhen an increasedblood volume stretches clase activity. cardiac muscle cells in the heart atrium, they releaseANF. Circulating ANF binds to ANF receptorson the surfaceof smooth musclecellssurroundingblood vessels,inducing activation of their guanylyl cyclaseactivity and formation of cGMP. Subsequentactivationof protein kinaseG causesdilation of the vesselby the mechanismdescribedabove.This vasodilationreducesblood pressureand countersthe stimulus that provoked the initial releaseof ANF.

G Protein-Coupled ReceptorsThat Activate PhospholipaseC r Simulation of someG protein-
nitric oxide synthase,and protein kinasesor phosphatases that control the activity of various transcription factors. r Stimulation of acetylcholine G protein-coupled receptors on endothelial cells induces an increase in cytosolic Ca2* and subsequentsynthesisof NO. After diffusing into surrounding smooth musclecells,NO activatesan intracellular guanylate cyclaseto synthesizecGMP. The resulting increasein cGMP leads to activation of protein kinase G, which triggers a pathway resulting to muscle relaxation and vasodilation(seeFigure 15-31). r cGMP is also produced in vascular smooth muscle cells by stimulation of cell-surfacereceptors that have intrinsic guanylatecyclaseactivity. Theseinclude receptorsfor atrial natriureticfactor (ANF).

of Cells IntegratingResponses Influences to Environmental

r IP3 triggers opening of IP3-gatedCa2* channels in the endoplasmic reticulum and elevation of cytosolic free Ca2*. In responseto elevatedcytosolic Ca2+, protein kinase C is recruited to the plasma membrane' where it is activatedby DAG (seeFigure 15-30).

no intracelJust as no cell lives in isolation from other cells' constantly All cells alone. functions iular signaling pathway including environment their from receive multiple signals gases such as and metabolites, levels, changesin hormone debehavioral to respond constantly cells o*yg..t. All body we section, In this infection. or injury to as mands as well in the demand variations to responses cellular consider the for the key metabolite glucose.Cellular responsesto changes in other nutrients and to oxygen' which are largely reflected in alterations in geneexpression'are covered in Chapter 7'

r A small rise in cytosolic Ca2* inducesa variety of cellular responsesincluding hormone secretion,contraction of muscle,and plateletaggregation(seeTable 15-3).

I n t e g r a t i o no f M u l t i p l e S e c o n dM e s s e n g e r s R e g u l a t e sG l Y c o g e n o l Y s i s

r TheseG proteins activatephospholipaseC, which generates two secondmessengers:diffusible IP3 and membranebound DAG (seeFigure 1,5-29).

One way for cells to respond appropriately to a complex . The Ca2*/calmodulin complex regulatesthe activity of to more many different proteins,including cAMP phosphodiesterase, environment is to senseand integrate its responses LFLUENCES T O E N V I R O N M E N T AI N OSF C E L L S I N T E G R A T I NRGE S P O N S E

657

than one signal. Again, the breakdown of glycogen to glucose (glycogenolysis)provides an excellent example. As described in Section 15.6, epinephrine stimulation of muscle and liver cells leads to a rise in the secondmessengercAMp, which promotes glycogenbreakdown (seeFigure 15-25a). In both muscle and liver cells, other second messengersalso produce the same cellular response. In muscle cells, stimulation by nerve impulses causesthe releaseof Ca2* ions from the sarcoplasmicieticulum and an increasein the cytosolic Ca2* concentration,which triggersmuscle contraction. The risein cytosolic Ca2* also activatesglycogen phosphorylasekinase (GPK), thereby stimulating the degradation of glycogento glucose1-phosphate,which fuels prolonged contraction. Recall that phosphorylation by cAMp-dependent protein kinase A also activatesglycogenphosphorylasekinase. Thus this key regulatory enzymein glycogenolysisis subject to both neural and hormonal regulation in muscle(Figure 15-32a). In liver cells, hormone-induced activation of the effector protein phospholipaseC also regulatesglycogenbreakdown by generating two second messengers,DAG and IP3. As we saw Section15.7,1P3inducesan increasein cytosolicCa2*, which activatesglycogenphosphorylasekinase as in musclecells,leading to glycogen degradation. Moreover, the combined effect of

.The dual regulation of glycogenphosphorylasekinase by Ca'- and protein kinase A in both muscle and liver results from its multimeric subunit structure (*gfE)+. The "ysubunit is the catalytic enzyme; the regulatory c and B subunits, which are similar in structure,are phosphorylatedby protein

> FIGURE 15-32Integratedregulationof glycogenolysis. (a)Neuronal stimulation of striated muscle cellsor epinephrine bindingto B-adrenergic receptors on theirsurfaces leadsto increased cytosolic concentration of the secondmessenqers Ca2*er cAMP,respectively. Thekeyregulatori glycogen enzyme phosphorylase (GpK)is kinase activated by Ca2"ionsandby phosphorylation by cAMP-dependent proteinkinase A (pKA)(b)In liver cells,hormonal stimulation of B-adrenergic receptors leadsto increased cytosolic concentrations of cAMp andtwo othersecondmessengers, diacylglycerol (DAG)andinositol1,4,5-trisphosphate (tp3) Enzymes aremarkedby whiteboxes. (+) : activation of enzyme (-) : inhibition activity;

kinase A; and the 6 subunit is calmodulin. Glycogen phosphorylase kinase is maximally active when Ca2* ions are bound to the calmodulin subunit and at least the o. subunit has beenphosphorylatedby protein kinaseA. In fact, binding of Ca't to the calmodulin subunit may be essentialto the enzymattc activity of glycogen phosphorylase kinase. Phosphorylation of the cr and B subunirs increasesthe affinity of the calmodulin subunit for Ca2*, enabling Ca2* ions to bind to the enzymeat the submicromolar Ca2* concentrationsfound in cells not stimulated by nerves. Thus increasesin the cytosolic concentration of Ca2* or of cAMP or of both induce incremental increasesin the activity of glycogenphosphorylasekinase.As a result of the elevatedlevel of cytosolic Ca2* after neuronal stimulation of musclecells,glycogenphosphorylase kinase will be active even if it is unphosphorylated; thus glycogen can be hydrolyzed to fuel conrinued muscle contraction in the absenceof hormone stimulation.

Insulinand GlucagonWork Together to Maintain a StableBlood GlucoseLevel During normal daily living the maintenanceof normal blood glucoseconcentrationsdependson the balance betweentwo peptide hormones, insulin and glucagon, which are made in distinct pancreatic islet cells and elicit different cellular responses.Insulin, which contains two polypeptide chains linked by disulfide bonds, is synthesizedby the p cells in the islets; glucagon, a monomeric peptide, is produced by the o islet cells.Insulin reducesthe level of blood glucose,whereas glucagon increasesblood glucose.The availability of blood glucoseis regulated during periods of abundance(following a meal) or scarcity (following fasting) by the adiustmenr of insulin and glucagon concentrationsin the blood.

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C E L Ls T G N A L T NrG: s T G N A LT R A N s D U C T o NA N D S H o R T - T E R M CELLULAR REspoNsEs

Glucose

After a meal, when blood glucose rises above its normal level of 5 mM, the pancreatic B cells respond to the rise in glucose(and amino acids)by releasinginsulin into the blood (Figure 15-33). The releasedinsulin circulatesin the blood and binds to insulin receptors present on many different kinds of cells,including muscleand adipocltes (fat-storing cells).The insulin receptor belongsto the classof receptorstermed receptor fyrosine kinases (RTKs), which we describein Chapter 16' It can transducesignalsthrough an intracellular pathway leading to the activation of protein kinase B. By an unknown mechanism, protein kinaseB triggersthe fusion of intracellular vesicles containing the glucose transporter GLUT4 with the plasma membrane(Figure15-34).The resultingtenfold increasein the number of GLUT4 moleculeson the cell surfaceincreasesglucoseinflux proportionallg thus lowering blood glucose' As the blood glucose level drops, insulin secretion and blood levelsdrop, and insulin receptorsare no longer being activated as strongly. In response,cell-surfaceGLUT4 is internalized by endocytosis,lowering the level of cell-surface GLUT4 and thus glucoseimport. Insulin stimulation of muscle cells also promotes the conversion of glucoseinto glycogen, and it enhancesthe degradation of glucoseto pyruvate. Insulin also acts on hepatocytes(liver cells)to inhibit glucose synthesis from smaller molecules' such as lactate and acetate, and to enhanceglycogen synthesisfrom glucose.The net effect of all these actions is to lower blood glucoseback to the fasting concentration of about 5 mM while storing the excessglucoseintracellularly as glycogenfor future use. If the blood glucoselevel falls below about 5 mM, for example due to suddenmuscular activity, reducedinsulin secretion from pancreatic B cells induces pancreatic ct cells to increasetheir secretionof glucagon into the blood. Like the

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15-33Secretion of insulinin responsetoarisein A FIGURE p cellsis intopancreatic blood glucose.Theentryof glucose ([) Because glucose the K. transpofter mediated bythe GLUT2 glucose from5 is-20 mM,a risein extracellular for glucose of GLUT2 increase a proportionate mM,characteristic of thefastingstate,causes of entry(seeFigure11-4) Theconversion in the rateof glucose glucose in an increase in the intopyruvate isthusaccelerated, resulting of ATPin the cytosol(El) Thebindingof ATPto ATPconcentration (B), thusreducing the K* channels closes thesechannels sensitive of the smalldepolarization effluxof K+ ionsfromthecellTheresulting (4) triggers plasma Ca2* theopening of voltage-sensitive membrane (Et) Theinfluxof Ca2*ionsraises Ca2thecytosolic channels secretory triggering thefusionof insulin-containing concentration, (6) of insulin withtheplasma membrane andthesecretion vesicles fromJ Q Henquin, 2000,Diabetes 49:1751 ] [Adapted

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FIGURE 15-34Insulinstimulationof fat a EXPERIMENTAL of GLUT4from intracellular cellsinducestranslocation fat cells to the plasmamembrane.Inthisexperiment, vesicles proteinwhoseN-terminal to express a chimeric wereengineered followedbythe sequence, to the GLUT4 endcorresponded to lightof Whena cellisexposed entirety of the GFPsequence yellow-green, indicating wavelength, GFPfluoresces theexciting cells(a),mostGLUT4 of GLUT4 withincellsIn resting the position to the plasma membranes thatarenot connected is in internal

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with images of the samecellaftertreatment membraneSuccessive i n c r e a sing t i m e , s h o w t h a t w i t h m i n u t e s 1 0 5 , a n d i n s u l ifno r 2 5 , fusewith the membranes of theseGLUT4-containing numbers to thecellsurface therebymovingGLUT4 plasma membrane, glucose fromthe bloodintothe (arrows) it to transport andenabling transporters GLUT4 insulin-responsive cellsalsocontain cell Muscle Biol 21:47851 Cell Mol al 2001, Bogan et J see J Bogan; , of [Courtesy

LFLUENCES T O E N V I R O N M E N T AI N OSF C E L L S I N T E G R A T I NRGE S P O N S E

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epinephrinereceptor,the glucagon receptor,found primarily on liver cells,is coupled to the Go, protein, whose effector protein is adenylyl cyclase.Glucagon stimulation of liver cells inducesa rise in cAMP, leading to activation of protein kinase A, which inhibits glycogen synthesisand promotes glycogenolysis, yielding glucose 1-phosphate(seeFigures 1S-25a and 15-32b). Liver cellsconvert glucose1-phosphateinto glucose, which is releasedinto the blood, thus raising blood glucose back toward its normal fastinelevel. Unfortunatelg these intricate and powerful control systemssometimesfail, causingserious,evenlife-threatening disease.Diabetes mellitus results from a deficiency in the amount of insulin releasedfrom the pancreasin responseto rising blood glucose(type I) or from a decreasein the ability of muscleand fat cellsto respond to insulin (type II). In both types, the regulation of blood glucoseis impaired, leading to persistent elevated blood glucose concentrations (hyperglycemia) and other possiblecomplications if left untreated. Type I diabetes is caused by an autoimmune process that destroys the insulin-producing B cells in the pancreas.Also called insulin-dependentdiabetes,this form of the diseaseis generallyresponsiveto insulin therapy.Most Americanswith diabetes mellitus have type II, or insulin-independent diabetes,but the underlying causeof this form of the disease is not well understood.Further identificat pathways that control energymetabolism vide insight into the pathophysiology of leading to new methods for its prevention

lntegrating Responsesof Cellsto Environmental Influences r Glycogen breakdown and synthesisis regulated by multiple second messengersinduced by neural or hormonal stimulation (seeFigure 1,5-32). r A rise in blood glucose stimulates the releaseof insulin from pancreatic B cells (see Figure 1S-33). Subsequent binding of insulin to its receptor on muscle cells and adipocytes leads to the activation of protein kinase B. which promotesglucoseuptake and glycogensynrhesis,resulting in a decreasein blood glucose. A lowering of blood glucose stimulates glucagon reasefrom pancreaticct cells.Binding of glucagonto its G protein-coupled receptor on liver cellspromotes glycogenolysis by the cAMP-triggered kinase cascade(similar to epinephrine stimulation under stress conditions) and an increasein blood glucose.

In this chapter we focused primarily on signal-transduction pathways activated by individual G protein-coupled receptors. However, even these relatively simple pathways presage the more complex situation within living cells. 650

C H A P T E R1 5

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Many G protein-coupled receptors form homodimers or heterodimers with other G protein-coupled receptors that bind ligands with different specificitiesand affinities. Much current researchis focused on determining the functions of thesedimeric receptorsin the body. With :720 membersin total, the G protein-coupled receptors represent the largest protein family in the human genome. Approximately half of these genes are thought to encode sensory receptors; of these the majority are in the olfactory systemand bind odorants. Of the remaining 360 G protein-receptors,the natural ligand has been identified for approximately 210 receptors,Ieaving 150 so-called orphan GPCRs, that is putative GPCRs without known cognateligands. Many of these orphan receptors are likely to bind heretofore unidentified signaling molecules, including new peptide hormones. G protein-coupled receptorsalready representthe largestclassof target moleculesfor drugs available in the clinic, and thereforeorphan GPCRsrepresenta fruitful resourcefor drug discoveryby the pharmaceuticalindustry. One approach that has proven fruitful in identifying ligands of orphan GPCRs involves expressingthe receptor genes in transfectedcellsand using them as a reporter systemto detect substancesin tissue extracts that activate signal-transduction pathways in thesecells.This approach has already led to stunning insights into human behavior. One example is two novel peptides,termed orexin-A and orexin-B (from the Greekorexis, meaning appetite), that were identified as the ligands for two orphan GPCRs.Further researchshowedthat the orexin geneis expressedonly in the hypothalamus, the part of the brain that regulatesfeeding. Injection of orexin into the brain ventricles causedanimals to eat more, and expressionof the orexin gene increasedmarkedly during fasting. Both of these findings are consistent with orexin's role in increasing appetite. Strikingly mice deficient for orexins suffer from narcolepsy, a disorder characterizedin humans by excessivedaytime sleepiness(for mice, nighttime sleepiness).Moreover, very recenr repofts suggest that the orexin system is dysfunctional in a majority of human narcolepsypatients: Orexin peptidescannot be detected in their cerebrospinal fluid (although there is no evidence of mutation in their orexin genes). These findings firmly link orexin neuropeptidesand their receptorsto both feedingbehavior and sleepin both animals and humans. One can only wonder about what other peptides and small-moleculehormonesremain to be discovered,and the insightsthat study of thesewill provide for our understanding of human metabolism,growth, and behavior.

KeyTerms adenylyl cyclase639

cAMP 634

adrenergicreceptors 636

competition assay629 desensitization531

agonist 629 arrcstin 645 attocrine 626

endocrine 525 functional expressionassay 631

calmodulin 555

glucagon 659

C E L LS I G N A L I N GI : S I G N A LT R A N S D U C T I OA NN D S H O R T - T E R M c E L L U L A RR E S P o N s E S

G protein-coupled rcceptors 624

rhodopsin 635

insulin 659

signal amplif ication 649

IP3/DAG pathway 554

signal transdtction 624 stimulatory G protein 539

muscarinic acetylcholine receptors 641

secondmessengers634

paracrine 626

transducin641 trimeric G proteins 534

phospholipaseC 539

visual adaptation 544

protein kinaseA 647 protein kinase C 657

Reviewthe Concepts 1. What common featuresare sharedby most of the different cell signaling systems? 2. Signalingby soluble extracellular moleculescan be classified as endocrine,paracrine,or autocrine.Describehow these three types of cellular signaling differ. Growth hormone is secretedfrom the pituitary, which is located at the base of the brain, and acts through growth hormone receptors located on the liver. Is this an example of endocrine, paracrine, or autocrine signaling?\7hy? 3, A ligand binds two different receptorswith a K6 value of 1.0-' M for receptor 1 and a K6 value of 1,0 " M for receptor 2. For which receptor does the ligand show the greater affinity? Calculate the fraction of receptors that have a bound ligand ([RL]/Rr) in the caseof receptor 1 and receptor2, if the concentrationof free ligand is 10-" M. 4. A study of the properties of cell-surfacereceptorscan be greatly enhanced by isolation or cloning of the cell-surface receptor.Describehow a cell-surfacereceptor can be isolated by affinity chromatography.How can you clone a cell-surface receptor using a functional expressionassay? 5. Signal-transducingtrimeric G proteins consistof three subunits designatedo, B, T. The G. subunit is a GTPase switch protein that cycles between active and inactive states depending upon whether it is bound to GTP or to GDP. Review the steps for ligand-induced activation of effector proteins mediated by the trimeric G proteins. Supposethat you have isolated a mutant G. subunit that has an increasedGTPaseactivity. S7hat effect would this mutation have on the G protein and the effector protein? 6. Explain how second messengerssuch as Ca2* and cAMP can transmit and amplify an extracellular signal. 7, The cholera toxin, produced by the bacterium Vibrio cholera, causes a watery diarrhea in infected individuals. What is the molecular basisfor this effect of choleratoxin? 8. Epinephrinebinds to both B-adrenergicand o-adrenergic receptors. Describe the opposite actions on the effector protein, adenylyl cyclase,elicited by the binding of epinephrine to these two types of receptors. Describe the effect of adding an agonist or antagonist to a B-adrenergicreceptor on the activity of adenylyl cyclase. 9. Both rhodopsin in vision and the muscarinic acetylcholine receptorsystemin cardiacmuscleare coupledto ion

channelsvia G proteins. Describethe similarities and differencesbetweenthesetwo systems' 10. In liver and muscle cells, epinephrine stimulates the releaseof glucosefrom glycogenby inhibiting glycogensynthesisand stimulating glycogenbreakdown. Outline the moleculareventsthat occur after epinephrinebinds to its receptor and the resultantincreasein the concentrationof intracellular cAMP. How are the cAMP levels returned to normal? Describe the eventsthat occur after cAMP levels decline. 11. Continuous exposure of a G. protein-coupled receptor to its ligand leads to a phenomenon known as desensitization. Describe several molecular mechanisms for receptor desensitization.How can a receptor be reset to its original 'Sfhat effect would a mutant receptor lacksensitizedstate? ing serineor threonine phosphorylation siteshave on a cell? 12. Visual adaptation and receptor desensitizationinvolve similar phosphorylation mechanisms.Describe how the B-adrenergicreceptorkinase(BARK) and rhodopsin kinase play important roles in theseprocesses.!7hat role doesdephosphorylation play in thesereactions? 13. Sometimescells need to localize the effects of signaling example is localsystemsto specificsubcellularregions. One '$7hat proteins are ization of cAMP signals in heart muscle. involved? How does this sYstemwork? 14. Inositol 1,4,5-trisphosphate(IP3) and diacylglycerol (DAG) are second messengermoleculesderived from the cleavageof the phosphatidylinositol 4,5-bisphosphate(PIP2) by activated phospholipaseC. Describethe role of IP3 in the releaseof Ca2* from the endoplasmic reticulum' How do cells replenishthe endoplasmicreticulum stores of Caz*? What is the principal function of DAG? 15. Recent research has identified a surprising molecular link between feeding behavior and sleep. Describe the signaling factors that may be shared by both systems.

Analyze the Data Mutations in trimeric G proteins can causemany diseasesin humans.Patientswith acromegalyoften havepituitary tumors that oversecretethe pituitary hormone called growth hormone (GH). A subsetof thesegrowth hormone (GH)- secretingpituitary tumors resultfrom mutationsin G proteins.GH-releasing hormone (GHRH) stimulatesGH releasefrom the pituitary by binding to GHRH receptors and stimulating adenylyl cyclase. Cloning and sequencingof the wild-type and mutant Go. gene

i > 200 a G o o

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ffi

GTP+iso GTP"yS Mutant K E YT E R M S

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from normal individuals and patients with the pituitary tumors revealeda missensemutation in the Go, genesequence. a. To investigatethe effect of the mutation on Go, activiry wild-type and mutant G*, cDNAs were transfectedinto cells that lack the Go, gene. These cells expressa B2-adrenergicreceptor,which can be activatedby isoproterenol,a B2-adrenergic receptor agonist. Membranes were isolated from transfected cells and assayedfor adenylyl cyclaseactivity in the presenceof GTP or the hydrolysis-resistantGTP analog, GTP-1S. From the figure above, what do you conclude about the effect of the mutation on Go, activity in the presence of GTP alone comparedwith GTP-1Salone or GTP plus isoproterenol(iso)? b. In the transfected cells described in part a, what would you predict would be the cAMP levels in cells transfected with the wild-type Go, and rhe mutant G.,? What effect might this have on the cells? c. To further characterizethe molecular defectcausedby this mutation, the intrinsic GTPaseactivity present in both wild-type and mutant Go, was assayed.Assaysfor GTpaseactivity showed that the mutation reduced the k..r_6.1p(catalysis rate constantfor GTP hydrolysis)from a wild-rypevalue of 4.1 min-1 to the mutant value of 0.1 min-1. Whai do you conclude about the effect of the mutation on the GTpase activiry present in the mutant Go. subunit? How do these GTpase resultsexplain the adenylylcyclaseresultsshown in part a?

References 1 5.2 Studying Cell-Surface Receptors receptorsin hemostasis, _ Coughlin, S. R. 2005. Protease-activated thrombosisand vascularbiology.J Thromb Haemost.3:1800-1814. . Gross,A., and H. F. Lodish.2006. Cellulartrafficking and degradation of erythropoietin and NESP./. Biol. Chem.28t2624-2U1. Simonsen,H., and H. F. Lodish. 1994. Cloning by function: expressioncloning in mammalian cells.Trendsphirmacol. Sci. l5:437441.

Filipek, S., et al. 2003. G protein-coupledreceptorrhodopsin: a prospectus.Ann. Reu.Physiol. 65:851,-879. Filipek, S., et al. 2003. The crystallographicmodel of rhodopsin and its use in studies of other G protein--coupled receptors.Ann. Reu.Biophys Biomol. Struc. 32:375-397. Hurley, J. H., and J. A. Grobler. 1997. Protein kinase C and phospholipaseC: bilayer interacrionsand regulation. Cwrr.Opin. Jtruc. btol. /:JJ /-)6). Nathans,J. 1,999. The evolution and physiologyof human color vision: insightsfrom moleculargeneticstudiesof visual pigments. Neuron 24:299-31.2. Palczewski,K. 2006. G protein-coupledreceptorrhodopsin. Ann. Rev. Biochem.75:743-767. Ramsey,I. S., M. Delling, and D. Clapham.2006. An introduction to TRP channels.Ann. Reu.Physiol.6S:619-647. Singer,W. D., H. A. Brown, and P. C. Sternweis.1997.Regulation of eukaryotic phosphatidylinositol-specific phospholipaseC and phospholipase D. Ann. Reu.Biocbem.66:475-509. 15.6 G Protein-Coupled Receptors That Activate or lnhibit Adenylyl Cyclase Browner,M., and R. Fletterick.1992. Phosphorylase:a biological transducer.TrendsBiochem.Sci. 17:66-71.. Hurley, J.H. 1999. Structure,mechanism,and regulation of mammalian adenylylcyclase.J. Biol. Chem.274:7 599-7602. Johnson,L. N. 1992. Glycogenphosphorylase:conrrol by phosphorylation and allostericeffectors.FASEBJ. 6: 227 4-2282. Taylor, S. S., et al. 2005. Dynamics of signalingby PKA. Bio chim. Biophys. Acta 1754:25-37 . Lefkowitz, R. J., and S. K. Shenoy.2005. Transductionof receptor signalsby B- arrestins.Science308:512-517. ShenoSS., and R. J. Lefkowitz. 2003. Multifaceted roles of B-arrestinsin the regulationof seven-membrane spannrngreceptor trafficking and signaling.Biochem./. 375:503-515. Smith, F. D., L. K. Langeberg,and J. D. Scott.2006. The where's and when's of kinase anchoring. Trends Biochem. Sci. 3l:31,6-323. 'Witters L. A., B. E. Kemp, and A. R. Means. 2005. Churesand ladders: the searchfor protein kinases that act on AMPK. Trends Biochem. Sci.3I:1,3-16. I7ong, W., and J. D. Scott.2004. AKAP signallingcomplexes:focal points in spaceand time. Nature Reu.Mol. Cell Biol. 5:959-970.

15.3 Highly Conserved Components of Intracellular Signal-Transduction Pathways

15.7 G Protein-Coupled Receptors That Activate Phospholipase C

Cabrera-Vera,T. M. et al.2003.Insights into G protein structure, function, and regulation.Endocr. Reu. 24:765-781. I. R., and A. \X/ittinghofer.2001,. The guaninenucleotide_ _Vetter, binding switch in three dimensions.ScienceZ9+I299-I304.

Carlton, J. G., and P.J. Cullen. 2005. Coincidencederectionin phosphoinositidesignaling.Trends Cell Biol. l5'540-547. Kahl, C. R., and A. R. Means. 2003. Regulationof cell cycle progressionby calcium/calmodulin-dependent pathways.Endocr. Reu.24:.719-736. Patterson,R., D. Boehning,and S. Snyder.2004. Inositol 1, 4, 5, trisphosphatereceptorsas signalintegrators.Ann. Reu.Biochem. 73:437465.

15.4 General Elements of G protein-Coupled Receptor

Systems Bourne, H. R. 1997. How receptorstalk to trimeric G proteins. Curr. Opin. Cell Biol. 921.34-1,42. Bourne,H. R. 2001. Receptoractivation:what doesthe rhodopsin structuretell us? Trezds Pharmacol. Sci.22:587-593. Farfe\,7 ., H. Bourne, and T. Iiri. 1999. The expanding spectrumof G protein diseases. New Eng. J. Med.340. 1.012_1020. Pierce,K. L., R. T. Premont, and R. J. Lefkowitz. 2002. Seventransmembranereceptors.Nature Reu.Mol. Cell Biol. 3:639-652. Oldham, \7. M., and H. Hamm. 2006. Structural basisof function in heterotrimericG proteins.Quart Reu.Biophys.40: (ln press). 15.5 G Protein-Coupled Receptors That Regutate lon Channels _ Chin, D., and A. R. Means. 2000. Calmodulin: a prototypical calcium sensor.Trends Cell Biol. l}:322-32g.

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15.8 lntegrating Responses of Cells to Environmental Influences Papin,J. A., et al. 2005. Reconstructionof cellular signalling networks and analysisof their properties.Nature Reu.Mol. Cel[ Biol. 6:99-11'1,. Rothman, D., M. Shults,and B. Imperiali. 2005. Chemical approachesfor investigatingphosphorylationin signaltransduction networks. TrendsCell Biol. 15:502-51.0. Taniguchi,C., B. Emanuelli,and C. R. Kahn. 2006. Critical nodes in signalling pathways: insights into insulin action. Nature Reu.Mol. Cell Biol.7:85-96. 'Watson, R. T., and J. Pessin.2006. Bridging the GAp between insulin signalingand GLUT4 translocarion.TrendsBiochem. Sci. 3l:21.5-222.

C E L LS I G N A L I N G I : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RcME L L U L A R REsPoNsEs

cLASSTC

EXPERIMENT

15

THEINFANCY OF SIGNALTRANSDUCTION-GTP OF cAMPSYNTHESIS STIMULATION M. Rodbellet al., 1971,J. Biol. Chem.246:1877

In the late 1960s the study of hormone action blossomedfollowing the discovery that cyclic adenosine monophosphate (cAMP) functioned as a second messenger, coupling the hormonemediated activation of a receptor to a cellular response.In setting up an experimental system to investigate the hormone-induced synthesis of cAMP, Martin Rodbell discoveredan important new player in intracellular signalingguanosine triphosphate (GTP).

Background The discoveryof GTP's role in regulating signal transduction began with studies on how glucagon and other hormones send a signal across the plasma membrane that eventually evokes a cellular response.At the outset of Rodbell's studies.it was known that binding of glucagon to specificreceptor proteins embeddedin the membrane stimulates production of cAMP. The formation of cAMP from ATP is catalyzed by a membrane-bound enzyme called adenyl cyclase.It had been proposed that the action of glucagon, and other cAMP-stimulating hormones, relied on additional molecular components that couple receptor activation to the production of cAMP. However, in studies with isolated fatcell membranes known as "ghosts," Rodbell and his coworkers were unable to provide any further insight into how glucagon binding leads to an increase in production of cAMP. Rodbell then began a seriesof studies with a newly developedcell-freesystem,purified rat liver membranes,which retained both membrane-bound and membraneassociatedproteins. Theseexperiments eventually led to the finding that GTP is required for the glucagon-induced stimulation of adenyl cyclase.

TheExperiment One of Rodbell's first goals was to characterizethe binding of glucagon to the glucagon receptor in the cell-free rat liver membrane system.First, purified rat liver membraneswere incubated with glucagon labeled with the radioactive isotope of iodine (12sI). Membranes were then separatedfrom the unbound [125I]glucagon by centrifugation. Once it was established that labeled glucagon would indeed bind to the purified rat liver cell membranes,the study went on to determine if this binding led directly to activation of adenyl cyclase and production of cAMP in the purified rat liver cell membranes, The production of cAMP in the cell-free system required the addition of ATP; the substrate for adenyl cyclase,Mg2*; and an AlP-regenerating system consisting of creatine kinase and phosphocreatine. Surprisingly, when the glucagon-binding experiment was repeated in the presenceof these additional factors, Rodbell observed a 50 percent decrease in glucagon binding. Full binding could be restored only when ATP was omitted from the reaction. This observation inspired an investigation of the effect of nucleoside triphosphates on the binding of glucagon to its receptor. It was shown that relatively high (i.e., millimolar) concentrationsof not only ATP but also uridine triphosphate (UTP) and cytidine triphosphate (CTP) reduced the binding of labeled glucagon. In contrast, the reduction of glucagon binding in the presenceof GTP occurred at far lower (micromolar) concentrations. Moreover, low concentrations of GTP were found to stimulate the dissociation of bound glucagon from the receptor.Taken together, these studies suggested that

GTP alters the glucagon receptor in a manner that lowers its affinity for glucagon. This decreasedaffinity both affects the ability of glucagon to bind to the receptor and encouragesthe dissociation of bound glucagon. The observation that GTP was involved in the action of glucagon led to a secondkey question: Can GTP also exert an affect on adenyl cyclase?Addressing this question experimentally required the addition of both ATP, as a substratefor adenyl cyclase,and GTP, as the factor being examined, to the purified rat liver membranes. However, the previous study had shown that the concentration of ATP required as a substrate for adenyl cyclase could affect glucagon binding. Might it also stimuIate adenyl cyclase?The concentration of ATP used in the experiment could not be reduced becauseATP was readily hydrolyzed by ATPases present in the rat liver membrane. To get around this dilemma, Rodbell replacedATP with an AMP analog, 5'-adenyl-imidodiphosphate (AMP-PNP), which can be converted to cAMP by adenyl cyclase,yet is resistant to hydrolysis by membrane ATPases.The critical experiment now could be performed. Purified rat liver membranes were treated with glucagon both in the presence and absence of GTP, and the production of cAMP from AMP-PNP was measured.The addition of GTP clearly stimulated the production of cAMP when comPared to glucagon alone (Figure 1) indicating that GTP affects not only the binding of glucagon to its receptor but also stimulates the activation of adenylyl cyclase.

Discussion Two key factors led Rodbell and his colleaguesto detect the role of GTP in signal transduction, whereas previous

T H E I N F A N C YO F S I G N A LT R A N S D U C T I O N _ G TSPT I M U L A T I O NO F C A M P S Y N T H E S I S

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< FIGURE 1 Effectof GTPon glucagon-stimulated cAMP productionfrom AMP-PNP by purifiedrat liver membranes. In the absence of GTI glucagon stimulates cAMPformation about twofoldoverthe basallevelin theabsence of addedhormoneWhen GTPalsoisadded,cAMPproduction increases anotherfivefold [Adaptedfrom M Rodbellet al , 1971,J Biol Chem 246:1877 ]

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studies had failed to do so. First. bv switching from fat-cellghosrsto the rat liver membrane system, the Rodbell researchersavoided contamination of their cell-freesystemwith GTP, a problem associatedwith the procedure for isolating ghosts.Suchcontamination would mask the effects of GTP on glucagon binding and activationofadenyl cyclase.Second, when AIP was first shown to influence glucagon binding, Rodbell did not simply acceptthe plausible explanation that ATP, the substrate for adenyl cyclase, also affectsbinding ofglucagon.Insread, he choseto test rhe effectson bindine of the other common nucleosidetriphosphates.Rodbell later noted that he knew commercial preparations of ATP often

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15

20

are contaminated with low concentrations of other nucleosidetriphosphates. The possibilityof contaminationsuggestedto him that small concentrations of GTP might exert large effects on glucagonbinding and the stimulationof adenylcyclase. This critical series of experiments stimulated a large number of studies on the role of GTP in hormone action. eventually leading to the discovery of G proteins, the GTP-binding proteins that couple certain receptors to the adenyl cyclase.Subsequentlyan enormous family of receptors that require G proteins to transduce their signals were identifiedin eukaryotesfrom yeast to humans. These G protein-coupled

receptorsare involved in the action of many hormones as well as in a number of other biological activities, including neurotransmissionand the immune response.It is now known that binding of ligands to their cognate G proteincoupled receptors stimulates the associated G proteins to bind GTP. This binding causestransduction of a signal that stimulates adenyl cyclaseto produce cAMP and also desensitizationof the receptor, which then releasesits ligand. Both of these affects were obs e r v e d i n R o d b e l l ' s e x p e r i m e n t so n glucagon action. For theseseminal observations, Rodbell was awarded the Nobel Prize for Physiology and Medicine in 1994.

C E L LS I G N A L I N G l : S I G N A LT R A N S D U C T I OANN D S H O R T - T E RCME L L U L A R E S p O N S E S

CHAPTER

M A Pk i n a s se i g n a l i nign e m b r y o n iDca y1 3 . 5m o u s el u n g ActivatedERKis detectedby a primaryantibodythat detects phosphorylated antibodyconjugated ERKfollowedby a secondary Bellusci, Saverio FITC@ StijnDelanghe, to greenfluorescing Denise Tefftand DavidWarburton.

cell's ability to respond to its environment is essential to its survival. Short-term responsesto environmental stimuli, which can occur rapidly and are usually reversible,most often result from modification of existing proteins, as detailed in Chapter 15. Longer term responses, which are discussedin this chapter, are usually the result of changesin transcription of genes.Transcription is influenced by chromatin structure and the cell's complement of transcription factors and other proteins (Chapter 7). Thesedetermine which genesthe cell can potentially transcribe at any given time. \7e think of theseproperties as the cell's "memory" determined by its history and responseto previous signals. But many key regulatory transcription factors are held in an inactive state in the cytosol or nucleus and become activated in responseto external signals.In this chapter we focus on how ligandsthat bind to cell-surfacereceptorstrigger activation of specifictranscription factors that, in turn, determine the precisepattern of cellular geneexpresslon. Extracellular signalsthat induce long-term responsesaffect many aspectsof cell function: division, differentiation, and even communication with other cells. Alterations in these signaling pathways cause many human diseases,including cancer,diabetes,and immune defects.In addition to the crucial roles external signalsplay in development,signals are essentialin enabling differentiated cells to respond to their environment by changing their shape, metabolism, or movement. For example, one type of transcription factor (NF-rB) ultimately impacts expressionof more than 150 genesinvolved in the immune responseto infection; NF-rcB is activated by many protein hormones that act on immune systemcells. Another family of extracellular signaling mole-

II: CELLSIGNALING SIGNALING THAT PATHWAYS GENE CONTROL ACTIVITY

cules, the cytokines, is involved in maintaining approprlate levels of blood cells such as erythrocytes (red blood cells), leukocytes(white blood cells),and platelets. In order to illustrate the variety of mechanismsused to

OUTLINE 1 6 . 1 TGFBReceptorsand the DirectActivation of Smads 16.2

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Phosphorylation and degradation o f i n h i b i t o ro f NF-KBin cytosol

thentranslocate intothe nucleus andregulate the activity of nuclear TFs(c,d) In otherpathways, activeTFsarereleased fromcytosolic (e,f) or by proteolysis multiprotein complexes (g,h).Somereceptor classes cantriggermorethanoneintracellular pathway, asshownin Figure16-2 lAfterA H Brivanlou andJ,Darnell, 2OOZ, Science 295:813 I

p A T H W A y s r H A T c o N T R o L G E N EA c l v t r y C E L LS I G N A L | N Gi l : s T G N A L | N G

The kinasemay be an intrinsic part of the receptorprotein or be tightly bound to the receptor.In either case,kinase activity is activated by ligand binding, resulting-directly or indirectly-in activation of specific transcription factors located in the cytosol (Figure 1,6-1a,b).Inother pathways, receptor stimulation leads to activation of cytosolic protein kinasesthat translocate into the nucleus and phosphorylate specificnucleartranscriptionfactors (Figure16-1c,d).Binding of ligand to receptors for other types of signaling proteins causesdisassemblyof multiprotein complexesin the cytosol, releasingtranscription factors that then translocateinto the nucleus (Figure 16-1e,f).In still other signalingparhways, proteolytic cleavageof an inhibitor or the receptor itself r e l e a s e sa n a c t i v e t r a n s c r i p t i o n f a c t o r ( F i g u r e 1 6 - 1 g , h ) . Importantly, all of thesepathways are highly regulated,often by negativefeedback,in order to control the leveland duration of the signal'seffectson cellular geneexpression. :100 differenttypes A typical mammaliancell expresses of cell-surfacereceptors,many of which activate rhe sameor similar signal-transduction pathways.As shown in Figure 16-2, several classesof receptors can transduce signals by more than one pathway, and some pathways are activated to a greaterextent in certain cells than others.Moreover, many genesare regulatedby multiple transcription factors, each of which can be activated by one or more extracellular signals. Especiallyduring early development,such "cross talk" be-

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tween signaling pathways and the resultant sequentialalterations in the pattern of gene expressioneventually can become so extensivethat the cell assumesa different developmental fate. The receivingcell's prior history and regulatory state can alter the effect of a signal; the same signal applied to different cells will elicit distinct responses. The pathways we discussin this chapter have been conservedthroughout evolution and operate in much the same manner in flies, worms, and man. The substantialhomology exhibited among many proteins in these signaling pathways has enabledresearchersto employ a variety of experimental approachesand systemsto identify and study the function of extracellularsignalingmolecules,receptors,and intracellular signal-transductionproteins.For instance,the secretedsignaling protein Hedgehog(Hh) and its receptorwere first identified in Drosophila mutants with developmentaldefects.Subsequently,the human and mouse homologs of theseproteins were cloned and shown to participate in a number of important signalingeventsduring differentiation.Abnormal activation of the Hh pathway occursin severalhuman tumors. The examplesexplored in this chapter illustrate the importance of studying signalingpathways both genetically-in flies, mice, worms, yeasts,and other organisms-and biochemically. Most of the discussion in this chapter is organized in terms of individual signalingpathways. That is, we consider the signaling molecules,their receptors,the intracellular

STAT proteins

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|

(seeFigure16-1).Manyreceptor to the nucleus translocate cytosol (RTKs), kinases receptor tyrosine receptors, cytokine including classes, signals by morethan cantransduce receptors, andG protein-coupled B;PKC: A; p63: proteinkinase onepathwayp14 : proteinkinase protein k i n a sC e ; P L C: p h o s p h o l i p aCs.e

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signal-transductionpathway(s), the regulated transcription factors, and regulation of the pathway itself for each of the receptorclassesshown in Figure 1,6-1,.\nChapters2l and22,we examine how extracellular signalsaffect generegulation during severalcrucial developmentalstages,and in particular how cells integrate the responsesto multiple signals.In Chapter 25 we illustrate how abnormalities in severalsignal-transduction pathways describedin this chapter can lead to cancer.

TGFBReceptors and the Direct Activationof Smads 'We

begin our survey of signaling systemsthat control gene activity with one of the simplest: one family of signaling molecules(the TGFB superfamily) binds to its receptors(the TGFB receptors)and activatesone classof transcription factors (the Smads),which are located in the cytosol; activated Smads then move into the nucleus to regulate transcription (seeFigure 16-1a). Unlike many of the other signaling systems presentedin this chapter, the TGFB recepror activates only one type of transcription factor) and the transcription factor is activated by only one type of receptor.However, in spite of its simplicitg the TGFB pathway can have widely diverseeffectsin different types of cells becausedifferent members of the TGFB superfamily activate different members of the TGFB receptor family that activate different membersof the Smad class of transcriprion factors. Additionally, the sameactivatedSmadprotein will partner with different transcription factors in different cell types and thus activate different setsof genesin thesecells. The transforming growth factor p (TGFp) superfamily includesa number of relatedextracellularsignalingmolecules that play widespreadroles in regulating developmentin both invertebratesand vertebrates.One member of this superfamlIy, bone morphogeneticprotein (BMP), initially was identified by its ability to induce bone formation in cultured cells. Now called BMP7, it is usedclinically to strengthenbone after severefractures. Of the numerous BMP proteins subsequently recognized,many help induce key steps in development, including formation of mesoderm and the earliest blood-forming cells.Most have nothing to do with bones. Another member of the TGFB superfamily, now called TGFB-1, was identified on the basisof its ability to induce a malignant phenotype in certain cultured mammalian cells ("transforming growth factor"). However, the three human TGFB isoforms that are known all potently preuent proliferation of most mammalian cells by inducing synthesisof proteins that inhibit the cell cycle. TGFB is produced by many cells in the body and inhibits growth both of the secreting cell (autocrine signaling) and neighboring (paracrine signaling) cells. Loss of TGFB receptorsor any of severalintracellular signal-transductionproteins in the TGFB pathway, thereby releasingcells from this growth inhibition, occurs frequently in human tumors. TGFB proteins also promore expression of cell-adhesionmoleculesand extracellular-matrixmolecules, which play important roles in tissue organization 668

C H A P T E R1 5

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(Chapter 19). A Drosophila homolog of TGFB, called Dpp protein, participatesin dorsal-ventralpatterning in fly embryos. Other mammalian members of the TGFB superfamily, the activins and inhibins, affectearly developmentof the genital tract. rWe consider such developmentally important TGFb proteins in Chapter 22. Despitethe complexity of cellular effectsinduced by various members of the TGFB superfamily the signaling pathway is basicallya simple one (seeFigure 16-2a). Once activated, receptorsfor theseligands directly phosphorylate and activate a particular type of transcription factor. The responseof a given cell to this activated transcription factor dependson the constellation of other transcription factors it already contains. In this section, we will progress sequentially through the TGFB pathway, considering first the family of signal molecules,then the TGFB receptors and their discovery.Next we presentinformation about how thesereceptors activate Smad transcription factors and the feedback loops that regulate signaling by this pathway. The role that TGFB plays in cancer completesour examination of TGFPSmad signaling.

A T G F FS i g n a l i n gM o l e c u l el s F o r m e d by Cleavageof an lnactivePrecursor Most animal cell types produce and secretemembers of the TGFB superfamily in an inactive form that is stored nearby in the extracellular matrix. Releaseof the active form from the matrix by protease digestion or inactivation of an inhibitor leads to quick mobilization of the signal already in place-an important feature of many signalingpathways. In humans TGFB consistsof three isoforms, TGFB-1, TGFB-2, and TGFB-3, each encoded by a unique gene and expressedin both a tissue-specificand developmentallyregulated fashion. Each TGFB isoform is synthesizedas part of a larger dimeric precursor, linked by a disulfide bond, that contains a pro-domain (often called LAP). After the precursor is secreted,LAP is cleavedoff but remains noncovalently bound to the mature TGFB via interactions betweenspecific four amino acid sequencesin each polypeptide. Most secreted TGFB is stored in the extracellular matrix as a latent, inactive complex containing the cleaved TGFB precursor and a disulfidelinked protein called latent TGFB-binding protein (LTBP). Binding of LAP by the matrix protein thrombospondin triggers releaseof mature, active dimeric TGFP. Alternatively digestion of the binding proteins by serum proteasesor by metalloproteasespresentin the matrix 'J,6-3a). can result in activation of TGFB (Figure The monomeric form of TGFB growth factors contalns three conservedintramolecular disulfide linkages. An additional cysteine in the center of each monomer links TGFB monomers into functional homodimers and heterodimers (Figure L6-3b). Much of the sequencevariation among different TGFB proteins is observedin the N-terminal regions, the loops joining the B strands, and the o. helices.Different heterodimeric combinations may increasethe functional diversity of these proteins beyond that generated by differencesin the primary sequenceof the monomer.

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16-3 Formationand structureof TGFpsuperfamily A FIGURE (a)StepIl: Dimeric of signalingmolecules. TGFBprecursors form inside formisshownin the the cell,although onlythe monomeric top diagramSoonafterbeingsecreted, a precursor molecule is cleaved, butthe pro-domain, oftencalledLAP, andthe matureTGFB remainnoncovalently interactions boundby specific betweenLSKL a n dR K P K a m i n oa c i ds e q u e n c er se,s p e c t i v ei nl ya, c o m p l etxh a t protein(LTBP, alsocontains latentTGFB-binding orange). Mature monomeric TGFP(blueandgreen)contains sixconserved cysteine (yellow residues circles), whichformthreeintrachain disulfide bonds; (shownin second a singledisulfide bondconnects two monomers resulting diagram). Theentirecomplex fromcleavage isstoredin the

RadioactiveTaggingWas Usedto ldentify TGFpReceptors Researchersfirst identified the TGFB signaling molecule as a growth inhibitory factor,but to understandthe way it worked, they had to find the receptorsto which it bound. The logic of how they went about their searchis representativeof typical biochemical approachesto identifying receptors. Investigators first reactedthe purified growth factor with the radioisotope iodine-1ZS1125i;under"conditionssuch that the iodine

TGFB matrixStepZ: Maturehomo-or heterodimeric intracellular by bindingof theextracellular fromthiscomplex canbe released (TSP-1) in the sequence to the LSKL matrixproteinthrombospondin-1 candigestthe binding serumproteases LAPproteinAlternatively, of mature proteins, activeTGFP(b)Inthisribbondiagram releasing areshownin greenandblue.DisulfideTGFBdimelthetwo subunits (yellowandred)areshownin ball-and-stick residues linkedcysteine (red)in eachmonomer linkages disulfide form.Thethreeintrachain (a) to degradation whichisresistant lPart domain, forma cystine-knot 14:627 andDevel. 2000,Genes ' andJ E andY-G Chen, seeJ Massagu6 Factor Rev11:59 Growth 2000,Cytokine andM Poczatek, Murphy-Ullrich (b) 257:369 1 992, Scrence Daopin et al l Part fromS ,

covalently bonds to exposed tyrosine resrrdues-effectively taggingthem with a radioactivelabel. The "'I-labeled TGFB protein was incubatedwith cultured cells,and the incubation mixture then was treated with a chemical agent that covalently cross-linkedthe labeled TGFB to its receptorson the l2sl-labeled TGFB-receptor cell surface. Purification of the complexesrevealedthree different polypeptideswith apparent molecular weights of 55, 85, and 280 kDa, referredto as types RI, RII, and RIII TGFp receptors'respectively. T G F BR E C E P T O RASN D T H E D I R E C TA C T I V A T I O NO F S M A D S

669

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< FIGURE 16-4 TGFp/Smad signalingpathway.Steplll: In (Rlll), somecells,TGFBbindsto thetypelllTGFBreceptor which increases theconcentration of TGFBnearthe cellsurface andalso (Rll). presents TGFB to thetypell receptor StepIId: In othercells, TGFBbindsdirectly phosphorylated to Rll,a constitutively andactive kinaseStep[: Ligand-bound Rllrecruits andphosphorylates the juxtamembrane (Rl),whichdoesnot segment of thetypeI receptor directly Thisreleases bindTGFB. the inhibition of Rlkinase activity thatotherwise is imposed bythesegment of Rlbetween the membrane anditskinase domainStepB: Activated Rlthen phosphorylates Smad3(shownhere)or anotherR-Smad, causing a conformational changethatunmasks itsnuclear-localization signal (NLS). StepZl: Twophosphorylated molecules of Smad3interact (Smad4), with a co-Smad whichis not phosphorylated, andwith importinF (lmp-p), forminga largecytosolic complexStepsg and translocates intothe nucleus, Ran.GTP @: Aftertheentirecomplex causes dissociation of lmp-Basdiscussed in Chapter13.Stepfl:A nuclear transcription factor(e.9.,TFE3) thenassociates with the Smad3/Smad4 complex, formingan activation complex that geometry cooperatively bindsin a precise to regulatory sequences of a targetgene.Shownat the bottomistheactivation complex for the g e n ee n c o d i npgl a s m i n o g e an c t i v a t oi nr h i b i t o(rP A l - 1[)S eZ e Xiao et al, 2000,J BiolChem275:23425; J Massagu6 andD Wotton, 2000, EMBO J 19:1745; X Huaetal, 1999, ProcNat'lAcadSciUSA 96:13130; andA Moustakas andC -H Heldin, 2002,Genes Devel16:1867 l

ine and threonine residuesin a highly conservedsequenceof the RI subunit adjacent to the cytosolic face of the plasma membrane, thereby activating the RI kinase activiry. Nucleus

ActivatedTGFFReceptorsPhosphorylateSmad TranscriptionFactors

Smad3-P

Figure16-4 (stepsIl and [)depicts the relationshipand function of the three TGFB receptor proteins. The most abundant, RIII, is a cell-surfaceproteoglycan, also called pglycan. RIII, a monomeric transmembrane protein, binds and concentratesmature TGFB moleculesnear the cell surface, facllitating their binding to RII receptors. The type I and type II receptors are dimeric transmembrane proteins with serine/threoninekinases as part of their cytosolic domains. RII exhibits constitutive kinase activity; that is, it is active even when not bound to TGFB. Binding of TGFB induces the formation of complexes containing two copies each of RI and RII. An RII subunit then phosphorylatesser570

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Researchersidentified the transcription factors downstream from TGFB receptorsin Drosopbila from geneticstudiesusing mutant fruit flies. These transcription factors in Drosophila and the related vertebrate proteins are now called Smads.Three types of Smad proteins function in the TGFB signaling pathway: R-Smads (receptor-regulated Smads),co-Smads,and l-Smads (inhibitory Smads). As illustrated in Figure 16-4, an R-Smad (Smad2 or Smad3) contains two domains, MH1. and MH2, separated by a flexible linker region. The N-terminal MH1 domain contains the specific DNA-binding segment and also a sequence called the nuclear-localization signal (NLS). NLSs are presentin virtually all transcription factors found in the cytosol and are required for their transport into the nucleus (Chapter13). \fhen R-Smadsare in their inacive, nonphosphorylated state, however,the NLS is masked and the MH1 and MH2 domains associatein such a way that they cannot bind to DNA or to a co-Smad.Phosphorylation of three serine residuesnear the C-terminus of an R-Smad by activated type I TGFB receptors separatesthe domains, permitting binding of importin B (seeFigure 13-35) to the NLS, which enablesentranceof the Smad into the nucleus.

c E L L s l c N A L l N G t t : S t G N A L t N GP A T H W A y sr H A T c o N T R o L G E N EA c l v t r y

Simultaneouslya complex containing two molecules of Smad3 (or Smad2) and one molecule of a co-Smad (Smad4) forms in the cytosol. This complex is stabilizedby binding of two phosphorylatedserinesin each Smad3 to phosphoserinebinding sitesin both the Smad3and the Smad4MH2 domains. The bound importin B then mediatestranslocation of the heteromeric R-Smad/co-Smadcomplexes into the nucleus. After importin B dissociatesinside the nucleus,the Smad2/Smad4or Smad3/Smad4complexesbind to other transcription factors to activate transcription of specifictarget genes. Vithin the nucleus R-Smads are continuously being dephosphorylated, which results in the dissociation of the RSmad/co-Smadcomplex and export of theseSmadsfrom the nucleus.Becauseof this continuous nucleocytoplasmicshuttling of the Smads,the concentration of active Smadswithin the nucleus closely reflects the levels of activated TGFB receptors on the cell surface. Virtually all mammalian cells secreteat least one TGFB isoform, and most have TGFB receptors on their surface. However, becausedifferent types of cells contain different sets of transcription factors with which the activated Smadscan bind, the cellular responsesinduced by TGFB vary among cell types.In epithelialcellsand fibroblasts,for example,TGFB induces expression not only of extracellular-matrix proteins (e.g., collagens)but also of proteins that inhibit serum proteases,which otherwise would degrade these extracellularmatrix proteins.This inhibition stabilizesthe matrix, allowing cells to form stable tissues.The inhibitory proteins include plasminogen activator inhibitor 1 (PAI-1). Transcription of the PAI-1 gene requiresformation of a complex of the transcription factor TFE3 with the Smad3/Smad4complex and binding of all theseproteins to specificsequenceswithin the regulatory region of the PAI-1 gene(seeFigure 16-4, bottom). By partnering with other transcription factors,Smad2/Smad4 and Smad3iSmad4complexespromote expressionof proteins such as p15, which arreststhe cell cycle at the G1 stageand thus blocks cell proliferation (Chapter20). As just discussed,binding of any one TGFB isoform to its specific receptors leads to phosphorylation of Smad2 or Smad3 followed by formation of Smad2/Smad4 or Smad3/Smad4complexes,and eventually transcriptional activation of specifictargetgenes(e.g.,the PAl-1 gene).On the other hand, BMP proteins, which also belong to the TGFB superfamily,bind to and activate a different set of receptors that are similar to the TGFB RI and RII proteins but phosphorylate Smadl (rather than Smad2 or Smad3). Smadl then dimerizeswith Smad4, and the Smadl/Smad4 complex activates different transcriptional responsesthan those induced by Smad2/Smad4or Smad3/Smad4.

NegativeFeedbackLoopsRegulate TGFp/SmaS d ignaling Most signalingpathwaysare modulatedin sucha way that the responseto a growth factor or other signalingmoleculeis decreased(or occasionallyincreased)with time; this enablesthe fine-tuned control of cellular responses. TGFB/Smad pathways are regulated by severalintracellular proteins, in-

15-5 Model of Ski-mediateddown-regulationof A FIGURE Smad function. Skirepresses Smadtranscription-activating domain theSki-binding to Smad4. Since functionby bindingdirectly for binding with the domainrequired overlaps significantly on Smad4 the normal bindingof Skidisrupts tailof Smad3, the phosphorylated Additionally, Skialsorecruits andSmad4. between Smad3 interactions in turn mSin3A to mSin3A; whichbindsdirectly the proteinN-CoR, (HDAC), that promotes an enzyme histone deacetylase with interacts (Chapter activation transcription 7) As a result, deacetylation histone isshutdown. by Smadcomplexes byTGFBandmediated induced andDeu14:.65,] [SeeK Luo,2004,Curr.OpinGenet. cluding tvvocytosolicproteinscalledSaoNand SAr(Ski stands for "Sloan-KetteringCancer lnstitute"). Theseproteins were originally identified as cancer-causingoncoproteins because in they causeabnormal cell proliferation when overexpressed this cultured primary fibroblast cells. How they accomplish was not understooduntil yearslater when SnoN and Ski were found to bind to both Smad4 and phosphorylatedSmad3 after TGFB stimulation.SnoN and Ski do not preventformation of Smad3/Smad4complexes or affect the ability of the Smad complexesto bind to DNA control regions.Rather,they block transcription activation by the bound Smad complexes, thereby rendering cells resistant to the growth-inhibitory effects of TGFB (Figure 16-5). Interestingly, stimulation by TGFp causesthe rapid degradationof Ski and SnoN' but after a few hours, expression of both Ski and SnoN becomes strongly induced via mechanismsnot yet understood.The increasedlevelsof theseproteins are thought to dampen longterm signalingeffectsdue to continued exposureto TGFB. Among the proteins induced after TGFB stimulation are the I-Smads,especiallySmad7. SmadT blocks the ability of activated type I receptors (RI) to phosphorylate R-Smads proteins, and it may also target TGFB receptorsfor degradation. In theseways Smad7,like Ski and SnoN, participatesin negative feedback loops: Its induction inhibits intracellular signalingby long-term exposureto the stimulating hormone' In later sectionswe see how signaling by other cell-surface receptorsis also restrained by negativefeedbackloops.

L o s so f T G F pS i g n a l i n gP l a y sa K e y R o l e in Cancer Many human tumors contain inactivating mutations in either TGFB receptorsor Smad proteins, and thus are resistant to growth inhibition by TGFB (seeFigure 25-24). T G F P R E C E P T O RASN D T H E D I R E C TA C T I V A T I O NO F S M A D S

671

Most human pancreatic cancers, for instance, conrarn a deletion in the geneencoding Smad4 and thus cannot induce p15 and other cell-cycleinhibitors in responseto TGFB. In fact, Smad4was originally called DPC (deletedin pancreatic cancer).Retinoblastoma,colon and gastriccancer,hepatoma, and some T- and B-cell malignanciesare also unresponsive to TGFB growth inhibition. This loss of responsiveness correlates with loss of type I or type II TGFP receprors; responsiveness to TGFB can be restored by recombinant expressionof the "missing" protein. Mutations in Smad2 also commonly occur in several types of human tumors. Not only is TGFB signaling essentialfor controlling cell proliferation, as theseexamplesshow, but it also causessome cells to differentiate along specificpathways, as discussed in Chapter 22. I

TGFp Receptorsand the Direct Activation of Smads r TGFB is producedas an inactiveprecursorthat is stored in the extracellular matrix. Severalmechanismscan release the active, mature dimeric growth factor (seeFigure 16-3). r Stimulation by TGFB leads to activation of the intrinsic serine/threoninekinase activity in the cytosolic domain of the type I (RI) receptor,which then phosphorylatesan RSmad, exposing a nuclear-localizationsignal. r After phosphorylated R-Smad binds a co-Smad, the resulting complex translocatesinto the nucleus,where it interacts with various transcription factors to induce expression of target genes(seeFigure 16-4). r Oncoproteins (e.g., Ski and SnoN) and I-Smads (e.g., SmadT)act as negativeregulators of TGFB signaling. r TGFB signaling generallyinhibits cell proliferation. Loss of various components of the signaling pathway contributes to abnormal cell proliferation and malignancy.

CytokineReceptors and the JAK/STAT Pathway 'S7e turn now to another type of signaling pathway that Ieads to long-term genetic effects by activation of transcription factors. The signal moleculesin this pathway, the cytokines, play many important roles in growth and differentiation of cells, especiallyblood and immune sysrem cells. Like the TGFB pathway just described,the cytokine pathway involves only a few steps.The cytosolic domain of all cytokine receptors tightly binds a member of a family of cytosolic protein tyrosine kinases, the JAK kinases. Activated JAK kinases,in turn, directly phosphorylateand activate transcription factors that are members of the S?HT (SignalTransduction and Activation of Transcription) family. Activated cytokine receptors activate additional pathways (see Figure 1,6-2b) rhat are also activated by other receptor classes.However, the JAK/STAT pathway describedin this sectionis initiated mainly by activation of 672

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cytokine receptors,although it can be activated by other receptors. 'We begin by discussingthe cytokine family of signaling molecules and the cytokine receptors. Next, we use an experimental approach to explore how the JAK/STAT pathway was discovered.Then we consider the details of how a JAK protein activatesa STAT transcription factor, followed by a discussion of how cytokine signaling paths are regulated. This section concludeswith a biological application exploring the regulation of red blood cells in the human body.

C y t o k i n e sI n fl u e n c eD e v e l o p m e n t of Many CellTypes The cytokines form a family of relatively small, secretedsignaling molecules (generallycontaining about 160 amino acids) that control many aspectsof growth and differentiation of specific types of cells. During pregnancy,for example, the cytokine prolactin induces epithelial cells lining the immature ductules of the mammary gland to differentiate into the acinar cells that produce milk proteins and secrere them into the ducts. Other cytokines, the interleukins, are essentialfor proliferation and functioning of T cells and antibody-producing B cells of the immune system.Another family of cytokines, the interferons, are produced and secreted by certain cell types following virus infection. The secreted interferons act on nearby cellsto induce enzymesthat render these cells more resistant to virus infection. The role of interleukins and interferons in immune responsesare covered in Chapter 24. Many cytokines induce formation of important types of blood cells. For instance,granulocyte colony srimu, lating factor (G-CSF)inducesa particular type of progenitor cell in the bone marrow to divide severaltimes and then differentiateinto granulocytes,the type of white blood cell that inactivatesbacteria and other pathogens.Becausemany cancer therapiesreduce granulocyte formation by the body, GCSF often is administeredto patients to stimulate proliferation and differentiation of granulocyte progenitor cells,thus restoring the normal level of granulocytesin the blood. Thrombopoietin, a "cousin" of G-CSF,similarly acts on megakaryocyteprogenitors to divide and differentiate into megakaryocytes.These then fragment into the cell pieces called platelets,which are critical for blood clotting. I Another cytokine, erythropoietin (Epo), triggers production of erythrocytes (red blood cells) by inducing the proliferation and differentiation of erythroid progenitor cells in the bone marrow (Figure 16-6). Erythropoietin is synthesized by kidney cells that monitor the concentration of oxygen in the blood. A drop in blood oxygen signifies a lower than optimal level of erythrocytes,whose major function is to transport oxygen complexed to hemoglobin. By means of the oxygen-sensitivetranscription factor HIF-1ct, the kidney cells respond to low oxygen by synthesizingmore erythropoietin and secretingit into the blood. As the level of erythropoietin rises, more and more erythroid progenitors are

pA c E L Ls T G N A L T N r :Gs T G N A L T N GT H W A y rsH A T c o N T R 9 LG E N EA c l v r r y

Hematoooieticstem cell

Epo receptors Progenitorsof other types of blood cells

Erythroidprogenitor(CFU-E)

*"r0"|

r-

-l-l

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Epo

coo-

coo-

Membranesurface

AAAA .4,4 A A A N N,.N t I I l. .1..1..1.J .t .l. l. t J t I I Mature red cells

16-6 Erythropoietin and formationof red blood A FIGURE progenitor cells(erythrocytes).Erythroid cells,calledcolony-forming (CFU-E), arederived fromhematopoietic stemcells, unitserythroid whrchalsogiveriseto progenitors of otherbloodcelltypes.Inthe (Epo), CFU-E cellsundergoapoptosis. absence of erythropoietin induces of on a CFU-E transcription Binding of Epoto itsreceptors proteins prevent programmed geneswhoseencoded cell several allowing the cellto surviveOtherEpo-induced death(apoptosis), program proteins of threeto fiveterminal triggerthe developmental proteins are TheEporeceptor andothermembrane celldivisions lf CFU-E cells differentiation lostfromthesecellsastheyundergo ( e . 9 .c, o n t a i n i n g a r ec u l t u r ewd i t hE p oi n a s e m i s o lm i de d i u m methylcellulose), daughter cellscannotmoveaway,andthuseach produces a colonyof 30-100erythroid cells,henceitsname CFU-E Blood98:3261 et al, 2OO1, ) lSeeM Socolovsky

saved from death, allowing each to produce -50 or so erythrocytes in a period of only a few days. In this way, the body can respond to the loss of blood by acceleratingthe production of erythrocytes.

CytokineReceptorsHave SimilarStructures a n d A c t i v a t eS i m i l a rS i g n a l i n gP a t h w a y s Strikingln all cytokines have a similar tertiary structure, consistingof four long conserveda helicesfolded together in a specific orientation. The structural homology among

16-7 Structureof erythropoietinbound to an a FIGURE (Epo)containsfour erythropoietinreceptor.Erythropoietin arrangement. thatarefoldedin a particular longcthelices conserved (EpoR) isa dimerof identical receptor erythropoietin Theactivated of isconstructed domainof eachmonomer theextracellular subunits; p strands foldedin sevenconserved eachcontaining two subdomains on two of the helices of residues Sidechains fashion. a characteristic on the whileresidues in Epocontactloopson oneEpoRmonomer, in a second loop segments the same bindto two otherEpohelices in a receptor the dimeric stabilizing thereby monomer, receptor andtheir of othercytokines Thestructures conformation. specific etal, fromR 5 Syed to EpoandEpoR. aresimilar receptors [Adapted 1l 395:51 1998,Nature cytokines is evidencethat they all evolved from a common ancestral protein. Likewise, the various cytokine receptors undoubtedly evolved from a single common ancestor as all cytokine receptorshave similar structures.Their extracellular domains are constructed of two subdomains' each of which contains sevenconservedB strands folded together in a characteristicfashion. The interaction of one erythropoietin molecule with two identical erythropoietin receptor (EpoR) proteins, depicted in Figure 16-7, exemplifies the binding of a cytokine to its receptor. Whether or not a cell responds to a particular cytokine depends simply on whether or not it expressesthe corresponding (cognate)receptor'Although all cytokine receptors activate similar intracellular signal-transduction pathways' the responseof any particular cell to a cytokine signal depends on the cell's constellation of transcription factors, chromatin structures,and other proteins relating to the developmental history of the cell. If receptorsfor prolactin or thrombopoietin, for example, are expressedexperimentally in an erythroid progenitor cell' the cell will respond to these cytokines by dividing and differentiating into erythrocytes, not into mammary cells or megakaryocytes. Figure 16-8 summarizesthe intracellular signaling pathways activated when the EpoR binds erythropoietin' Stimulation of other cytokine receptors by their specific ligands C Y T O K I N ER E C E P T O RASN D T H E J A K / s T A TP A T H W A Y

.

673

(a) STATs

(bl GRB2or Shc

Transcriptionalactivation

--+

(c) PhospholipaseC",+

(d) Pl-3kinase

+

Ras +

MAP kinase +

Transcriptional activationor repression

trlevatlOnol Vaz++

Transcriptionalactivationor repression Modificationof other cellularproteins

protein kinaseB --+

activationor repression I:tn::''o:'onul Modificationof other cellularproteins

FIGURE 16-8 Overviewof signal-transduction pathways triggeredby the erythropoietinreceptor.Erythropoietin (Epo) bindsto theerythropoietin (EpoR), receptor activating theassociated JAKkinaseFourmajorpathways cantransduce a signafromthe phosphorylated activated, EpoR-JAK complexEachpathway ultimately regulates transcription of different setsof genes(a)Inthemostdirect pathway, discussed in th s section, thetranscription factorSTAT5 is phosphorylated andactivated directly in the cytosol(b)Binding of

adapterproteins(GRB2or Shc)to an activatedEpoRleadsto activation of the Ras/MAPkinasepathway(Section16 4) (c, d) Two phosphornositidepathwaysare triggeredby recruitmentof phospholipase C" and Pl-3kinaseto the membrane(Section16 5) followingactivation of EpoR Elevatedlevelsof Ca2* and activatedproteinkinaseB also modulatethe activityof cytosolicproteinsthat are not involvedin controlof transcription

activatessimilar pathways. All these pathways eventually lead to activation of transcription factors, causing an increaseor decreasein expressionof particular target genes. Here we focus on the JAK/STAT pathway; the other pathw a y s a r e d i s c u s s e idn l a r e rs e c t i o n s .

but cannot be phosphorylated. In erythroid cells, expression of this mutant JAK2 in greater than normal amounrs totally blocks EpoR signaling, because the mutant JAK2 binds to the majority of cytokine receprors, prevenring binding and functioning of the wild-type JAK2 protein. This type of mutation, referred to as dominant-negative, causes loss of function even in cells that carry copies of the wild-type gene (Chapter 5). Once the JAK kinases become activated, they phosphorylate several tyrosine residues on the cytosolic domain of

JAK KinasesActivate STATTranscriptionFactors To understandhow JAK and STAT proteins function, we examine the pathway downstreamof the erythropoietinreceptor (EpoR), one of the best-understoodcytokine signaling pathways. A JAK2 kinase is tightly bound to the cytosolic domain of all cytokine receprors(seeFigure 16-1b). Like the three other members of the JAK family of kinases,JAK2 containsan N-terminal receptor-bindingdomain, a C-terminal kinase domain that is normally poorly active catalytically, and a middle domain that regulateskinase activity by an unknown mechanism.JAK2, erythropoietin, and the EpoR are all required for formation of adult-type erythrocytes,which normally beginsat day 12 of embryonicdevelopment in mice.As Figure 16-9 shows,embryonicmice lacking functional genes encoding the EpoR cannot form adult-type eryrhrocytesand eventually die owing to the inability to transport oxygen to the fetal organs. Mice lacking functionalgenesencodingEpo or JAK2 show similar blocks in fetal development. As already nored, erythropoietin binds simultaneouslyto the extracellulardomains of two EpoR monomers on the cell surface(seeFigure16-7).As a resuh,the associared JAKs are brought closeenough together so that one can phosphorylate the other on a critical tyrosine in a region of the protein called the actiuationlip (Ftgure16-10).As with other kinases,phosphorylation of the activation lip leads to a conformational change that reducesthe K., for ATP or the substrateto be phosphorylated,thus increasingkinase activity. One pieceof evidencefor this activation mechanismcomes from study of a mutant JAK2 in which the critical tyrosine is mutated to phenylalanine.The mutant JAK2 binds normally to the EpoR 674

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+/+

-l-

a EXPERIMENTA FLI G U R E1 6 - 9 S t u d i e sw i t h m u t a n t m i c e reveal that the erythropoietin receptor (EpoR) is essential for development of erythrocytes. Mice in which both allelesof the g e n ee n c o d i n gE p o Ra r e " k n o c k e do u t " d e v e l o pn o r m a l l yu n t i l e m b r y o n i dc a y 1 3 ,w h e n t h e yb e g i nt o d i e o f a n e m i ad u e t o t h e l a c k of erythrocyte-mediated transportof oxygento the fetal organs The red organ in the wild-typeembryos(+/+) is the fetal liver,the major siteof erythrocyteproductionat this developmental stage The absenceof color in the mutant embryos(-/-) indicatesthe absence of erythrocytes containinghemoglobinOtherwisethe mutant e m b r y o sa p p e a rn o r m a l ,i n d i c a t i ntgh a t t h e m a i nf u n c t i o no f t h e EpoRin earlymousedevelopmentis to supportproductionof erythrocytes[FromH Wu et al , 1995,Ce//83:59]

c E L Ls r c N A L I N rG: s T G N A L TpNAGT H W A yrsH A T c o N T R o LG E N EA c r v r r y

LigandLigand binding sites

B o u n dl i g a n d

a

Transmembrane o helix

Activation lip JAK

olN"

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Active JAK

z

E Cytokinereceptors without bound ligand

E Phosphorylation of additional tyrosineresidues

D i m e r i z a t i o an n d phosphorylationof activationlip tyrosines

16-10Generalstructureand activationof cytokine A FIGURE receptors Thecytosolic domainof cytokine tightlyand receptors. of with a separate ln the absence irreversibly associates JAKkinase. forma homodimer are ligand(tr), the receptors but theJAKkinases poorlyactiveLigandbindingcauses that a conformational change whichthen domains, the associated JAKkinase bringstogether phosphorylate residue in the activation eachotheron a tyrosine

the receptor (seeFigure 16-1,0,B). Severalof thesephosphotyrosine residuesthen serveas binding sitesfor SH2 domains, which are part of many signal-transductionproteins, including the STAT group of transcription factors. The SH2 domain derived its full name, the Src Domology 2 domain, from its homology with a region in the prototypical Src cytosolic tyrosine kinase encoded by the src gene. (Src is an acronym for sarcoma, and a mutant form of the cellular src gene was found in chickens with sarcomas,as Chapter 25 details.)The three-dimensionalstructuresof SH2 domains in different proteins are very similar, but each binds to a distinct sequenceof amino acids surrounding a phosphotyrosine residue.The unique amino acid sequenceof each SH2 domain determinesthe specific phosphotyrosine residuesit binds (Figure 1.6-11\.Variations in the hydrophobic socket in the SH2 domains of different STATs and other signaltransductionproteinsallow them to bind to phosphotyrosines adjacentto different sequences,accounting for differencesin their binding partners. All STAT proteins contain an N-terminal DNA-binding domain, an SH2 domain that binds to a specificphosphotyrosine in a cytokine receptor'scytosolic domain, and a Cterminal domain with a critical tyrosine residue. Once a STAT is bound to the receptor, the C-terminal tyrosine is phosphorylatedby an associatedJAK kinase (Figure 1'6-1'2a1. This arrangement ensuresthat in a particular cell only those STAT proteins with an SH2 domain that can bind to a particular receptor protein will be activated. The erythropoietin recepto! for example, activates STAT5 but not STATs 1, 2, 3, or 4. A phosphorylatedSTAT dissociates spontaneously from the receptor, and two phosphorylated

causes the lipto moveout of the kinase lip(E). Phosphorylation the abilityof ATPandthe protein site,thusrncreasing catalytic several thenphosphorylates kinase to bind.Theactivated substrate (E). resulting The domain cytosolic receptor's in the residues tyrosine STAT phosphotyrosines functionasdockingsitesfor inactive proteins that factorsandothersignal-transduction transcription containSH2or PTBdomatns

*q''...

15-11 Surfacemodelof a SH2domainboundto a FIGURE peptide'Thepeptideboundbythis phosphotyrosine-containing (gray)isshownin spacefill. kinase tyrosine from Src SH2domain contatning to shorttargetpeptides TheSH2domainbindsstrongly (TyrO and phosphotyrosine coresequence: four-residue a critical (lle3). acid(Glu2)-isoleucine acid(Glu1)-glutamic OPO3-)-glutamic "plu9"-the two-pronged of a rnsertion the resembles Binding sidechainsof the peptide-intoa andisoleucine phosphotyrosine residues "socket"in theSH2domainThetwo glutamate two-pronged the of the SH2domainbetween areboundto siteson the surface peptide colored are the target on residues two socketsNonbinding Cell72:7791 etal, 1993, green[See G Waksman SN D T H E J A K / s T A T P A T H W A Y C Y T O K I N ER E C E P T O RA

.

675

(a)

Epo

< FIGURE 16-12 Activationand structureof STATproteins. (a)Phosphorylation proteinsStep[: Folanddimerization of STAT (seeFigure16-10), lowingactivation of a cytokine receptor an inactive monomeric STAT transcription factorbindsto a phosphotyrosine in the receptor, bringingthe STAT closeto the activeJAKassociated with the receptor TheJAKthenphosphorylates the C-terminal tyrosine in the STAT. StepsEl and B: Phosphorylated STATs spontaneously dissociate fromthe receptor andspontaneously dimerize. Because the STAT homodimer hastwo phosphotyrosine-SH2 domaininteractions, whereas the receptor-STAT complex isstabilized by onlyone phosphorylated suchinteraction, STATs tendnotto rebindto the receptorStep@: TheSTAT dimermovesintothe nucleus, whereit canbindto promoter sequences andactivate transcription of target (b)Ribbon genes. diagram of the STATldrmerboundto DNA(black). TheSTAT1 dimerformsa C-shaped clamparoundDNAthatisstabilizedby reciprocal andhighlyspecific interactions between the SH2 domain(purple) of onemonomer andthe phosphorylated tyrosine (yellow) residue on the C-terminal segment of the otherThephosphotyrosine-binding siteof the SH2domainin eachmonomer is coupled structurally to the DNA-binding domain(magenta), suggesting a potential rolefor the SH2-phosphotyrosine interaction in thestabilization (b)afterX Chen of DNAinteracting elementsIpart et al, 1998,Cell93:827 l

Epo receptor

Active JAK kinase

E Into nucleus; b i n d sD N A and activates tra nscription (b) S H 2d o m a i n Tyrosine

S H 2d o m a i n Tyrosine Poo

DNAbinding domain

STAT proteins form a dimer in which the SH2 domain on each binds to the phosphotyrosinein the other. Because dimerization also exposesthe nuclear-localizationsignal (NLS), STAT dimers move into the nucleus,where they bind to specific enhancers(DNA regulatory sequences)controlIing target genes(Figure 1.6-12b). Different STATsactivaredifferent genesin different cells. As just noted, stimulation of erythroid progenitor cells by erythropoietin (Epo) leads to activation of STAT5. The major protein induced by active STAT5 is Bcl-x1, which prevents the programmed cell death, or apoptosis,of theseprogenitors, allowing them to proliferate and differentiate into

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erythroid cells (seeFigure 1,6-6).In the normal state.when Epo levelsare low, bone marrow stem cellscontinuously create progenitor erythroid cells that are quickly destroyed. This energeticallyexpensive process allows the body to respond to the need for more erythrocytes very quickly in responseto a rise in Epo levels.Indeed, mice lacking STAT5 are highly anemic becausemany of the erythroid progenitors undergo apoptosis even in the presenceof high erythropoietin levels.Suchmutant mice produce some erythrocytesand thus survive, becausethe erythropoietin receptor is linked to other anti-apoptotic pathways that do not involve STAT proteins (seeFigure 16-8).

C E L LS I G N A L T N G i l : s t G N A L | N Gp A T H W A y s r H A T c o N T R o L G E N EA c l v t r y

ComplementationGeneticsRevealedThat JAK and STATProteinsTransduceCytokineSignals Researchershave long known about cytokine signaling molecules,and once radioactive tagging (or expressioncloning) was applied to cytokine-responsivecells,it did not take long to find the cytokine receptors, as describedin the previous section for TGFB receptors. However, without new techniques researchershad no way to explore the intracellular signalingpathways that transducecytokine signalsfor many years.A new type of functional geneticscreening(functional complementation) led researchersto both JAK proteins and STAT transcription factors. In the first part of thesestudies,a bacterial reporter gene encoding guanine phosphoribosyl transferase(GPRT) was linked to an upstream promoter that is activated by the antiviral cytokine interferon. The resulting construct was introduced into cultured mammalian cells that were genetically deficient in the human homolog HGPRT. Either GPRT or HGPRT must be expressedfor a cell to incorporate purines in the culture medium into ribonucleotidesand then into DNA or RNA. As shown in Figure 1.6-1.3a,HGPRTnegativecells carrying the reporter gene respondedto interferon treatment by expressingGPRI thus acquiring the ability to grow in HAT medium. Cells lacking GPRT or HGPRT cannot grow in HAT medium since synthesisof purines by the cells is blocked by aminopterin (the A in HAI); thus DNA synthesisby the cells is dependenton incorporation of purinesfrom the culture medium (seeFigure 9-36). Simulta-

neously the cells acquired sensitivity to killing by the nonnatural purine analog 6-thioguanine,which is convertedinto the correspondingribonucleotide by GPRT; incorporation of this purine into DNA in place of guanosineeventuallycauses cell death. The reporter cells were then heavily treated with mutagens in an attempt to inactivate both alleles of the genes encoding critical signal-transduction proteins in the interferon signalingpathway. Researcherslooked for mutant cells that expressedthe interferon receptor (as evidencedby the cell's ability to bind radioactive interferon) but did not express GPRT in response to interferon and thus survived killing by 6-thioguaninewhen cellswere cultured in the presence of interferon (Figure 1'6-13b). After many such interferon-nonrespondingmutant cell lines were obtained, they were used to screena genomic or cDNA library for the wildtype genes that complemented the mutated genes in nonresponding cells, a technique called functional complementation (seeFigure 5-18). In this case, mutant cells expressingthe corresponding recombinant wild-type gene grew on HAT medium and were sensitiveto killing by 6-thioguaninein the presenceof interferon. That is, they actedlike wild-type cells. Cloning of the genesidentified by this procedure led to recognition of two key signal-transductionproteins that are activated by the cytokine interferon: a JAK tyrosine kinase and a STAT transcription factor. Subsequentwork showed that one (sometimestwo) of the four human JAK proteins and at least one of severalSTAT proteins are involved in signaling downstream from all cytokine receptors'

Interferon-responsive promoter

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(b) HGPRT-cells (+ reporter gene) defective for interferon signaling

+ Wild type gene that reslores In t e r l e r o n responsrveness

FIGURE 16-13 Mutagenizedcellscarryinga A EXPERIMENTAL as essential reportergene were usedto identify JAKsand STATs genewasconstructed proteins.A reporter signal-transduction promoter upstream of the of an rnterferon-responsive consisting in the purinesalvage geneencoding GPRT, a keyenzyme bacterial of thisconstruct pathway(seeFigure 9-36)(a)Introduction g GPRT i n t om a m m a l i ac ne l l sl a c k i ntgh em a m m a l i ahno m o l o H yielded cellsthatgrewin HATmediumandwerekilledby reporter of interferon. in the presence but not theabsence 6-thioquanine

cellswith cellswith a mutagen, (b)Following of reporter treatment do not induce by interferon pathwayinitiated in thesignaling defects thetoxic andthuscannotincorporate to interferon in response HGPRT by responsiveness of interferon Restoration purine6-thioguanine. genes identified DNA clones wild-type with complementation functional et al, R McKendry Seethetextfor details. [See JAKsandSTATs encoding 1 l9 9 3 , N a t u r e 5 ;a t l i n g e t, a c lS A 8 8 : 1 1 4D5W l c a d . 5U 1 g g,1P r o cN a t 'A 3 6 5 : 1 6 6 ;a n d G S t a r ka n d A G u d k o v 1 9 9 9 ,H u m a nM o l G e n e t 8 : 1 9 2 5l

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( a l Short-termregulation:JAK2 deactivation

< FIGURE 16-14Two mechanisms for terminatingsignal transductionfrom the erythropoietinreceptor(EpoR), (a)Short-term regulation: SHP1, phosphatase, a phosphotyrosine is present in an inactive formin unstimulated cells.Binding of an SH2 domainin SHPlto a particular phosphotyrosine in theactivated receptor unmasks itsphosphatase catalytic siteandpositions it nearthe phosphorylated tyrosine in the lip regionof JAK2.Removal of the phosphate f romthistyrosine inactivates theJAKkinase. (b)Long-term regulation: proteins, SOCS whoseexpresston ts proteins induced by STAT in erythropoietin-stimulated erythroid c e l l si ,n h i b iot r p e r m a n e nttel yr m i n a tsei g n a l i nogv e rl o n g etri m e periods, Binding of SOCS to phosphotyrosine residues on EpoR or JAK2blocksbindingof othersignaling (/eft). proteins TheSOCS boxcanalsotargetproteins suchasiAK2for degradation bythe ubiquitin-proteasome pathway(riSht). Similar mechanisms regulate signaling fromothercytokine receptors fromS lpart(a)adapted Constantinescu etal, 1999,Trends Endocrin Metabol10:1 8 part(b) adapted fromB T.KileandW 5 Alexander, 2001 , CellMot.LifeSci58:1l

by SHPl phosphatase Epo

SH2 domains

Phosphatase domain

(b) Long-termregulation:signal blockingand protein degradationby SOCSproteins

t I I I I

SOCS protein SH2 domain

Recruitment of E3 ubiquitin ligase

SOCS box

S i g n a l i n gf r o m C y t o k i n eR e c e p t o r tss R e g u l a t e d b y N e g a t i v eS i g n a l s Signal-inducedtranscription of target genesfor too long a period can be as dangerousfor the cell as too little induction. Thus cells must be able to turn off a signaling parhway quickly unlessthe extracellular signal remains continuously present. In various progenitor cells, two classesof proteins serveto dampen signaling from cytokine receptors,one over the short term (minutes)and the other over longer periods of time (hoursto days). Short-Term Regulation by SHpt, a phosphotyrosine Phosphatase Mutant mice lacking a protein calledSHpl die becauseof excessproduction of erythrocytesand severalother types of blood cells. Analysis of thesemutant mice offered the first suggestionthar SHP1, a phosphotyrosinephosphatase, negativelyregulatessignaling from severaltypes of cyt;kine receptorsin severalrypesof progenitorcells. 678

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SHP1 dampens cytokine signaling by binding to a cytokine receptor and inactivating the associatedJAK protein, as is depictedin Figure 1,6-14a.Inaddition to a phosphatase catalytic domain, SHPl has two SH2 domains.When cells are in the resting state,unstimulatedby a cytokine, one of the SH2 domains in SHP1 physically binds to and inactivates the catalytic site in the phosphatase domain. In the stimulated state, however, this blocking SH2 domain binds to a specificphosphotyrosineresiduein the activatedreceptor. The conformational changethat accompaniesthis binding unmasks the SHP1 catalytic site and also brings it adjacent to the phosphotyrosine residue in the activation lip of the JAK associatedwith the receptor. By removing this phosphate, SHP1 inactivates the JAK, so that it can no longer phosphorylatethe receptoror other substrates(e.g., STATs) unless additional cytokine molecules bind to cellsurfacereceptors,initiating a new round of signaling. Long-Term Regulation by SOCSProteins In a classicexample of negative feedback, among the geneswhose transcription is induced by STAT proteins are rhose encoding a classof small proteins, termed SOCSproteins, that terminate signaling from cytokine receptors. These negative regulators act in two ways (Figure I6-14b). First, the SH2 domain in severalSOCS proteins binds to phosphotyrosines on an activated receptor, prevenring binding of other SH2containing signaling proteins (e.g., STAIs) and thus inhibiting receptor signaling. One SOCS protein, SOCS-1, also binds to the critical phosphotyrosinein the activation lip of activatedJAK2 kinase, thereby inhibiting its catalytic activity. Second,all SOCS proteins contain a domain, called the SOCS box, that recruits components of E3 ubiquitin ligases (seeFigure 3-29). As a result of binding SOCS-I, for instance,JAK2 becomespolyubiquitinated and then degraded in proteasomes,thus permanently turning off all JAK2mediated signaling pathways. The observation that proteasome inhibitors prolong JAK2 signal transduction supports this mechanism.

C E L LS I G N A L | N Gi l : s t c N A L | N G p A T H W A y s r H A T c o N T R o L G E N EA c l v t r y

Studies with cultured mammalian cells have shown that the receptor for growth hormone, which belongs to the cytokine receptor superfamily, is down regulated by another SOCS protein, SOCS-2.Strikingly, mice deficient in SOCS-2 grow significantly larger than their wild-type counterparts and have long bone lengths and proportionate enlargementof most organs.Thus SOCS proteins play an essentialnegativerole in regulating intracellular signaling from the receptorsfor erythropoietin, growth hormone, and other cytokines.

Mutant ErythropoietinReceptorThat Cannot Be TurnedOff Leadsto IncreasedNumbers of Erythrocytes In normal adult men and women the percentageof erythrocytesin the blood (the hematocrit) is maintainedvery closeto 4547 percent.A drop in the hematocrit results in increased production of erythropoietin by the kidney, and the elevated erythropoietin level causesmore erythroid progenitors to undergo terminal proliferation and differentiation into mature erythrocytes, soon restoring the hematocrit to its normal level. In endurance sports, such as cross-country skiing, where oxygen transport to the musclesmay becomelimiting, an excessof erythrocytes may confer a competitive advantage. For this reason, use of supplementalerythropoietin to increasethe hematocrit above the normal level is banned in all international athletic competitions, and athletesare regularly tested for elevatedhematocrit and for the presenceof commercial recombinant erythroDoietinin their urine. Supplementalerythropoietinnot only confersa possible competitiveadvantagebut also can be dangerous. Too many erythrocytes can cause the blood to become sluggish and clot in small blood vessels,especiallyin the brain. Severalathleteswho doped themselveswith erythropoietin have died of a stroke while exercising. Discovery of a mutant, unregulatederythropoietin receptor (EpoR) explaineda suspicioussituation in which a winner of three gold medals in Olympic cross-countryskiing was found to have a hematocrit above 60 percent.Testing for erythropoietin in his blood and urine, however, revealed lower-than-normal amounts. Subsequent DNA analysis showed that the athlete was heterozygousfor a mutation in the geneencodingthe erythropoietin receptor. The mutant allele encoded a truncated receptor missing severalof the tyrosinesthat normally becomephosphorylated after stimulation by erythropoietin. As a consequence, the mutant receptor was able to activate STAT5 and other signaling proteins normally, but was unable to bind the negativelyacting SHPl phosphatase,which usually terminatessignaling(seeFigure 1,6-74a).Thus the very low level of erythropoietin produced by this athlete induced prolonged intracellular signaling in his erythroid progenitor cells,resulting in production of higher-than-normalnumbers of erythrocytes.This example vividly illustratesthe fine level of control over signaling from the erythropoietin receptor in the human bodv. I

Cytokine Receptors and the JAK/STATPathway r All cytokines are constructed of four ct helices that are folded in a characteristicarrangement. r Erythropoietin, a cytokine secretedby kidney cells, prevents apoptosis and promotes proliferation and differentiation of erythroid progenitor cells in the bone marrow. An excessof erythropoietin or mutations in its receptor that prevent down-regulation result in production of elevated numbers of erythrocytes. r All cytokine receptorsare closely associatedwith a JAK protein tyrosine kinase, which can activate severaldownstream signaling pathways leading to changesin transcription of target genesor in the activity of proteins that do not regulatetranscription(seeFigure 16-8). r The JAICSTAT pathway operatesdownstream of all cytokine receptors.STAI monomers bound to receptors are phosphorylatedby receptor-associated JAKs, then dimerize and move to the nucleus,where they activate transcription (seeFigure 1,6-1'2).Signalingfrom cytokine receptorsis terminated by the phosphotyrosine phosphatase SHP1 and severalSOCSproteins(seeFigure 1,6-1'4).

ReceptorTyrosineKinases 'We

now turn our attention to another large and important classof cell-surfacereceptors-the receptor tyrosine kinases (RTKs)-that regulatemany aspectsof cell proliferation and differentiation, cell survival' and cellular metabolism. The signaling moleculesthat activate RTKs are soluble or membrane-bound peptide or protein hormones including many that were initially identified as growth factors for specific types of cells. These RTK ligands include nerve growth factor (NGF), platelet-derivedgrowth factor (PDGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF). Many RTKs and their ligands were identified in studies on human cancers associatedwith mutant forms of growthfactor receptorsthat stimulate proliferation evenin the absence of growth factor. Other RTKs have been uncovered during analysis of developmentalmutations that lead to blocks in differentiation of certain cell types in C. elegans,Drosophila, and the mouse. Like cytokine receptors' RTKs signal through a protein tyrosine kinase. However' unlike cytokine receptors,which associatewith a separatecytosolic kinase protein (a JAK)' RTKs have an intrinsic kinase as part of their cytosolic domain. Ligand-induced dimerization and activation of an RTK stimulates its tyrosine kinase activity' which triggers several intracellular signal-transductionpathways (seeFigure 1.6-2). In this section we discuss how ligand binding leads to activation of the RTKs; subsequentlywe consider how the numbers of cell-surfaceRTKs are controlled. In the following section we examine one pathway that is triggered by virtually every RTK' the Ras/MAP kinase pathway. R E C E P T OTRY R O S I N EK I N A S E S

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L i g a n dB i n d i n gL e a d st o P h o s p h o r y l a t i o n a n d A c t i v a t i o no f I n t r i n s i cK i n a s ei n R T K s All RTKs have three essentialcomponents: an extracellular domain containing a ligand-binding site, a singlehydropho, bic transmembranecr helix, and a cytosolic domain that includes a region with protein tyrosine kinase activity. Most RTKs are monomeric, and ligand binding to the extracellular domain induces formation of receptor dimers. Some monomeric ligands for RTKs, including fibroblast growth factor (FGF), bind tightly to heparan sulfate, a negatively charged polysaccharidecomponent of the extracellular matrix (Chapter 19); this associationenhancesligand binding to the monomeric receptor and formation of a dimeric receptor-ligand complex (Figure 16-15). The ligands for some RTKs are dimeric; their binding brings rwo recepror monomers together directly. Yet other RTKs, such as the insulin receptor, form disulfide-linked dimers even in the absenceof hormone; binding of ligand to this type of RTK

( a ) S i d ev i e w

alters its conformation in such a way that the receptor becomes activated. Regardlessof the mechanism by which a ligand binds and locks an RTK into a functional dimeric stare, the next step is universal. In the resting, unstimulated state, the intrinsic kinase activity of an RTK is very low (Figure 16-16, step n). In the ligand-bound dimeric receptor,however,the kinase in one subunit phosphorylatesone tyrosine residuein the activation lip of the catalytic site in the other subunit (step Z). This leads to a conformational change that facilitates binding of ATP in some receptors (e.g., insulin receptor) and binding of protein substratesin other receptors (e.g.,FGF receptor).The resultingenhancedkinaseactivity then phosphorylates additional tyrosine residuesin the cytosolicdomain of the receptor(stepB). This ligand-induced activation of RTK kinase activity is analogousto rhe activation of the JAK kinases associatedwith cytokine receptors (seeFigure 16-10).The differenceresidesin the location of the kinase catalytic site, which is within the cytosolic domain of RTKs, but within a separate,associated,cytosolic JAK kinasein the caseof cytokine receptors.

Overexpression of HER2,a ReceptorTyrosine K i n a s eO , c c u r si n S o m eB r e a s tC a n c e r s Four receptor tyrosine kinases (RTKs) participate in signaling by the many members of the epidermal growth factor (EGF) family of signaling molecules.In humans, the four members of the HER (Duman epidermal growth factor /eceptor) family are denotedHER1, 2,3, and 4. HERl directly binds three EGF family members: EGF, heparinbinding EGF (HB-EGF), and tumor-derivedgrowth factor alpha (TGF-cr). Binding of any of these ligands to the extracellulardomain of a HERl monomer leads to homodimerization of the HER1 extracellular domain (Figure 16-17). Binding of EGF triggers a conformational change in the receptor extracellular domain so that it "clamps" down on the ligand. This pushes out a loop located between the two EGF-binding domains, and interactions ber w e e nt h e t w o e x t e n d e d( " a c t i v a t e d " ) l o o p s e g m e n t sa l l o w

Membrane surface (b) Top-downview Heparan sulfate

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< FIGURE 16-15Structureof the fibroblastgrowth factor (FGF)receptor,stabilizedby heparansulfate.Shownhereare sideandtop-down viewsof thecomplex comprising theextracellular (FGFR) domains of two FGFreceptor (green monomers andblue), two (white), boundFGFmolecules andtwo shortheparan sulfate chains (purple), whichbindtightlyto FGF(a)Inthesideview,theupper domainof onereceptor (blue)isseensituated monomer behindthat of theother(green); theplaneof theplasma membrane isat the bottomA smallsegment of theextracellular domain whosestructure rsnot knownconnects to themembrane-spanning o-helical segment of eachof thetwo receptor (notshown)that protrude monomers downwards (b)Inthetopview,theneparan intothemembrane sulfate chains areseenthreading between andmakingnumerous contacts with the upperdomains of bothreceptor monomers. These interactions promote bindingof the ligandto thereceptor andreceptor dimerization. fromJ Schlessinger [Adapted etal, 2OOO, MolCeilG:743 ]

C E L LS I G N A L I N Gl l : S I G N A L I N GP A T H W A Y ST H A T C O N T R O LG E N EA C T t V t T y

Ligandb i n d i n gs i t e s

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a D i m e r i z a t i o an n d phosphorylationof activationlip tYrosines

16-16Generalstructureand activationof receptor FIGURE a contains Thecytosolic domainof RTKs tyrosinekinases(RTKs). of ligand proteintyrosine kinase catalytic site In theabsence (tr), RTKs with poorlyactivekinasesLigand existasmonomers formation thatpromotes change a conformational bindingcauses together two poorlyactive receptor, bringing dimerrc of a functional

formation of the receptor dimer. Dimerization of HER1 leadsto activation of the receptor'skinaseactivity through phosphorylation of the activation lip in the kinase cytosolic domain (seeFigure 16-16). (a)

Exterior

E Phosphorylation of additional tyrosine residues

in the residue eachotheron a tyrosine thatthenphosphorylate kinases the lip to moveout of causes lip (E). Phosphorylation activation to site,thusallowingATPor a proteinsubstrate the kinasecatalytic residues tyrosine phosphorylates other then kinase bind Theactivated phosphotyrosines domain(E). Theresulting cytosolic in the receptor's proteins signal-transduction functionasdockingsitesfor various

Two other membersof the EGF familS neuregulins1 and (NRG1 and NRG2) bind to both HER3 and HER4; HB2 to HER4. Importantly' HER2 does not dibinds also EGF rectly bind a ligand, but exists on the membrane in a preactivated conformation with the loop segment protruding outward and the ligand-binding domains in close proximity (Figure 16-18a).HER2 signalsby forming heterocomplexes with ligand-bound HER1, HER3, or HER4 and facilitates signalingby all EGF family members(Figure16-18b).HER3 l"ikr a functional kinase domain; after binding a ligand it dimerizes with HER2 and becomesphosphorylated by the HER2 kinase.This activatesdownstreamsignal-transduction oathwavs.

(b)

a human of HER1, dimerization 16-17Ligand-induced < FIGURE (a)Schematic depiction receptorfor epidermalgrowth factor (EGF). isa which HER1, of domains andtransmembrane of the extracellular to a monomeric molecule EGF of one Binding kinase. tyrosine receptor between of a looplocated in thestructure an alteration causes receptor t h et w o E G F - b i n d idnogm a i n sD. i m e r i z a t i o fnt w o i d e n t i c al i lg a n d occurs in the planeof the membrane boundreceptormonomers "activated" loop the two between interactions through primarily protein bound to HERl (b) the dimeric of Structure segments. a memberof the EGFfamily' growthfactorcr(TGFct), transforming domainsareshownin white(left)and extracellular Thereceotor's domainisshownin redasan alpha thetransmembrane blue(right); TGFct in detailThetwo smaller known not is its structure helixbut betweenthe arecoloredgreen.Notethe interaction molecules in the two receptormonomerslPart(a) "activated"loopsegments Part(b)fromI Garrett ger,2OO2' Cell1'10:669 fromJ Schlessin adapted | etal, 2002,Cell11O:763 R E C E P T OTRY R O S I N EK I N A S E S

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A rcr A xa-ecr

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A EGF A HB-EGF A tcF-a

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HER4HER2

FIGURE 15-18The HERfamilyof receptorsand their ligands.Humans express fourreceptor tyrosine kinases-denoted HER1, 2, 3, and4-that bindepidermal growthfactor(EGF) and otherEGFfamilymembers(a)Asshown,the HERproteins differentially bindEGF, heparin-binding EGf(le-f df), rumorgrowthfactoralpha(TGFct), derived andneuregulins 1 and2 (NRG1 andNRG2). NotethatHER2, whichdoesnotdirectly binda ligand, exists in the plasma surface membrane in a pre-acttvated state

indicatedby red hook (b) Ligand-bound HERl can form acttvated homodrmersbound togetherby loop segments(redhooks),as d e t a i l e di n F i g u r e1 6 - 1 7 H E R 2f o r m sh e t e r o d i m e w r si t h l i g a n d b o u n dH E R 1H , E R 3a, n d H E R 4a n d f a c i l i t a t essi g n a l i n b gy a l lE G F f a m i l ym e m b e r sH E R 3l a c k sa f u n c t i o n akl i n a s ed o m a i na n d c a n signalonlywhen complexed wlth HER2lAfterN E Hynes andH A Lane, 2005,NatureRev.Cancer5i341(erratumin ivatureRer Cancer5:5g0),and A B SinghandR C Harris, 2005,CellSignal17(Oct.):1 I 83 l

Normal epithelial cells express a small amount of HER2 protein on their plasma membranesin a tissuespecificpattern. In tumor cells, errors in DNA replication often result in multiple copies of a gene on a single chromosome,an alterationknown as geneamplification (Chapter 25). Amplification of the HER2 gene occurs rn approx_ imately 25 percent of breast cancer patients, resulting in overexpressionof HER2 protein in the tumor cells. Breast cancer patients with HER2 overexpressionhave a worse prognosis, including shortened survival, than do patienrs without this abnormality. As Figure 16-18 emplasizes, overexpressionof HER2 makes the tumor cells sensitiveto growth stimulation by low levels of any member of the EGF family of growth facrors,levelsthat would not stimulate proliferation of cells with normal HER2 levels. Discovery of the role of HER2 overexpressionin certain breast cancersled researchersto develop monoclonal antibodies

specific for the HER2 protein. These have proven to be effective therapies for those breast cancer patients in which HER2 is overexpressed, reducing recurrence by about 50% in these patients. I

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C o n s e r v e dD o m a i n sA r e l m p o r t a n t f o r B i n d i n gS i g n a l - T r a n s d u c t i po rno t e i n s to Activated Receptors As in signaling by cytokine receptors,certain phosphotyrosine residuesin activated receptor tyrosine kinases (RTKs) serve as docking sites for proteins involved in downstream signal transduction. These phosphotyrosineresiduesbind PTB domains as well as SH2 domains,which we encountered in discussingthe JAK/STAT pathway (seeFigure 16-12). Both of thesedomains are presentin a large arcayof intracellular signal-transductionproteins that couple activated RTKs and

C E L LS I G N A L I N Gl l : S t c N A L t N Gp A T H W A Y ST H A T C O N T R O LG E N EA C T t V t T y

cytokine receptorsto downstream components of signaltransductionpathways. Once bound to an activated receptor, some signaltransduction proteins are phosphorylatedby the receptorassociatedkinaseto achievetheir activeform. Many enzymes that function in signal-transductionpathways are present in the cytosol in unstimulated cells.Binding to an activated receptor positions these enzymesnear their substrates,which are localized in the plasma membrane; the modified substrate then triggers a downstream signal-transductionpathway. Severalcytokine receptors(e.g.,the IL-4 receptor) and RTKs (e.g., the insulin receptor) bind multidocking proteins, hke IRS-1,via a PTB domain in the docking protein (Figure1,6-19). The activatedreceptorthen phosphorylatesthe bound docking protein, forming many phosphotyrosinesthat in turn serve as docking sitesfor SH2-containing signalingproteins. Some of theseproteins in turn may also be phosphorylated by the activated receptor. As noted earlier,each SH2 domain binds to a distinct sequence of amino acids surrounding a phosphotyrosine residue. The unique amino acid sequenceof each SH2 domain determines which amino acid sequencecontaining a phosphotyrosine it will bind. This specificity plays an important role in determining which signal-transductionproteins bind to which receptors.The SH2 domain of the Src tyrosine kinase for example, binds strongly to any peptide

Activated RTK

S i g n a l i npgr o t e i n s of intracellularsignal-transduction 16-19Recruitment A FIGURE proteinsto the cellmembraneby bindingto phosphotyrosine proteins.Cytosolic residuesin receptorsor receptor-associated domains canbindto proteins or PTB(maroon) with SH2(purple) (shownhere)or phosphotyrosine RTKs residues in activated specific proteins ln somecases, thesesrgnal-transduction receptors cytokrne intrinsic or associated by the receptors thenarephosphorylated and RTKs proteintyrosine kinase, enhancing theiractivityCertain proteins to suchasIRS-1 receptors utilizemultidocking cytokine proteins and thatarerecruited increase the numberof signaling IRS-1 phosphorylation of a receptor-bound Subsequent activated dockingsitesfor SH2kinase creates additional by the receptor c o n t a i n i nsgi g n a l i npgr o t e i n s

containing a critical four-residuecore sequence:phosphotyrosine-glutamic acid-glutamic acid-isoleucine (see Figure 16-11). Thesefour amino acids make intimate contact with the peptide-bindingsite in the Src SH2 domain. Binding resemblesthe insertion of a two-pronged "plug"-the phosphotyrosine and isoleucineside chains of the peptide-into a iwo-pronged "socket" in the SH2 domain. The two glutamic acids fit snugly onto the surfaceof the SH2 domain between the phosphotyrosinesocket and the hydrophobic socketthat acceptsthe isoleucineresidue.The binding specificityof SH2 domains is largely determined by residuesC-terminal to the phosphotyrosinein atargetpeptide. In contrast, the binding ipecificity of PTB domains is determined by the specific residues on the N-terminal side of a phosphotyrosine residue. Sometimesa PTB domain binds to a target peptide even if the tyrosine is not phosphorylated.

o f R T KS i g n a l i n gO c c u r s Down-regulation by Endocytosisand LysosomalDegradation We have alreadyseenseveralways that signal-transduction pathways are controlled. Intracellular proteins such as Ski and SOCS negatively regulate their respectivesignaltransductionpathways after their expressionis induced by TGFB or cytokines. Phosphorylation of receptors and downstream signalingproteins is reversedby the carefully Here we discusstwo recontrolled action of phosphatases. RTK signaling is restrained: which by lated mechanisms receptor-ligand of surface endocytosis ligand-induced receptor or liginternalized of the and sorting complexes, Thus treatment of degradation' for lysosome the and 1o number of the reduces hours for several ligand with cells available cell-surfacereceptors such that they no longer resoond to that concentration of hormone. This prevents inappropriate prolonged receptor activity' but under these conditions cellsusually will respondif the hormone level is increasedfurther. In the absenceof epidermal growth factor (EGF), for instance,cell-surfaceHER1 receptors for this ligand are relatively long-lived, with an averagehalf-life of 10 to 15 hours' To a large measurethis is becausethe unbound receptorsare internalized via clathrin-coatedpits into endosomesat a relatively slow rate, on averageonce every 30 minutes, and are returned rapidly to the plasma membrane. Following binding of an EGF ligand, the rate of endocytosisof HER1 is inireased :10 fold, and only about half of the internalized receptorsreturn to the plasma membrane, dependingon the cell type; the rest are degradedin lysosomes.Thus each time a HERI-EGF complex is internalized, via the process termed receptor-mediated endocytosis (see Figure 14-29), the receptor has about a 50 percent chance of being degraded. E*pottt.. of a fibroblast cell to high levels of EGF for severalhours induces severalrounds of endocytosis,resulting in degradation of most cell-surfacereceptor molecules and thus a reduction in the cell's sensitivity to EGF In this way, prolonged treatment with EGF desensitizesthe cell to that level of hormone' though the cell may respond if the level of EGF is increasedfurther. R E C E P T OTRY R O S I N EK I N A S E S

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HER1 mutants that lack kinase activity do not undergo acceleratedendocytosisin the presenceof ligand. It is likely that ligand-induced activation of the kinase activity in normal HERl inducesa conformational changein the cytosolic tail, exposinga sorting motif that facilitatesreceptor recruirment into clathrin-coated pits and subsequentinternalization of the receptor-ligand complex. Despite extensivestudy of mutant HER1 cytosolic domains, the identity of these "sorting motifs" is controversial, and most likely multiple motifs function to enhanceendocytosis.Interestinglg internalized receptorscan continue to signal from endosomesor other intracellular comparrmentsbefore their degradationas evidencedby their binding to signalingproteins such as Grb2, and SOS,which are discussedin the next sectron. After internalization,some cell-surfacereceptors(e.g., the LDL cholesterol receptor) are efficiently recycled to the surface (seeFigure 14-29). In contrast, the fraction of activated HER1 receptorsthat are sorted to lysosomescan vary from 20 to 80 percent in different cell types. There is a strong correlation between monoubiquitination of the HERl cytosolicdomain by c-Cbl, an E3 ubiquitin ligase(see Figure 3-29), and HER1 degradation. c-Cbl contains an EGFR-binding domain, which binds directly to phosphorylated EGF receptors,and a RING finger domain, which recruits ubiquitin-conjugatingenzymesand mediatestransferof ubiquitin to the receptor.The ubiquitin functionsas a,,tag,, on the receptor that stimulatesits incorporation from endosomes into multivesicular bodies (seeFigure 14-33) that ultimately are degradedinside lysosomes.A role for c-Cbl in EGF receptor trafficking emergedfrom genetic studiesin C. elegans,which establishedthat c-Cbl negativelyregulatesthe nematode EGF receptor (Let-23), probably by inducing its degradation.Similarly knockout mice lacking c-Cbl show hyperproliferation of mammary gland epithelia, consistent with a role of c-Cbl as a negativeregulatorof EGF signaling. Experiments with mutant cell lines demonstratethat internalization of RTKs plays an important role in regulating cellular responsesto EGF and other growth factors. For instance,a mutation in the EGF receptor (HER1) that orevents it from beingincorporatcdinto coaredpits makesit resistant to receptor-mediated (ligand-induced)endocytosis.As a result, this mutation leadsto substantiallyabove-normalnumbers of EGF receptorson cells and thus increasedsensitivity of cells to EGF as a mitogenic signal. Such mutant cells are prone to EGF-induced transformation into tumor cells. Interestingly,the other EGF family receptors-HER2, HER3, and HER4-do not undergo ligand-induced internalization, an observation that emphasizeshow each receptor evolved to be regulatedin its own approprraremanner.

ReceptorTyrosine Kinases r Receptor tyrosine kinases(RTKs), which bind to peptide and protein hormones, may exist as preformed dimers or dimerize during binding to ligands. Ligand binding triggers formation of functional dimeric receprorsand phtsphory-

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lation of the activation lip in the intrinsic protein tyrosine kinases,enhancingtheir catalytic activity (seeFigure 16-16). The activatedreceptoralso phosphorylatestyrosine residues in the receptor cytosolic domain and in other protein substrates. r Humans expressfour RTKs that bind different members of the epidermal growth factor family of signaling molecules (see Figure 16-18). One of these receptors, HER2, does not bind ligand; it forms active heterodimers with ligand-bound monomers of the other three HER proreins. Overexpressionof HER2 is implicated in about 25 percent of breastcancers. r Short peptide sequencescontaining phosphotyrosine residuesare bound by SH2 and PTB domains, which are found in many signal-transducingproteins. Such proteinprotein interactions are important in many signaling pathways. r Endocytosis of receptor-hormone complexes and their degradationin lysosomesis a principal way of reducing the number of receptor tyrosine kinasesand cytokine receptors on the cell surface,thus decreasingthe sensitivity of cellsto many peptidehormones.

Activationof Rasand MAP KinasePathways Almost all receptor tyrosine kinases can activate the Ras/MAP kinase pathway (seeFigure 16-2c). The Ras protein, a monomeric (small) G protein, belongsto the GTpase superfamilyof intracellular switch proteins (seeFigure 15-8). Activated Ras promotes formation, at the membrane, of signal-transductioncomplexescontaining three sequentially acting protein kinases.This kinase cascadeculminatesin activation of certain membersof the MAP kinase family, which can translocate into the nucleus and phosphorylate many different proteins. Among the target proteins for MAP kinase are transcription factors that regulate expressionof proteins with important roles in the cell cycle and in differentiation. Many cytokine receptors also can activate the Ras/MAP kinase pathway (seeFigure 16-2b). Moreover, different types of extracellular signals often activate different signaling pathways that result in activation of different membersof the MAP kinase family. Becausean activating mutation in a RTK, Ras, or a protein in the MAP kinase cascadeis found in almost all types of human tumors, the RTI(Ras/MAP kinase pathway has been subjectedto extensivestudy and a great deal is known about the componentsof this pathway. !7e begin our discussion by reviewing how Ras cyclesbetweenrhe active and inactive state. We then describe how Ras is activated and passesa signal to the MAP kinase parhway. Finally we examine recent studiesindicating that both yeastsand cells of higher eukaryotes contain multiple MAP kinase pathways, and consider the ways in which cells keep different MAp

c E L L s t c N A L t N G i l : s t G N A L t N Gp A T H W A y s r H A T c o N T R o L G E N EA c l v t r y

kinase pathways separatefrom one another through the use of scaffold proteins.

Ras,a GTPaseSwitch Protein,CyclesBetween Active and InactiveStates Like the Go subunits in trimeric G proteins, Ras alternates between an active on state with a bound GTP and an inactive off statewith a bound GDP. As discussedin Chapter 15, trimeric G proteins are directly linked to G protein-coupled receptors(GPCRs)on the cell surfaceand transducesignals, usually via the Go subunit, to various effectors such as adenylyl cyclase.In contrast, Ras is not directly linked to cell-surfacereceptors. The activity of the Ras protein is regulated by several factors. Ras activation is accelerated by a guanine nucleotide-exchange factor (GEF), which binds to the Ras'GDP complex, causingdissociationof the bound GDP (seeFigure3-32). BecauseGTP is presentin cellsat a higher concentration than GDR GTP binds spontaneously to "empty" Ras molecules,with releaseof GEF and formation of the active Ras'GTP. Subsequenthydrolysis of the bound GTP to GDP deactivatesRas. Unlike the deactivation of of G*'GTR deactivationof Ras'GTP requiresthe assistance another protein, a GTPase-actiuatingprotein (GAPI. Binding of GAP to Ras'GTP acceleratesthe intrinsic GTPaseactivity of Ras by more than a hundredfold. Thus the average lifetime of a GTP bound to Ras is about 1 minute, which is much longer than the averagelifetime of a G.'GTP complex. In cells, GAP binds to specificphosphotyrosinesin activated RTKs, bringing it close enough to membrane-bound Ras'GTP to exert its acceleratingeffect on GTP hydrolysis. The actual hydrolysis of GTP is catalyzed by amino acids from both Ras and GAP. In particular, insertion of an arginine side chain on GAP into the Ras active site stabilizesan intermediate in the hydrolysis reaction. Ras (-176 amino acids) is smaller than Go proteins (:300 amino acids),but the GTP-binding domains of the two proteins have a similar structure (see Figure 15-8). Structural and biochemicalstudiesshow that Go also contains a GAP domain that increasesthe rate of GTP hydrolysis by G.. Becausethis domain is not presentin Ras, it has an intrinsicallvslower rate of GTP hvdrolvsis.

ReceptorTyrosineKinasesAre Linkedto Ras by Adapter Proteins The first indication that Ras functions downstream from RTKs in a common signaling pathway came from experiments in which cultured fibroblast cellswere induced to proliferate by treatment with a mixture of two protein hormones:platelet-derivedgrowth factor (PDGF) and epidermal growth factor (EGF). Microinjection of anti-Ras antibodies into these cells blocked cell proliferation' Conversely'injection of RasD,a constitutively active mutant Ras protein that hydrolyzesGTP very inefficiently and thus persistsin the active state,causedthe cellsto proliferate in the absenceof the growth factors. These findings are consistent with studies showing that addition of FGF to fibroblasts leads to a rapid increasein the proportion of Ras presentin the GTP-bound active form. However, an activated RTK (e.g', a ligand-bound EGF receptor) cannot directly activate Ras' Rather, two cytosolic proteins-GRB2 and Sos-must first be recruited to provide a link betweenthe receptor and Ras (Figure 1'6-20).An SH2 domain in GRB2 binds to a specificphosphotyrosineresidue in the activated receptor. GRB2 also contains two SH3 domains, which bind to and activate Sos.GRB2 thus functions as an adapter protein for many receptor tyrosine kinases.Sos is a guanine nucleotide-exchangeprotein (GEF), which catalyzesconversion of inactive GDP-bound Ras to the active GTP-bound form.

GeneticStudiesin Drosophilaldentified Key r o t e i n si n t h e R a s / M A P S i g n a l - T r a n s d u c iP ng KinasePathway

Our knowledge of the proteins involved in the RasiMAP kinasepathway came principally from geneticanalysesof mutant fruit flies (Drosophila) and worms (C. elegans)which were blocked at particular stagesof differentiation' To illustrate the power of this experimental approach, we consider developmentof a particular type of cell in the compound eye ol DrosoPhila. The compound eyeof the fly is composedof some 800 individual eyescalled ommatidia (Figure 1'6-21'a).Each ommatidium consistsof 22 cells,eight of which are photosensitive neurons called retinula, or R cells, designatedR1-R8 (Figure 16-21.b).An RTK called Seuenless(Seu)specifically reguMammalian Ras proteins have been studied in great developmentof the R7 cell and is not essentialfor any lates detail becausemutant Ras proteins are associated known function. In flies with a mutant seuenless(seu) other with many types of human cancer.Thesemutant proteins, gene,the R7 cell in each ommatidium does not form (Figwhich bind but cannot hydrolyze GTP, are permanentlyin ure 1,6-21c).Sincethe R7 photoreceptor is necessaryfor flies the "on" state and contribute to oncogenictransformation ( C h a p t e r 2 5 ) . D e t e r m i n a t i o n o f t h e t h r e e - d i m e n s i o n a l to seein ultraviolet light, mutants that lack functional R7 cells but are otherwisenormal are easilyisolated.Thereforefly R7 structure of the Ras-GAP complex and tests of mutant cells are an ideal geneticsystemto study cell development. forms of Ras explainedthe puzzling observationthat most During development of each ommatidium, a protein (RasD) proteins conRas active oncogenic,constitutively called Boss (Bride of Seuenless)is expressedon the surface tain a mutation at position 1,2.Replacementof the normal protein is the ligand of the R8 cell. This membrane-tethered glycine-12 with any other amino acid (except proline) R7 preneighboring blocks the functional binding of GAP and in essence for the Sev RTK on the surface of the photosensitive a into cursor cell, signaling it to develop "locks" Ras in the active GTP-bound state.I

A C T I V A T I O NO F R a s A N D M A P K I N A S EP A T H W A Y S

< FIGURE 16-20Activationof Rasfollowing ligandbindingto receptortyrosinekinases(RTKs).Thereceptors for epidermal growthfactor(EGF) andmanyothergrowthfactorsareRTKsThe cytosolic proteinGRB2bindsto a specif adapter ic phosphotyrosine on an activated, ligand-bound receptor andto the cytosolic Sos protein, bringing rt nearrtssubstrate, the inactive Ras.GDP The guaninenucleotide-exchange factor(GEF) activity of Sosthen promotes formation of activeRas.GTP Notethat Rasistethered to the membrane by a hydrophobic farnesyl anchor(seeFigure'l0-19) [ S e eJ S c h l e s s i n g e2r0, 0 0 , C e l l1 0 3 : 2 11 , a n d M A S i m o n ,2 O O OC, e l lj 0 3 : 1 3 ]

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neuron (Figure 16-22a).In mutant flies that do not express a functional Bossprotein or Sev RTK, interaction between the Bossand Sevproteins cannot occur,and no R7 cellsdevelop (Figure 16-22b); this is the origin of the name "Sevenless"for the RTK in the R7 cells. To identify intracellular signal-transducing proteins in the Sev RTK pathway, investigatorsproduced mutant flies expressinga temperature-sensitiveSev protein. \Vhen these flies were maintained at a permissivetemperature, all their ommatidia contained R7 cells; when they were maintained at a nonpermissivetemperature,no R7 cells developed.At a particular intermediate temperature, however, just enough of the Sev RTK was functional to mediate normal R7 development. The investigarorsreasonedthat at this intermediate temperature,the signaling pathway would becomedefective (and thus no R7 cells would develop) if the level of another protein involved in the pathway was reduced,thus reducing the activity of the overall pathway below the level required to form an R7 cell. A recessivemutarion affecting such a protein would have this effect because,in diploid organisms hke Drosophila, a heterozygote containing one wild-type and one mutant allele of a genewill produce half the normal amount of the gene product; hence, even if such a recessive mutation is in an essentialgene,the organism will usually be viable. However.a fly carryinga temperature-sensirive mutation in the seu gene and a second mutation affecting another protein in the signalingpathway would be expectedto lack R7 cells at the intermediatetemperature. By use of this screen,researchersidentified three genes encoding important proteins in the Sev pathway: an SH2containing adapter protein exhibiting 64 percent amino acid sequenceidentity to human GRB2; a guanine nucleotide-exchange factor called Sos (Son of Sevenless)exhibiting 45 percent identity with its mouse counterpart; and a Ras protein exhibiting 80 percent identity with its mammalian counterparts (see Figure 16-20). These three proteins later were found to function in other signalingpathways initiated by ligand binding to different RTKs and used at different times and placesin the developingfly. In subsequentstudies,researchersintroduced a mutant rasD geneinto fly embryos carrying the sevenlessmutation. As noted earlier, the rasDgene encodes a constitutive Ras protein that is presentin the active GTP-bound form even in the absenceof a hormone signal.Although no functional Sev RTK was expressedin thesedouble-mutants (seu ; rasD1.R7 cellsformed normally, indicating that presenceof an activated

c E L L S t G N A L | N Gi l : s T G N A L T NpGA T H W A y s r H A T c o N T R o L G E N EA c l v t r y

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R4 R2 R3 Toward eye surface

FfGURE 16-21The compoundeye of Drosophilamelanogaster. m u t a n ft l i e sv i e w e db y a s p e c i at el c h n i q uteh a tc a nd i s t i n g u i st hhe isindicated Theplaneof sectioning photoreceptors in an ommatidium. ( a )S c a n n i negl e c t r om l m a t i d tr ha a t n i c r o g r a psh o w r nign d i v i d uoam plane (b), of these the is out of R8 cell and the in arrows the blue (b) by views of a fruit fly eye Longitudinal and cutaway comprise the seenin the in thisplaneareeasily imagesThesevenphotoreceptors E a c ho f t h e s et u b u l asr t r u c t u r e co s n t a i nesi g h t ommatidium single (top),whereas in the mutant onlysixarevisible wild-type ommatidia p h o t o r e c e p t odr e s ,s i g n a t eRd1 - R 8w, h i c ha r el o n g ,c y l i n d r i c a l l y lackthe R7 mutation seven/ess with the Flies ommatidia bottom) the cells.R1-R6(yellow) extendthroughout shapedlight-sensitive (a) Development 1991 K Basler, E Hafen and from , eyes in their (brown) cell [Part is locatedtowardthe depthof the retina,whereasR7 Zipurs1 k y9,8 8 ' C e l l dS Rk e a n L 1 ( s u p)p: 1l 2 3P a r t ( b ) a d a p t e d f rRo emi n wherethe surfaceof the eye,and R8(blue)towardthe backside (c)courtesy of U Banerjee l 55:321Part of eyesfromwild-typeandseven/ess axonsexit (c)Comparison

Ras protein is sufficient for induction of R7-cell development (Figure16-22c).This finding, which is consistentwith the resultswith cultured fibroblastsdescribedearlier,supports the conclusion that activation of Ras is a principal step in intracellular signaling by most if not all RTKs.

B i n d i n go f S o sP r o t e i nt o I n a c t i v eR a sC a u s e s a C o n f o r m a t i o n aCl h a n g eT h a t A c t i v a t e sR a s In addition to its SH2 domain that binds to activated RTKs, the GRB2 adapter protein contains two SH3 domains, which bind to Sos,the Ras guanine nucleotide-exchangefac-

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tor (see Figure 16-20). Like phosphotyrosine-binding SH2 and PTB domains, SH3 domains are presentin a large number of proteins involved in intracellular signaling. Although structuresof variousSH3 domainsare the three-dimensional similar, their specific amino acid sequencesdiffer. The SH3 domains in GRB2 selectivelybind to proline-rich sequences in Sos;different SH3 domains in other proteins bind to proline-rich sequencesdistinct from those in Sos. Proline residuesplay two roles in the interaction between an SH3 domain in an adapter protein (e.g., GRB2) and a proline-richsequencein anotherprotein (e.g.,Sos).First' the proline-rich sequenceassumesan extended conformation

( c ) D o u b l em u t a n t \sev ; tlasu)

15-22Geneticstudiesrevealthat FIGURE < EXPERIMENTAL activationof Rasinducesdevelopmentof R7photoreceptorsin flies, of wild-type eye.(a)Duringlarvaldevelopment the Drosophila a cell-surface expresses ommatidium the RBcellin eachdeveloping of its thatbindsto theSevRTKon the surface protein, calledBoss, in changes induces interaction precursor This cell R7 neighboring cellinto of the precursor that resultin differentiation geneexpression with a mutationin the R7neuron(b)Infly embryos a functional (sev) cellscannotbindBossand gene,R7precursor seven/ess the intoR7cellsRather normally do not differentrate therefore pathway and developmental precursor cellenters n alternative (sev-,RasD) larvae becomesconecell.(c)Double-mutant eventually cell, in the R7precursor activeRas(RasD) a constitutively express of in the absence precursor cells R7 of differentiation whichinduces is Ras activated that shows finding This signal Boss-mediated the etal, of an R7cell lseeM A Simon induction sufficientto medrate 355:559 et al, 1992,Nature l andM E Fortini Celt67:701, 1991, A C T I V A T I O NO F R a s A N D M A P K I N A S EP A T H W A Y S

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for binding, and neighboring residuesconfer specificity to the binding. Following activation of an RTK (e.g., Sevenlessor the EGF receptor), a complex containing the activated receptor, GRB2, and Sosis formed on the cytosolic face of the plasma membrane (seeFigure 16-20). Complex formation depends on the ability of GRB2 to bind simultaneowslyto the receptor and to Sos.Thus receptor activation leads to relocalization of Sos from the cytosol to the membrane, bringing Sos near to its substrate,namely, membrane-boundRas.GDP. Binding of Sosto Ras.GDPleadsto conformationalchanges in the Switch I and Switch II segmentsof Ras, thereby opening the binding pocket for GDP so it can diffuse out (Figure 16-24).GTP then binds to and activatesRas.Binding of GTP to Ras, in turn, induces a specific conformation of Switch I and Switch II that allows Ras.GTP ro activate the first downstream protein kinase of the MAP kinasepathway. S H 3d o m a i n

A FIGURE 16-23Surfacemodelof an SH3domainboundto a target peptide.Theshort,proline-rich targetpeptideisshownas a space-filling modelInthistargetpeptde,two prolines (pro4and Pro7,darkblue)fit intobindingpockets on thesurface of the SH3 domain,Interactions (Argl,red),two other involving an arginrne (lightblue),andotherresidues prolines (green) in thetargetpeptide determine thespecificity of binding[After H yuetal, 1994, Cett 76:9331

that permits extensive contacts with the SH3 domain, thereby facilitating interaction. Second,a subset of these prolinesfit into binding "pockets" on the surfaceof the SH3 domain (Figure 16-23). Severalnonproline residuesalso in, teract with the SH3 domain and are responsiblefor determining the binding specificity.Hence the binding of proteins to SH3 and to SH2 domains follows a similar srraregy:certain residuesprovide the overall structural motif necessary

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FIGURE 15-24Structures of Rasboundto GDBSosprotein, and GTP.(a)In Ras.GDB the Switch| (green) andSwitchil (blue) segments do not directly interact with GDp(b)Oneo helix(orange) ln Sosbindsto bothswitchregions of Ras.GDp, leading to a massive conformational changein Ras,In effect,SospriesRasopenby displacing the SwitchI region, thereby allowingGDpto diffuseout (c)GTPisthoughtto bindto the Ras-Sos complex firstthroughits c H A P T E R1 6

Biochemical and genetic studies in yeasr, C. elegans, Drosophila, and mammals have revealeda highly conserved cascadeof protein kinases,culminating in MAP kinase, that operatesdownstream from activated Ras. Although activation of the kinase cascadedoes not yield the same biological results in all cells, a common set of sequentially acting kinasesdefinesthe MAP kinase pathway, as outlined in Figure 16-25. Active Ras'GTP binds to the N-terminal regulatory domain of Raf, a serine/threoninekinase, thereby activating it (step[). Hydrolysisof Ras.GTPto Ras.GDPreleasesactive Raf (step B), which phosphorylatesand thereby activates MEK (step 4). (A dual-specificityprotein kinase, MEK phosphorylatesits target proteins on both tyrosine and serine/threonineresidues.)Active MEK then phosohorvlates

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base(guanine); subsequent bindingof the GTpphosphates completes the interaction, Theresulting conformational changein SwitchI andSwitchll segments of Ras,allowingbothto bindto the GTP1 phosphate, displaces Sosandpromotes interaction of Ras.GTp (discussed with itseffectors later),SeeFigure15-8for another deprction ot Ras.GDP andRas.GTP fromp A Boriack-Sjodin lAdapted and.JKuriyan, 1998, Nature394:341 I

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14-3-3/ cells, 16-25Ras/MAPkinasepathway.In unstimulated A FIGURE gf a l i g a n d m o s tR a sr si n t h ei n a c t i vf eo r mw i t hb o u n dG D Pb; i n d i n o receptor leadsto formation of theactive to itsRTKor cytokine (step[; seealsoFigure16-20)Activated Ras Ras'GTP complex in stepsE-6, kinase cascade depicted trrggers thedownstream c u l m i n a t i nr nga c t i v a t i oonf M A Pk i n a s(eM A P KI)n u n s t i m u l a t e d it in an inactive cells,bindingof the 14-3-3proteinto Rafstabilizes domain Interaction regulatory of the RafN-terminal conformation of in dephosphorylation relieves thisinhibition, results with Ras.GTP of that bindRafto 14-3-3,andleadsto activation oneof the serines activityNotethat in contrast to manyotherprotein Rafkinase of the of Rafdoesnot dependon phosphorylation kinases, activation fromRaf,it lip Afterinactive Ras.GDP dissociates activation presumably receptors, fromactivated canbe reactivated by signals Seethe additional Rafmolecules to the membrane therebyrecruiting Regul 2001, Adv.Enzyme andU Rapp, textfor details[SeeE Kerkhoff Prog HormoneRes56:127;andM 41:261,J Avruchet al , 200'1,Recent et al , 2000,Biochem / 351:151l Yip-Schneider

and activates MAP kinase, another serine/threonine kinase

also known as ERK (step E). MAP kinase phosphorylates many different proteins, including nuclear transcription factors, that mediatecellularresponses(step6). Severaltypes of experimentshave demonstratedthat Raf. MEK. and MAP kinase lie downstream from Ras and have revealedthe sequentialorder of theseproteins in the pathway. For example, mutant Raf proteins missing the Nterminal regulatory domain are constitutively active and induce quiescentcultured cells to proliferate in the absenceof stimulation by growth factors. These mutant Raf proteins were initially identified in tumor cells; like the constitutively active RasDprotein, such mutant Raf proteins are said to be encoded by oncogenes,whose encoded proteins promote transformation of the cells in which they are expressed (Chapter25). Converselgculturedmammaliancellsthat express a mutant, nonfunctional Raf protein cannot be stimulated to proliferate uncontrollably by a constitutively active RasD protein. This finding establisheda link between the Raf and Ras proteins. In vitro binding studies further showed that the purified Ras'GTP complex binds directly to the N-terminal regulatory domain of Raf and activates its catalytic activity.

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That MAP kinase is activated in responseto Ras activation was demonstratedin quiescentcultured cells expressing a constitutively active RasD protein. In these cells activated MAP kinase is generated in the absenceof stimulation by growth-promoting hormones. More importantly, R7 photoreceptors develop normally in the developing eye of Drosophila mutants that lack a functional Ras or Raf protein but express a constitutively active MAP kinase. This finding indicatesthat activation of MAP kinase is sufficient to transmit a proliferation or differentiation signal normally initiated by ligand binding to a receptor tyrosine kinase such (seeFigure 1'6-221.Biochemicalstudiesshowed, as Sevenless howeve! that Raf cannot directly phosphorylate MAP kinaseor otherwiseactivateits activity. The final link in the kinase cascade activated by Ras'GTP emergedfrom studiesin which scientistsfractionated extracts of cultured cells searchingfor a kinase activity that could phosphorylateMAP kinaseand that was present only in cells stimulated with growth factors, not quiescent cells.This work led to identificationof MEK, a kinasethat specifically phosphorylates one threonine and one tyrosine residue on the activation lip of MAP kinase, thereby activating its catalytic activity. (The acronym MEK comesfrom MAP and ERK kinase.) Later studies showed that MEK binds to the C-terminalcatalyticdomain of Raf and is phosphorylated by the Raf serine/threoninekinase; this phosphorylation activatesthe catalytic activity of MEK. Hence, activation of Ras induces a kinase cascadethat includes -+ Raf Raf, MEK, and MAP kinase: activated RTK -+ Ras -+ MEK + MAP kinase' A C T I V A T I O NO F R a s A N D M A P K I N A S EP A T H W A Y S

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Activation of Raf Kinase The mechanismfor activatingRaf differs from that usedto activatemany other protein kinasesincluding MEK and MAP kinase. In a resting cell prior to stimuIation, Raf is presentin the cytosol in a conformation in which the N-terminal regulatory domain is bound to the kinase domain, thereby inhibiting its activity. This inactive conformarion is stabilizedby a dimer of the 14-3-3 protein, which binds phosphoserineresiduesin a number of important signalingproteins. Each 1.4-3-3monomer binds to a phosphoserineresiduein Raf, one to phosphoserine-2i9 in the N-terminal domain and the other to phosphoserine-62L(seeFigure 16-25). These interactions are thought to be essentialfor Raf to achievea conformational state such that it can bind to activated Ras. The binding of Ras.GTP,which is anchored ro the membrane,to the N-terminal domain of Raf relievesthe inhibition of Raf's kinase activity and also induces a conformational change in Raf that disrupts its associarionwith 14-3-3. Raf phosphoserine-259 thenis dephosphorylated(by an unknown phosphatase)and other serine or threonine residueson Raf becomephosphorylatedby yet other kinases.Thesereactions incrementallyincreasethe Raf kinase activity by mechanisms that are not fully understood. Activation of MAP Kinase Biochemical and x-ray crystallographic studies have provided a detailed picture of how phosphorylation activates MAP kinase. As in JAK kinases and receptor tyrosine kinases,the catalytic site in the inactive, unphosphorylatedform of MAP kinase is blocked by a stretch of amino acids, the activation lip (Figure 16-26a). Binding of MEK to MAP kinase destabilizesthe lip structure, resulting in exposure of tyrosine-185, which is buried in the inactive conformarion. Following phosphorylation of this critical tyrosine, MEK phosphorylatesthe neighboring threonine-183 (Figure1,6-26b). Both the phosphorylated tyrosine and the phosphorylated threonine residue in MAP kinase interact with additional amino acids, thereby conferring an altered conformation to the lip region, which in turn permits binding of ATP to the catalytic site. The phosphotyrosine residue (pY185) also plays a key role in binding specificsubstrate proteins to the surface of MAP kinase. phosphorylation promotes not only the catalytic activity of MAp kinase but also its dimerization. The dimeric form of MAp kinase (but not the monomeric form) can be translocatedto the nucleus, where it regulatesthe activity of many nuclear transcription factors.

MAP KinaseRegulatesthe Activity of Many TranscriptionFactorsControiling Early-Response Genes Addition of a growth factor (e.g., EGF or PDGF) to quiescent cultured mammalian cells causesa rapid increasein the expression of as many as 100 different genes. These are calledearly-responsegenesbecausethey are induced well before cells enter the S phase and replicate their DNA (see Chapter 20). One important early-response geneencodesthe transcription factor c-Fos.Togetherwith other transcription 690

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A FIGURE 16-26 Structuresof inactive,unphosphorylated MAP kinaseand the active,phosphorylatedform. (a)ln inactive MAPkinase (b)Phosphorylation the activation lip is notfullyexposed. (Y185) by MEKat tyrosine-185 (T183) andthreonine-183 leads to a markedconformational changein the activation lip.Thisactivating c h a n g ep r o m o t edsi m e r i z a t i o nf M A Pk i n a s a e n db i n d i n go f i t s substrates-ATP anditstargetproteins. phosphorylationA similar dependent mechanism activates JAKkinases, the intrinsic kinase activityof RTKs, andMEK.[AfterB.J.Canagarajah eral, 1997, Ce// 90:859.]

factors, such as c-Jun, c-Fos induces expression of many genes encoding proteins necessary for cells to progress through the cell cycle. Most RTKs that bind growth factors utilize the MAP kinase parhway to activate genesencoding proteins like c-Fos that in turn propel the cell through the cell cycle. The enhancer that regulates the c-fos gene contains a serum responseelement ('SRE/,so named becauseit is activated by many growth factors in serum. This complex enhancer contains DNA sequencesthat bind multiple transcription factors. Some of these are activated by MAp kinase; others by different protein kinases that function in other signalingpathways. As depictedin Figure 16-27, activated (phosphorylated) dimeric MAP kinase induces transcription of the c-fos gene by direct activation of one transcription factor, ternary complex factor (TCF), and indirect activation of another, seruln responsefactor (/SRF). In the cytosol, MAP kinasephosphorylatesand activatesa kinase called p90RSK,which translocatesto the nucleus where it phosphorylates a specific serine in SRF. After translocating to the nucleus, MAP kinase directly phosphorylates specific serinesin TCF. Association of phosphorylated TCF with two molecules of phosphorylated SRF forms an active trimeric factor that binds strongly to the SRE DNA segment. As evidencefor this model, abundant expressionin cultured mammalian cells of a mutant dominant-negativeTCF that lacks the serineresiduesphosphorylated by MAp kinase

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16-27Inductionof genetranscriptionby MAP > FIGURE phosphorylates kinase.Steps[-B: In thecytosol, MAPkinase and pgo*t*,whichthenmovesintothe nucleus the kinase and activates phosphorylates the SRFtranscription factorStepsZl andEl: After phosphorylates intothe nucleus, MAPkinase directly translocating factorTCFStep@: Phosphorylated TCFandSRF the transcription transcription of genes(e9,, c-fos)that acttogetherto stimulate in theirpromoterSeethetextfor details. containan SRE sequence R Marais etal, 1993, Cell73:381 eta1,1993,Mol , andV M Rivera [See

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Nucleus

G Protein-CoupledReceptorsTransmitSignals to MAP Kinasein YeastMating Pathways Although many MAP kinase pathways are initiated by RTKs or cytokine receptors,signalingfrom other receptorscan activate MAP kinase in different cell types of higher eukaryotes. Moreover, yeasts and other single-celledeukaryotes, which lack cytokine receptors or RTKs, do possessseveral MAP kinase pathways. To illustrate, we consider the mating pathway in S. cereuisiae,a well-studied example of a MAP kinase cascade linked to G protein-coupled receptors (GPCRs),in this casefor two secretedpeptide pheromones, theaandafactors. As discussedin Chapter 21, these pheromones control mating between haploid yeast cells of the opposite mating type, a or cr.An a haploid cell secretesthe a mating factor and has cell-surfacereceptorsfor the cr factor; an ct cell secretes the o.factor and has cell-surfacereceptorsfor the a factor (see Figure 21-19). Thus each type of cell recognizesthe mating factor produced by the opposite type. Activation of the MAP kinase pathway by either the a or o receptorsinduces transcription of genesthat inhibit progressionof the cell cycleand others that enable cells of opposite mating type to fuse together and ultimately form a diploid cell. Ligand binding to either of the two yeast pheromone GPCRs triggers the exchange of GTP for GDP on the Go subunit and dissociationof G.'GTP from the G9" complex. This activation processis identical to that for the GPCRsdiscussedin the previouschapter (seeFigure 15-13). In many mammalian GPCR-initiated pathways, the active Go transducesthe signal. In contrast, mutant studieshave shown that the dissociatedGs, complex mediates all the physiological responsesinduced by activation of the yeast pheromone receptors.For instance,in yeastcellsthat lack Go, the Gp, subunit is always free. Such cells can mate in the absenceof

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mating factors; that is, the mating responseis constitutively on. However, in cells defectivefor the GB or G" subunit' the mating pathway cannot be induced at all. If dissociatedG* were the transducer,in thesemutant cellsthe pathway would be expectedto be constitutively actlve. In yeast mating pathways, Gg, functions by triggering a kinase cascadethat is analogousto the one downstream from Ras. The components of this cascadewere uncovered mainly through analysesof mutants that possessfunctional a and cr receptorsand G proteins but are sterile (Sre)'or defectivein mating responses.The physicalinteractionsbetween through immunoprecipitation the componentswere assessed experiments with extracts of yeast cells and other types of studies.Basedon these studies,scientistshave proposed the kinase cascadeshown in Figure 16-28a. Free GB1,which is tethered to the membrane via the lipid bound to the "y subunit, binds the Ste5 protein, thus recruiting it and its bound kinasesto the plasma membrane. Ste5 has no obvious catalytic function and acts as a scaffold for assemblingother componentsin the cascade(Ste11,Ste7, and Fus3). Next' Ste20protein kinase, a protein localizedto the plasma membrane, phosphorylates and activates Ste11, a serine/threonine kinase analogousto Raf and other mammalian MEKK proteins. Activated Ste11 then phosphorylatesSte7, a dualspecificity MEK that then phosphorylates and activates A C T I V A T I O NO F R A SA N D M A P K I N A S EP A T H W A Y S

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A FIGURE 16-28YeastMAPkinasecascades in the mating and osmoregulatorypathways.In yeast,differentreceptors activate multipleMAPkinase pathways, two of whichareoutlined here(a)Matingpathway: Thereceptors for yeasta ando mating factorsarecoupled to the sametrimeric G proteinFollowing ligandbindinganddissociation of the G protein, the membranetethered Gp"subunitbindsthe Ste5scaffold to the plasma membrane Theresident Ste20kinase thenphosphorylates and activates Stel1,whichisanalogous to Rafandothermammalian (MEKK) MEKkinase proteinsThisinitiates a kinase cascade in w h i c ht h ef i n a cl o m p o n e nFt ,u s 3i,sf u n c t i o n a e l l yq u i v a l etnot (N/APK) MAPkinase in highereukaryotes LikeotherMApkinases, actrvated Fus3thentranslocates intothe nucleusThereit phosphorylates two proteins, Digl andDig2,relieving their

Fus3, a serine/threoninekinase equivalent to MAp kinase. After translocation to the nucleus,Fus3 phosphorylatesrwo proteins, Digl and Dig2, relieving their inhibition of the Ste12 transcription factor. Activated Ste12 in turn induces expression of proteins involved in mating-specific cellular resDonses. 692

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cHAPTER 16 |

inhibition of the Ste12transcription factor,allowingit to bindto DNA andinitiate transcription genes(b)Osmoregulatory of mating-type p a t h w a yT: w op l a s m m a e m b r a nper o t e i n sS,h o la n dM s b 1 a , re activated in an unknownmannerby exposure of yeastcellsto media of highosmotic strength. Activated Shol recruits the Pbs2scaffold p r o t e i nw, h i c hc o n t a i nasM E Kd o m a i nt ,o t h e p l a s mm a embrane At the plasmamembrane, Ste20phosphorylates andactivates S t e 1 1i,n i t i a t i nag k i n a s ce a s c a dteh a ta c t i v a t eHso g 1 a , MAP k i n a s eA. f t e rt r a n s l o c a t itnogt h e n u c l e u sH, o g l p h o s p h o r y l a t e s the Hotl transcription factor,allowingit to promotetranscription of genesencoding proteins thatcatalyze synthesis of proteins required for survival in highosmotic strength medialAfterM A Schwartz and H D lvadhani, 2004,AnnRev. Genet38:-/25, andN Dardandt\,4peter, 2006,BioEssays 28:146l

ScaffoldProteinsSeparateMultiple MAp Kinase Pathwaysin EukaryoticCells In addition to the MAP kinasesdiscussedabove, both yeasts and higher eukaryotic cells contain other members of the MAP kinase family. Mammalian MAP kinases include lun

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N-terminal kinases //NKs/ and p38 kinases,which become activated by various types of stresses;severalmembersof the MAP kinase family in yeastsare describedbelow. All members of the MAP kinase family are serine/threoninekinases that are activatedin the cytosol in responseto specificextracellular signals and then translocate to the nucleus. Activation of all known MAP kinasesrequires phosphorylation of both a tyrosine and a threonine residuein the lip region by a member of the MEK family of dual-specificitykinases (see Figure 16-26). Thus in all eukaryotic cells,binding of a wide variety of extracellular signaling molecules triggers highly conservedkinase cascadesculminating in activation of a particular MAP kinase. Current genetic and biochemical studies in the mouse and Drosophila are aimed at determining which MAP kinasesare required for mediating the responseto which signals in higher eukaryotes.This has already been accomplished in large part for the simpler organism S. cereuisiae. Each of the six MAP kinases encoded in the S. cereuisiae genomehas been assignedby geneticanalysesto specificsignaling pathways triggered by various extracellular signals, such as pheromones,high osmolarity, starvation, hypotonic shock, and carbon/nitrogen deprivation. Each of theseMAP kinasesmediatesvery specificcellular responses,as exemplified by Fus3 in the mating pathway and Hogl in the osmoregulatory pathway (seeFigure 16-28). In both yeasts and higher eukaryotic cells, different MAP kinase cascadesshare some common components. For instance,Ste11 functions in the three yeast signaling pathways that regulate mating, the responseto high osmotic strengthconditions, and filamentousgrowth, which is induced by starvation.Nevertheless,each pathway activates its own MAP kinase: Fus3 in the mating pathway, Hogl in the osmoregulatorypathway, and Kssl in the filamentation pathway. Similarln in mammalian cells, common upstream signal-transducingproteins participate in activating multiple JNK kinases. Once the sharing of componentsamong different MAP kinase pathways was recognized, researcherswondered how the specificity of the cellular responsesto particular signalscould be achieved.Studieswith yeast provided the initial evidencethat pathway-specific scaffold proteins enable the signal-transducingkinasesin a particular pathway to interact with one another but not with kinasesin other pathways. For example,the scaffoldprotein Ste5stabilizes a large complex that includes Ste11 and other kinasesin the mating pathway; similarly Pbs2 binds Ste11 and other kinasesin the osmoregulatorypathway (seeFigure 16-28). I n e a c h p a t h w a y i n w h i c h S t e 1 1 p a r t i c i p a t e s ,i t i s c o n strainedwithin a large complex that forms in responseto a specific extracellular signal, and signaling downstream from Ste11is restrictedto the complex in which it is localized. As a result, exposureof yeast cells to mating factors induces activation of a single MAP kinase, Fus3, whereas exposure to a high osmolarity induces activation of a different MAP kinase,Hog1. S c a f f o l d sf o r M A P k i n a s e p a t h w a y s a r e w e l l d o c u mented in yeast,fl5 and worm cells, but their presencein

mammalian cells has been difficult to demonstrate. Perhaps the best-documentedscaffold protein is Ksr (frinase suppressorof Ras), which binds both MEK and MAP kinase. Loss of the Drosophila Ksr homolog blocks signaling by a constitutively active Ras protein' suggestinga positive role for Ksr in the Ras/MAP kinasepathway in fly cells.Although knockout mice that lack Ksr are phenotypically normal, activation of MAP kinase by growth factors or cytokines is lower than normal in severaltypes of cells in these animals. This finding suggeststhat Ksr functions as a scaffold that enhances but is not essential for Ras/MAP kinase signalingin mammalian cells. Other proteins also have been found to bind to specificmammalian MAP kinases.Thus the signal specificityof different MAP kinases in animal cells may arise from their association with various scaffold-like proteins, but much additional researchis neededto test this possibility.

T h e R a s / M A PK i n a s eP a t h w a yC a nI n d u c e D i v e r s eC e l l u l a rR e s p o n s e s The Ras/MAP kinase pathway can be activated in many if not all vertebratecells by a wide variety of receptor tyrosine kinases (RTKs). In particular, signaling through this pathway is used repeatedlyin the course of development'yet the outcome in regard to cell-fatespecificationvariesin different tissues.\7hy does one cell respond by dividing, another by differentiating, and still another by dying? If there is no specificity beyond the ligand and receptor,an activated Ras might substitutefor any signal. In fact' activated Ras can do so in many cell types. In one DNA microarray study of fibroblasts,for instance,the sameset of geneswas transcriptionally induced by platelet-derivedgrowth factor (PDGF) and by fibroblast growth factor (FGF)' suggestingthat exposure to either signaling molecule had similar effects. The PDGF receptor and the FGF receptor are both receptor tyrosine kinases,and the binding of ligand to either receptor can activateRas. Although severalmechanismsfor producing diverse cellular responsesto a particular signaling molecule have been uncovered, here we focus on two: (1) the strength or duration of the signal governsthe nature of the response;and (2) different intracellular pathways are activated by the samereceptor in different cell types. Differences in Signal Strength or Duration Evidence supporting the use of the first mechanismcomes from studies with PCL2 cells, a cultured cell line capable of differentiating into adipocytes or neurons. Nerve growth factor (NGF) promotes the formation of neurons, whereas epidermal growth factor (EGF) promotes the formation of adipocytes.Strengtheningthe EGF signal by prolonging exDosureto it causesneuronal differentiation. Although both NCp and EGF are RTK ligands,NGF is a much stronger activator of the Ras/MAP kinase pathway than is EGF. The EGF receptor can apparently activate this pathway only after prolongedstimulation. A C T I V A T I O NO F R a s A N D M A P K I N A S EP A T H W A Y S

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Differences in Downstream Pathways Signalingthrough cell type-specificpathways downstream of an RTK has been demonstratedin C. elegans.In worms, EGF signalsinduce at leastfive distinct responses,eachone in a different type of cell. Four of the five responsesare mediated by the common Ras/MAP kinasepathway; the fifth, hermaphroditeovulation, employsa different downsrreampathway in which the second messengerinositol 1,4,5-trisphosphate(IP3) is generated. Binding of IP3 to its receptor,an IP3-gatedCa2* channel, in the endoplasmicreticulum (ER) membraneleadsto the release of stored Ca2* from the ER (seeFigure 15-30). The rise in cytosolic Ca2* then triggersovulation. This alternativepathway was discoveredwith a geneticscreenthat implicated the IP3 receptor in EGF signaling-a good example of how a mutation in an unexpectedgenecan lead to a discovery.

Activation of Ras and MAP Kinase Pathways r Ras is an intracellular GTPase switch protein that acts downstream from most RTKs. Like G., Ras cyclesbetween an inactive GDP-bound form and an acrive GTP-bound form. Ras cycling requiresthe assistanceof two proteins: a guanine nucleotide-exchangefactor (GEF) and a GTpaseactivatingprotein (GAP). r RTKs are linked indirectly to Ras via rwo proteins: GRB2, an adapter protein, and Sos,which has GEF activity (seeFigure 16-20). r The SH2 domain in GRB2 binds to a phosphotyrosinein activated RTKs, while its two SH3 domains bind Sos, thereby bringing Sos close ro membrane-boundRas.GDp and activating its nucleotide-exchangeactivity. r Binding of Sos to inactive Ras causesa large conformational change that permits releaseof GDP and binding of GTP, forming active Ras (seeFigure 16-24). GAP, which acceleratesGTP hydrolysis,is localizednear Ras.GTp by binding to activated RTKs. r Activated Ras triggers a kinase cascadein which Raf. MEK, and MAP kinase are sequentially phosphorylated and thus activated. Activated MAP kinase dimerizes and translocatesto the nucleus (seeFigure 16-25). r Activation of MAP kinase following stimulation of a growth factor receptor leads to phosphorylation and activation of two transcription factors, which associateinto a trimeric complex that promotes transcription of various early-response genes(seeFigure 16-27). r Different extracellular signalsinduce activation of different MAP kinase pathways, which regulate diverse cellular processes. r The upstream components of MAP kinase cascadesassembleinto large pathway-specificcomplexesstabilized by scaffold proteins (seeFigure 16-28). This assuresthat activation of one pathway by a particular extracellular signal does not lead to activation of other pathways containing sharedcomponents.

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Phosphoinositides as SignalTransducers In previous sections,we have seenhow signal transduction from cytokine receptors and receptor tyrosine kinases (RTKs) beginswith formation of multiprotein complexesassociatedwith the plasma membrane (seeFigures 16-12 and 1,6-20).Here we discusshow these same receptors initiate signaling pathways that involve membrane-boundphosphorylated inositol lipids, collectively referred to as phosphoinositides. Many of these pathways have short-term effects on cell metabolism and all have long-term effectson the pattern of gene expression. We begin with the branch of the phosphoinositide pathway that also is mediated by G protein-coupled receptors and then consider another branch that is not sharedwith thesereceDtors.

Phospholipase Cr ls Activated by SomeRTKs and CytokineReceptors As discussedin Chapter 15, hormonal stimulation of some G protein-coupled receptors leads to activation of phospholipaseC (PLC), specificallythe B isoform (PLCB).This membrane-associatedenzyme then cleavesphosphatidylinositol 4,S-bisphosphate(PIP2)to generatetwo important second messengers,l,2-diacylglycerol (DAG) and inositol 1,4,5trisphosphate (IP3).Signalingvia the IP3/DAG pathway described in Chapter 15 leads to an increasein cytosolic Ca2* and to activationof protein kinaseC (seeFigure15-30).This pathway has both short-term effectson cell metabolism and movement and long-term effectson geneexpression. Many RTKs and cytokine receptorsalso can initiate the IP3/DAG pathway by activating another isoform of phospholipase C, the 1 isoform (PLC"). The SH2 domains of PLC" bind to specificphosphotyrosineson the activated receptors, thus positioning the enzymeclose to its membranebound substrate, phosphatidyl inositol 4,5-bisphosphate (PIP2).In addition, the kinase activity associatedwith receptor activation phosphorylatestyrosine residueson the bound PLC", enhancingits hydrolase activity. Thus activated RTKs and cytokine receptorspromote PLC, activity in two ways: by localizing the enzymeto the membrane and by phosphorylating it.

R e c r u i t m e not f P l - 3K i n a s et o H o r m o n e StimulatedReceptorsLeadsto Synthesis o f P h o s p h o r y l a t eP dhosphatidylinositols Many activated RTKs and cytokine receptors initiate another phosphoinositide pathway by recruiting the enzyme phosphatidylinositol-3(PI-3) kinase to the membrane. In some cells, this P1-3 hinasepathway can trigger cell division and prevent programmed cell death (apoptosis),thus assuring cell survival. In other cells, this pathway inducesspecific changesin cell metabolism. PI-3 kinase was first identified in studiesof the polyoma virus, a DNA virus that transforms certain mammalian cells

p A T H W A y s r H A T c o N T R o L G E N EA c l v t r y c E L L S I G N A L | N Gi l : S t G N A L T N G

to uncontrolled growth. Transformation requires several viral-encoded oncoproteins, including one termed "middle T. " In an attempt to discoverhow middle T functions, investigators uncovered PI-3 kinase protein in partially purified preparations of middle I suggestinga specific interaction between the two. Then they set out to determine how PI-3 kinase might affect cell behavior. 'S7hen an inactive, dominant-negativeversion of PI-3 kinase was expressedin polyoma virus-transformed cells, it inhibited the uncontrolled cell proliferation characteristicof

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virus-transformed cells. This finding suggestedthat the normal kinase is important in certain signaling pathways essential for cell proliferation or for the prevention of apoptosis. Subsequentwork showed that PI-3 kinasesparticipate in many signalingpathways related to cell growth and apoptosis.Of the nine PI-3 kinasehomologsencodedby the human genome,the best characterizedcontains a p1,10subunit with catalytic activity and a p85 subunit with an SH2 phosphotyrosine-bindingdomain. PI-3 kinaseis recruitedto the plasmamembraneby binding of its SH2 domain to phosphotyrosineson the cytosolic domain of many activated RTKs and cytokine receptors.This recruitment of PI-3 kinaseto the plasma membranepositions its catalyticdomain near its phosphoinositidesubstrateson the cytosolicfaceof the plasmamembrane,leadingto formation of PI 3,4-bisphosphateor PI 3,4,5-trisphosphate(Figure 16-29)' By acting as docking sites for various signal-transducing proteins, these membrane-bound PI 3-phosphatesin turn transducesignalsdownstreamin severalimportant pathways.

Accumulationof Pl 3'PhosPhates i n t h e P l a s m aM e m b r a n eL e a d s to Activationof SeveralKinases Many protein kinasesbecomeactivated by binding to phosphatidyl inositol 3-phosphatesin the plasma membrane. In turn, these kinases affect the activity of many cellular proteins. One important kinase that binds to PI 3-phosphatesis protein kinase B (PKB), a serine/threoninekinase that is also called Akt. Besidesits kinase domain, protein kinase B also contains a PH domain that can tightly bind the 3-phosphate in both PI 3,4-bisphosphateand PI 3,4,5-trisphosphate'In unstimulated,restingcells,the level of both thesecompounds is low, and protein kinase B is presentin the cytosol in an inactive form (Figure 16-30). Following hormone stimulation and the resulting rise in PI 3-phosphates'protein kinase B binds to them and becomeslocalized at the plasma membrane. Binding of protein kinase B to PI 3-phosphatesnot only recruits the enzyme to the plasma membrane but also releasesinhibition of the catalytic site by the PH domain. Maximal activation of protein kinaseB, however,dependson recruitment of two other kinases:PDK1 and PDK2. PDK1 is recruited to the plasma membrane via binding of its own PH domain to PI 3-phosphates.Both membraneassociatedprotein kinase B and PDK1 can diffuse in the plane of the membrane, bringing them close enough that PDK1 can phosphorylate protein kinase B on a critical

316-29Generationof phosphatidylinositol < FIGURE (Pl-3kinase) kinase phosphatidylinositol-3 phosphates. Theenzyme tyrosine receptor by manyactivated to the membrane isrecruited addedby The3-phosphate (RTKs) receptors andcytokine kinases is or Pl3,4,5-trisphosphate, to yieldPl3,4-bisphosphate thisenzyme, proteins, suchasthe PH signal-transduction a bindingsitefor various alsoisthesubstrate B.Pl4,5-bisphosphate domainof proteinkinase (see Rameh andL C Cantlev, L 1 Figure 5-29) phospholipase C [See of 1999, J Biol Chem 274:8347 | P H O S P H O I N O s I T I DAESSS I G N A LT R A N S D U C E R S .

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FIGURE 15-30Recruitment and activationof protein groupsserveasdocking 3-phosphate siteson the plasma kinaseB (PKB)in Pl-3kinasepathways.In unstrmulated cells membrane for the PHdomainof PKB(E) andanotherkinase, (n), PKBisin thecytosol with itsPHdomainboundto thecatalytic PDK1. Fullactivation phosphorylation of PKBrequires bothin the krnase domain,inhibiting itsactivityHormone stimulation leadsto activation lipby PDKlandat theC-terminus by a second kinase, activation of Pl-3kinase andsubsequent formation of PDK2(B) [Adapted fromA Toker andA Newton, 2000,Cetl103:185, phosphatidylinositol (Pl)3-phosphates (seeFigure 16-29)The andS Sarbassov etal, 2005,CurrOpinCellBiol.17:5961

threonine residue in its activation lip-yet another example of kinaseactivationby phosphorylation.Phosphorylationof a secondserine,not in the lip segment,by PDK2 is necessary for maximal protein kinase B activity (seeFigure 16-30). Similar to the regulationof Raf activity (seeFigure 16-25), releaseof an inhibitory domain and phosphorylation by other kinasesregulate the activity of protein kinase B.

Activated ProteinKinaseB Induces M a n y C e l l u l a rR e s p o n s e s Once fully activated, protein kinase B can dissociatefrom the plasma membrane and phosphorylate its many rarger proteins, which have a wide range of effectson cell behavior. Although activation of PKB takes only 5-10 minutes, its effectscan last as long as severalhours. Promotion of Cell Survival In many cellsactivatedprotein kinaseB directly phosphorylatesand inactivatespro-apoptotic proteins such as Bad, a short-term effect that prevenrs acrivation of an apoptoticpathway leadingto cell death (Chapter 21). Activated protein kinase B also promotes survival of many cultured cells by phosphorylating the Forkhead transcription factor FOXO 3A on multiple serine/threonine residues,thereby reducing its pro-apoptotic effect and contributing to cell survival. In the absenceof growth facrors,FOXO 34 is unphosphorylated and mainly localizesto the nucleus,where it activates transcriptionof severalgenesencodingpro-apoptoticproteins. When growth factors are added to the cells, protein kinase B 696

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becomesactiveand phosphorylatesFOXO 3A..This allows the cytosolicphosphoserine-binding protein 14-3-3to bind FOXO 34 and thus sequesterit in the cytosol.(Recallthat 14-3-3 also retains phosphorylatedRaf protein in an inactive state in the cytosol;seeFigure 16-25.)\Tithdrawal of growth factor leads to inactivation of protein kinase B and dephosphorylationof FOXO 3A.,thus favoring its accumulationin the nucleusand transcription of apoptosis-inducinggenes.A FOXO 34' mutant in which the three serinetarget residuesfor protein kinase B are mutated to alaninesis "constitutively active" and initiatesapoptosisevenin the presenceof activatedprotein kinase B. This finding demonstratesthe importanceof FOXO 34 and protein kinase B in controlling apoptosis of cultured cells. Deregulationof protein kinaseB is implicatedin the pathogenesisboth of cancerand diabetes. Promotion of Glucose Uptake and Storage by Insulin As we learned in Chapter 15, insulin acts on muscle, liver, and fat cellsto lower the level of blood glucoseby increasing its uptake from the blood. In muscle and liver, insulin also promotes storageof glucoseas glycogen.The insulin receptor is a dimeric receptor tyrosine kinase that triggers the Ras/MAP kinase pathway, leading to changesin gene expression.Insulin stimulation also can initiate the PI-3 kinase/protein kinase B pathway. The resultant, activated protein kinase B exerts several short-term effects that lower blood glucose and promote glycogen synthesis.The principal shorr-term effect is increasedimport of glucoseby fat and muscle cells. The GLUT4 glucose transporter is normally retained in intracellularmembranevesiclesby a protein called AS160.

c E L L S I G N A L | N Gi l : S I G N A L | N Gp A T H W A y s r H A T c o N T R O L G E N EA c l v t r y

Activated protein kinase B phosphorylatesAS160; through mechanismsthat are not fully understood this causesmovement of GLUT4 to the cell surface (seeFigure 15-34). The resulting increasedinflux of glucose into these cells lowers blood glucoselevels. In both liver and muscle,insulin stimulation also leadsto short-term activation of glycogen synthase(GS), which synthesizesglycogen from UDP-glucose (seeFigure 15-24). ln resting cells (i.e., in the absenceof insulin), glycogensynthasekinase3 (GSK3)is activeand phosphorylatesglycogen synthase,thereby blocking its activity. In insulin-stimulated cells, activated protein kinase B phosphorylatesand thereby inactivatesGSK3, relieving the GSK3-mediatedinhibition of glycogen synthaseand promoting glycogen synthesis.As a result, the intracellular concentration of glucose and its metabolites is reduced,stimulating glucoseuptake from the blood. This insulin-dependenteffect representsanother mechanismfor reducing the blood glucoselevel.

The Pl-3KinasePathway ls Negatively Regulatedby PTENPhosphatase Like virtually all intracellular signalingevents,phosphoryl a t i o n b y P I - 3 k i n a s e i s r e v e r s i b l e .T h e r e l e v a n t p h o s phatase, termed PTEN phosphatase, has an unusually broad specificity.Although PTEN can remove phosphate groups attachedto serine,threonine, and tyrosine residues in proteins, its ability to remove the 3-phosphatefrom PI 3,4,5-trisphosphateis thought to be its major function in c e l l s . O v e r e x p r e s s i o no f P T E N i n c u l t u r e d m a m m a l i a n cells promotes apoptosisby reducing the level of PI 3,4,5trisphosphateand hencethe activation and anti-apoptotic effect of protein kinase B. F:E In multiple typesof advancedhuman cancers,the PTEN geneis deleted.The resulting loss of PTEN protein conI.| tributesto the uncontrolledgrowth of cells.Indeed,cellslacking PTEN have elevatedlevelsof PI 3,4,5-trisphosphateand PKB activity. Sinceprotein kinase B exerts an anti-apoptotic effect, loss of PTEN indirectly reducesthe programmed cell death that is the normal fate of many cells.In certain cells, such as neuronal stem cells, absenceof PTEN not only prevents apoptosis but also leads to stimulation of cell-cycle progressionand an enhancedrate of proliferation. Knockout mice lacking PTEN have big brains with excessnumbers of neurons,attestingto PTEN's importancein control of normal develooment.I

Phosphoinositidesas Signal Transducers r Many RTKs and cytokine receptors can initiate the IP3/DAG signaling pathway by activating phospholipase C^y(PLCI), a different PLC isoform than the one activated by G protein-coupled receptors. r Activated RTKs and cytokine receptorsalso can initiate another phosphoinositidepathway by binding PI-3 kinases,

thereby allowing the enzymesaccessto their membranebound phosphoinositidesubstrates,which then become phosphorylated at the 3 position (seeFigure 1'6-29). r The PH domain in various proteins binds to PI 3phosphates, forming signaling complexes associatedwith the plasmamembrane. r Protein kinase B (PKB) becomespartially activated by binding to PI 3-phosphates.Its full activation requires phosphorylation by another kinase, PDK1' which also is recruited to the membrane by binding to PI 3-phosphates and by a secondkinase, PDK2, (seeFigure 16-30). r Activated protein kinase B promotes survival of many cells by directly phosphorylating and inactivating several pro-apoptotic proteins and by phosphorylating and inactivating a transcription factor that otherwise inducessynthesis of pro-apoptoticproteins. r Activation of the insulin receptor,a receptor tyrosine kinase, on fat and muscle cells initiates the PI-3 kinase pathway. The resulting activatedprotein kinase B promotes glucoseuptake and glycogensynthesis. r Signalingvia the PI-3 kinasepathway is terminated by the PTEN phosphatase,which hydrolyzesthe 3-phosphatein PI 3-phosphates.Loss of PTEN, a common occurrencein human tumors, promotes cell survival and proliferatron.

Activationof GeneTranscriPtion Cell-surface by Seven-Spanning Receptors Chapter 15 focused on intracellular signal-transduction pathways initiated by ligand binding to G protein-coupled receptors (GPCRs). These seven-spanningreceptorsoften have short-term (secondsto minutes) effectson cell metabolism, primarily by modulating the activity of preexisting enzymesor other proteins. However, GPCR signaling pathways also can have long-term effects (hours to days) owing to activation or repressionof genetranscription. \7e have already seenhow yeast G protein-coupled receptors for mating factors activatea MAP kinase pathway leading to longterm changesin geneexpression(seeFigure 16-28a).lnthe first part of this section, we discusstwo other ways that G protein-coupled receptors affect gene expression.The first mechanism operatesthrough phosphorylation of transcription factors by protein kinase A, which is activated downstream of G,-coupled receptors (see Figure 16-2d). The secondmechanismacts through binding of arrestin to many ligand-occupiedG protein-coupled receptors and subsequent binding of enzymesin the MAP kinase and other pathways. In the remainder of this section, we consider two other classesof seven-spanningreceptors-those that bind Wnt and Hedgehog,two protein signalsthat play key roles in development(seeFigure 16-2e,f).Although similar in structure to G protein-coupled receptors,the receptors for'S7nt and

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Hedgehog do not activate G proteins. Thesepathways have been elucidatedmainly through geneticanalysisof developmental mutants in Drosophila but are operative in humans as well. Activation of these receptorsleads to expressionof key genesrequired for a cell to acquire a new identify or fate. In Chapter 22, we explore the roles of thesereceptorsin several key developmental pathways and also illustrate how these signaling pathways interact with others, activated by different receptors, to specify the precise fate of many cells during development.

C R E BL i n k sc A M Pa n d P r o t e i nK i n a s eA to Activation of GeneTranscription In mammalian cells, an elevation in the cytosolic cAMp Ievel results in activation of protein kinase A (pKA), leading to many different rypes of short-term responsesin different cell types (seeTable 15-2). One of the most important short-term PKA-mediated effects is activation of g l y c o g e n o l y s i si n l i v e r a n d m u s c l e , i n c r e a s i n gt h e l e v e l of glucosein the blood (seeFigure 15-25a).Activation of protein kinase A also stimulates the expressionof many genes,Ieading to long-term effects on the cells that often enhancethe short-term effectsof activated Drotein kinase A. 698

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< FIGURE 16-31Activationof CREB transcriptionfactor following ligandbindingto Gsprotein-coupled receptors. ([) leadsto activation Receptor stimulation of proteinkinase A, PKA(U ) Catalytic (El) subunits of PKAtranslocate to the nucleus andtherephosphorylate andactivate the CREB transcription factor (@) Phosphorylated CREB associates with the co-activator (E) to stimulate CBP/P300 various targetgenescontrolled by the CREregulatory elementSeethetextfor details. K A Leeand [See N Masson, 1993,Biochim.Biophys Acta1174:221 , andDparkeretal , 1996,Mol CellBiol 16(2):6941

For instance,in liver cells,protein kinaseA inducesexpression of several enzymes involved in converting three-carbon compounds such as pyruvate (Figure 12-3) to glucose, thus increasingthe level of glucosein the blood. AII genes regulated by protein kinase A contain a cisacting DNA sequence,the cAMP-responseelement (CRE), that binds the phosphorylated form of a transcription factor called CRE-binding (CREB) protein, which is found only in the nucleus. As detailed in Chapter 15, binding of neurotransmitters and hormones to G, protein-coupled receptors resultsin the releaseof the active catalytic subunit of protein kinase A. Some of the catalytic subunits then translocate to the nucleusand phosphorylate serine-133on CREB protein. PhosphorylatedCREB protein binds to CRE-containing target genesand also binds to a co-actiuator termed CBP/300, which links CREB to the basal transcriptional machinery, thereby permitting CREB to stimulate transcription (Figure 15-3 1 ). As discussedin Chapter 7 , other signal-regulated transcription factors rely on CBP/P300to exert their activating effect. Thus this co-activator plays an important role in integrating signals from multiple signaling pathways that regulategenetranscription.

GPCR-Bound ArrestinActivatesSeveral K i n a s eC a s c a d e s In higher organisms,activation of the MAP kinase pathway is often triggered by G protein-coupled receptors (GPCRs). As we discussedin Chapter 15, B-arrestin binds to phosphorylated serinesin the cytosolic domain of activated G protein-coupled receptors and desensitizescells to further hormone stimulation in two ways: by inhibiting activation of a Go protein and by promoting endocytosis of the GPCR-arrestin complex. The GPCR-arrestin complex also acts as a scaffold for binding and activating severalcytosolic kinases (seeFigure 15-27). These include c-Src, a cytosolic protein tyrosine kinase that activatesthe MAP kinase pathway and other pathways leading to transcription of genes neededfor cell division. A complex of three arrestin-bound proteins, including a Jun N-terminal kinase (JNK-1), iniriates a kinase cascade that ultimately activates the c-Jun transcription factor. Activated c*Jun promotes expressionof certain growth-promoting enzymesand other proteins that help cells respond to stresses.

pA c E L Ls T G N A L T N r :Gs T G N A L T N GT H W A y rsH A T c o N T R 9 LG E N EA c r v r r y

Wnt SignalsTriggerReleaseof a Transcription Factorfrom CytosolicProteinComplex Like G protein-coupled receptors, the receptors for Wnt proteins span the plasma membrane seventimes, but there the similarity ends. The first vertebrate Wnt gene ro be discovered, the mouse Wnt-1 gene,attracted notice becauseit was overexpressedin certain mammary cancers.Subsequent work showed that overexpressionwas causedby insertion of a mouse mammary tumor virus (MMTV) provirus near the Wnt-l. gene. Hence $7nt-1 is a proto-oncogene, a normal cellular gene whose inappropriate expressionpromotes the onset of cancer (Chapter 25). The word -il/nt is an amalgamation of wingless,the corresponding fly gene,with int for the retrovirusintegrationsite in mouse. Activation of the V/nt pathway controls numerous crirical developmental events, such as brain development, limb patterning, and organogenesis.A major role for'S7nt signal ing in bone formation was revealed by the finding that mutations in Wnt pathway components affect bone density in humans. Wnt signaling is now known to control formation of osteoblasts(bone-formingcells).AdditionallS Wnt signals are important in controlling stem cells (Chapter 21) and in many other aspectsof development(Chapter22). Disturbancesin signaling through the Wnt pathway are associated with various human cancers, particularly colon cancer ( C h a p t e r2 5 ) . Becauseof the conservation of the Wnt signaling pathway in metazoan evolution, genetic studies rn Drosophila and C. elegans,studies of mouse proto-oncogenesand tumor-suppressorgenes,and studiesof cell-junctioncomponents have all contributed to identifying various pathway components.Wnt proteins, the extracellular signaling moleculesin the pathway,are modified by addition of a palmitate

group near their N termini. This hydrophobic group is thought to tether'S7nt proteins to the plasma membrane of 'Wnt secretingcells, thus limiting their range of action to adjacent cells. Wnt proteins act through two cell-surfacereceptor proteins: Frizzled (Fz), which contains seventransmembrane o. helices and directly binds Wnt; and a co-receptor designatedLRP, which appearsto associatewith Frizzled in '$fnt signal-dependentmanner (seeFigure 1.6-2e1.Mutaa tions in the genesencoding Wnt proteins, Frizzled, or LRP (called Arrow in Drosophila) all have similar effects on the developmentof embryos. According to a current model of the Wnt patbway, the central player in intracellular Wnt signal transduction is calledp-catenin in vertebratesand Armadillo in Drosophila. This multi-talented protein functions both as a transcriptional activator and as a membrane-cytoskeletonlinker protein (seeFigure 1.9-1.2).In the absenceof a'Wnt signal,Bcatenin is phosphorylated by a complex containing glycogen synthasekinase 3 (GSK3), the same protein kinase that functions in regulation of blood glucose(Section16.5); the adenomatosispolyposis coli (APC) protein, an important human tumor suppressor;and Axin, a scaffold protein. PhosphorylatedB-cateninis then ubiquitinated and degraded in proteasomes(Figurel6-32a). In the presenceof I7nt, Axin binds to the cytosolic domain of the LRP co-receptor.This binding disrupts the complex containing GSK3 and B-catenin,preventsphosphorylation of B-catenin by GSK3, and stabilizes B-catenin in the cytosol (Figure 16-22b). \fnt-induced stabilization of B-cateninalso requiresthe Dishevelled(Dsh) protein, which is bound to the cytosolic domain of the receptor Frizzled (Fz).The freed B-catenintranslocatesto the nucleuswhere it associateswith the TCF transcription factor to control

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< FIGURE 16-32Wnt signalingpathway. (a)Inthe absence isfoundin a of Wnt,B-catenin protein), APC,and complex with Axin(ascaffold whichphosphorylates the kinase GSK3, P-catenin, l e a d i n tgo i t sd e g r a d a t i oTnh eA x i n - m e d i a t e d phosphorylation facilitates formation of thiscomplex factorof of B-catenin by GSK3by an estimated >20,000TheTCFtranscription factorin thenucleus alteredby of targetgenesunless actsasa repressor Wnt signaling(b)Binding of Wnt to itsreceptor (Fz),triggersphosphorylation of the LRP Frizzled andthus co-receptor by GSK3andanotherkinase, allowssubsequent bindingof Axin Thisdisrupts preventi ng complex, theAxin-APC-G5K3-B-catenin phosphorylation by GSK3andleads of B-catenin t o a c c u m u l a t i o fnB - c a t e n i n t h e c e l l A f t e r mayact translocation to the nucleus, B-catenin targetgenesor,alternatively, withTCFto activate and cause theexportof TCFfromthe nucleus possibly in the cytosol.[AfterR Nusse, itsactivation 438i747; seealsoTheWntGeneHomepage, 2005,Nature www stanfordedu/-rnusse/wntwindowhtml l

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expressionof particular target genes.(Recall that TCF also functions in the MAP kinase pathway; see Figure 16-27.) Among the Wnt target genes are many that also control Wnt signaling, indicating a high degreeof feedback regulation. The importance of B-catenin stability and location means that Wnt signals affect a critical balance between the three pools of B-catenin in the cell: the cytoskeleton, cytosol, and nucleus. \7nt signaling also requires binding to cell-surfaceproteoglycans.A proteoglycan consistsof a core protein bound to glycosaminoglycan(GAG) chains such as heparin sulfate and chondroitin sulfate (seeFigure 19-29). Evidencefor the participation of proteoglycansin'Wnt signaling comes from Drosophila sugarless/sgf mutants, which lack a key enzyme neededto synthesizeheparin and chondroitin sulfate. These mutants have greatly depressedlevels of Wingless, the fly '!fnt protein, and exhibit other phenotypesassociatedwith defectsin Wnt signaling. Mutations in two other fly genes, dally and dally-like, both of which encode core proteins of cell-surfaceproteoglycans,also are associatedwith defective 'Wnt signaling in Drosophila. How proteoglycansfacilitate Wnt signaling is unknown, but perhaps binding of Wnt to specificglycosaminoglycanchains is required for it to bind to its receptor Fz or co-receptorLRP. This mechanismwould be analogousto the binding of fibroblast growth factor (FGF) to heparan sulfate,which enhancesbinding of FGF to its receptor tyrosine kinase (seeFigure 16-15).

many tissuesand organs. Mutations in componentsof the Hedgehog signaling pathway have been implicated in human birth defects such as cyclopia, a single eye resulting from union of the right and left brain primordia, and in multiple forms of cancer.I Processing of Hh Precursor Protein Hedgehogis formed from a precursor protein with autoproteolytic activity that enablesthe protein to cut itself in half. The cleavageproduces an N-terminal fragment, which is subsequentlysecretedto signal ro other cells,and a C-terminal fragment, which is degraded.As shown in Figure 16-33, cleavageof the precursor is accompanied by covalent addition of the lipid cholesterolto the new carboxyl terminus of the N-terminal fragment. The C-terminal domain of the precursor,which catalyzesthis reaction, is found in

Hh precursor

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H e d g e h o gS i g n a l i n gR e l i e v e sR e p r e s s i o n of TargetGenes The Hedgehog (Hh) pathway is similar to the lfnt pathway in that two membrane proteins, one with seven membranespanning segments,are required to receive and transduce a signal (seeFigure 16-2f). The Hh pathway also involvesdisassembly of an intracellular complex containing a transcription factor, Iike the Sfnt pathway. However, in contrast to Wnt signaling, the Hh protein, the extracellularsignalin the pathway, is synthesizedas a precursorthat is cleaved,and the two membrane proteins involved in Hh signalingare thought to move betweenthe plasma membraneand intracellular vesicles. Although Hedgehogis a secretedprotein, it moves only a short distance from a signaling cell, on the order of 1-20 cells, and is bound by receptorson receivingcells. Thus Hh signals,like \fnt signals,have quite localized effects.As Hh diffuses away from secreting cells, however, its concentration decreases.As we learn in Chapter 22, different Hh concentrationsinduce different fates in receivingcells: Cells that receivea large amount of Hh turn on certain genesand form certain structures;cellsthat receivea smaller amount turn on different genes and thus form different structures. Signals that induce different cell fates dependingon their concentration are referred to as morphogens.During development,the production of Hedgehog and other morphogens is tightly regulatedin time and space. Hedgehog signaling, which is conservedthroughout the animal kingdom, functions in the formation of

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A FIGURE 16-33Processing of Hedgehog(Hh)precursor protein.Cellssynthesize a 45-kDaHh precursor, whichundergoes a nucleophilic attackbythe thiolsidechainof cysteine 258(Cys-258) glycine carbonof the adjacent residue 257(Gly-257), on the carbonyl forminga high-energy thioester intermediate. An enzymic activity in the C-terminal domainthencatalyzes theformation of an esterbond groupof cholesterol andglycine 257, between the B-3hydroxyl intotwo fragmentsTheN-terminal cleaving the precursor signaling (blue)retains fragment thecholesterol moietyandisalsomodified by groupto the N-terminus, theadditionof a palmitoyl Thisprocessing isthoughtto occurmostlyintracellularly Thetwo hydrophobrc processed anchors maytetherthe secreted, Hh proteinto the plasma membrane. fromJ A Porter etal,1996,Science2T4:2551 lAdapted

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other proteins and may promote the linkage of theseproteins to membranesby a similar autoproteolpic mechanism. A second modification to Hedgehog, the addition of a palmitoyl group to the N-terminus, makes the protein even more hydrophobic. Together,the two attached hydrophobic groups may causesecretedHedgehogto bind nonspecifically and reversiblyto cell plasma membranes,thereby limiting its diffusion and thus its range of action in tissues.Spatial restriction plays a crucial role in constraining the effects of powerful inductive signalslike Hh. Recall that the palmitoyl group also is added to Wnt proteins and likely also causes ril/nt to bind reversibly to cells, thereby restricting'Sfnt signaling to cellsadjacentto the signalingcell. Hh Pathway in Drosophila GeneticstudiesinDrosophila tndicate that two membrane proteins, Smoothened(Smo) and Patched(Ptc), arerequired to receiveand transducea Hedgehog signal to the cell interior. Smoothenedhas sevenmembranespanning a helicesand is related in sequenceto the ITnt receptor Fz. Patchedis predictedto contain 12 transmembranea helices and is most similar structurallyto the Niemann-PickC1 (NPC1) protein, a member of the ABC superfamilyof mem-

brane proteins (seeThble 11-3). The NPC1 protein, which probably functions as an AlP-powered pump, is necessaryfor normal intracellular movement of sterols and other substances through vesicle-traffickingpathways. In humans, mutations in the NPC1 gene cause a rar% autosomal recessivedisorder marked by defectsin movements of late endosomesand in the handling of cholesterolin endosomesand lysosomes.A related protein,NPC1L1, is the major cholesteroluptaketransporterin the mammalian intestine.Patchedlikely evolved from a NPC1like ancestor,since NPC1 but not Patchedis clearly present in yeast.This may be an exampleof how a cell componentneeded for fundamental cell metabolism was adapted as a component of a developmentalsignaling pathway. Duplication of the npcl genewould have beenfollowed by divergentevolution of one of the copies. Figure 16-34 depicts a current model of the Hedgehog (Hh) pathway, based largely on work in Drosophila. Evidence supporting this model comes from study of fly embryos with loss-of-function mutations in the hedgehog (hh) or smoothened (smo) genes.Both types of mutant embryos have very similar developmentalphenotypes.Moreover, both the bh ar'd smo genesare required to activate transcription

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A FIGURE 15-34 Hedgehog(Hh)signalingpathway.(a)Inthe (Ptc)proteininhibits (Smo), absence of Hh,Patched Smoothened whichis present in the membrane largely of internal vesicles A (Fu),a kinase, (Cos2), complex containing Fused Costal-2 a kinesin(Ci),a zinc-f relatedmotorprotein, andCubitisinterruptus inger transcrrption factor,bindsto microtubules Ci isphosphorylated in a proteinkinase series of stepsrnvolving glycogen A (PKA), synthase , dc a s e ikni n a s 1 k i n a s3e ( G S K 3a) n t ei ids e ( C K 1 )T h ep h o s p h o r y l aC thenproteolytically cleaved rna process requiring the proteinSlimb pathway, andthe ubiquitin/proteasome generating the fragment Ci75,whichf unctions asa transcriptional repressor of Hh-responsive

somePtc genes(b)In the presence of Hh,Hh bindsto Ptc,causing (notshown)andrelieving the compartments to moveto internal is membrane, inhibition of Smo Smothenmovesto the plasma fromdegradation phosphorylated, bindsCos2,andisstabilized phosphorylated, andmost extensively BothFuandCos2become frommicrotubules. isdissociated complex importantly the Fu/Cos2lCi modified Ci, alternately of a full-length, Thisleadsto thestabilization with in conjunction activator asa transcriptional whichfunctions in compartments protein(CBP)Theexactmembrane CREB-binding to Hhandfunctionareunknown[After whichPtcandSmorespond Dev.2Q:399 lVA Price, 2006,Genes l

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of the same target genes(e.g.,patched and wingless)during embryonic development.In contrast, loss-of-function mutations in the patched (ptc) geneproduce a quite different phenotype, one similar to the effect of flooding the embryo with Hedgehog protein. Thus Patched appearsto antagonizethe actions of Hedgehog and vice versat These findings suggest that, in the absenceof Hedgehog, Patched repressestarget genes by inhibiting a signaling pathway needed for gene activation. The additional observation that Smoothened is required for the transcription of target genes in mutants Iacking patched function, places Smoothened downstream of Patched in the Hh pathway. The evidenceindicates that Hedgehog binds directly to Patched and prevents Patched from blocking Smoothenedaction, thus activating the transcription of target genes. Immunostaining of appropriate Drosophila embryonic cells with antibodies to Hh, Ptc, and Smo has shown that in the absenceof Hedgehog, Patchedis enriched in the plasma membrane, but Smoothened is in internal vesicle membranes. Following binding of Hedgehog to Patched, both proteins move from the cell surface into internal vesicles, while Smoothenedmoves from internal vesiclesto the surface. The similarity of Patched to transporter proteins suggests that in the absenceof Hh binding, it either pumps a small-moleculeinhibitor toward Smoothened or pumps an activator away from it. This mechanismis supported by the finding that a number of natural and synthetic small molecules bind to and regulate Smo activity. In the absence of the Hh signal, the cytosolic protein complex in the Hh pathway consistsof three proteins (see Figure 16-34a):Fused(Fu), a serine-threoninekinase;Costal2 (Cos2), a microtubule-associatedkinesin-like protein; and Cubitis interruptus (Ci), a transcription factor. This complex is bound to microtubules in the cytosol. Phosphorylation of Ci by at least three kinasescausesbinding of the Slimb protein. Slimb in turn directs ubiquitination of Ci and its targering to proteasomes,where Ci undergoesproteolytic cleavage. A resulting Ci fragment, designatedCi75, translocatesto the nucleusand repressesexpressionof Hh target genes. Binding of Hedgehogto Patchedinhibits Ptc activity, perhaps by blocking a pumping process,which relievesthe inhibition of Smoothened(seeFigure 16-34b).Severalresponses are triggered by Hh binding: SomePatchedis internalizedby the receiving cells; Smoothened is phosphorylated by two protein kinases and moves to the plasma membrane; and phosphorylation of Fu and Cos2 increases.In addition, the complex of Fu, Cos2, and Ci dissociatesfrom microtubules, and Cos2 becomesassociatedwith the C-terminal tail of Smoothened. The resulting disruption of the Fus/Cos2iCi complex causes a reduction in both phosphorylation and cleavageof Ci. As a result, a modified form of full-length Ci is generatedand translocatesto the nucleuswhere it binds to the transcriptional co-activator CREB-binding protein (CBP), promoting the expressionof target genes. Hh Pathway in Mammals The Hh signalingpathway in mammals sharesmany featureswith the Drosophila pathway, but there are also some striking differences.First. mammalian

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genomescontain three hh genesand two ptc genes,which are expresseddifferentially among various tissues.Second,mammals expressthree Gli transcription factors that divide up the rolesof the singleCi proteininDrosophila.Thfud,thereappears to be no mammalian Cos2 ortholog, and the role of possibleFu orthologs is unclear. The most fascinating aspectof the mammalian Hh pathway is the newly recognized involvement of intraflagellar transport (IFT) proteins. IFT proteins are required to move materials within flagella and cilia, long plasma membraneenvelopedstructuresthat protrude from the cell surface.The roles of the abundant cilia in the trachea in moving materials along the tracheal surface and of flagella in sperm locomotion are well known (Chapter 18). Most cells, however, have a single immotile cilium called the primary cilium. The function of a primary cilium has been rather mysterious,but there is increasingevidencefor its involvement in signal transduction, especiallyin the mammalian Hh signaling pathway. For example, mutations that eliminate IFT function causeinduction of Hh pathway target genes,similar to the effect of inactivating mutations in Patched.In addition, several components of the Hh pathway, including Smoothened,are located,in part, in primary cilia. The primary cilium may be a signal-transductioncenter, and its functions may substitute for the apparently missing Cos2 kinesin-like protein that is found in the Drosophila Hh pathway. The absenceof IFT proteins in Hh signaling in Drosophila provides some support for this substitution hypothesis. 'We have aheady seenexamplesof how intracellular trafficking of many cell-surfacereceptors affects their level on the plasma membrane and thus their signaling capabilities. How Cos2 in flies and IFT proteins in mammals contribute to trafficking of Smoothenedis still unknown. Clearly,much remains to be learned about the complex relationships between receptor signal transduction and protein trafficking within cells. Regulation of Hh Signaling Feedbackcontrol of the Hh pathway is important becauseunrestrained Hh signaling can cause cancerous overgrowth or formation of the wrong cell types.In Drosophila, one of the genesinduced by the Hh signal is patched. The subsequentincreasein expression of Patched antagonizesthe Hh signalin large measureby reducing the pool of active Smoothenedprotein. Thus the system is buffered: If during developmenttoo much Hh signal is made, a consequent increasein Patched will compensate;if too little Hh signal is made. the amount of Patchedis decreased.

Activation of Gene Transcriptionby Seven-Spanning Cell-SurfaceReceptors r Downstream of activated G protein-coupled receptors, signal-inducedactivation of protein kinase A (PKA) often leads to phosphorylation of nuclear CREB protein, which together with the CBP/300 co-activator stimulates transcription of many targetgenes(seeFigure 1,6-31,).

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r The GPCR-arrestin complex activates several cytosolic kinases, initiating cascadesthat lead to transcriptional activation of many genescontrolling cell growth (seeFigure 15-27). r Both Hedgehog and lfnt signal proteins contain lipid anchorsthat can tether them to cell membranes.thereby reducing their signaling range. r Wnt signalsact through two cell-surfaceproteins, the receptor Fizzled and co-receptor Lrp, and an intracellular complex containing B-catenin (seeFigure 16-32). Binding of \fnt promotes the stability and nuclear localization of B-catenin,which either directly or indirectly promotes acrivation of the TCF transcription factor. r The Hedgehog signal also acts through two cell-surface proteins, Smoothenedand Patched,and an intracellular complex containing the Cubitis interruptus (Ci) transcription factor (seeFigure 16-34). An activating form of Ci is generated in the presence of Hedgehog; a repressing Ci fragment is generated in the absenceof Hedgehog. Both Patchedand Smoothenedchangetheir subcellularlocation in responseto Hedgehog binding to Patched.

PathwaysThat Involve Signal-lnduced ProteinCleavage All the signalingpathways discussedso far are reversibleand thus can be turned off relatively quickly if the signal is removed. In this section, we discuss several essentiallyirreversible pathways in which a component is proteolytically cleaved.First we examine the NF-rB pathway in which an inactive transcription factor is sequesteredin the cytosol bound to an inhibitor (see Figure 16-19l; several stressinducing conditions causeimmediate degradation of the inhibitor enabling cells to respond immediately and vigorously by activating genetranscription. Next we consider signaling pathways involving protein cleavageoutside of the cell by members of the matrix metalloprotease (MMP) family. ln the Notch/Delta pathway (seeFigure 16-1h), for instance, extracellular MMP cleavageof the receptor is followed by its cleavagewithin the plasma membrane by a different protease.This pathway determinesthe fates of many types of cells during development. Many growth factors, including members of the epidermal growth factor (EGF) familS are made as membrane-spanningprecursors; cleavageof these proteins by matrix metalloproteasesreleasesthe active growth factor into the extracellular medium. This process goesawry in many cancersand may lead to an often fatal enlargement of the heart. Inappropriate MMP cleavageof yet another membrane-spanningprotein has been implicated in the pathology of Alzheimer's disease.We conclude our discussion by describingthe intramembrane cleavageof a transcription factor precursor within the Golgi membrane in responseto low cholesterollevels.This pathway is essentialfor maintainingthe proper balanceof cholesteroland phospholipids for constructingcell membranes(Chapter 10).

Degradationof an Inhibitor ProteinActivates the NF-rBTranscriptionFactors The examplesin previous sections,like TGFB receptorsand MAP kinases,have demonstrated the importance of signalinduced phosphorylation in modulating the activity of many transcription factors. Another mechanism for regulating transcription factor activity in responseto extracellular signals was revealedin studieswith both mammalian cells and Drosophila. This mechanism, which involves phosphorylation and subsequentubiquitin-mediated degradation of an inhibitor protein, is exemplified by the NF-rB transcription factor. NF-rcBis rapidly activatedin mammalian immune-system cells in responseto bacterial and viral infection, inflammation, and a number of other stressfulsituations, such as ionizing radiation. As we learn in Chapter 24, the NF-rcB pathway is activated in some cells of the immune system when components of bacterial or fungal cell walls bind to certain Toll-like receptorson the cell surface.This pathway is also activated by so-calledinflammatory cytokines, such as tumor necrosisfactor alpha /TNFc/ and interleukin 1 (IL1), that are releasedby nearby cells in responseto infection. In all cases,binding of ligand to its receptor induces assembly of a multiprotein complex in the cytosol. Formation of this complex triggers a signaling pathway that results in activation of the NF-rB transcription factor. NF-rcBwas originally discoveredon the basisof its transcriptional activation of the gene encoding the light chain of antibodies (immunoglobulins) in B cells.It is now thought to be the master transcriptional regulator of the immune system in mammals. Although flies do not make antibodies, NF-rB homologs in Drosophila induce synthesisof a large number of secretedantimicrobial peptides in responseto bacterial and viral infection (Chapter 24). This phenomenon indicates that the NF-rB regulatory system has been conservedduring evolution and is more than half a billion years old. Biochemicalstudiesin mammalian cells and geneticstudies in flies have provided important insights into the operation of the NF-rcB pathway. The two subunits of heterodimericNF-rB (p65 and p50) sharea regionof homology at their N-termini that is required for their dimerization and binding to DNA. In cells that are not undergoing a stressor responding to signs of an infection, NF-rB is sequesteredin an inactive state in the cytosol by direct binding to an inhibitor called l-rcB. A single molecule of I-rB binds to the paired N-terminal domains of the p50/p65 heterodimer, thereby masking their nuclear-localizationsignals.The protein kinase complex termed I-xB kinase is the point of convergenceof all of the extracellular signalsthat activate NFrcB.Within minutes of stimulation of the cell by an infectious agent or inflammatory cytokine, I-rB kinase becomesactivated and phosphorylatestwo N-terminal serineresidueson I-rcB(Figure L6-35, steps n and El). An E3 ubiquitin ligase then binds to these phosphoserinesand polyubiquitinates IrcB, triggering its immediate degradation by a proteasome (stepsS and Zl ). In cellsexpressingmutant forms of I-rcBin

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FIGURE 16-35NF-rBsignalingpathway.In resting polyubiquitination cells,the and4: Subsequent of l-rBtargetsit for dimeric transcription factorNF-rB,composed of p50andp65 degradation by proteasomes StepE: Theremoval of l-rB unmasks (NLS) subunits, issequestered in thecytosol, boundto the inhibitor l-rBo the nuclear-localization signals in bothsubunits of NF-rB, Step[: Activation of thetrimericl-rcB kinaseisstimulated by many allowingtheirtranslocation to the nucleusStep6: Inthe nucleus, a g e n t isn c l u d i nvgi r u si n f e c t i o n i o, n i z i nrga d i a t i o b n i,n d i n o gf t h e NF-rcB activates transcription of numerous targetgenes,including the proinflammatory cytokines TNFaor lL-1to theirrespective receptors, geneencoding l-rBct,whichactsto terminate signaling, andgenes or activation of anyof several Toll-like receptors by components of encoding various inflammatory whichpromotesignaling cytokines, invading bacteria or fungi.Step[: l-rcB kinase thenphosphorylates [SeeR Khush et al, 2001,Trends lmmunol22:260, andJ-LLuoetal, 2005, the inhibitor l-rc8a, whichthenbindsan E3ubiquitin ligaseSteps! J Clinlnvest115:26251 which these two serineshave been changed to alanine, and thus cannot be phosphorylated, NF-rcBis permanently inactive, demonstrating that phosphorylation of I-rB is essential for pathway activation. The degradation of I-rB exposesthe nuclear-localization signals on NF-rB, which then translocatesinro the nucleus and activatestranscription of a multitude of target genes(see Figure 16-35 , stepsI and 6 ). Despite its activation by proteolysis,NF-rB signaling eventually is turned off by a negative feedback loop, since one of the geneswhose transcription is immediately induced by NF-rcB encodes I-rcB. The resulting increasedlevelsof the I-rB protein bind active NFrB in the nucleusand return it to the cyrosol. In many immune-system cells, NF-rcB stimulates transcription of more than 150 genes,including those encoding cytokines and chemokines;the latter attract other immunesystemcells and fibroblasts to sitesof infection. NF-rB also promotes expression of receptor proteins that enable neutrophils (a type of white blood cell) to migrate from the blood into the underlying tissue (see Figure 19-36). In 704

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addition, NF-rB stimulatesexpressionof iNOS, the inducible isoform of the enzyme that produces nitric oxide, which is toxic to bacterial cells, as well as expressionof severalantiapoptotic proteins, which prevent cell death. Thus this single transcription factor coordinates and activatesthe body's defenseeither directly by respondingto pathogensand stressor indirectly by responding to signaling molecules released from other infected or wounded tissuesand cells. Besidesits roles in inflammation and immunity, NF-rB plays a key role during mammalian development. For instance, NF-rcB is essentialfor survival of developing liver cells.Mouse embryos that cannot expressone of the I-rcBkinase subunits die at mid-gestation due to liver degeneration caused by excessiveapoptosis of cells that would normally survive. As we will see in Chapter 20, phosphorylationdependentdegradationof a cyclin kinase-dependentinhibitor plays a central role in regulating progressionthrough the cell cycle in S. cereuisiae.It seemslikely that phosphorylationdependentprotein degradationmay emergeas a common regulatory mechanismin many different cellular processes.

C E L LS I G N A L I N G ll: SIGNAL|NG PATHWAYS T H A TC O N T R O L G E N EA C T t V t T y

Ligand-ActivatedNotch ls CleavedTwice, Releasing nactor a T r a n s c r i p t i oF Both the receptorcalledNotch and its ligand Delta are single-passtransmembraneproteins found on the cell surface. Notch also has other ligands,including Serrate,but the molecular mechanismsof activation are the samewith each ligand. Delta binds to Notch, activating Notch so that it undergoes two cleavageevents; these result in releaseof the Notch cytosolic domain, which functions as a transcription factor. For activation to occut Notch and Delta must be located in the membranesof adiacent cells. Their location in different cellsis essential,becausethey participate in a highly conservedand important differentiation processin both invertebratesand vertebrates,called lateral inhibition. In this process,adjacent and developmentallyequivalentcells assume completely different fates. In effect, one cell in a group of equivalent cells instructs the others around it to choose a different fate. This process,discussedin detail in Chapter 22, is particularly important in preventing too many nerve precursor cells forming from an undifferentiated layer of epithelial cells. Notch protein is synthesizedas a monomericmembrane protein in the endoplasmicreticulum. In the Golgi complex,

it undergoesa proteolytic cleavagethat generatesan extracellular subunit and a transmembrane-cytosolicsubunit; the two subunits remain noncovalently associatedwith each other in the absenceof interaction with Delta residing on another cell. Following binding of Delta, the Notch protein on the respondingcell undergoestwo proteolytic cleavagesin a proscribed order (Figure 1'6-36).The first is catalyzedby ADAM 10, a matrix metalloprotease.(The name ADAM stands for A Disintegrin and Metalloprotease. Disintegrin refersto an ADAM domain that binds integrins (Chapter 19) and disrupts cell-matrix interactions.) The second cleavage occurs within the hydrophobic membrane-spanningregion of Notch, and is catalyzedby a four-protein transmembrane complex termed y-secretase.This cleavage releasesthe Notch cytosolic segment,which immediately translocatesto the nucleus where it affects transcription of various target genes.Such signal-inducedregulated intramembrane proteolysis (RIP) also occurs in the responseof cells to low cholesterol (seebelow) and to the presenceof unfolded proteins in the endoplasmicreticulum (Chapter13). The 1-secretasecomplex contains a protein termed presenilin 1 and three other essentialsubunits, aph-1, pen-2, and nicastrin. Presenilin 1 (PS1) was first identified as the

CELL SIGNALING

Cytosol

EGFrepeats Notchbinding domain

Deltabinding domain

Extracellular space

Cytosol

--Y,,

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FIGURE 16-36 Notch/Deltasignalingpathway.Inthe absence cellis of Delta,theextracellular subunitof Notchon a responding noncovalently associated with itstransmembrane-cytosolic subunit W h e nN o t c hb i n d st o i t sl i g a n dD e l t ao n a n a d j a c e ns ti g n a l i ncge l l (steptr), Notchisfirstcleaved bythe matrixmetalloprotease ADAM 1 0 ,w h i c hi sb o u n dt o t h em e m b r a nree, l e a s i nt hgee x t r a c e l l u l a r (stepEl) 1-Secretase, Notchsegment a complex of four membrane proteins protease presenilin 1,thenassociates including the presumed

an intramembrane portionof Notchandcatalyzes with the remarning of Notch(stepB) segment the cytosolic thatreleases cleavage interacts thisNotchsegment to the nucleus, translocation Following genes that in of expression to affect factors transcription with several of cellfateduringdevelopment the determination turn influence andS 100:391, andD Seals (step4) [See etal, 2000,Ce// M S Brown C o u r t n e i d g e2, 0 0 3 , G e n e sD e v . 1 7 2 1|

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product of a genethat commonly is mutated in patients with an early-onset autosomal dominant form of Alzheimer's disease.Studies on cells lacking nicastrin revealed why tsecretasecan only cleaveproteins that have first beencleaved by an ADAM or other matrix metalloprotease. Nicastrin binds to the N-terminal extracellular stumo of the membrane protein that is generatedby the firsi protease (see Figure 16-36). Without this stump, nicastrin and thus the entire T-secretase complex cannot interact with its target protein. Iil7eexamine the role of ADAM proteins and ^y-secretase in the developmentof Alzheimer's diseaselater. ln Drosophila, the released intracellular segment of Notch forms a complex with a DNA-binding protein called Suppressorof Hairless,or Su(H). This complex stimulates transcription of many geneswhose net effect is to influence the determination of cell fate during development. One of the proteins increased in this manner is Notch itself, and Delta production is correspondingly reduced (see Figure 22-42). As we seein Chapter 22, reciprocalregulation of the receptor and ligand in this fashion is an essentialfeature of the interaction between initially equivalent cells that causes them to assumedifferent cell fates. Further studies have revealed several lines of evidence that the Notch pathway is carefully tuned with many builtin checksand balances.Genetic studiesin Drosophila led to the discovery of the Fringe (Fng) protein, a glycosyl transferase that influences the activity of Notch. ln the transGolgi network (Chapter 14), Fringe adds fucose residuesto a region in the extracellular domain of Notch. This modification biasesNotch toward greater sensitivity to its Delta Iigand than to its Serrateligand. In vertebrates,three colorfully named proteins that are related to Fringe (Lunatic fringe, Manic fringe, and Radical fringe) alter the relative sensitivity of Notch to its three ligands (Delta, Jaggedl, and Jagged2).In flies and in mammais, the biases imposed by Fng proteins influence developmentaloutcomesbecauseFng modifies Notch in some cells but not others.

Matrix MetalloproteasesCatalyzeCleavage of Many SignalingProteinsfrom the CellSurface Many signalingmoleculesare synthesizedas transmembrane proteins whose signal domain extends into the extracellular space.Suchsignalingproteins are often biologicallyacive bur can signalonly by binding ro receptorson adjacentcells.Delta is a good example of such a membrane-boundsignalthat has very localized effects. However, many growth factors and other protein signals are synthesizedas transmembrane precursors whose cleavagereleasesthe soluble, active signaling moleculeinto the extracellularspace.The human genomeencodes19 metalloproteases in the ADAM family and many are involved in cleaving the precursorsof signaling proteins just outsidetheir transmembranesegment.This ADAM-mediated proteolysis of such precursors is similar to the cleavageof Notch by ADAM 10 (seeFigure 16-36) except that the reIeasedextracellularsegmenrhas signalingactivity.ADAM activity and hencethe releaseof active signaling proteins must be tightly regulatedby the cell, but how this happensis not yet 706

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clear. A breakdown in the mechanismsfor regulating ADAM proteasescan lead to abnormal cell proliferation. Medically important examplesof the regulatedcleavage of signal protein precursors are members of the EGF family, including EGR HB-EGF, TGF-a, NRG1, and NRG2 (seeFigure 16-18). The increasedactivity of one or more ADAMs that is seen in many cancerscan promote cancer development in two ways. First, heightened ADAM activity can lead to high levels of extracellular EGF family growth factors that stimulate secretingcells (autocrine signaling) or adjacentcells (paracrinesignaling)to proliferate inappropriately. Second, by destroying components of the extracellular matrix, increased ADAM activity is thought to facilitate metastasis,the movement of tumor cells to other sites in the body. ADAM proteasesalso are an important factor in heart disease.As we learned in the last chapter, activation by adrenalineof B-adrenergicreceptorsin heart musclecauses glycogenolysisand an increasein the rate of muscle contraction. Prolongedtreatmentof heart musclecellswith epinephrine, however, leads to activation of ADAM 9 by an unknown mechanism. This matrix metalloprotease cleaves the transmembrane precursor of HB-EGF. The released HB-EGF then binds to EGF receptorson rhe signalingheart muscle cells and stimulates their inappropriate growth. This excessiveproliferation can lead to an enlarged but weakenedheart-a condition known as cardiac hyoertrophy, which may causeearly death. I

InappropriateCleavageof Amyloid Precursor ProteinCan Leadto Alzheimer'sDisease Alzheimer's diseaseis another disorder marked by the inappropriate activity of matrix metalloproteases.A major pathologic changeassociatedwith Alzheimer'sdisease is accumulation in the brain of amyloid plaques containing aggregatesof a small peptide containing 42 residuestermed ABa2.This peptide is derived by proteolytic cleavageof amyloid precursor protein (APP), a transmembranecell-surface protein of still mysteriousfunction expressedby neurons. Like Notch protein, APP undergoes one extracellular cleavageand one intramembrane cleavage (Figure 16-37). First, the extracellulardomain is cleavedat one of two sitesin the extracellular domain: by ADAM 10 or ADAM 17 (often collectively calleda-secretase)or by another matrix metalloproteasetermed B-secretase.In either case,y-secretasethen catalyzesa secondcleavageat the same intramembrane site, releasingthe same APP cytosolic domain but different small peptides in the two pathways. The pathway initiated by osecretasegeneratesa 26-residuepeptide that apparently does no harm. In contrast, the pathway initiated by B-secretase generatesthe pathologic ABa2 peptide, which spontaneously forms oligomers and then the larger amyloid plaques found in the brain of patientswith Alzheimer'sdisease. APP was recognizedas a major player in Alzheimer's diseasethrough a genetic analysisof the small percentage of patients with a family history of the disease.Many had

p A T H W A y s r H A T c o N T R o L G E N EA c l v t r y C E L LS I G N A L I N Gt t : S t G N A L T N G

Cytosol A FIGURE 15-37 Proteolyticcleavageof APPand Alzheimer's (ADAM proteolytic disease.(leff) Sequential cleavage by a-secretase (Z) produces 10 or ADAM17)(tr) and"y-secretase an innocuous peptide membrane-embedded of 26 aminoacids(Righf) in Cleavage (tr)followedbycleavage theextracellular domainby B-secretase within (E) generates the membrane by 1-secretase the42-residue ABo2 peptidethatspontaneously formsoligomers andthenthe largeramyloid

In plaques disease. with Alzheimers foundin the brainof patients intothe segmentof APPis released the cytosolic both pathways andC.Haass, 5 Lichtenthaler but itsfunctionisnot known.[See cytosol, 2004,Curr. andB DeStroopet andV Wilquet l Clintnvest113:1384, 2OO4, 14:582InsetO |SM/Phototakel OpinNeurobiol

mutations in the APP protein, and intriguingly these mutations are clusteredaround the cleavagesitesof o-, B-, or 1secretasedepictedin Figure 16-37. Other casesof familial Alzheimer's diseaseinvolve missensemutations in presenilin 1, a subunit of 1-secretase, that enhancesthe formation of the ABa2 peptide, leading to plaque formation and eventuallyto the death of neurons.I

maintain appropriate lipid levels by regulating their supply and utilization of lipids. Coordinate regulation of the metabolism of phospholipids and cholesterol is necessaryto maintain the proper composition of membranes.Regulated intramembraneproteolysis,which occurs in the Notch pathway, also plays an important role in the cellular responseto low cholesterollevels. As we learned in Chapter 1.4, low-density lipoprotein (LDL) is rich in cholesteroland functions in transporting cholesterolwithin the circulation (seeFigure 1'4-27).Both the cholesterol biosynthetic pathway (see Figure 1,0-26) and cellular levels of LDL receptors are down-regulated when cholesterolis in excess.Since LDL is imported into cells via receptor-mediated endocytosis (see Figure 1429). a decreasein the number of LDL receptors leads to reduced cellular import of cholesterol. Both cholesterol biosynthesisand import are regulated at the level of gene transcription. For example, when cultured cells are incubated with increasingconcentrationsof LDL, the level and the activity of HMG-CoA reductase'the rate-controlling enzymein cholesterol biosynthesis is suppressed,whereas the activity of acyl:cholesterolacyl transferase (ACAT), which converts cholesterol into the esterified storage form, is increased. Cholesterol-dependenttranscriptional regulation often dependson 1O-base-pairsterol regulatory elements/SREs), or SRE half-sites,in the promoters of regulatedtarget genes. (These SREs differ from the serum responseelementsthat control many early-responsegenes' as discussedin Section 16.4.) The interaction of cholesterol-dependenttranscription factors called SRE-binding proteins (SREBPs) with theseresponseelementsmodulates the expressionof the target genes.The SREBP-mediatedpathway beginsin the membranes of the endoplasmic reticulum (ER) and includes at least two other proteins besidesSREBP' 'S7hen cells have adequateconcentrations of cholesterol, SREBPis found in the ER membrane complexed with SCAP

^y-Secretase catalyzesthe regulatedintramembranecleavage of over 100 cell-surfaceproteins, including Notch (see Figure 16-36). Evidence supporting the involvement of the presenilin 1 subunit of ^y-secretase in Notch signaling came from geneticstudiesin the roundworm C. elegans.Mutations in the worm homolog of presenilin 1 causeddevelopmental defects similar to those caused by Notch mutations. Later work showed that mammalian Notch does not undergo signal-induced intramembrane proteolysis in mouse neuronal cells geneticallymissing presenilin 1. But whether presenilin 1 is the actual 1-secretaseproteaseor an essentialcofactor of '$Tithin the "real" proteaseis not yet certain. its membranespanning segments,presenilin t has two aspartate residues in a configuration that resemblesthat of the two aspartates in the active site of water-soluble "aspartyl proteases," and mutation of either of theseaspartateresiduesin presenilin 1 abolishesits ability to stimulate cleavageof Notch. Current data are thus consistentwith the notion that presenilin 1 is the proteasethat cleavesNotch, APP, and many other proteins within their transmembranesegments.

RegulatedIntramembraneProteolysisof SREBP Releasesa Trans€riptionFactorThat Acts to M a i n t a i nP h o s p h o l i p i d a n d C h o l e s t e r oLl e v e l s A cell would soon face a crisis if it did not have enough lipids to make adequate amounts of membranesor had so much cholesterol that large crystals formed and damaged cellular structures.To prevent such disastrousevents,cells normally

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Insig-1(2)

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FIGURE16-38 Cholesterol-sensitivecontrol of SREBP activation. Thecellularpool of cholesterol is monitoredby the combinedactionof insig-1(2) proteins and SCABboth transmembrane locatedin the ERmembrane(a)When cholesterol levelsare high, insig-1(2) bindsto the sterol-sensing domainin SCABanchoringthe SCAP-SREBP complexin the ERmembrane(b)Thedissociation of insig-1(2) from SCAPat low cholesterol levelsallowsthe SCAP-SREBp

complexto moveto the Golgicomplexby vesicular transportIn the Golgi,the sequential cleavage of SREBP by the site 1 and site2 (S1B52P)releases proteases the N-terminalbHLHdomainof SREBP (nSREBP), After this released domain,callednuclearSREBP translocates into the nucleus,it controlsthe transcription of genescontainingsterol regulatoryelements(SREs) in their promoters.lAdapted fromI F.Osborne, 2001,Genes Devel.15:1873, seeT.Yanget al , 2002,Ce//110:489l

(SREBPcleavage-activating protein), insig-1 (or its closehomolog insig-2),and perhapsother proteins (Figure 76-38a). SREBPhas three distinct domains: an N-terminal cytosolic domain, containing a basic helix-loop-helix(bHLH) DNAbinding motif (see Figure 7-26), that functions as a transcription factor when cleaved from the rest of SREBP; a central membrane-anchoringdomain containing rwo transmembrane o helices;and a C-terminal cytosolic regulatory domain. SCAP has eight transmembranea helicesand a Iarge C-terminal cytosolic domain that interacts with the regulatory domain of SREBP.Five of the transmembranehelices in SCAP form a sterol-sensingdomain similar to that in HMG CoA reductase(seeSection 10.3). When the sterolsensingdomain in SCAPis bound to cholesterol,the protein also binds to insig-1(2).When insig-1(2)is tightly bound to the SCAP-cholesterolcomplex, it blocks the binding of SCAP to coat proteins on COPII vesicles,thereby preventing incorporation of the SCAP/SREBPcomplex into ER-to-Golgi transport vesicles(seeChapter 14). Thus the cholesteroldependentbinding of insig to the SCAP-cholesterol-SREBP complex traps that complex in the ER. -il/hen cellular cholesterol levels drop. some of the cholesterolbound to SCAP is released.Consequentl5insig-1(2) no longer binds to the cholesterol-freeSCAR and the

SCAP-SREBPcomplex moves from the ER to the Golgi apparatus via COPII vesicles(Figure 16-38b). In the Golgi, SREBP is cleaved sequentiallyat two sites by two membrane-boundproteases,S1Pand 52P,additionalexamplesof regulatedintramembraneproteolysis.The secondcleavage at site 2 releasesthe N-terminal bHlH-containing domain into the cytosol. This fragment, called nSREBP (nwclear SREBP/, is rapidly translocatedinto the nucleus.There it activates transcription of genescontaining sterol regulatory elements (SREs)in their promoters, such as those encoding the LDL receptor and HMG-CoA reductase.Thus a reduction in cellular cholesterol,by activating the insigl (2)/SCAP/SREBP pathway, triggers expressionof genesencoding proteins that both import cholesterolinto the cell (LDL receptor) and synthesize cholesterol from small precursor molecules (HMGCoA reductase). After cleavageof SREBPin the Golgi, SCAP apparently recyclesback to the ER where it can interacrwith insig-1(2) and another intact SREBPmolecule.High-level transcription of SRE-controlledgenesrequires the ongoing generation of new nSREBP becauseit is degraded fairly rapidly by the ubiquitin-mediatedproteasomalpathway (Chapter 3). The rapid generation and degradation of nSREBPhelp cells respond quickly to changesin levelsof intracellular cholesterol.

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Under some circumstances(e.g., during cell growth), cells need an increasedsupply of all the essentialmembrane lipids and their fatty acid precursors(coordinate regulation). But cells sometimes need greater amounts of some lipids, such as cholesterol to make steroid hormones, than others, such as phospholipids (differential regulation). The complex regulation of lipid metabolism characteristic of higher eukaryotes depends in large part on abundant transcription factors, including multiple SREBPs,that control the expression of proteins involved in lipid metabolism. For example, various SREBPsregulatethe transcription of genesencoding many proteins participating in the cellular uptake of lipids (e.g., the LDL receptor) and most enzymesin the pathways for synthesizing cholesterol, fatty acids, triglycerides, and phospholipids. Mammals express three known isoforms of SREBP:SREBP-1aand SREBP-1c,which are generatedfrom alternatively spliced RNAs produced from the same gene, and SREBP-2,which is encodedby a different gene.Together these protease-regulatedtranscription factors control the availability not only of cholesterolbut also of fatty acids and the triglyceridesand phospholipids made from fatty acids.In mammalian cells, SREBP-1aand SREBP-1cexert a greater influence on fatty acid metabolism than on cholesterol metabolism, whereasthe reverseis the casefor SREBP-2. Becausethe risk for atherosclerotic diseaseis directly proportional to the plasma levelsof LDL cholesterol ( t h e s o - c a l l e d" b a d " c h o l e s t e r o l )a n d i n v e r s e l yp r o p o r tional to those of HDL cholesterol,a major public health goal has been to lower LDL and raise HDL cholesterol levels. The most successful drugs for controlling the LDL:HDL ratio are the statins,which causereductionsin plasma LDL. As discussedin Chapter 10, thesedrugs bind to HMG-CoA reductase and directly inhibit its activitS thereby lowering cholesterolbiosynthesisand the pool of cholesterolin the liver. Activation of SREBPin responseto this cholesteroldepletion promotes increasedsynthesisof HMG-CoA reductaseand the LDL receptor. Of greatest importance here is the resulting increasednumbers of hepatic LDL receptors,which mediate increasedimport of LDL cholesterolfrom the plasma and thus lower the level of LDL cholesterolin the circulation. Statinsalso appearto inhibit atherosclerosisby suppressingthe inflammation that triggers the process. Although the mechanism of this inhibition is not well understood,it apparentlycontributes to the atheroprotective effect of statins. I

Pathways That Involve Signal-lnduced Protein Cleavage r The NF-rcB transcription factor regulates many genes that permit cells to respond to infection and inflammation. r In unstimulated cells, NF-rcB is localized to the cytosol, bound to the inhibitor protein I-rB. In responseto many types of extracellular signals, phosphorylation-dependent ubiquitination and degradation of I-rB in proteasomes

releasesactive NF-rcB, which translocates to the nucleus (seeFigure 1,6-35). r Upon binding to its ligand Delta on the surfaceof an adjacent cell, the receptor Notch protein undergoestwo proteolytic cleavages(seeFigure 15-36). The releasedNotch cytosolic segment then translocates into the nucleus and modulates transcription of target genes critical in determining cell fate during development. r Cleavageof membrane-boundprecursorsof membersof the EGF family of signaling molecules is catalyzed by ADAM proteases.Inappropriate cleavageof these precursors can result in abnormal cell proliferation, potentially leading to cancer,cardiachypertrophy, and other diseases. r 1-Secretase,which catalyzesthe regulated intramembrane proteolysis of Notch, also participates in the cleavage of amyloid precursor protein (APP) into a peptide that forms plaquescharacteristicof Alzheimer'sdisease(seeFigure L6-37\. r In the insig-1(2)/SCAP/SREBP pathway, the active nSREBP transcription factor is released from the Golgi membrane by intramembrane proteolysis when cellular cholesterolis low (seeFigure 15-38). It then stimulatesthe expressionof genesencodingproteins that function in cholesterol biosynthesis(e.g.,HMG-CoA reductase)and cellular import of cholesterol (e.g., LDL receptor). When cholesterol is high, SREBP is retained in the ER membrane complexed with insig-1(2) and SCAP'

The confluence of genetics,biochemistry' and structural biology has given us an increasinglydetailed view of how signals are transmitted from the cell surface and transduced into changesin cellular behavior' The multitude of different extracellular signals, receptors for them, and intracellular signal-transduction pathways fall into a relatively small number of classes,and one major goal is to understand how similar signaling pathways often regulate very different cellular processes.For instance,STATS activatesvery different sets of genesin erythroid precursor cells, following stimulation of the erythropoietin receptor'than in mammary epitheIial cells,following stimulation of the prolactin receptor.Presumably STAT5 binds to different groups of transcription factors in these and other cell types, but the nature of these proteins and how they collaborate to induce cell-specificpatterns of geneexpressionremain to be uncovered. Conversely activation of the same signal-transduction component in the samecell through different receptorsoften elicits different cellular responses.One commonly held view is that the duration of activation of the MAP kinase and other signaling pathways affects the pattern of gene expression. But how this specificity is determined remains an outstanding question in signal transduction. Genetic and molecular studies in flies, worms' and mice will contribute to our understanding of the interplay between different pathway P E R S P E C T I VF EO S RT H E F U T U R E

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components and the underlying regulatory principles controlling specificity in multicellular organisms. Researchershave determinedthe three-dimensionalstructures of various signaling proteins during the past several years, permitting more detailed analysis of several signaltransduction pathways. The molecular structuresof different kinases,for example, exhibit striking similarities and important variations that impart to them novel regulatory features. The activity of severalkinases,such as Raf and protein kinase B (PKB), is controlled by inhibitory domains as well as by multiple phosphorylationscatalyzedby severalother kinases. Our understanding of how the activity of these and other kinasesis preciselyregulated ro meet the cell's needswill require additional strucural and cell biological studies. Abnormalities in signal rransduction underlie many different diseases,including the majority of cancersand many inflammatory conditions. Detailed knowledge of the signaling pathways involved and the strucrureof their protein components will continue to provide important molecular clues for the design of specific therapies.Deipite the close structural reIationshipbetweendifferentsignalingmolecules(e.g.,kinases), recent studies suggestthat inhibitors selectivefor specific subclassescan be designed.In many tumors of epithelialorigin, the EGF receptor has undergonea mutation that increasesrts activity. Remarkablya small-moleculedrug (IressarM) inhibits the kinase activity of the mutanr EGF receptor but has no effect on the normal EGF receptor or other receprors. Thus the drug slows cancergrowth only in patients with this particular mutation. Similarlg monoclonal antibodiesor decoyreceptors(soluble proteins that conrain the ligand-bindingdomain of a receptor and thus sequester the ligand) that prevent pro-inflammatory cytokines like IL-1 and TNFct from binding to their cognate receptors are now being used in treatment of severalinflammatory diseasessuch as arthritis.

KeyTerms activation lip 674 adapter protein 685 constitutive 570

phosphoinositides 694 PI-3 kinasepathway594 presenllrn L/u)

cytokines 672 erythropoietin 672

protein kinaseB (PKB) 695

Hedgehog (Hh) pathway 700 HER family 680 insig-1 (2 )/SCAPiSREBP pathway 708 JAK/STM pathway 672 kinase cascade684 MAP kinase 684

NF-rcB pathway 703 Notch/Delta pathw ay 703 nuclear-localizationsignal (NLS) 670 C H A P T E R1 6

receptor tyrosine kinases (RTKs) 679 regulated intramembrane proteolysis 705 scaffold proteins 693 SH2 domains 625

matrix metalloprotease (MMP) famrly 703

710

PTEN phosphatase692 Ras protein 584

|

Smads 570 SRE-binding protein (SREBP)707 transforming growth factor

668 B (TGFB) lwnt pathway 699

Review the Concepts L Binding of TGFB to its receptorscan elicit a variery of responsesin different cell types.For example,TGFB inducesplasminogen activator inhibitor in epithelial cells and specific immunoglobulins in B cells.In both cell types, Smad3 is activated. Given the conservationof the signalingpathway, what accounts for the diversity of the responseto TGFB in various cell types? 2. How is the signal generatedby binding of TGFB to cellsurface receptors transmitted to the nucleus where changes in target geneexpressionoccur? 3. Name three featurescommon to the activation of cytokine receptors and receptor tyrosine kinases. Name one difference with respectto the enzymatic activity of thesereceptors. 4. The intracellular eventsthat proceed when erythropoietin binds to its cell-surfacereceptorare well-characterizedexamples of cell-signalingpathways that activate gene expression.What molecule translocatesfrom the cytosol to the nucleus after (a) JAK2 activatesSTAT5 and (b) GRB2 binds to the Epo receptor? 5. Once an activatedsignalingpathway has elicited the proper changesin target gene expression,the pathway must be inactivated. Otherwise,pathologic consequences may result, as exemplified by persistentgrowth factor-initiated signaling in many cancers.Many signaling pathways possessintrinsic negative feedback by which a downstream event in a pathway turns off an upstream event. Describethe negative feedbackthat downregulatessignalsinduced by (a) TGFB and (b) erythropoietin. 6. Even though GRB2 lacks intrinsic enzymatic activity, it is an essentialcomponent of the epidermal growth factor (EGF) signalingpathway that activatesMAP kinase.'What is the function of GRB2? What role do the SH2 and SH3 domains play in the function of GRB2? Many other signaling proteins possessSH2 domains. What determinesthe specificity of SH2 interactions with other molecules? 7. A mutation in the Ras protein rendersRas constitutively 'S7hat active (RasD). is constitutive activation? How is constitutively active Ras cancer-promoting? What type of mutation might render the following proteins constitutively active:(a) Smad3;(b) MAP kinase;and (c) NF-rcB? 8. The enzyme Ste11 participates in several distinct MAP kinase signaling pathways in the budding yeast S. cereuisiae. What is the substratefor Ste11in the mating factor signaling pathway? -Whena yeast cell is stimulated by mating factor, what prevents induction of osmolytes required for survival in high osmotic strength media since Ste11 also parricipates in the MAP kinase pathway initiated by high osmolarity? 9. Describethe evenrsrequired for full activation of protein kinase B. Name two effectsof insulin mediated by protein kinaseB in musclecells. 10. Describe the function of the PTEN phosphatasein the PI-3 kinase signalingpathway. \Why is a loss-of-functionmutation in PTEN cancer-promoting?Predict the effect of constitutively active PTEN on cell growth and survival. 11. Activation of protein kinase A (PKA) can have both 'What short- and long-term effects in cells. are some of the long-term effectsof activated PKA in liver cells?Sfhar pathway mediatestheseeffects?

C E L LS I G N A L | N Gi l : S T G N A L T NpGA T H W A y s r H A T c o N T R o L G E N EA c T | V t r y

b. ERK5 is a MAP kinase previously shown to be activated when phosphorylatedby MEK5. Vhen ERK5 is phosphorylated by MEK5, its migration on a polyacrylamide gel is retarded. In another experiment, HEK293 cells were transfected with a plasmid encoding ERK5 along with plasmids encoding MEK5, MEKK2, MEKK2 and MEK5, or MEKK2 and MEKSAA. MEKSAA is a mutant, inactive version of MEK5 that functions as a dominant-negative.Expressionof MEKSAA in HEK293 cellspreventssignalingthrough active' endogenousMEK5. Lysates of transfected cells were analyzed by'Westernblotting with an antibody againstrecombinant ERK5. From the data in part (b) of the figure' what can we conclude about the role of MEKK2 in the activation of ERKS? How do the data obtained when cells are cotransfectedwith ERKS, MEKK2, and MEKSAA help to elucidate 15. What biochemicalreaction is catalyzedby 1-secretase? the order of participants in this kinase cascade? \fhat is the role of 1-secretasein transducing the signal inducedby the binding of Delta to its receptor,Notch? How are mutations in presenilin 1, one of the subunits of 1-secretase, References thought to contribute to Alzheimer's disease?

12. Similar to Wnt proteins, the extracellular signaling protein Hedgehog can remain anchored to cell memhranes. !(hat modifications to Hedgehog enableit to be membranebound? \Why is this property useful? 13. Intraflagellar transport (IFT) proteins are required to move materials within cilia and flagella. Most cells have a single immobile cilium called the primary cilium. What evidence shows the primary cilium may be involved in signal transduction? 'Why is the signalingpathway that activatesNF-rB con14. sidered to be relatively irreversible compared with cytokine or RTK signaling pathways? Nonetheless,NF-rcB signaling must be down-regulated eventually.How is the NF-rcB signaling pathway turned off?

Analyze the Data G. Johnson and colleagueshave analyzedthe MAP kinase cascadein which MEKK2 participates in mammalian cells. By a yeast two-hybrid screen(seeChapter 7), MEKK2 was found to bind MEK5, which can phosphorylate a MAP kinase. To elucidate the signaling pathway transduced by MEKK2 in vivo, the following studieswere performed in human embryonickidney (HEK293) cellsin culture. a. HEK293 cellswere transfectedwith a plasmid encoding recombinant, taggedMEKK2, along with a plasmid encoding MEK5 or a control vector that did not encode a protein (mock). Recombinant MEK5 was precipitated from the cell extract by absorptionto a specificantibody.The immunoprecipitated material was then resolvedby polyacrylamidegel electrophoresis, transferred to a membrane, and examined by Western blotting with an antibody that recognized tagged MEKK2. The resultsare shown in part (a) of the figure below. -il/hat information about this MAP kinase cascadedo we learn from this experiment?Do the data in part (a) of the figure prove that MEKK2 activatesMEK5, or vice versa?

(u) $oo

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Pathway and the JAK/sTAT 16.2CytokineReceptors LevS D. E., and J. E. Darnell, 1r.2002. Stats:transcriptional control and biological impact' Nature Reu.Mol. Cell Biol. 3:651-62. Naka, T., et al. 2005. Negativeregulation of cytokine and TLR signalingsby SOCSand others.Adu. Immunol' 8726t-122. Ozaki. K.. and W. Leonard.2002. Cytokine and cytokine receptor pleiotropy and redundancy.J. Biol. Chem.277:29355-29358' 'Wornold, S., and D. Hilton. 2004. Inhibitors of cytokine signal transduction.J. Biol, Chem. 279:821'-824. Yoshimura,A.2006. Signaltransductionof inflammatory cytokinesand tumor development.CancerSci. 97:439447.

Kinases 16.3ReceptorTYrosine 2005. Cellular Eswarakumar,V. P.,L Lax, and J' Schlessinger. signalingby fibroblast growth factor receptors.Cytohine 6 Growth Factor Reu.16:139-149. Hubbard, S. R. 2004. Juxtamembraneautoinhibition in receptor tyrosine kinases.Nature Reu.Molec. Cell Biol. 5:464471'. Hynes. N. E., and H. A. Lane. 2005. ERB receptorsand cancer: the complexity of targeted inhibitors. Nature Reu. Cancer 5:Jr+l-JJ+.

+- MEKK2

o5e(6* o5e(Sts (b) ose( sr.."." ls"-" ;"**t -,&

16.1 TGFB Receptors and the Direct Activation of Smads Luo, K. 2004. Ski and SnoN: negativeregulatorsof TGF-B signaling. Curr. Opin. Genet. Deuel. 14:65-70. Massagu6,J., J. Seoane,and D. Womon.2005. Smadtranscription factors. Genes(t Deuel. 19:2783-281'0' Moustakas,A., S. Souchelnytskyi,and C. Heldin. 2001. Smad regulationin TGF-beta signaltransduction./. Cell Sci' t14:4359-4369. Xu, L. and J. Massagu6.2004. Nucleocytoplasmicshuttling of signal transdu cers.Nature Reu.Mol. Cell Biol. 5:209-219 '

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ERK5 ehorpr'orylated

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ERKS

Mohammadi, M., S. K. Olsen, and O. A. Ibrahimi. 2005. Structural basisfor fibroblast growth factor receptoractivation. Cytokine and Growth Factor Reu. 16:t07-1'37. Schwartz,M. A. and H. Madhani' 2004' Principlesof MAP kiAnn. Reu' cereuisiaenasesignalingspecificityrn Saccbaromyces Genet. 382725-748. Simon, M. 2000. Receptortyrosine kinases:specificoutcomes from generalsignals.Cell lO3:13-15. Singh,A. B., and R. C' Harris. 2005. Autocrine' paracrineandjuxtacrine signalingby EGFR ligands. Cell Signaling7721'1'83-It93' REFERENCES

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, I(ile5 H. S. 2003. Trafficking of the ErbB receptorsand its influenceon signaling.Exp. Cell Res.284:78-88. 'Wiley, H. S., S. Y. Shvartsman,and D. A. Lauffenburger.2003. Computational modeling of the EGF-receptorsystem:a plradigm for systemsbiology. Trends Cell Biol. 13;4-50. 16.4 Activation of Ras and MAp Kinase pathways _ Avruch, J., et al. 2001. Ras activation of the Raf kinase:tyrosine kinaserecruitmentof the MAP kinasecascade.Recentprog. Horm. Res.56:127-155. Bhattacharyya,R. P.,er a\.2006. The Ste5scaffold allostericallv modulatessignalingoutput of the yeastmating pathw ay.Science 3ll:822-826. Dard, N., and M. Peter.2006. Scaffoldproteinsin MAp kinase signaling:more than simple passiveactivatingplatforms. BioLssays 28:L46-1.56. Elion, E., M. Qi, and W. Chen. 2005. Signalingspecificiryin ye^st.Science307:687-688. Gastel,M. 2006. MAPKAP kinases-MK5-1y76'5 company, three'sa crowd. Nature Reu.Molec. Cell Biol.7z211-224. Huse,M., and J. Kuriyan.2002.The conformationalplasticitv of protein kinases. Cell 1092275-282. _Kerkhoff, E., and U. Rapp. 2001. The Ras-Rafrelationship:an unfinished puzzle.Adu. Enz. Regul. 4l:261-267 . Kolch, \7. 2005. Coordinating ERK/MAPK signallingthrough scaffoldsand inhibitors Nature Reu.Molec. Cett E;ol. 1zSZZ-SS1 . Murphy, T,.O., and J. Bienis.2005. MAPK signal specificity:the right place at the right time. Trends Biochem. Sci. 31268-27 5 .' Tzivion, G., and J. Avruch. 2002. 14-3-3 proreins:active cofactors in.cellularregulation by serine/threoninephosphorylation.J. Biol. Chem. 277:3061-3064.

16.5Phosphoinositides as SignalTransducers Brazil, D., Z.-Z.Yang, and B. A. Hemmings.2004. Advancesin protein kinaseB signalling:AKTion on multiple fronts. Trends Biochem. Sci. 29:23 3-242. Cantley,L.2002. The phosphoinositide3-kinasepathway.Science 296:1655-1657. Carlton, J. G., and P.J. Cullen. 2005. Coincidencedetectionin phosphoinositidesignaling.TrendsCell Biol. t5:540-547. Kahl, C. R., and A. R. Means. 2003. Regulationof cell cycle progressionby calcium,/calmodulin-dependent pathways. Endocr. Reu.24:71.9-736. Michell, R. H., et aL.2006.Phosphatidylinositol 3,5-bisphosphate: metabolismand cellular functions. TrendsBiochem. Scl.it:Si-63. Niggli, V. 2005. Regulationof protein activiriesby phosphoinositidephosphates.Ann. Reu.Cell Deuel. Blol.2I:57-79.' Patterson,R., D. Boehning,and S. Snyder.2004.Inositol 1,4,5, trisphosphatereceptorsas signalintegrators.Ann. Reu.Biochem.' 73:437-465. Toker,A., and A. Newton. 2000. Cellular signaling:pivoting aroundPDK-1. Cel/ 103:185-188

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16.6Activationof GeneTranscription by Seven-Spanning Cell-Surface Receptors Gordon, M. D., and R. Nusse.2006. I(nt signaling:multiple pathways,multiple receptors,and multiple transcription factors./. Bio l. Chem. 281:22429-2243 3. Logan, C. Y., and R. Nusse.2004. The Wnt signalingpathway in developmentand disease.Ann. Reu.Cell Deuel.Biol.20z 781-810. Lum, L., and P.A. Beachy.2004. The Hedgehogresponsenerwork: sensors,switches,and routers. Science304:1755-1759. Mann, R. K., and P.A. Beachy.2004. Novel lipid modifications of secreted proteinsignals.Ann. Reu.Biochem.73891,-923. Moon, R. T., et al. 2002.The promiseand perils of Wnt signaling through beta-catenin.Science296:1.644-1.646. Nusse,R. 2005. Relaysat the membrane:the \7nt signaling pathway.Nature 438:747-7 49. Price,M. 4.2006. CKI, there'smore than one: caseinkinaseI family membersin Wnt and Hedgehogsignaling.Genes(y Deuel. 20:399410. Seto,E. S. and Bellen,H.2004. The ins and outs of rVinsless signaling.TrendsCell Biol. l4:45-53 Singla,V., and J. F. Reiter.2006. The primary cilium as the cell's antenna:signalingat a sensoryorganelle.Science3132629-633. 16.7 Pathways That Involve Signal-lnduced

ProteinCleavage Brown, M., J. Ye, R. Rawson, and J. Goldstein.2000. Regulated intramembraneproreolysis:a control mechanismconservedfrom bacteriato humans. Cell 10O:397-398. De Strooper,B. 2005. Nicastrin: gatekeeperof the 1-secretase complex. Cell 122':318-320. Goldstein,J. L., R. A. DeBose-Boyd, and M. S. Brown. 2005. Protein sensorsfor membranesterols.Cel/ L24:35-46. Lazarov,V. K., et aL.2006.Electron microscopicstructureof purified, activeJ-secretase revealsan aqueousintramembranechamber and two pores.Proc. Nat'l. Acad. Sci.tlSA 703:6889-6894. . Lichtenthaler,S. F., and C. Haass.2004. Amyloid at the cutting edge:activation of o-secretase preventsamyloidogenesis in an Alzheimer diseasemousemodel./. Clin. Inuest. 113:1,384-1387. Luo, J, !.,H. Kamata,and M. Karin. 2005. IKK,TNF-rBsignaling: . balancing life and death-a new approacn ro cancer therapy.j. Clli. Inuest. ll 5 :2625-2632. Seals,D., and S. A. Courtneidge.2003. The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes6 Deuel. L7:7-30. 'Weihofen, A., and B. Martoglio. 2003. Intramembrane-cleaving proteases.TrendsCell Biol. 13:7L-78. Wilquet, V., and B. De Strooper.2004. Amyloid-Deraprecursor protein processingin neurodegeneration. Curr. Opin. Neurobiol. 14:582-588. Yamamoto,Y., and R. B. Gaynor.2004. IrcBkinases:key regulators of the NF-rB pathway. TrendsBiochem. Sci. 29:72-79.

C E L LS | G N A L | N Gi l : S | G N A L | N Gp A T H W A y s r H A T c o N T R o L G E N EA c I V t r y

CHAPTER

CELLORGANIZATION AND MOVEMENTI: MICROFILAMENTS phalloidin, a drugthat Migratingcellstainedwith fluorescent of K RottnerandJ,V Small] specifically bindsF-actin[Courtesy

fhen we look through a microscopeat the wonderful diversity of cells in nature, the variety of cell \n/ V Y shapesand movementswe can discern is astonishing. At first wi may notice that some cells,such as vertebrate sperm, ciliates such as Tetrahymena, or flagellates such as Chlamydomonas swim rapidly, propelled by cilia and flagella. Other cells, such as amebasand human macrophages, move more sedatelS propelled not by external appendages but by coordinated movement of the cell itself. We also might notice that some cells in tissuesattach to one another, forming a pavementlikesheet,whereasother cells-neurons' for example-have long processes,up to 3 ft in length, and make selectivecontacts between cells. Looking more closely at the internal organization of cells, we see that organelles have characteristiclocations; for example, the Golgi apparatus is generallynear the central nucleus.How is this diversity of shape,cellular organization, and motility achieved?Why is it important for cells to have a distinct shape and clear internal organization? Let us first consider two examplesof cells with very different functions and organizations. The epithelial cellsthat line the intestine form a tight, pavementlike layer of brick-shaped cells, known as an epithelium (Figure1.7-|a,b). Their function is to import nutrients(suchas glucose)acrossthe apical (top) plasma membraneand export them acrossthe basolateral(bottom-side)plasma membrane into the bloodstream. To perform this directional transport, the apical and basolateralplasma membranesof epithelial cells must have different protein compositions. Epithelial cells are attachedand sealedtogetherby cellularjunctions(Chapter19), which also separatethe apical and basolateraldomains.This

I

A

separation allows the cell to place the correct transport proteins in the plasma membranesof the two surfaces.In addition,

teins to the correct membrane surface. Also consider macrophages'a type of white blood cell whose iob it is to seekout infectious agentsand destroy them by phagocytosis.Bacteria releasechemicalsthat attract the -uitoph"g. and guide it to the infection. As the macrophage crawls along the chemical gradient, twisting and turning to

OUTLINE "17.1 Microfilamentsand Actin Structures

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17.2

Dynamicsof Actin Filaments

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17.3

Mechanismsof Actin FilamentAssembly

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CellularStructures728 17.4 Organizationof Actin-Based 17.5

Myosins:Actin-BasedMotor Proteins

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A FIGURE 17-1 Overviewof the cytoskeletons of an epithelial celland a migratingcell.(a)Transmrssion electron micrograph of a thinsection of an epithelial cellfromthesmallintestine. (b)Epithelial cellsarehighlypolarized, withdistinct apicalandbasolateral domains. An intestinal epithelial celltransport nutrients intothecellthrough theapicaldomainandout across (c)Scanning the basolateral domain. electron micrograph of a migrating cell.Thethinleading edge(also

knownasa lamellipodium) at thefrontcanbe seen,with the large cellbodybehindit. (d)A migrating cell,suchasa fibrobtast or a macrophage, hasmorphologically distinct domains, with a leading edgeat the front Microfilaments areindicated in red,microtubules in green,andintermediate filaments in darkblue Theposition of the (lightblueoval)isalsoshownlpart(a).Courtesy nucleus of Mark photoLibrary Mooseker; Part(b),Courtesy of Science l

get to the bacteriaand phagocytosethem, it has to constantly reorganizeits cell locomotion machinery.As we will see,its internal motile machinery must be oriented in one direction as it crawls (Figure17-1c,d).

the cytoskeleton.The cytoskeletonextendsthroughout the cell and is attached to the plasma membrane and internal organelles,so providing a framework for cellular organizatton. The term cytoskeleton does not imply a fixed structure like a skeleton. In fact, the cytoskeletoncan be very dynamic, with componentscapableof reorganization in less than a minute, or it can be quite stable for several hours. As a result, the lengths and dynamics of filaments can vary greatly, filaments can be assembled into diverse types of structures,and they can be regulatedlocally in the cell. The cytoskeletonis composedof three major filament systems (Figure17-2), aIl of which are organizedand regulatedin time and space.Eachfilament sysremis composedof a polymer

cell division and then set up the machinery ro segregare their organellesalong that axis. A . _cell'sshape and its functional polarity are provided by a three-dimensionalfilamentousprotein network called

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CHAPTER 17

I

C E L LO R G A N T Z A T T O A N D M O V E M E N Tt : M T C R O F T L A M E N T S

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cellasseenby immunofluorescence in thesamecultured systems filamentprotein, andan intermediate of actin,tubulin, microscopy V Small of J ] respectively. [Courtesy

of assembledsubunits. The subunits that make up the filaments undergo regulated assemblyand disassembly,giving the cell the flexibility to lay down or disassembledifferent types of structures as needed.Microfilaments are polymers of the protein actin organized into functional bundles and networks by actin-binding proteins. Microfilaments are especially important in the organization of the plasma membrane,including surface structures such as microvilli. Microfilaments can function on their own or serveas tracks for ATP-powered myosin motor proteins, which provide a contractile function (as in muscle) or ferry cargo along microfilaments. Microtubules are long tubes formed by the protein tubulin and organized by miproteins. They often extend throughout crotubule-associated the cell, providing an organizational framework for associated organelles and structural support to cilia and flagella. They also make up the structureof the mitotic spindle,the machine for separatingduplicatedchromosomesat mitosis. Molecular motors called kinesins and dyneins transport cargo along microtubules and are also powered by ATP hydrolysis. Intermediate filaments are tissue-specificfilamentous structures providing a number of different functions, including structural support to the nuclear membrane, structural integrity to cells in tissues, and structural and barrier functions in skin, hair, and nails. No known motors use intermediate filaments as tracks. As we can seein Figure 17-1, cellscan construct very different arrangementsof their cytoskeletons.To establish these from soluble facarrangements,cellsmust sensesignals----either tors bathing the cell, adjacentcells,or the extracellularmatrixand interpretthem (Figure17-3).Thesesignalsare detectedby cell-surfacereceptorsthat activatesignaltransduction pathways that convergeon factors that regulatecytoskeletalorganization.

The importance of the cytoskeletonfor normal cell function and motility is evident when a defect in a cytoskeletal component-or in cytoskeletalregulation-causes a disease. For example, about 1 in 500 people has a defect that affects

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movemenr, andmovement of organelles andcontraction function' 17-3 Cellsignalingregulatescytoskeleton A FIGURE the from signals external sense to receptors cell-surface Cellsuse are Thesesignals factors. matrix,othercells,or soluble extracellular specific andactivate membrane the plasma across transmitted frommore integrated pathways. Signals-often signaling cytosolic to cytoskeleton of the the organization to receptor-lead thanone organelle aswellasto determine provide cellswith theirshape, cells signals, of external In theabsence andmovement. distribution polarized manner in a not but structure, theirinternal stillorganize

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the contractileapparatusof the heart, which resultsin cardiomyopathiesvarying in degreeof severity.Many diseases of the red blood cell affect the cytoskeletal components that support thesecells'plasmamembrane.Metastaticcancer cells exhibit unregulatedmotility, breaking away from their tissue of origin and migrating to new locations to form new coloniesof uncontrolled growth. In this and the following chapter,we discussthe structure, function, and regulation of the cytoskeleton.Ve will seehow a cell arrangesits cytoskeletonro determinecell shapeand polarity, to provide organization and motility to its organelles, and to be the structural framework for such processesas cell swimming and cell crawling. We will discusshow cellsassemble the three different filament systemsand how signal-transduction pathways regulate these structures both locally and globally. Our focus in this chapter is on microfilaments and actin-basedstructures.Although we initially examine the cytoskeletal systemsseparately,in the next chapter we will see that microfilamentscooperatewith microtubulesand intermediate filamentsin the normal functioning of cells.

of slender,fingerlike cell-surfaceprojections called microvilli but can also be in a less-orderednetwork, known as the cell cortex, beneath the plasma membrane, where they provide support and organization. In epithelial cells, microfilaments form a contractile band around the cell, the adberens beh, that is intimately associatedwith adherensjunctions (Chapter 19) to provide strength to the epithelium. In migrating cells, a network of microfilaments is found at the front of the cell in the leading edge, or lamellipodium, which can also have protruding bundles of filaments called filopodia. Many cells have contractile microfilaments called stress fibers, which attach to the substratum through specialized regions called focal adhesions or focal contacts. At a late stage of cell division, after all the organelleshave been duplicated and segregated, a contractile ring forms and constricts to generatetwo daughter cells in a processknown as cytokinesis.The electron micrograph in Figure 17-4b shows microfilaments in microvilli. Different arrangemenrs of microfilaments can coexist within a single cell, as shown in Figure 17-4c,in this casea migrating fibroblast. The basic building block of microfilaments is actin, a protein that has the remarkable property of being able to reversibly assembleinto a polarized filament with functionally distinct ends. These filaments are then molded into various structures by actin-binding proteins. Cells use actin filaments ln many ways: in a structural role, by harnessingthe power of actin polymerization to do work, or as tracks for myosin motors. In this section, we look at the actin protein itself and the filaments into which it assembles.

Microfilaments and Actin Structures Microfilaments can assembleinto many different types of structureswithin a cell (Figure 1,7-4).Each of these structures underliesparticular cellular functions.Microfilaments can exist as a tight bundle of filaments making up the core

Microvilli

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Adherensbelt

Filopodia

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Cell cortex

Stressfibers

Contractile ring

A FIGURE17-4 Examplesof microfilament-basedstructures. (a) In eachpanel,microfilaments are depictedin red (b) Electron micrographof the apicalregionof a polarizedepithelialcell,showino the bundlesof actinfilamentsthat make up the coresof the microviiii 716

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(c)A cellmovingto the top, stainedfor actinwith fluorescent phalloidin,a drug that specifically bindsF-actinNote how many differentorganizations can existin one cell tpart(b),courtesy of N Hirokawa; Part(c)courtesy of J V Smalll

C E L L' R G A N r z A T r oANN D M . ' E M E N Tr : M T G R o F T L A M E N T S

A c t i n l s A n c i e n t ,A b u n d a n t ,a n d H i g h l yC o n s e r v e d Actin is an abundantintracellularprotein in eukaryoticcells.In musclecells,for example,actin comprises10 percentby weight of the total cell protein; even in nonmuscle cells, actin makes up 1-5 percentofthe cellularprotein. The cytosolicconcentration of actin in nonmusclecellsrangesfrom 0.1 to 0.5 mM; in special structures such as microvilli, however, the local actin concentration can be as high as 5 mM. To grasp how much actin cells contain, consider a typical liver cell, which has 2 x 10a insulin receptormoleculesbut approximately5 X 108,or half a billion, actin molecules.Becausethey form structures that extend across large parts of the cell interior, cytoskeletal proteins are among the most abundantproteinsin a cell. A moderate-sizedprotein with a molecular weight of 43,000, actin is encoded by a large, highly conservedgene family. Actin arose from an ancestralbacterial gene, which then evolvedas eukaryoticcellsbecamespecialized.Somesingle-celledorganisms,such as rod-shapedbacteria,yeasts,and amebas,have one or two ancestralactin genes,whereasmulticellular organismsoften contain multiple actin genes.For instance,humanshavesix actin genes,which encodeisoforms of the protein, and some plants have more than 60 actin genes, although most are pseudogenes,which do not encodefunctional actin proteins. In vertebrates,four actin isoforms are presentin various musclecells,and two isoforms,B-actin and

y-actin,are found in nonmusclecells. Thesesix isoforms differ at only about 25 of the 375 residuesin the completeprotein. Although these differences among isoforms seem minor, the isoforms have different functions: a-actin is associatedwith contractile structures, y-actifi accounts for filaments in stress fibers, and B-actin is enriched in the cell cortex and leading edgeof motile cells. Sequencingof actins from different sources has revealedthat they are among the most conservedproteins in a cell, comparablewith histones,the structural proteins of chromatin (Chapter 6). The protein sequencesof actins from amebas and from animals are identical at 80 percent of the amino acid positionsdespiteabout a billion yearsof evolution.

G-ActinMonomersAssembleinto Long, HelicalF-ActinPolYmers Actin exists as a globular monomer called G-actin and as a filamentous polymer calledF-actin, which is a linear chain of G-actin subunits. (The microfilaments visualized in a cell by electron microscopy are F-actin filaments plus any bound proteins.) Each actin molecule contains a Mg'- ion complexed with either ATP or ADP' The importance of the interionrrersion between the ATP and the ADP forms of actin in the assemblyof the cytoskeletonis discussedlater. Although G-actin appears globular in the electron microscope, x-ray crystallographic analysis reveals that it is separatedinto two lobes by a deep cleft (Figure 17-5a). The

(c)

(b)

(a)

(-) end

ATPbinding cleft

1 I 1 I

3 6n m

36 nm N-terminus C-terminus

(+)end

17-5 Structures of monomericG-actinand F-actin FIGURE r i.n5gx 5 5 x 3 5 f i l a m e n t s(.a )M o d eol f a c t i nm o n o m e r ( m e a s u 5 nm)showsit isdividedby a centralcleftintotwo approximately l-lV ATP(red) numbered equally sizedlobesandfoursubdomains, (theyellow lobes both contacts bindsat the bottomof thecleftand lie i i n s u b d o m aIi n b a l lr e p r e s e nMt sg 2 * )T h eN -a n dC - t e r m i n (b) An actinfilamentappears of subunitsOne astwo strands (14in eachstrand,rndicated by of 28 subunits repeating unitconsists * for onestrand), of 72 nm TheATP-binding covering a distance

(top)in allactinsubunits in the in the samedirection cleftisoriented is the cleft binding exposed with an filament Theendof a filament. (-) end;theopposite endisthe(+) end (c)In the electron appearaslong, actinfrlaments stained negatively microscope, of the of beadedsubunitsBecause andtwistedstrands flexible, (7-nm and diameter) thinner alternately twist,thefilamentappears C E Schutt (arrows) (9-nm diameter) [Part(a)adaptedfrom thicker Part(c)courtesy of M Rozycki O;courtesy 365:81 et al, 1993,Nature o I K L r a r ql M I C R O F I L A M E NA TN S D A C T I NS T R U C T U R E S .

717

(+)end

lobes and the cleft compose the ATPase fold, the site where ATP and Mg2* are bound. In actin, rhe floor of the cleft acts as a hinge that allows the lobesto flex relativeto eachother. When ATP or ADP is bound ro G,acrin, the nucleotide affects the conformation of the molecule; in fact, without a bound nucleotide, G-actin denatures very quickly. The addition of cations-Mg'*, K*, or Na+-to a solution of Gactin will induce the polymerizatton of G-actin into F-actin filaments. The processis reversible:F-actin depolymerizes into G-actin when the ionic strength of the solution is lowered. The F-actin filaments that form in vitro are indistinguishablefrom microfilamentsisolated from cells, indicating that actin alone makes up the filamentous structure of microfilaments. When negativelystainedwith uranyl acetatefor electron microscopy F-actin appearsas a twisted string whose diameter varies berween 7 and 9 nm (Figure 77-5c). From the results of x-ray diffraction studiesof actin filaments and from the actin monomer structureshown in Figure 17-5a, scientists have produced a model of an actin filament in which the subunits are organized in a helical structure (Figure 17-Sb). In this arrangement,the filament can be consideredas two helical strands wound around each other. Each subunit in the structure contacts one subunit above, one beloq and two on the side in the other strand. The subunits in a single strand wind around the back of the other strand and repeat after 72 nm or 14 actin subunits. Since there ,.. i*o strands, the actin filamenr appears ro repeat every 36 nm (Figure17-5b).

F - A c t i nH a sS t r u c t u r aal n d F u n c t i o n apl o l a r i t y All subunits in an actin filament point toward the same end of the filament. Consequently,a filament exhibits polarity; that is, one end differs from the other. As we will see,one end of the filament is favored for the addition of a c t i n s u b u n i t sa n d i s d e s i g n a t e dt h e ( + ) e n d , w h e r e a st h e o t h e r e n d i s f a v o r e d f o r d i s s o c i a t i o n ,d e s i g n a t e dt h e ( - ) end. At the (+) end, the ATP-bindine cleft of the terminal actin subunit contacrs the neighboring subunit, whereas on the (-) end, the cleft is exposedro the surrounding sol u t i o n ( F i g u r el 7 - 5 b ) . Without the atomic resolution afforded by x-ray crystallography,the cleft in an actin subunit and therefore the polarity of a filament are not detectable.However. the oolarity of actin filaments can be demonstratedby electron

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< EXPERIMENTAL FIGURE 17-5 Decoration demonstrates the polarityof an actinfilament.Myosin S 1h e a dd o m a i nbsi n dt o a c t i ns u b u n i ti n s a particular o r i e n t a t i oW n h e nb o u n dt o a l lt h es u b u n i ti sn a f i l a m e n t , S 1a p p e a rt so s p i r aal r o u n d t h ef i l a m e nTt .h i sc o a t i n o gf myosin headsproduces a series of arrowheadlike (arrows), decorations mosteasily seenat thewideviewsof thefilamentThepolarity in decoration defines a pointed (*) endanda barbed(+) end;theformercorresponds to thetop of the modelin Figure17-5 [Courtesyof R Craig ]

microscopy in "decoration" experiments, which exploit the ability of the protein myosin to bind specifically to actin filaments. In this type of experiment, an excessof myosin S1, the actin-bindingglobular head domain of myosin, is mixed with actin filaments and binding is permitted to take place. Myosin attachesto the sidesof a filament with a slight tilt. When all the actin subunirs are b o u n d b y m y o s i n , t h e f i l a m e n t a p p e a r sc o a t e d ( ' . d e c o rated") with arrowheadsthat all point toward one end of t h e f i l a m e n t ( F i g u r e1 7 - 6 ) . The ability of the myosin 51 head to bind and decorate F-actin is very useful experimentally-it has allowed researchersto identify the polarity of actin filaments, both in vitro and in cells.The arrowheadpoints to the (-) end, and so the (-) end is often calledthe "pointed" end of an actin filament;the (+ ) end is known as the "barbed" end. Because myosin binds to actin filaments and not to microtubules or intermediate filaments, arrowhead decoration is one criterion by which actin filaments can be definitively identified among the other cytoskeletalfibers in electron micrographs of thin-sectionedcells.

Microf ilaments and Actin Structures r Microfilaments can be assembledinto diverse structures, many associatedwith the plasma membrane (see Figtre 17-4). r Actin, the basic building block of microfilamenrs, is a major protein of eukaryotic cells and is highly conserved. r Actin can reversibly assembleinto filaments that consist of two helicesof actin subunits. r The actin subunits in a filament are all oriented in the samedirection, with the nucleotide-bindingsite exposedon the (-) end (seeFigure 17-5).

Dynamicsof Actin Filaments The actin cytoskeletonis not a static, unchanging structure consisting of bundles and networks of filaments. Although microfilaments may be relatively static in some structures,in others they are shrinking or growing in length. Thesechanges in the organization ol actin filaments can generateforcesthat causelarge changesin the shapeof a cell or drive intracellular

C E L LO R G A N T Z A T I OANN D M O V E M E N Tt : M T C R O F T L A M E N T S

movements.In this section,we consider the mechanismand regulation of actin polymerization, which is largely responsible for the dynamic nature of the cytoskeleton-

Actin Polymerizationin Vitro Proceedsin ThreeSteps The in vitro polymerization of G-actin (for Globular, or monomeric actin) to form F-actin filaments can be monitored by viscometry sedimentation,fluorescencespectroscopy,or fluorescencemicroscopy (Chapter 9). When actin filaments become long enough to becomeentangled,the viscosity of the solution increases,which is measuredas a decreasein its flow rate in a viscometer.The basisof the sedimentationassayis the ability of ultracentrifugation(100,0009 for 30 minutes) to sediment F-actin but not G-actin. The third assaymakes use of Gactin covalently labeledwith a fluorescentdye; the fluorescence spectrum of the labeled G-actin monomer changeswhen it is polymerized into F-actin. Finallg growth of the fluorescently

labeled filaments can be imaged with fluorescencevideo microscopy. These assaysare useful for kinetic studies of actin polymerization and for characterizationof actin-binding proteins to determine how they affect actin dynamics or how they crossJink actin filaments. The mechanismof actin assemblyhas beenstudiedextensively.Remarkably,one can purify G-actin at a high protein concentration without it forming filaments-provided it is maintained in a buffer with ATP and low levels of cations. However. as we saw above, if the cation level is increased (e.g., to 100 mM K+ and 2 mM Mg'*), G-actin will polymerize, with the kinetics of the reaction depending on the starting concentrationof G-actin. The polymerization of pure G-actin in vitro proceedsin three sequentialphases(Figure 17-7a). The first, nucleation phaseis marked by alag period in which G-actin subunits combine into short, unstable oligomers. When the oligomer reaches three subunits in length, it can act as a stableseed,or nucleus'which in the second, elongation phaserapidly increasesin filament length by

llll+ Animation:Actin Polymerization (a)

e

e

o e o e + ee G-actin

o Nuc

(l

e

o )

-)/

o

Nucleus

^,r/:

-+

(ef.ia

eo

F-actin

e

I

Steady state

Elongation

Nucleation

(+l end

(c)

--+l![<- Nucleation

Elongation--+lk-

Steadystate---------->l

--+ll
Steadystate-->l

q

q

q)

c 0)

E

E o @

Time 17-7 Polymerizationof G-actinin vitro occursin A FIGURE phase, ATP-G-actin three phases.(a)In the initialnucleation (red)slowlyformstablecomplexes of actin(purple) monomers phasebythe rnthesecond Thesenucleiarerapidlyelongated filament Inthethirdphase, both ends of the to additionof subunits statewith monomeric arein a steadv the endsof actinfilaments

ilme

reaction (b) Timecourse of the in vitropolymerization G-actin. the nucleation, with period associated the initiallag reveals (c) actin stable short lf some state. phase, steady and elongation to actas areaddedat the startof the reaction f ragments f ilament withoutanylagperiod' proceeds immediately elongation nuclei,

D Y N A M I C SO F A C T I N F I L A M E N T S

719

q q

o

Cc

Total actin concentration ( m o n o m e ra n d f i l a m e n t s )

FIGURE17-8 Concentration of actin determines filament formation. The criticalconcentration (C.)is the concentration of G - a c t i nm o n o m e r si n e q u i l i b r i u m w i t h a c t i nf i l a m e n t sA t m o n o m e r concentrations below the C., no polymerization takesplace When polymerization is inducedat monomerconcentrations abovethe C., filamentsassembleuntil steadystateis reachedand the monomer concentration fallsto C.

the addition of actin monomers to both of its ends.As F-actin filaments groq the concentration of G-acun monomers decreasesuntil equilibrium is reached berween filaments and monomers. In this third, steady-statephase, G-actin monomersexchangewith subunitsat the filament ends,but there is no net changein the total massof filaments.The kinetrc curves in Figure 17-7c show that the lag period is due to nucleationsinceit can be eliminatedby the addition of a small number of F-actinnuclei to the solution of G-actin. How much G,actin is neededto spontaneouslyform a filament? If ATP-G-actin at various concentrationsis placed under polymerizingconditions,below a certainconcentrarion, filaments do not assemble(Figure 17-8). Above this concentration, filaments are formed, and when steady state is reached,the addition of free subunitsis balancedby the dissociation of subunitsfrom filament endsto yield a mixture of filamentsand monomers.The concentrationat which filaments are formed is known as the overall critical concentration,C,. Below C., filaments will not form; above C., filaments form. At steadystate,the concentrationof monomeric actin remains at the critical concentration(Figure 17-8).

A c t i n F i l a m e n t sG r o w F a s t e ra t ( + ) E n d s T h a na t ( - ) E n d s S7esaw earlier that myosin S1 head decoration expenmenrs revealan inherent structuralpolarity of F-actin (seeFigure 176).lf freeAfP-G-actin is addedto a preexistingmyosin-dec, orated filament, the two ends grow at very different rates (Figure17-9).ln fact, for a given free ATp-G-actin concentration, the rate of addition of ATP-G-actin is nearly 10 times fasterat one end,the (+) end,than at the (-) end.The rate of addition is, of course,determinedby the concentrationof free ATP-G-actin. Kinetic experimentshave shown that the rate

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EXPERIMENTAL FIGURE 17-9 Thetwo endsof a myosindecoratedactinfilamentgrow unequally. Whenshortactin filaments aredecorated with a myosin S'1headandthenusedto nucleate actinpolymerization, the resulting actinsubunjts addmuch moreefficiently to the(+) endthanthe(-) endof the nucleating filamentThisresultindicates thatG-actin monomers areaddedmuch fasteratthe (+) endthanat the(-) end [Courtesyof I pollard ]

of addition at the (+ ) end is about 12 yt"M-rs-1and at the (- ) end about 1.3 pM-1s-t lFigure 17-I0a). This meansthat if 1 pM free ATP-G-actin is addedto preformed filaments,on average12 subunitswill be added to the (+) end everysecond, whereasonly 1.3 will be addedat the (-) end everysecond. What about the rate of loss of subunits from each end? By contrast, the rates of dissociationof ATP-G-actin subunits from the two endsare quite similar,about 1.4 s- 1 from the ( + ) end and 0.8 s-l from the (-) end. Sincethis dissociationis simply the rate at which subunits leave ends, it does not depend on the free ATP-G-actin concentration. \fhat implications does this have for actin dynamics? First let's considerjust one end, the (+) end. As we noted above,the rate of addition dependson rhe freeATP- G-actin concentration,whereasthe rate of loss of subunitsdoesnot. Thus subunitswill be added at high free ATP-G-actin concentrations, but as the concentration is lowered, a point will be reached at which the rate of addition is balanced by the rate of loss and no net growth occurs at that end. This is called the critical concentration C* , for the ( + ) end, and we can calculate it by setting the rate of assemblyequal to the rate of disassembly.Thus at the critical concentration, the rate of assemblyis C+. times the measuredrate of addition of 12 pM-1 s-r, whereasthe rate of disassembly is independent of the free actin concentration, namely 1.4 s-1. Setting theseequalro eachother yieldsa C*. of 0.12 pM for the (+ ) end. Above this free ATP-G-actin concentration,subunits add to the (+ ) end and net growth occurs,whereasbelow it, there is a net loss of subunitsand shrinkageoccurs. Now let'sconsiderjust the (- ) end. Becausethe rate of addition is much lower, 1.3 pM-l s-1,yet the rate of dissociation is about the same,0.8 s-1, the critical concentrationC-. at the (-) end is calculatedto be about 0.8 s-1/1.3pM-t s-1, that is, 0.6 pM. Thus at lessthan 0.6 pM freeATP-G-actin,

C E L LO R G A N I Z A T T OANN D M O V E M E N Tl : M T C R O F T L A M E N T S

(a)

(-l end

(+lend

ADP-actin

ADP-P1-actin

ATP-actin <------->

1.3pM-t 5-t

12 PM-t 5-t

+

<------> 0.8s-1

1 . 4s - 1

C-" = 0.60 PM

C * " = 0 . 1 2P M

(+l

(-l

(b)

-l

17-10 ATP-actin subunitsadd faster at the (+) end A FIGURE than the (-) end, resultingin a lower criticalconcentration and treadmillingat steadystate.(a)Therateof additionof ATP-G-actin ismuchfasterat the (+) endthanat the(-) end, issimilar. Thisdifference whereas the rateof dissociation of G-actin in a lowercritical concentration atthe (+) end.At steadv results

at the (+) end,givingrise isaddedpreferentially state,ATP-actin andregions ATP-actin filament containing the to a shortregionof towardthe(-) end.(b)At andADP-actin ADP-P1-actin containing to the(+) end, addpreferentially subunits state,ATP-G-actin steady (-) end,givingriseto fromthe disassemble subunits whileADP-actin of subunits treadmilling

say,0.3pM, the (-) end will lose subunits.But at this concentration, the (+) end will grow, since 0.3 pM is above C*.. Becausethe critical concentrations are different, at steady state the free ATP-G-actin will be intermediate between C*" and C=",so the (+ ) end will grow and the (- ) end will lose subunits. This phenomenon is known as treadmilling (seeFigure 1.7-1.0b). The ability of actin filaments to treadmill is powered by hydrolysisof ATP.'WhenATP-G-actin binds to a (*) end, ATP is hydrolyzed to ADP and P;. The P1is slowly released from the subunits in the filament, so that the filament becomes asymmetric,with ATP-actin subunits at the (+) end of the filament followed by a region with ADP-P1-actinand ADP-actin subunits toward the (-) end (Figure 17-1'0a), During hydrolysis of ATP and subsequentrelease of P1 from subunits in a filament, actin undergoes a conformational change that is responsible for the different association and dissociation rates at the two ends. Here we have consideredonly the kinetics of ATP-G-actin, but in reality it is ADP-G-actin that dissociatesfrom the (-) end. Our analysisalso relies on a plentiful supply of ATP-G-actin, which, as we will see,turns out to be the casein vivo. Thus actin can use the power generated by hydrolysis of ATP to treadmill and treadmilling filaments can do work in vivo, as we will seelater.

pure actin in vitro under physiologicalconditions. Consistent with a treadmilling model, growth of actin filaments in vivo only ever occurs at the (+) end. How is enhanced treadmilling achieved,and how does the cell rechargethe ADPactin dissociatingfrom the (- ) end to ATP-actin for assembly at the (*) end? Two different actin-binding proteins make important contributions to theseprocesses. The first is profilin, a small protein that binds G-actin on the side opposite to the nucleotide-binding cleft. When profilin binds ADP-actin, it opens the cleft and greatly enhancesthe loss of ADP, which is replaced by the more abundant cellular AIP, yielding a profilin-ATP-actin complex. This complex cannot bind to the (- ) end becauseprofilin blocks the siteson Gactin for (- ) end assembly.However, the profilin-AT?-actin complex can bind efficiently to the (+) end' and then profilin dissociatesafter the new actin subunit is bound (Figuret7-1'1'), This does not enhance treadmilling but provides a supply of ATP-actin from releasedADP-actin; as a consequence,essentially all the G-actin in a cell has bound ATP' Profilin has another important property: it can bind proteins with sequencesrich in proline residuesat the sametime as binding actin. \Wewill see later (Figure 1'7-1'4)how this property is important in actin filament assembly' Cofilin is also a small protein, but it binds specificallyto F-actin in which the subunits contain ADP, which are the older subunits in the filament toward the (-) end. Cofilin binds by bridging two actin monomers and inducing a small changein the twist of the filament. This small twist destabilizes the filament, breaking it into short pieces' By breaking the filament in this way, cofilin generatesmany more free (- ) ends and therefore greatly enhancesthe disassemblyof

Actin FilamentTreadmillingls Acceleratedby P r o f i l i na n d C o f i l i n Measurementsof the rate of actin treadmilling in vivo show that it can be severaltimes higher than can be achievedwith

D Y N A M I C SO F A C T I N F I L A M E N T S

721

(+) end

(-) end

Actin-ATP Actin-ADP-P;

Actin-ADP

€

Thymosin-po

ADP

in such a way that it inhibits addition of the actin subunit to either end of the filament. Thymosin-F+ can be very plentiful; for example, in human blood platelets. These discoid-shapedcell fragments are very abundant in the blood, and when activatedduring blood clotting, they undergo a burst of actin assembly.Plateletsare rich in actin: they are estimated to have a total concentration of about 550 pM actin, of which about 220 pM is in the unpolymerized form. They also contain about 500 pM thymosinBa, which sequestersmuch of the free actin. However, as in any protein-protein interaction, free actin and free thymosin-Ba are in a dynamic equilibrium with the actin-thymosin-Ba.If some of the free actin is used up for polymerization, more actin-thymosin-Ba will dissociate,providing more free actin for polymerization (see Figure 1,7-1,1). Thus thymosin-Ba behaves as a buffer of unpolymerized actin for when it is needed.

ATP

C a p p i n gP r o t e i n sB l o c kA s s e m b l ya n d D i s a s s e m b layt A c t i n F i l a m e n tE n d s

ADP-actin ATP-actin V

Profilin

@ Cofilin Thymosin-Ba

A FIGURE 17-11Actin-bindingproteinsregulatethe rate of assemblyand disassembly as well as the availabilityof G-actin for polymerization. ln the profilincycle(tr), profilinbindsADpG-actin andcatalyzes the exchange of ADpfor ATpTheATP-Gactin-profilin complex caneitherdeliver actinto the (+) endof a filamentor dissociate. ln thecofilincycle(Z), cofilinbinds preferentially to filaments containing ADp-actin, inducing themto fragment andthusenhancing depolymerization by makingmore filamentends Inthethymosin-B+ cycle(B), G-actin isboundby thymosin-Ba, sequestering it frompolymerization As thefreeG-actin concentration is loweredby polymerization, G-actin-thymosin-B4 dissociates to makeavailable freeG-actin for polvmerization.

the (-) end of the filament (seeFigure 1,7-11).The released ADP-actin subunits are then rechargedby profilin and added to the (+)end as describedabove.In this wa5 profilin and cofilin can enhancetreadmilling in vitro more than tenfold, up to levelsseenin vivo.

The treadmilling and dynamics of actin filaments are regulated in cells by capping proteins that specificallybind to the ends of the filaments. If this were not the case, actin filaments would continue to grow and disassemblein an uncontrolled manner.As one might expect, two classesof proteins have been discovered:ones that bind the (+) end and ones that bind the (-) end (Figure1,7-1,2). A protein known as CapZ, consisting of two closely related subunits,binds with a very high affinity (-0.1 nM) to the (+ ) end of actin filaments,therebyinhibiting subunit addition or loss.The concentrationof CapZ in cellsis sene r a l l y s u f f i c i e n tr o r a p i d l y c a p a n y n e * l y f o r m e d ( + ) e ; d s . So how can filaments grow at their (+) ends?At least two mechanisms regulate the activity of CapZ. First, the capping activity of CapZ is inhibited by the regulatory lipid

(+lend --------\

Thymosin-paProvidesa Reservoirof Actin for Polymerization It has long been known that cells often have a very large pool of unpolymerized actin, sometimesas much as half the actin in the cell. Sincecellular actin levelscan be as high as 100-400 pM, this means that there can be 50-200 pM unpolymerized actin in cells. Since the critical concentration in vitro is about 0.2 pM, why doesn't all this actin polymerize?The answer lies, at least in part, in the presenceof actin monomer sequesteringproteins. One of these is thymosin-Ba,a small prorein that binds to ATp-G-actin

722

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C; = O.OpVt

(-)end

Tropomodulin

A FIGURE 17-12Cappingproteinsblockassemblyand disassembly at filamentends.CapZblocks the (+) end,whichis wherefilaments grow,so itsfunctionisto limitgrowthat normally the(+) end,A cappingproteinsuchastropomodulin blocks(-) ends, wherefilamentdisassembly normally occurs; thusthe majorfunction of tropomodulin isto stabilize filaments.

C E L LO R G A N T Z A T T O A N D M O V E M E N Tl : M I C R O F I L A M E N T S

PI(4,5,)P2,found in the plasma membrane (Chapter 16). Second, recent work has shown that certain regulatory proteins are able to bind the (+) end and simultaneously protect it from CapZ while still allowing assemblythere. Thus cells have evolved an elaboratemechanismto block assemblyof actin filaments at their (+ ) ends except when and where assemblyis needed. Another protein, called tropomodwlin, binds to the (-) end of actin filaments,also inhibiting assemblyand disassembly. This protein is found predominantly in cells in which actin filaments need to be highly stabilized.Two exampleswe will encounterlater in this chapter are the short actin filaments in the cortex of the red blood cell and the actin filamentsin muscle. As we will see,in both casestropomodulin works with another protein, tropomyosin, that lies along the filament to stabilize it. Tropomodulin binds to both tropomyosin and actin at the (- ) end to greatly stabilizethe filament. In addition to CapZ, another classof proteins can cap the (+ ) endsof actin filaments.Theseproteinsalso can severactrn filaments. One member of this family, gelsolin, is regulated by increasedlevelsof Ca2*. On binding Ca2+,gelsolinundergoes a conformational changethat allows it to bind to the side of an actin filament and then insert itself betweensubunitsof the helix, thereby breakingthe filament. It then remainsbound to the (+)end, generatinga new (-) end that can disassemble. Actin cross-linkingproteins can turn a solution of filaments into a gel, and under conditions of elevatedCa2+,gelsolincan break the filaments and turn it into a sol; hencerts name.

Dynamics of Actin Filaments r The rate-limiting stepin actin assemblyis the formation of a short actin oligomer (nucleus)that can then be elongated into filaments. r The critical concentration (C.) is the concentration of free G-actin at which the assemblyonto a filament end is balancedby loss from that end. r I7hen the concentration of G-actin is above the C., the filament end will grow; when it is lessthan the C., the filament will shrink (seeFigure 17-8). r AIP-G-actin addsmuch fasterat the (+ ) end than the (- ) end, resulting in a lower critical concentration at the (* ) end than at the (-) end. r At steady state, actin subunits treadmill through a filament. ATP-actin is added at the (+) end, ATP is then hydrolyzed to ADP and P;, P; is lost, and ADP-actin dissociates from the (-)end. r The length and rate of turnover of actin filaments is regulatedby specializedactin-bindingproteins(seeFigure 1711). Profilin enhancesthe exchangeof ADP for AIP on Gactin; cofilin enhancesthe rate of lossof ADP-actin from the filament (-) end, and thymosin-Babinds G-actin to provide reserveactin when it is needed.Capping proteins bind to filament ends, blocking assemblyand disassembly.

of Actin Mechanisms FilamentAssembly The rate-limiting step of actin polymerization in vitro is the formation of an initial actin nucleus from which a filament can grow (seeFigure 17-7a). In cells, this inherent property of actin is used as a control point to determine where actin filaments are assembled-this is how the different actin assemblieswithin a single cell are generated(seeFigures 17-1 and 17-4). Two classesof actin nucleating proteins, the formin protein family and the Arp2/3 complex, nucleate actin assembly under the control of signal-transduction pathways. Moreover, they nucleatethe assemblyof different actin organizations: formins lead to the assembly of long actin filaments, whereas the Arp2l3 complex leads to branched networks. Ve will discusseach separatelyand see how the power of actin polymerization can power motile in a cell. processes

F o r m i n sA s s e m b l eU n b r a n c h e dF i l a m e n t s Formins are found in essentiallyall eukaryoticcellsas quite a diverse family of proteins: seven different classesare presentin vertebrates.Although diverse,all formin family members have two adjacent domains in common, the socalled FH1 and FH2 domains (formin-homology domains l and2). Two FH2 domains, which provide the basic nucleating function of formins, associateto form a doughnutshaped complex (Figure 17-13a). This complex has the ability to nucleateactin assemblyby binding two actin subunits, holding them so that the (+)end is toward the FH2 domains. The nascentfilament can now grow at the (+) end while the FH2 domain dimer remains attached.How can it do this? As we saw earlier,an actin filament can be thought of as two intertwined strands of subunits. The FH2 dimer can bind to the two terminal subunits. It then probably rocks betweenthe two end subunits,letting go of one to allow addition of a new subunit and then binding the newly added subunit and freeing up spacefor the addition of another subunit to the other strand. In this way, rocking betweenthe two subunitson the end, it can remain aftached while simultaneously allowing growth at the (+ ) e n d ( s e eF i g u r e1 7 - 1 ' 3 a ) . The FH1 domain adjacent to the FH2 domain also makes an important contribution to actin filament growth (Figure 1,7-14).This domain is rich in proline residuesthat are sitesfor the binding of severalprofilin molecules.'Wediscussedearlier how profilin can exchangethe ADP nucleotide on G-actin to generateprofilin-ATP-actin. The FH1 domain behavesas a landing site to increasethe local concentratron of profilin-G-actin-ATP complexes.In a way that is not yet completely understood, thesecomplexesare provided to the FH2 domain to add actin to the (+) end of the filament, thereby allowing rapid assemblyto occur between the FH2 domain dimer on the growing filament (Figure 17-14). Since the formin allows addition of actin subunits to the (+) end, long filaments with a formin at their (+ ) end are generated (seeFigure t7-13b).In this manner' formins nucleate actin O F A C T I N F I L A M E N TA S S E M B L Y MECHANISMS

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Animation: Elongationof Actin Filamentby Formin FH2 Dimer {lltl

f o r m i n F H 2d o m a i n

< FIGURE 17-13Actin nucleationby the formin FH2domain. ( a )F o r m i nhsa v ea d o m a i nc,a l l e F d H 2t,h a tc a nf o r ma d i m e ar n d n u c l e a taes s e m bTl yh ed i m e b r i n d st w o a c t i ns u b u n i t(st r ) , a n d ,b y r o c k i nbga c ka n df o r t h( E - 4 ) , c a na l l o wi n s e r t i oonf a d d i t i o n a l s u b u n i tbse t w e etnh eF H 2d o m a i n a n dt h e( + ) e n do f t h eg r o w i n g f i l a m e nTt ,h eF H 2d o m a i np r o t e c t sh e( + ) f r o mc a p p i nbgyc a p p i n g p r o t e i n (sb )T h eF H 2d o m a i n o f a f o r m i nw a sl a b e l ew d i t hc o l l o i d a l g o l d( b l a c dk o t )a n du s e dt o n u c l e a taes s e m bol yf a n a c t i nf i l a m e n t Theresulting filamentwasvisualized by electron microscopy after staining with uranylacetateFormins assemble longunbranched (b)fromD Pruyne filaments[Part etal, 2007,Science 297.612 ]

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FIGURE17-14 Regulation of formins by an intramolecular interaction. At leastone of the sevenformin classes found in vertebrates is regulatedby an intramolecular interactionThe inactive formin is activatedby bindingmembrane-bound activeRho-GTpto i t s R h ob i n d i n gd o m a i n( R B D )r,e s u l t i n g in exposuro ef the FH2 domainw , h i c hc a nt h e n n u c l e a t teh e a s s e m b loyf a n e w f i l a m e n tA , ll f o r m i n sh a v ea n F H 1d o m a i na d j a c e ntto t h e F H 2d o m a r nt:n e proline-rich FH1domainis a sitefor recruitmentof profilin-ATp-Gactincomplexes that can then be "fed" into the growing (+) end F o rs i m p l i c i toyf r e p r e s e n t a t i oans, i n g l ef o r m i np r o t e i ni s s h o w n ,b u t .l a s s h o w ni n F i g u r e 7 - 1 3 ,t h e p r o t e i nf u n c t i o n sa s a 0 t m e rt o nucleateactinassembly

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assemblyand have the remarkableability to remain bound to the (+) end while also allowing rapid assemblythere.To ensurethe continued growth of the filament, formins bind to the (+ ) end in sucha way that precludesbinding of a (+ ) end cappingprotein suchas CapZ, which would normally terminate assembly. F o r m i n a c t i v i t y h a s t o b e r e g u l a t e d .A t l e a s t s o m e formins exist in a folded inactive conformation as a result of an interaction between the first half of the protein and the C-terminal tail. These formins are activated by membrane-bound Rho-GTP, a Ras-relatedsmall GTPase (Figure 17-14). Thus when Rho is switched from the inactive Rho-GDP form into its activated Rho-GTP state, it can bind and activate the formin. Recentstudieshaveshown that formins are responsiblefor the assemblyof long actin filaments such as those found in stressfibers and in the contractilering during cytokinesis(see Ftgure 17-4). The actin-nucleatingrole of formins was only discoveredrecently,so the rolesperformedby this diverseprotein family are only now beinguncovered.Sincethereare many different formin classesin animals,it is likely that formins will be found to assembleadditional actin-basedstrucrures.

T h e A r p 2 l 3 C o m p l e xN u c l e a t e sB r a n c h e d F i l a m e n tA s s e m b l y The Arp2l3 complex is a protein machine consisting of seven subunits, two of which are actin-related proteins

C E L LO R G A N T Z A T T O A N D M O V E M E N TI M I C R O F I L AEMN T S

("Arp"), explaining its name. It is found in essentiallyall eukaryotes, including plants, yeasts, and animal cells. The Arp2/3 complex alone is a very poor nucleator when added into an actin assemblyassay.To be active, Arp2l3 needsto bind both a regulatory protein, an example of which is WASp (Viskott-Aldrich syndrome protein), and a preformed actin filament (Figure 17-15]l.Thus if you add into an actin assembly assay activated rVASp and preformed actin filaments, Arp2l3 is a potent nucleator. How does the Arp2l3 complex nucleate filaments? When it binds to the side of F-actin in the presenceof an activator, it changes conformation so that the two actin-related proteins, Arp2 and Arp3, resemblethe (+ ) end of an actin filament (Figure 1.7-1.5a). This provides a template for the assemblyof a new filament with a free (+) end. This new (+) end then grows as long as ATP-G-actin is available or until it is capped by a (+ ) end-cappingprotein such as CapZ. The angle between

the old filament and the new one is 70' (Figure 17-1.5b). This is also the angle observed experimentally in branched filaments at the leading edge of motile cells, which is believed to be formed by the action of the activated Arp2l3 complex (Figure 1'7-1'5c).As we discussin the next section, the Arp2l3 complex can be used to drive actin polymerization to power intracellular motility. Actin nucleation by the Arp2l3 complex is exquisitely controlled, and the WASp protein is part of that regulatory process.WASp exists in a folded inactive conformation, so that the Arp2l3 activation domain at the carboxy-terminal end of the protein is not available (Figure 17-1'6).One mechanism to activate the protein involves the small Ras-related GTP-binding protein Cdc42 (Figure 17-1'6; Section 17.7), which in the GTP statebinds to and opensSfASp,making the acidic activation domain accessibleto Arp2l3. \fASp also has an actin-monomer-binding site adjacent to the C-terminal

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17-15Actin nucleationby the Arp2l3 complex.(a)To A FIGURE nucleate actinassembly efficiently, theArp2l3complex bindsto the sideof an actinfilament(tr) andto an activator suchasthe a C-terminal domainof theWASpprotein(U ) Thisinduces changein Arp2/3sothatthecomplex cannow bind conformational in a conformation corresponding to a free(+) end two actinsubunits (E), andassembly occursTheArp2l3branchmakesa

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(b)Averaged thefilaments. 70"anglebetween characteristic of Arp2l3at an micrographs electron from several rmagecompiled edge,with (c)lmageof actinfilaments in the leading actinbranch. filaments. branched of individual lPart andcoloring a magnification Part(b) 281:10589 J Biol.Chem (a)adaptedfromA E Kelleyetal ,2006, Part(c)fromT M Svitkina Biol.3:e383 etal, 2006,PLoS fromC Egile 145:1009 Biol 1999, J Cell Borisy, G G l and

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Exterior

by the Arp2l3 complex. Second,it can hold on to the end of the newly formed filament. Third, it can protect the (+ ) end of the growing filament from capping by CapZ. The assembling filament then pusheson the bacterium. Since the filament is embedded in the cytoskeletal matrix of the cell, it is stationary,and so the bacterialcell moves forward (ahead of the polymerizing actin). Researchershave reconstituted Listeria motility in the test tube using purified proteins to seewhat the minimal requirementsfor Listeria motility are. Remarkably, the bacteria will move when just four proteins are added: ATP-G-actin, the Arp2l3 complex, CapZ, and cofilin (Figure17-17b,c). We havediscussed the role of actin and Arp2l3, but for what are CapZ and cofilin needed?As we have seenearlier, CapZ rapidly caps the free (+) end of actin filaments, so when a growing filament no longer con(-) end tributes to bacterial movement, it is rapidly capped and inhibited from further elongation. In this way, assemblyoccurs A FIGURE 17-16Regulationof the Arp2l3complexby WASp. only adjacentto the bacterium where ActA is stimulating the TheWASpisinactive dueto an intramolecular interaction, thereby masking theactivation kp2l3 complex. Cofilin is necessaryto acceleratethe disasdomain.On binding the membrane-bound (a member activesmallG-protein Cdc42-GTP of the Rhofamily) sembly of the (-) end of the actin filament to regenerateacrin throughthe Rhobindingdomain(RBD), the intermolecular interaction to keep the polymerization cycle going (seeFigure 17-11). isrelieved, exposing theacidicdomain(purple) for activation of the This minimal rate of motility can be increasedby the presArp2l3complexWASpalsohasa G-actin bindingregion(brown) that ence of other proteins, such as VASP and profilin, as menaidsin actinnucleation of theactivated Arp2l3complex tioned above. To move inside cells, the Listeria bacterium, as well as other opportunistic pathogens such as the Shigella species Arp2/3 activation domain that facilitatesthe first srepsin nuthat causedysentery,has hijacked a normal, regulated cellucleation of a new filament (Figure 17-76). Iar processinvolved in cell locomotion. As we discussin more detail later (Section17.7), moving cells have a thin sheet of cytoplasm that protrudes from the front of the cell I n t r a c e l l u l aM r o v e m e n t sC a n B e P o w e r e db y called the leading edge (seeFigure 17-4 and 17-15c).This Actin Polymerization thin sheet of cytoplasm consists of a dense meshwork of How can actin polymerization be harnessedto do work? actin filaments that are continually elongating at the front of As we have seen,actin polymerization involvesthe hydrolthe cell to push the membrane forward. Factors in the leadysis of actin-MP to actin-ADR which allows actin ro grow ing edgemembrane activate the Arp2l3 complex to nucleate p r e f e r e n t i a l l ya t t h e ( + ) e n d a n d d i s a s s e m b l e at the (-) thesefilaments. Thus the power of actin assemblypushesthe end. If an actin filament were to becomefixed in the meshmembrane forward to contribute to cell locomotron. work of the cytoskeletonand you could bind and ride on The power of actin assemblyis also used during endocythe assembling(+) end, you would be transported across tosis. As we discussedin Chapter 14, endocytosisinvolves the cell. This is just what the intracellular bacterialparasite the pinching in of the plasma membrane to make an endoListeria monocytogenes does.The study of Listeria motilcytic vesiclethat is transported into the cell. Recent studies ity was, in fact, the way the function of the Arp2l3 protein have shown that after clathrin-coatedvesiclespinch off from was discovered. the membrane, they are driven into the cytoplasm, powered Listeria is a food-borne pathogen that causesmild gasby a rapid and very short-lived burst (a few secondsin duratrointestinal symptoms in most adults but can be fatal to the tion) of actin polymerization, in a manner very similar to elderly or immunocompromised individuals. Ir can enter anListeria motility. imal cellsand divide in the cytoplasm. To move from one cell to another in the animal, it moves around the cell by polyToxinsThat Perturbthe Pool of Actin Monomers merizing actin into a comet tail like the plume behind a Are Usefulfor StudyingActin Dynamics rocket (Figure1,7-17a,b), and when it runs into the plasma membrane,it pushesits way into the adjacentcell to infect it. Certain fungi and spongeshave developedtoxins that target How does it recruit the host cell actin to move around? the polymerization cycle of actin and are therefore toxic to Listeria has on its surface a protein called ActA, which can animal cells. Two types of toxins have been characterized. bind and activatethe Arp2/3 complex (Figure 17-17c).The The first class is representedby two unrelated toxins, ActA protein also binds a protein known as VASP,which has cytochalasin D and latrunculin, which promote the depolythree important properties. First, VASP has a proline-rich merization of filaments, though by different mechanisms. region that can bind profilin-ATP-actin for enhancingATpCytochalasin D, a fungal alkaloid, depolymerizesactin filaactin assemblyinto the newly formed barbed end generated ments by binding to the (+ ) end of F-actin, where it blocks P l a s m am e m b r a n e

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C E L LO R G A N T Z A T T O A N D M O V E M E N Tt : M T C R O F T L A M E N T S

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phasemicrographshowsbacteria(black),behindwhich are the FIGURE 17-17Listeriautilizesthe powerof EXPERIMENTAL actintails (c)A modelof how LtsferiamovesusingJUSI for intracellular movement.(a)Fluorescence phase-dense actinpolymerization to the bacterial four proteinsThe ActA proteinon the cellsurfaceactivatesthe of a cultured cellstained with an antibody microscopy from preexisting Arp2l3 complexto nucleatenew filamentassembly protein(red)andf luorescent phalloidin F-actin to localize surface (+) by CapZ until capped grow end at their Filaments "comet (green)Behind filaments isan actin tail"thatpropels eachbacterium enhances which of cofilin, the action through is recycled Actin polymerization When the bacterium forwardby actin the bacterium at the (-) end of the filamentsln this way, depolymerization out intoa membrane, it pushes the membrane runsintotheplasma polymerization is confinedto the backof the bacterium,which cell likea f ilopodium, whichprotrudes intoa neighboring structure Part(b) propelsit forward [Part(a)courtesy andI Michison of J Theriot (b)Listeria andlust in vitrowith bacteria motilitycanbe reconstituted 401'613 Nature 1999, et al T P Loisel l from , The ATP-G-actin, Arp2l3complex, CapZ,andcofilin, fourproteins:

further addition of subunits.Latrunculin,a toxin secretedby sponges,binds and sequestersG-actin, inhibiting it from adding to a filament end. Exposureto either toxin thus increasesthe monomer pool. When cytochalasinor latrunculin and is addedto live cells,the actin cytoskeletondisassembles cell movements such as locomotion and cytokinesis are inhibited.Theseobservationswere among the first to implicate actin filamentsin cell motility. Latrunculin is especially

useful becauseit binds actin monomers and preventsany new actin assembly.Thus if you add latrunculin to a cell, the rate at which actin-basedstructures disappear reflects their normal rate of turnover. This has revealedthat some structures have half-times of much less than a minute, whereas others are much more stable.For example, it has beenfound that the leadingedgeof motile cellsturns over every30-180 seconds,whereasstressfibers turn over every 5-10 minutes. ASSEMBLY I M E C H A N I S MO S F A C T I NF I L A M E N T

727

In contrast, the monomer-polymer equilibrium is shifted in the direction of filaments by jasplakinolide,another sponge toxin, and by phalloidin, which is isolated from Amanita phalloides (the "angel of death" mushroom). Jasplakinolideenhancesnucleation by binding and stabilizing actin dimers and thereby lowering the critical concentration.Phalloidin poisons a cell by binding at the interface between subunits in F-actin, locking adjacent subunits together and preventing actin filaments from depolymerizing. Even when actin is diluted below its critical concentration,phalloidin-stabilizedfilaments will not depolymerize. Fluorescent-labeledphalloidin, which binds only to F-actin, is commonly used to stain actin filaments for light microscopy (seeFigure 17-4).

Mechanismsof Actin Filament Assembly r Actin assemblyis nucleated by two classesof proteins: formins nucleatethe assemblyof unbranchedfilaments (see Figure 77-13), whereas the Arp2l3 complex nucleatesthe assemblyof branchedactin nerworks (seeFigure 17-15). The activities of formins and Arp2l3 are regulated by signal-transduction pathways. r Functionally different actin-based structures are assembledby formins and Arp2l3 nucleators. Formins drive the assembly of stress fibers and the contractile ring, whereas Arp2l3 nucleates the assembly of branched actin filaments found in the leading edge of motile cells. r The power of actin polymerization can be harnessedto do work, as is seen in the Arp2l3-dependent intracellular movement of pathogenicbacteria (seeFigure 17-17) and inward movement of endocytic vesicles. r Severaltoxins affect the dynamics of actin polymerizationl some, such as latrunculin, bind and sequesteractin monomers, whereasothers, such as phalloidin, stabilizefilamentous actin. FluorescentlyIabeled phalloidin is useful for staining actin filaments.

Organizationof Actin-Based CellularStructures So far in this chapter, we have seenthat actin filaments can be assembledinto a wide variery of different arrangementsand how many associatedproteinsnucleateactin assemblyand regulate filament turnover. Dozens of proteins in a vertebrate cell organize these filaments into diverse functional structures. Here we discussjust a few of theseproteins,giving examplesof typical typesof actin crosslinking proteinsfound in cells,and also discussthe proteins involved in making functional links betweenactin and membraneproteins. Onetscinating problem, about which very little is known, is how cellsassembledif-

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ferent actin-based structures within the same cytoplasm of a cell. Someof this organization must be due to local regulation, a topic we come to at the end of the chapter.

C r o s s - L i n k i nPgr o t e i n sO r g a n i z eA c t i n F i l a m e n t s into Bundlesor Networks \7hen one assemblesactin filaments in a test tube, they form a tangled network. However, in cells, actin filaments are found in a variety of structures, such as the highly ordered filament bundles in microvilli or the meshwork char* acteristicof the leading edge (seeFigure 17-4a). Thesedifferent organizations are determined by the presenceof actin cross-linking proteins. To be able to organize actin, an actin cross-linking protein must have two F-actin-binding sites (Figure 17-18). C r o s s - l i n k i n g o f F - a c t i n c a n b e a c h i e v e db y h a v i n g two actin-binding siteswithin a single polypeptide,as with fimbrin, a protein found in microvilli to build bundles of filaments all having rhe same polarity. Other actin cross-linking proteins have a single actin-binding site in a polypeptide chain and then associateto form dimers to bring together two actin-binding sites. These dimeric cross-linking proteins can assembleto have a rigid rod connecting the two binding sites,as happens with a-actinin, which also holds parallel actin filaments but farther apart than fimbrin. Another protein, called spectrin, is a tetramer with two actin-binding sites; spectrin spans an even greater distance between actin filaments and makes networks under the plasma membrane (seeFigure 17-19). Other types of cross-linking proteins, such as filamin, have a highly flexible region between the two binding sites,functioning like a molecular leaf spring, so they can make stabilizing cross-linksbetween filaments in a meshw o r k ( F i g u r e 1 7 - 1 . 8 ) ,a s i s f o u n d i n t h e l e a d i n g e d g e o f motile cells. The Arp2l3 complex, which we discussedin terms of its ability to nucleate actin filament assembly,is also an important cross-linking protein, attaching the (- ) end of one filament to the side of another filament (see Figure17-1,5).

A d a p t o r P r o t e i n sL i n kA c t i n F i l a m e n t s to Membranes To contribute to the structure of cells and also harnessthe power of actin polymerization, actin filaments are attached to membranesor are associatedwith intracellular structures. Actin filaments are especiallyabundant in the cell cortex underlying the plasma membrane, to which they give support. Actin filaments can interact with membraneseither laterally or at their end. Our first example of actin filaments attached to membranes is the human erythrocyte-the red blood cell. The erythrocyte consists essentiallyof plasma membrane enclosing a high concentration of the protein hemoglobin to

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cell-surfacestructures such as microvilli and membrane ruffles. If we think of a microvillus, it is clear that it must have an end-on attachment at the tip and lateral attach'What ments down its length. is the orientation of actin filaments in microvilli? Decoration of microvillar filaments by the 51 fragment of myosin show that it is the (* ) end at the tip (Figure 17-19c). Moreover, when fluorescentactin is added to a cell, it is incorporatedat the tip of a microvillus, showing that not only is the (+) end there but that actin filament assemblyoccurs there. At presenrit is not known how actin filaments are attachedat the microvillus tip, but a likely candidateis a formin protein. This (+ ) end orientation of actin filaments with respectto the plasma membrane is almost universally found-not just in microvilli but also, for example,in the leadingedgeof motile cells.The lateral attachmentsto the plasma membraneare believedto be provided, at leastin part, by the ERM (ezrinradixin-moesin) family of proteins. These are regulated 'When proteins that exist in a folded, inactive form. activated, often by the membrane-boundregulatory lipid PIP2 (phosphatidylinositol(4,5)P2)and subsequentphosphorylation, F-actin and membrane-protein-bindingsites of the ERM protein are exposed to provide a lateral linkage to actin filaments (Figure 17-19d). At the plasma membrane, ERM proteins can link the actin filaments directly or indirectly through scaffolding proteins to the cytoplasmic d o m a i n o f m e m b r a n ep r o t e i n s . The two types of actin membrane linkages we have discusseddo not involve areas of the plasma membrane directly attachedto other cells or the extracellularmatrix. In contrast, highly specializedregions of the plasma membrane of epithelial cells, called adherensjunctions, make contactsbetweencells(seeFigure 1,7-1,b). Other specialized regions of association,called focal adhesions,mediate attachmentof cellsto the extracellularmatrix. Thesespecialized types of attachmentsin turn connect to the cytoskeleton and are describedin more detail when we discusscell migration (Section17.7) and cells in the context of tissues ( C h a p t e r1 9 ) . Muscular dystrophies are genetic diseases often characterizedby the progressiveweakening of skeletal muscle. One of these geneticdiseases,Duchenne muscular dystrophy, affects the protein dystrophin, whose gene is located on the X-chromosome, and so the disease is much more prevalent in males. Dystrophin is a modular protein whose function is to link the cortical actin network of muscle cells to a complex of membrane proteins that link to the extracellular matrix. Thus dystrophin has an N-terminal actin-binding domain, followed by a series of spectrinlike repeats and terminating in a domain that binds the transmembrane dystroglycan complex to the extracellular matrix protein laminin (seeFigure 17-18a). In the absenceof dystrophin, the plasma membrane of muscle cells becomesweakened by cycles of muscle contraction and eventually ruptures, resulting in death of the muscle myofibril. I

Organization of Actin-BasedCellular Structures r Actin filaments are organized by cross-linking proteins that have two F-actin-binding sites. Actin cross-linking proteins can be long or short, rigid or flexible, depending on the type of structure involved (seeFigure 1,7-1'8). r Actin filaments are attachedlaterally to the plasma membrane by specificclassesof proteins, such as are seenin the red blood cell or in cell-surfacestructuressuch as microvilli (seeFigure 17-19). r The (* ) end of actin filaments can also be attached to membranes,with assemblymediated betweenthe filament end and the membrane. r Severaldiseaseshave beentraced to defectsin the microfilament-basedcortical cytoskeleton that underlies the olasmamembrane.

Myosins:Actin-Based Motor Proteins We have discussedhow actin polymerization nucleatedby the Lrp2l3 complex can be harnessedto do work. In addition to motility, cellshave a large family actin-polymerization-based of motor proteins, called myosins,that can move along actin filaments powered by ATP hydrolysis. The first myosin discovered,myosin II, was isolated from skeletalmuscle.For a long time, biologists thought that this was the only type of myosin found in nature. However, they then discoveredother types of myosins and beganto ask how many different functional classesmight exist. Today we know that there are several different classesof myosins,in addition to the myosin II of skeletalmuscle,that provide a motor function. The other classesof myosin provide a myriad of functions, such as moving organellesand other structures around cells as well as contributing to cell migration. Indeed,with the discoveryand analysis of all these actin-basedmotors and the corresponding microtubule-basedmotors describedin the next chapter, the former relatively static view of a cell has been replaced with the realization that it is incredibly dynamic-more like an organizedbut busy freeway systemwith motors busily ferrying componentsaround. To begin to understand myosins, we first discusstheir general domain organization. Armed with this information, we explore the diversity of myosins in different organisms and describein more detail some of those that are common in eukaryotes.Myosins have the amaztngability to convert the energyreleasedby ATP hydrolysis into mechanicalwork. All myosins convert ATP hydrolysis into work, yet different myosins can perform very different types of functions. For example, many moleculesof myosin II pull together on actin filaments to bring about muscle contraction, whereas myosin V binds to vesicularcargo to transport it along actin filaments. To understand how such diversefunctions can be

MOTORPROTEINS M Y O S I N SA : CTIN-BASED

731

accommodatedby one type of motor mechanism,we will investigatethe basic mechanismof how the energyreleasedby ATP hydrolysis is convertedinto work and then seehow this mechanism is modified to tailor the properties of specific myosin classesfor their specificfunctions.

M y o s i n sH a v eH e a d ,N e c k ,a n d T a i lD o m a i n s w i t h D i s t i n c tF u n c t i o n s Much of what we know about myosins comes from studies of myosin II from skeletalmuscle.In skeletalmuscle,myosin II is assembledinto so-calledbipolar thick filaments containing hundreds of individual myosin II proteins (Figure 1.7-20a)with oppositeorientationsin eachhalf of the bipolar filament. Thesemyosin filaments interdigitate with actin thin filaments to bring about muscle contraction. We will

discusshow this systemworks in a later section,but here we investigatethe properties of the myosin itself. It is possible to dissolve the myosin thick filament in a solution of ATP and high salt. The resulting solublemyosin II protein consists of six polypeptides-two associatedheavy chains and four light chains.Two light chains associatewith the "neck" region of eachheavychain (Figure1.7-20b).The soluble myosin has ATPase activity, reflecting its ability to power movementsby hydrolysis of ATP. But which domain of myosin is responsiblefor this activity? To identify functional domains in a protein, a standard approach is to cleave it into fragments with specific proteases and ask which fragmentshave the activity. Solublemyosin II can be cleaved into two pieces by gentle treatment with the protease chymotrypsin to yield two fragments, one called heavy mero-myosin (HMM: mero means "Dart of") and the other

(a)

( c ) H e a da n d n e c kd o m a i n

( b ) M y o s i nl l Head Neck

Tail

Regulatory l i g h tc h a i n

Essential

Nucleotideb i n d i n gs i t e

H e a v yc h a i n s

Regulatory l i g h tc h a i n

FIGURE 17-20Structureof myosinll. (a)Organization of myosin ll rnfilaments isolated fromskeletal muscleMyosin ll assembles intobipolar filaments in whichthetailsformtheshaftof thefilament withheads exposed. Extraction of bipolar filaments wrthhighsaltand ATPdisassembles thefilament intoindividual myosin ll molecules (b)Myosin ll molecules (lrghtblue) consist of two identical heavy chains (green andfourlightchains andblue)Thetailof theheavy chains formsa coiled-coil to dimerize; theneckregion of eachheavy chainhas two lightchainsassociated with it. Limitedproteolytic cleavage of 732

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myosinll generates tailfragments-LMMand S2-and the S'1motor domain (c)Three-dimensional modelof a singleS'1headdomainshows that lt hasa curved,elongatedshapeand is bisectedby a cleft.The pocketlieson one sideof thiscleft,and the actinnucleotide-binding bindingsitelieson the othersidenearthe tip of the head Wrapped aroundthe shaftof the ct-helical neckaretwo lightchainsThesechains stiffenthe neckso that it can act as a leverarm for the head Shown hereisthe ADP-bound conformation

C E L LO R G A N I Z A T I OANN D M O V E M E N Tt : M T C R O F T L A M E N T S

llll+ Animation:In Vitro Motility MyosinAssay 51 head of myosrn

light meromyosin (LMM) (Figure 17-20b). The heavy meromyosin can be further cleavedwith the proreasepapain to yield subfragment 1 (S1) and subfragment2 (52). By analyzing the properties of the various fragments-S1, 52, and LMM-it was found that the intrinsic ATPase activity of myosin residesin the 51 fragment, as doesan F-actin-binding site.Moreover, it was found that the MPase activity of the 51 fragment was greatly enhanced by the presenceof filamentous actin, so it is said to have an actin-actiuatedATPase actiuity, which is a hallmark of all myosins.The 51 fragment of myosin II consistsof the head and neck domains, whereas the 52 and LMM regionsmake up the tail domain (Figure 1720b). X-ray crystallographic analysis of the head and neck domains revealedits shape,the positions of the light chains, and the locations of the ATP-binding and actin-binding sites. The elongatedmyosin head is attached at one end to the ahelical neck (Figure 17-20c1.Two light-chain moleculeslie at the baseof the head,wrapped around the neck like C-clamps. In this position, the light chains stiffen the neck region. How much of myosin II is necessaryand sufficient for "motor" activity? To answer this question, one needsa simple in vitro motility assay.In one such assay,the slidingfilament assay,myosin moleculesare tethered to a coverslip to which is added stabilized fluorescence-labeledactin filaments. Becausethe myosin moleculesare tethered,they cannot slide; thus any force generatedby interaction of myosin headswith actin filaments forces the filaments to move relative to the myosin (Figure17-21a).If ATP is present,added actin filaments can be seento glide along the surface of the coverslip; if ATP is absent, no filament movement is observed.Using this assay,one can show that the 51 head of myosin II is sufficient to bring about movement of actin filaments. This movement is caused by the tethered myosin 51 fragments (bound to the coverslip) trying to "move" toward the (+ ) end of a filament; thus the filaments move with

17-21 Sliding-filament assayis FIGURE < EXPERIMENTAL usedto detect myosin-poweredmovement.(a)After myosin excess of a glasscoverslip, molecules areadsorbedontothe surface the coverslip thenisplacedmyosin-side unboundmyosinisremoved; throughwhichsolutions downon a glassslideto forma chamber madevisible andstableby of actinfilaments, canflow.A solution phalloidin, isallowedto flow into with rhodamine-labeled staining (Thecoverslip fromits isshowninverted in the diagram thechamber. to seethe to makeit easier on theflow chamber orientation positions of ATBthe myosinheads of the molecules ) Inthe presence in illustrated bythe mechanism walktowardthe (+) endof filaments walkingof the myosin tailsareimmobilized, Figure17-24Because of individual Movement of thefilaments. headscauses sliding lightmicroscope. in a fluorescence filaments canbe observed (b)Thesephotographs of threeactinfilaments showthe positions (numbered recorded by video intervals 1,2, 3) at 3O-second from canbe determined Therateof filamentmovement microscopy. andS Kron] of M Footer suchrecordingslPart(b)courtesy

the (-) end leading. The rate at which myosin moves an actin filament can be determined from video camera recordings of sliding-filament assays(Figure 1'7-21'b). All myosins have a domain related to the 51 domain of myosin II, which is responsiblefor their motor activity. The tail domain doesnot contribute to motility but rather defines what is moved by the S1-relateddomain. Thus, as might be expected,the tail domains can be very different and are tailored to bind specificcargoes.

M y o s i n sM a k e U p a L a r g eF a m i l yo f Motor Proteins Mechanochemical Since all myosins have related S1-motor domains with considerable similarity in primary amino acid sequence,it is possible to determine how many myosin genes, and how many different classes of myosins, exist in a sequenced genome. There are about 40 myosin genes in the human genome (Figure 1,7-22),nine in Drosophila, and five in the budding yeast. Computer analysis of the sequencerelationships between the myosin head domains suggeststhat about 20 distinct classesof myosins have evolved in eukaryotes' with greater sequencesimilarity within a classthan between' As indicated in Figure 17-22, the geneticbasis for some diseaseshas beentraced to genesencoding myosins. All myosin head domains convert ATP hydrolysis into mechanicalwork using the samegeneralmechanism.However, as we will see, subtle differences in this mechanism can have profound effects on the functional properties of different classesof myosin. How do thesedifferent classesrelateto tail domains? Amazingly if one takes just the protein sequencesof the tail domains of the myosins and uses this information to place them in classes,they fall into the same groupings as the motor domains. This implies that motor domains with specific properties have co-evolved with specific classesof tail MOTORPROTEINS M Y O S I N SA : CTIN-BASED

733

< FIGURE 17-22The myosinsuperfamilyin humans.Computer analysis of the relatedness of S1headdomains of allof the approximately 40 myosins encoded bythe humangenomeEach myosinisindicated by a line,with the lengthof the lineindicating phylogenetic relationships distance Thusmyosins connected by short linesareclosely related, whereas thoseseparated by longerlinesare moredistantly relatedAmongthesemyosins arethreeclassesmyosins l, ll,andV-widely represented amongeukaryotes, with othershavingmorespecialized functionsIndicated areexamples in whichlossof a specific myosin causes a disease andmodified [Redrawn f r o m R E C h e n e y , 2 0 0 1M , o l B i o l C e l l1 2 . l 8 O l

domains, which makes a lot of sense,suggestingthat each classof myosin has evolved to carry out a specificfunction. In every case that has been tested so far, myosins move toward the (+ ) end of an acrin filament-with one exception, myosin VI. This remarkable myosin has an insert in its head

domain to make it work in the opposite direction, and so motility is toward the (-) end of an actin filament. Myosin VI in animal cellsis believedto contribute to endocytosisby moving the endocyticvesiclesalong actin filamentsaway from the plasma membrane.Recallthat membrane-associated actin filamentshave their (+ ) endstoward the membrane,so a motor directedtoward the (- ) end would take them away from the membranetoward the centerof the cell. Among all these different classesof myosins are three especiallywell-studied ones, which are commonly found in animals and fungi: the so-called myosin I, myosin 1l and myosin V families (Figure 17-23). In humans, eight genes '14 encode heavy chains for the myosin I family, for the myosin II family, and three for the myosin V family. The myosin II class assemblesinto bipolar filaments, which is important for its involvement in contractile functions; indeed, this is the only class of myosins involved in contractile functions. The laree number of members in this

Step size

Function

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Membrane association, endocytosis

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FIGURE17-23 Three common classesof myosin. MyosinI consistof a headdomainwith a variablenumberof light chains associated with the neckdomain Membersof the myosinI classare the only myosinsto havea singleheaddomain Someof these myosinsare believedto associate directlywith membranesthrough lipid interactions. Myosinlls havetwo headdomainsand two liqht

734

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Organelle transport

chainsperneckandarethe onlyclass thatcanassemble intobipolar filaments. MyosinVshavetwo headdomains andsixlightchainsper (brownbox)on organelles, neckTheybindspecific receptors which theytransport, All myosins in thesethreeclasses movetowardthe (+) endof actinfilaments

C E L LO R G A N T Z A T T O A N D M O V E M E N Tt : M | C R O F | L A M E N T 5

rt !f eod."rt: MyosinMovementAgainstActin Filaments Animation:Myosin-Actin Cross'Bridge Cycle ( a ) T h i c kF i l a m e n t

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< FfGURE17-24 The myosinhead usesATPto pull on an actin of ATBthe myosinheadisfirmlyattached filament.(a)Inthe absence in living Although thisstateisveryshort-lived to theactinfilament. in death(rigor stiffness for muscle muscle, it isthe stateresponsible fromthe mortis)Step(tr): On bindingATBthe myosinheadreleases (Z): ADP andP, the ATP to hydrolyzes The head Step actinfilament. to the neckThis a rotationin the headwith respect whichinduces aselastic "cockedstate"storesthe energyreleased by ATPhydrolysis springStep(B): Myosinin the "cocked"state likea stretched energy, headcouples bindsactinStep(4): Whenboundto actinthemyosin energyto movethe actin of the elastic of P;with release release moving the filamentThisisknownasthe "powerstroke"asit involves Step neckdomain. to theendof the myosin with respect actinfilament asADPisreleased (E): Theheadremains tightlyboundto thefilament (b) of models Molecular head. by the is bound fresh ATP andbefore in "cocking" headinvolved in the myosin changes theconformational (lowerpanel)The andduringthepowerstroke thehead(upperpanel) therestof the areshownin darkblueandgreen; lightchains myosin is redlpart(a) and actin light blue, in myosinheadandneckarecolored a d a p t e d f r o mR D V a l ea n d R A M i l l i g a n2, O O 2 , S c i e n c e 2 8 8 : 8P8a r t ( b )b a s e d o n G e e v ea s n d H o l m e s2 0 0 5 ( u n p u b l i s h e d ) l

M O T O RP R O T E I N S M Y O S I N SA : CTIN-BASED

735

classreflects the need for myosin IIs with the slightly different contractile properties seen in different muscles (e.g., skeletal, cardiac, and various types of smooth muscle) as well as in nonmusclecells. The myosin II class is the only one that assemblesinto bipolar filaments. All myosin II members have a relatively short neck domain, with two light chainsper heavychain.The myosin I class is quite large, has a variable number of light chains associatedwith the neck region, and is the only one in which heavy chains are not associatedthrough their tail domains and so are single-headed. The large sizeand diversity of the myosin I class suggeststhat these myosins perform many functions, most of which remain to be determined,but some membersof this family connectactin filamentsto membranes, and others are implicated in endocytosis.Members of the myosin V classhave two heavy chains, giving a motor with two heads,long neck regionswith six light chains each, and tail regionsthat dimerize and terminate in domains that bind to specificorganellesto be transported.

ConformationalChangesin the Myosin Head CoupleATPHydrolysisto Movement The results of studies of muscle contraction provided the first evidencethat myosin headsslideor walk along actin filaments. Unraveling the mechanism of muscle contraction was greatly aided by the development of in vitro motility assaysand singlemolecule force measurements.On the basis of information obtained with these techniques and the three-dimensional structure of the myosin head, researchersdevelopeda general model for how myosin harnessesthe energy releasedby ATP hydrolysis to move along an actin filament (Figure 1,7-24 on page735).Becauseall myosinsare thought to usethe samebasic mechanism to generatemovement, we will ignore whether the myosin tail is bound to a vesicleor is part of a thick filament, as it is in muscle.One important aspectof this model is that the hydrolysisof a singleATP moleculeis coupledto each step taken by a myosin molecule along an actin filament. A question that has intrigued biologists is how myosin can convert the chemical energy releasedby ATP hydrolysis into mechanicalwork. It has beenknown for a long time that the 51 head of myosin is an AIPase, having the ability to hydrolyze ATP into ADP and P;. Biochemical analysisreveals how this works (Figure17-24a).In the absenceof ATp, the head of myosin binds very tightly to F-actin. When ATp binds, the affinity of the head for F-actin is greatly reduced and releasesfrom actin. The myosin head then hydrolyzesthe ATP, and the hydrolysis products, ADP and P1, remain bound. The energyprovided by the hydrolysisof ATP induces a conformation change in the head that results in the head domain rotating with respectto the neck. This is known as the "cocked" positionof the head (Figure17-24b).In the absenceof F-actin, releaseof P; is exceptionallyslow, the slowest part of the ATPase cycle. However, in the presenceof actin, the head binds F-actin tightly, inducing both releaseof P; and rotation of the head back to its original position, thus moving the actin filament relative to the neck domain (Figure 17-24b).In this way, binding to F-actin inducesthe move736

.

c H A p r E R1 7 |

EXPERIMENTAL FIGURE 17-25 The lengthof the myosintl neckdomaindeterminesthe rate of movement.Totestthe leverarmmodelof myosin movement, investigators usedrecombinant DNA techniques to makemyosin headsattached to different-length neck domainsTherateat whichtheymoveon actinfilaments was determined. Thelongertheleverarm,thefasterthe myosin moves, supporting theproposed mechanism. fromK A Ruppel andJ [Redrawn A Spudich, 1996, Annu ReuCellMol.Biol.12:543-5731

ment of the head and releaseof Pi, thereby coupling the two processes.This step is known as the power stroke. The head remains bound until the ADP leavesand a fresh ATP binds the head, releasingit from the filament. The cycle then repeats,and the myosin can move again againstthe filament. How is hydrolysis of ATP in the nucleotide-binding pocket converted into force? The results of structural studies of myosin in the presenceof nucleotides, and nucleotide analogs that mimic the various steps in the cycle, indicate that the binding and hydrolysis of a nucleoride cause a small conformational change in the head domain. This small movement is amplified by a "converter" region at the base of the head acting like a fulcrum and causing the leverlike neck to rotate. This rotation is amplified by the rodlike lever arm, which constitutesthe neck domain, so the actin filament moves by a few nanometers(Figure 17-24b). This model makes a strong prediction: the distance a myosin head moves actin during hydrolysis of one ATP-the myosin step size-should be proportional to the length of the neck domain. To test this, mutant myosin moleculeswere constructedwith different-lengthneck domains and the rate at which they moved down an actin filament was determined. Remarkably there is an excellentcorrespondence betweenthe length of the neck domain and the rate of movement (Figure 17-25).

Myosin HeadsTakeDiscreteStepsAlong Actin Filaments The most critical feature of myosin is its ability ro generarea force that powers movements.Researchershave usedoptical traps to measure the forces generated by single myosin molecules (Figure 17-26). In this approach, myosin is

C E L Lo R G A N r z A T r oAN N D M o v E M E N rT: M T c R o F T L A M E N T S

immobilized on beadsat a low density.An actin filament, held between two optical traps, is lowered toward the bead until it contacts the myosin molecule. When ATP is added, the myosin pulls on the actin filament. Using a mechanicalfeedback mechanismcontrolled by a computer, one can measure the distancepulled and the forces and duration of the movement (Figure 17-26). The resultsof optical trap studiesshow that myosin II does not interact with the actin filament continuously but rather binds, moves, and releasesit. In fact, myosin II spendson averageonly about 10 percent of each ATPase cycle in contact with F-actin-it is said to have a duty ratio of 10 percent. This will be important later when we consider that in muscle, hundreds of myosin heads pull on actin filaments, so that at any one time, 10 percent of the headsare engagedto provide a smooth contraction. 'When myosin II does contact F-actin, it takes discrete steps,approximately5-15 nm long (Figure1.7-27),and generates 3-5 piconewtons (pN) of force, approximately the same force as that exerted by gravity on a single bacterium. If we now look at a similar optical trap experiment with myosin V, the curves look completely different (Figure 1,7-27).Now we can easilydiscernclear stepsof about 35 nm in length. This larger step sizereflectsthe longer neck domain-the lever arm-of myosin V. Moreover, we seethat the motor takes many sequentialsteps without releasing from the actin-it is said to move processiuely.This is because its ATPase cycle is modified to have a much higher duty ratio (>70 percent)by slowing the rate of ADP release; thus the head remains in contact with the actin filament for a much larger percentageof the cycle. Sincea single myosin V moleculehas two heads,a duty ratio of )50 percentensures that one head is in contact at all times as it moves down an actin filament so that it does not fall off.

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17-27Measuringthe stepsizeand FIGURE EXPERIMENTAL to the Using an opticaltrapsetupsimilar processivity of myosins. the analyzed have investigators 1 7-26, in Figure described one As V (bottomtrace). of myosinll (toptrace)andmyosin behavior shownbythe peaksin thetrace,myosinll takeserraticsmallsteps andthen moves, (5-15 nm),whichmeansit bindstheactinfilament, V myosin By contrast, motor. nonprocessive a letsgo lt istherefore takesclear36-nmstepsoneaftertheother,so it hasa stepsizeof is,it doesnot let 90 of the 36 nm andis highlyprocessive-that (b)from Part (a)fromFiner 368:113 etal,1994,Nature actinfilamentIPart Acad Sci.USA97:94821 ProcNat'l M Riefetal,20OO,

M y o s i nV W a l k sH a n d O v e r H a n d D o w n an Actin Filament

17-25Opticaltrap determinesthe stepsizeand A FIGURE forcegeneratedby a singlemyosinmolecule.In an opticaltrap, on a laserisfocused by a lightmicroscope the beamof an infrared light), Iatexbead(oranyotherobjectthatdoesnot absorbinfrared andholdsthe beadin thecenterof the beamThe whichcaptures or by increasing of theforceholdingthe beadisadjusted strength an the rntensity of the laserbeam ln thisexperiment, decreasing two ootrcal trapsTheactinfilamentis actinfilamentisheldbetween of thenlowered ontoanotherbeadwith a diluteconcentration a myosinmolecules attached to it lf the actinfilamentencounters will pullon the in the presence of ATBthe myostn myosinmolecule to measure boththe whichallowsthe investigators actinfilament, takes forcegenerated andthestepsizethe myosin

The next question is, how do the two heads of myosin V work together to move down a filament? One model proposes that the two heads walk down a filament hand over hand with alternately leading heads (Figure'J-7-28a). An alternative possibility is an inchworm model, in which the leading head takes a step' the second head is pulled up behind it, and then the leading head takes another step (Figure 1,7-28b).How can one distinguish between these models? In the inchworm model, each individual head takes 36-nm steps,whereasin the walking model' eachtakes 72-nm steps.Scientistshave managed to attach a fluorescentprobe to just one neck region of myosin V and watch it walk down an actin filament: it takes 72-nm steps (Figure \7-28c), and so it walks hand over hand down a filament (72 nm is the MOTORPROTEINS M Y O S I N SA : CTIN-BASED

737

(a) Hand over hand

the properties one would expect for a motor designed to transport cargo along an actin filament.

on neck

Myosins: Actin-Based Motor Proteins r Myosins are actin-based motors powered by ATP hydrolysis. r Myosins have a motor head domain, a lever-arm neck domain, and a cargo-binding tail domain (seeFigure 17-20).

(b) Inchworm

r There are many classesof myosin, with three classes present in many eukaryotes: myosin I has a single head domain, myosin II has two headsand assemblesinto bipolar filaments, and myosin V has two headsand doesnot assemblein filaments (seeFigure 17-23).

Labelon n

r Myosins convert ATP hydrolysis to mechanicalwork by amplifying a small conformational change in their head through their neck domain when the head is bound to F-actin (seeFigure 1,7-24). (c)

r Myosin heads take discrete steps along an actin filament, which can be small (5-15 nm) and nonprocessivein the case of myosin II or large (36 nm) and processivefor myosin V.

Myosin-PoweredMovements

E 500 o

o

L

0102030405060 T i m e( s ) A EXPERIMENTAT FTGURE 17-28MyosinV hasa stepsizeof 36 nm, yet eachhead movesin 72-nmsteps,so it moveshand over hand.Twomodelsfor myosinV movement downa filament havebeensuggested. (a)Inthe hand-over-hand model,onehead bindsan actinfilament, andtheotherthenswingsaroundandbinds a site72 nm ahead.(b)In the inchworm model,the leading head moves36 nm,thenthe laggingheadmovesup behindit, allowing the leading headto takeanother36-nmstep.(c)MyosinV labeled with a fluorescent tag on justone headcanbe seento havea step sizeof 72 nm.Thusmyosrn V walkshandoverhand.[Adapted fromA Yildiz et al, 2003,Science 300:2061 l step size for each head; the step size for the double-headed motor is 35 nm). IThy is the step size of myosin V so large? If we compare its step size of 35 nm to the structure of ihe actin filament, we see that it is the same as the length between helical repeats in the filament (seeFigures 17-5 and 17-28a), so myosin V stepsbetweenequivalent binding sites as it walks down one side of an acin filament. Myosin V has presumably evolved to take large steps the size of the helical repeat of actin and to do this very processivelyso it rarely dissociatesfrom an actin filament. These are exactly 738

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We have akeady discussedhow myosins have head and neck 'We domains responsible for their motor properties. now come to the tail regions, which define the cargoes that myosins move. The function of many of the newly discovered classes of myosins found in metazoans is not yet known. Below, we give just two examples where we have a good idea of what myosins do. Our first example is skeletal muscle,which is where myosin II was discovered.In muscle, many myosin II heads joined together in bipolar filaments, each with a short duty cycle, work together to bring about contraction. Similarly organized contractile machineries function in the contraction of smooth muscle and in stress fibers and the contractile ring during cytokinesis. We then turn to the myosin V class, which has a long duty cycle to allow thesemyosins to transport cargoesover relatively long distanceswithout dissociatingfrom actin filaments.

Myosin Thick Filamentsand Actin Thin Filamentsin SkeletalMuscleSlidePastOne Another During Contraction Muscle cells have evolved to carry out one highly specialized function-contraction. Muscle contractions must occur quickly and repetitivelg and they must occur through long distancesand with enough force to move large loads. A typical skeletal muscle cell is cylindrical, large (140 mm in length and 10-50 trr,min width), and multinucleated (containing as many as 100 nuclei) (Figure 17-29a). Within the muscle cell are many myofibrils consisting of a regular repeating array of a specializedstructure called a sarcomere (Figure 77-Z9b). A

C E L LO R G A N T Z A T T OANN D M O V E M E N Tl : M T C R O F T L A M E N T S

17-29 Structureof the skeletalmusclesarcomere. < FIGURE of fibersmadeof bundles (a)Skeletal of muscle muscles consist which a bundleof myofibrils, cells.Eachcellcontains multinucleated called structures contractile of repeating of thousands consist in muscle (b)Electron of mousestriated micrograph sarcomeres. of theZ either side On sarcomere. one showing longitudinal section, of actinthin entirely I bands,composed disksarethe lightlystained extendfrombothsidesof theZ disk thinfilaments filamentsThese in theA thickfilaments myosin with thedark-stained to interdigitate filaments actin myosin and (c) of of the arrangement band. Diagram (b)courtesy of S P Dadoune ] in a sarcomere [Part

Muscles

B u n d l eo f m u s c l ef i b e r s

Multinucleated m u s c l ec e l l

Myofibril

Nuclei

Sarcomere

The thick filaments are composedof myosin II bipolar filaments, in which the heads on each half of the filament have opposite orientations(seeFigure 1'7-20a).The thin actin filaments are assembledwith their ( + ) endsembeddedin a densely staining structure known as the Z disk (Figute 1'7-29b),so that the rwo setsof actin filaments in a sarcomerehave opposite orientations (Figure 17-30). To understand how a muscle contracts, consider the interactions between one myosin head (among the hundreds in a thick filament) and a thin (actin) filament, as diagrammed in Figure 1'7-24'Duting these cyclical interactions, also called the cross-bridgecycle,the hydrolysis of ATP is coupled to the movement of a myosin head toward the Z disk, which correspondsto the (+ ) end of the thin filament. Becausethe thick filament is bipolar, the action of the myosin heads at opposite ends of the thick filament draws the thin filaments toward the center of the thick filament and therefore toward the center of the sarcomere(Figure 17-30). This movement shortens the sarcomereuntil the ends of the thick filaments abut the Z disk. Contraction of an intact muscle results from the activity of hundreds of myosin headson a single Relaxed Actin

i _ (c)

Z disk | band ----)(-

A band ---------------><-

Myosin

Actin

I Z disk | band -

Z disk Contracted

f

+nre,ca'?.

M y o s i nf i l a m e n t s Actin filaments

Actin filaments

sarcomere,which is about2 pm long in restingmuscle,shortens by about 70 percentof its length during contraction. EIectron microscopy and biochemical analysishave shown that each sarcomerecontains two major types of frlaments:tbick filaments,composedof myosin II, and thin filaments,containing actin and associatedproteins (Figure 17-29c).

modelof contractionin 17-30The sliding-filament A FIGURE andthinactin myosin thick of The arrangement striatedmuscle. In the stateisshownin thetop diagram. in the relaxed filaments fromthe presence of ATPandCa2*,the myosinheadsextending walktowardthe (+) endsof thethinfilaments thickfilaments at theZ disks(purple), areanchored thethinfilaments Because towardthe centerof filaments pulls actin the myosin of movement state,as itslengthin the contracted shortening the sarcomere, shownrnthe bottomdiagram M Y O 5 I N - P O W E R EM DO V E M E N T S

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thick filament, amplified by the hundreds of thick and thin filaments in a sarcomereand thousands of sarcomeresin a muscle fiber. \Wecan now seewhy myosin II is both nonprocessive and needsto have a short duty cycle (seeFigure 17-27): each head pulls a short distance on the actin filament and then lets go to allow other heads to pull, and so many heads working together allow the smooth contraction of the sarcomere. The heart is an amazing contractile organ-it contracts without interruption about 3 million times a year, or a fifth of a billion times in a lifetime. The musclecellsof the heart contain contractile machinery very similar to that of skeletal muscle except that they are mono- and bi-nucleated cells. In eachcell, the end sarcomeresinsert into structuresat the olasma membrane called intercalated disks, which link the cells into a contractile chain. Since heart muscle cells are only generated early in human life, they cannot be replacedin responseto damage,such as occurs during a heart attack. Many different mutations in proteins of the heart contractile machinery give rise to hyp ertrop h i c cardiomy opath ies-thickening of the heart wall muscle, which compromises its function. For example, many

mutations in other comoonents of includingactin,myosin light chains, and structural componentssuch as t

S k e l e t aM l u s c l el s S t r u c t u r e db y S t a b i l i z i n ga n d ScaffoldingProteins The structure of the sarcomereis maintained by a number of accessoryproteins (Figure 17-31). The actin filaments are stabilized on their (+) ends by CapZ and on their (-) ends by tropomodulin. A giant protein known as nebulin extends along the thin actin filament all the way from the Z disk to tropomodulin, to which it binds. Nebulin consistsof repeating domains that bind ro the actin in the filament, and it is believed that the number of actin binding repeats, and therefore the length of nebulin, determinesthe length of the thin filaments. Another giant protein, called titin (becauseitis so large), has its head associatedwith the Z disk and extends to the middle of the thick filament, where another titin molecule extends to the subsequentZ disk. Titin is believedto be an elasticmolecule that holds the thick filaments in the middle of the sarcomere and also prevents overstretching to ensure that the thick filaments remain interdigitated between the thin filaments.

Contractionof SkeletalMusclels Regulatedby Ca2*and Actin-Bindingproteins Like many cellular processes,skeletalmuscle contraction is initiated by an increasein the cytosolic Ca2* concentration.As described in Chapter 11, the Ca2* concentration of the cytosol is normally kept low, below 0.1 ir.M. In skeletalmusclecells,a low 740

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A FIGURE 17-31 Accessoryproteinsfound in skeletalmuscle. Tostabilize the actinfilaments, CapZcapsthe(+) endof thethin filaments at theZ disk,whereas tropomodulin capsthe (-) end The giantproteintitinextends throughthethickfilaments andattaches to theZ disk.Nebulin bindsactinsubunits anddetermines the lenqth o f t h et h i nf i l a m e n t . cytosolic Ca2* level is maintained primarily by a unique Ca2* AT?asethat continually pumps Ca2* ions from the cytosol containing the myofibrils into the sarcoplasmicreticulum (SR), a specializedendoplasmic reticulum of the muscle cells (Figure 1,7-32).This activiryestablishes a reservoirof Ca2* in the SR. The arrival of a nerve impulse (or action potential; see Chapter 23) at a neuromuscular junction triggers an action potential in the muscle-cellplasma membrane (also known as the sarcolemma). The action potential travels down invaginations of the plasma membrane known as transuerse tubules, which penetratethe cell to lie around each myofibril (Figure 17-32). The arrival of the action potential in the transversetubules stimulates the opening of voltage-gated Ca2* channelsin the SR membran., ard th. ensuing ..l.ur. of Caz* from the SR raisesthe cytosolic Ca2* concJntration in the myofibrils. This elevatedCa2* concentrarion induces a change in two accessoryproteins, tropomyosin and troponin, which are bound to the actin thin filaments and normally block myosin binding. The changein position of these proteins on the thin filaments in turn permits the myosinactin interactions and hence contraction. This type of regulation is very rapid and is known as thin filament regulation. Tropomyosin (TM) is a ropelike molecule, about 40 nm in length, that binds ro seven actin subunits in an actin fllament. TM moleculesare strung together head to tail, forming a continuous chain along each side of the actin thin filament (Figure 17-33a, b). Associatedwith each tropomyosin is /roponin (TN), a complex of three subunits, TN-l TN-I, and TN-C. Troponin-C is the calcium-binding subunit of troponin. TN-C controls the position of TM on the surfaceof an actin filament through the TN-I and TN-T subunits. Under the control of Ca2* and TN, TM can occupy rwo positions on a thin filament-switching from a state of muscle relaxation to contraction. In the absenceof Ca2* (the relaxed state), TM blocks myosin's interaction with F-actin and the

C E L Lo R G A N r z A T r oANN D M o v E M E N Tr : M T G R o F T L A M E N T S

(a) Myofibrils

Nucleus

S a r c o o l a s m irce t i c u l u m

Sarcolemma reticulumregulatesthe 17-32The sarcoplasmic A FfGURE levelof free Ca2*in myofibrils.(a)Whena nerveimpulse istransmitted downa cell,the actionpotential a muscle stimulates withthe plasma whichiscontinuous tubule(yellow), transverse (sarcolemma), of Ca2*fromthe leading to release membrane

(b)Thin-section intothe myofibrils reticulum sarcoplasmic adjacent showingthe intimate muscle, of skeletal micrograph electron (b) f iberslPart to the muscle reticulum of thesarcoplasmic relationship August Library & Video lmage ASCB C Franzini-Armstrong KR, fromPorter

muscle is relaxed. Binding of Ca2* ions to TN-C triggers movementof TM to a new site on the filament,therebyexposing the myosin-bindingsiteson actin (Figure17-33b).Thus, at Ca2* concentrationsgreaterthan 1 pM, the inhibition exerted bv the TM-TN complex is relievedand contractionoccurs'The iat*-d.pe.rd.n, .y.ling between relaxation and contraction statesin skeletalmuscleis summarizedin Figure 17-33c.

cle cells contain severaltypes of related contractile bundles composedof actin and myosin II filaments' which are similar to skeletalmusclefibers but much lesswell organized.Moreover,they lack the troponin regulatory systemand are instead regulatedby myosin phosphorylation,as we will discusslater' In epithelial cells, contractile bundles are most commonly found as an adherensbelt, which encirclesthe inner surfaceof the cell at the level of the adherensiunction (seeFigure 1'7-4a), and are important in maintaining the integrity of the epithelium. Stressfibers, which are seenalong the lower surfacesof cells cultured on artificial (glassor plastic) surfacesor in extracellular matrices, are a second type of contractile bundle (see Figure 17-4a, c) important in cell adhesion, especiallyon

A c t i n a n d M y o s i nl l F o r mC o n t r a c t i l eB u n d l e s i n N o n m u s c l eC e l l s In skeletal muscle, actin thin filaments and myosin II thick filaments can assembleinto contractile structures.Nonmus(a)

a s c bo r g l u? / p 4 0 41c o l l1 , 8 3: l at: http://cellimage 2 0 0 6 : F N Dl -4 A v a i l a b l e

(b)

(c) Skeletalmuscle

Contraction Actin.TM.TN-Ca2+

T r o p o n i n( T N ) 4g1;nollvl oTN Relaxation

Tropomyosin (TM)

thin-filamentregulationof 17-33Ca2+-dependent FIGURE (a)Modelof thetropomyosinskeletalmusclecontraction. isa clublike on a thinfrlamentTroponin complex troponinregulatory tropomyosin protern that isboundto the longa-helical complex reconstructions electron-microscopic molecule(b)Three-dimensional helix(yellow) on a thinfilamentfromscallop of thetropomyosin in the relaxed state(/eft)shiftsto a new muscleTropomyosin

(nElht) whenthe position(arrow)in the stateinducingcontraction myosinexposes movement This increases Ca2*concentration isnot shownin this bindingsites(red)on actin.(Troponin in bothstates') boundto tropomyosin but remalns representation by Ca2* contraction muscle of skeletal (c)Summary of the regulation P and R Craig, (b) Lehman, W from bindingto troponin[Part adapted Vibert P of l 123:313;courtesy 1993,Nature Vibert, M Y O S I N - P O W E R EM DO V E M E N T S

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Contractile

Chromosome

flng

A EXPERIMENTAL FTGURE 17-34 Fluorescentantibodies reveal the localization of myosinI and myosinll duringcytokinesis. (a)Diagram of a cellgoingthroughcytokinesis, showing the mitotic (microtubules spindle green,chromosomes blue)andthecontractile ringwithactinfilaments (red)(b)Fluorescence micrograph of a

Dictyostelium amebaduringcytokinesis reveals that myosinll (red)is concentrated in the cleavage furrow,whereasmyosin| (green)is localized at the polesof thecell Thecellwasstained withantibodies specific for myosin I andmyosin ll,witheachantibody preparation linkedto a differentfluorescent dye [Courtesy of y Fukui ]

deformablesubstrates.The ends of stressfibers terminate ar integrin-containing focal adhesions,special structures that aftach a cell to the underlyingsubstratum(seeFigure 17-39). Circumferential belts and stressfibers contain severalproteins found in the contractile apparatusof smooth muscleand exhibit some organizationalfeaturesresemblingthose of muscle sarcomeres.A third type of contractile bundle, referredto as a contractile ring, is a transient structure that assemblesat rhe eauator of a dividing cell, encircling the cell midway berween rhe poles of the mitotic spindle(FigureI7-34a). As division of the cltoplasm (cytokinesis)proceeds,the diameter of the contractile ring decreases, and so the cell is pinched into two parts by a deepeningcleavagefurrow. Dividing cellsstainedwith antibodies againstmyosin I and myosin II show thar myosin II is localized to the contractile ring, whereas myosin I is at the distal regions,where it links the actin cortex to the plasmamembrane (Figure17-34b).This localizationindicatesthat myosin II, but not myosin I, takes part in cytokinesis. Cells in which the myosin II gene has been deletedare unable to undergo cytokinesis.Instead, thesecells form a multinucleated syncytium becausecytokinesis,but not nuclear division, is inhibited.

the contraction-inducing state in the presenceof Ca2* and the relaxed state in its absence.In contrast, smooth muscle contraction is regulated by the cycling of myosin II between on and off states.Myosin II cycling, and thus contraction o{ smooth muscle and nonmusclecells, is regulatedin response to many extracellularsignalingmolecules. Contraction of vertebratesmooth muscleis regulatedprimarily by a pathway in which the myosin regulatory light chain (LC) associatedwith the myosin II neck domain (see Figure 17 -20b) undergoesphosphorylation and dephosphorylation. \fhen the regulatorylight chain is not phosphorylated, the myosin II ATPasecycle is inactive. The smooth muscle contractswhen the regulatoryLC is phosphorylatedby the en-

M y o s i n - D e p e n d e nMt e c h a n i s m R s egulate C o n t r a c t i o ni n S m o o t hM u s c l ea n d N o n m u s c l eC e l l s

containslarge, loosely alignedconrractilebundlesthar resem_ ble the contracile bundles in epithelial cells. The contractile

742

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Fig^ure3-31). Calcium first binds to calmodulin, and the Ca'* lcalmodulin complex then binds to myosin LC kinase and activatesit. This mode of regulationrelieson the diffusion of Caz* over greaterdistancesthan in sarcomeresand on the action of protein kinases,so contraction is much slower in smooth musclethan in skeletalmuscle.Becausethis regulation involves myosin, it is known as thick filament regulation. The role of activated myosin LC kinase can be demonstrated by microinjecting a kinase inhibitor into smooth muscle cells. Even though the inhibitor does not block the rise in the cytosolic Ca2* level that follows the arrival of a nerve impulse, injected cells cannot contract. Unlike skeletal muscle, which is stimulated to contract solely by nerve impulses, smooth muscle cells and nonmuscle cells are regulated by many types of external signals in addition to nervous stimuli. For example, norepinephrine, angiotensin,endothelin, histamine, and other signaling molecules can modulate or induce the contraction of smooth muscle or elicit changesin the shape and adhesion of nonmuscle cells by triggering various signal-transduction

C E L Lo R G A N r z A T r o AN N D M o v E M E N rT: M T G R o F T L A M E N T s

Gontraction

ca2*I

s MLC kinase+ CaM MLC kinase-CaM-Ca2+s Ca2'I (inactive) (active)

MLC phosphatase

Relaxation for mechanism 17-35 Myosinphosphorylation A FIGURE smooth regulatingsmooth musclecontraction.In vertebrate lightchain(LC)by phosphorylation of the myosinregulatory muscle, At Ca2* contraction. activates myosinLCkinase Ca2*-dependent

anda is inactive, <10-6M, the myosinLCkinase concentrations on Ca2*for activity, whichisnot dependent LCphosphatase, myosin relaxation' muscle LC,causing the myosin dephosphorylates

pathways. Someof thesepathways lead to an increasein the cytosolic Ca2* level; as previously described,this increase can stimulate myosin activity by activating myosin LC kinase (see Figure 17-35). As we will discuss beloq other pathways activate Rho kinase, which is also able to activate myosin activity by phosphorylating the regulatory light chain, although in a Ca2*-independentmanner.

filaments. In the next chapter we discusshow they can work together with microtubule motors to bring about transport of organelles.Although not a lot is known about their functions in mammalian cells, myosin V motors are not unimportant: defectsin a specificmyosin V protein can causesesuch as seizures(seeFigure 17-22)' vere diseases, Much more is known about myosin V motors in more

VesiclesAre CarriedAlong Myosin-V-Bound Actin Filaments In contrast to the contractile functions of myosin II filaments, the myosin V family of proteins are the most processive myosin motors known and transport car9o down actin

llil+ Animation:Movementof MultipleCargoesby MyosinV in Yeast 17-36 Myosin Vs carry many different cargoesin < FIGURE (usedin cerevisiae budding yeast.(a)TheyeastSaccharomyces Secretory grows budding. by wine) and beer, makingbread, to aboutthe intothe bud,whichswells aretransported vesicles and sizeof the mothercell.Thecellsthen9o throughcytokinesis showing bud (b) of a medium-sized eachdivideagain Diagram (5V)down actincables vesicles how myosinVstransportsecretory (purple) budtip andbud at the located formins by nucleated sucnas organelles, Vsarealsousedto segregate neck Myosins peroxisomes, (theyeastequivalent of a lysosome), thevacuole andeven (ER), network(TGN), trans-Golgi reticulum endoplasmic of end the binds V also Myosin bud into the mRNAs selected for in preparation to orientthe nucleus microtubules cytoplasmic CellBiol' Ann'Rev' el al,2OO4, fromD Pruyne mitosis.lAdapted

Bud

'.:

\.;;= (b)

20:559 l

(+ Vacuole

M O V E ME N T S MYOSN I .POWERED

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Moving cytoplasm

Moving cytoplasm

ER

(--

{+}

Nonmoving cortical cytoplasm { '

P l a s m am e m b r a n e

Actin filaments Chloroplast

C e l lw a l l

A FIGURE 17-37Cytoplasmic streamingin cylindricalgiant algae. (a)Thecenterof a Nitella cellisfilledwith a singlelargewater-filled vacuole, whichissurrounded bya layerof moving (blue cytoplasm arrows). A nonmoving layerof cortical cytoplasm filledwith chloroplasts liesjustundertheplasma (enlarged membrane in bottomfigure). On theinnersideof thislayerarebundles of stationary (red), actinfilaments alloriented with thesamepolarityA motorprotein(blue), a plant

myosin V,carries partsof theendoplasmic (ER) reticulum alongtheactin filamentsThemovement of the ERnetworkpropels theenttrevtscous cytoplasm, including organelles thatareenmeshed in theERnetwork. (b)Electron micrograph of thecortical cytoplasm showinga largevesicle connected to an underlying bundleof actinfilamentsThisvesicle, which ispartof theERnetwork,contacts thestationary actinfilaments and movesalongthembya myosinmotorproteinlpart(b)fromB Kachar.]

all the organelleshave to be distributed betweenthe mother and daughter cells. RemarkablS myosin Vs in yeast provide the transport system for segregationof many organelles, includingperoxisomes,lysosomes(alsoknown as vacuoles). endoplasmicreticulum, and the trans-Golgi network and even transport the ends of microtubules and some specific messengerRNAs into the bud (Figure I7-36b).Ifhereas budding yeastusesmyosin V and polari zed actinfilaments in the transport of many organellei, animal cells, which are much larger,employ microtubules and their morors ro rransport many of theseorganellesover relatively long distances. We discussthesetransport mechanismsin the next chapter. Perhapsthe most dramatic use of myosin Vs is seenin the giant green algae, such as Nitella and Chara. In these

This gradientin the rate of flow is most easilyexplainedif the motor generatingthe flow lies at the membrane. In electron micrographs,bundles of actin filaments can be seen aligned along the length of the cell, lying acrosschloroplastsembedded at the membrane.Attached to the actin bundlesare vesiclesof the ER network. The bulk cytosol is propelled by myosin attachedto parts of the ER adjacentto the actin filaments.The flow rare of the cytosolinNitella is at least 15 times as fast as the movementproduced by any other known myosin.

_Closeinspectionof objectscaught in the flowing cytosol, such as the endoplasmicreticulum (ER) and other membrane_ bounded vesicles,shows that the velocity of streaming increasesfrom the cell center(zerovelocity)to the cell periphery. 744

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Myosin-Powered Movements In skeletalmuscle,contractilemyofibrils are composedof ousands of repeating units called sarcomeres.Each sarcomereconsistsof two interdigitatingfilament types:myosln thick filamentsand actin thin filaments (seeFigure 17-29). Skeletalmusclecontractioninvolvesthe ATp-dependent iding of myosin thick filaments along acin thin filaments to shorten the sarcomereand hence the myofibril (seeFigure 17-30). r The ends of the actin thin filaments in skeletalmuscleare stabilizedby CapZ at the (+) end and by tropomodulin at

c E L L o R G A N t z A T t o NA N D M o v E M E N TI M I C R O F I L AEMN T S

Migration of FishKeratinocyte #' viU"o:Mechanisms of a FishKeratinocyte in the Lamellipodium Video:Actin Filaments with begins 17-38Stepsin cellmovement.Movement > FIGURE edgeof fromthe leading of oneor morelamellipodia the extension by focal adhereto thesubstratum thecell(n); somelamellipodia (El). in the cell bodyflows bulk of the cytoplasm Then the adhesions at the rearof thecell(B) Thetrailing forwarddueto contraction untilthetail attached to thesubstratum edgeof thecellremains intothe cellbodyDuringthis andretracts detaches eventually membrane cycleinternalizes cycle, the endocytic cytoskeleton-based to thefront them cell and transports the rear of the integrins at and (4) for reusein makinqnewadhesions of the cell(arrows)

Focal adhesion

Directionof movement .€

Extension

I

Lamellipodium

@ Adrresion New adhesion

the (-) end. Two large proteins, nebulin associatedwith the thin filaments and titin with the thick filaments, also contribute to skeletalmuscle organization. r Skeletal muscle contraction is subject to thin filament regulation. At low levels of free Ca", the muscle is relaxed and tropomyosin blocks the interactionof myosin and F-actin.At elevatedlevelsof freeCaz* ,the troponin complex associatedwith tropomyosin binds Ca'* and moves the tropomyosin to uncover the myosin-binding siteson actin, allowing contraction(seeFigure 1'7-33). r Smooth and nonmusclecells have contractile bundles of actin and myosin filaments, with a similar organization to skeletalmuscle but lesswell ordered. r Contractile bundles are subject to thick filament regulation. A myosin light chain is phosphorylated by myosin light-chain kinase, which activates myosin and hence induces contraction. The myosin light-chain kinase is activated by binding Ca2*-calmodulin when the free Ca2* concentration rises (seeFigure 17-35). r Myosin V transports cargo by walking processively along actin filaments.

CellMigration:Signaling and Chemotaxis

E

Translocation

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and endocyticrecycling De-adhesion

Cellbody movement+

Old adhesion

the slow but constant migration of endothelial cells that Iine the blood vessels.The inappropriate migration of cancer cells away from their normal tissueresults in metastasis' Cell migration is initiated by the formation of a large, broad membrane protrusion at the leading edge of a cell' Video microscopy revealsthat a maior feature of this movement is the polymerization of actin at the membrane' Actin filaments ai the leading edge are rapidly cross-linked into bundles and networks in a protruding region, called a lamel-

'We

have now examined the different mechanismsused by cells to createmovement-from the assemblyof actin filaments and the formation of actin-filament bundles and networks to the contraction of bundlesof actin and myosin and the transport of organellesby myosin moleculesalong actin filaments.Some of thesemechanismsconstitute the major processeswhereby cells generatethe forces needed to migrate. Cell migration resultsfrom the coordinationof motions generatedin different parts of a cell, integratedwith a directedendocyticcycle. The study of cell migration is imponant to many fields of biology and medicine. For example, an essentialfeature of animal development is the migration of specific cells along predeterminedpaths. Epithelial cells in an adult animal migrate to heal a wound, and white blood cells migrate to sites of infection. Less obvious are the continual slow migration of intestinal epithelial cells along the villi in the intestineand

at how cells employ the various force-generatingprocesses surface.\7e also consider the role of sigto move ".ror, " in coordinating and integrating the actions naling pathways of the -ytoskeleton, a maior focus of current research'

Cell Migration CoordinatesForceGeneration w i t h C e l lA d h e s i o na n d M e m b r a n eR e c y c l i n g

AND CHEMOTAXIS ' C E L LM I G R A T I O Ns:I G N A L | N G

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Video:Actin Dynamicsin a MigratingFibroblast (b) Leading edge

Stressfibers

Stressfibers

L e a d i n ge d g e

FIGURE 17-39 Actin-basedstructuresinvolvedin cell locomotion.(a)Localization of actinin a fibroblast expresstng (b)Diagram GFP-actin. of theclasses of microfilaments involved in cell migration. Themeshwork of actinfilaments in the leading edge advances thecellforward.contractile fibersin thecellconexsqueeze thecellbodyforward,andstress fibersterminating in focaladhesions alsopullthebulkof thecellbodyup astherearadhesions arereleased.

(c)Thestructure of focaladhesions involves the attachment of the endsof stress fibersthroughintegrins to theunderlying extracellular matrixFocal adhesions alsocontain manysignaling molecules important for celllocomotion(d)Thedynamic actinmeshwork in the leading edgeisnucleated bytheArp2l3complex andemploys the samesetof factorsthatcontrolassembly anddisassemblv of actinfilaments in theLsten'a tail(seeFiqure17-17).

Membrane Extension The network of actin filaments at the leading edge is a type of cellular engine that pushesthe membrane forward by an actin-polymerization-basedmech-

New membrane at the leading edge is probably supplied by exocytosis of membrane internalized by endocytosisat the rear, as diagrammed in Figure 17-39, step E.

spectto the substratum,the front membraneis pushedout as the filaments elongate. SimilarlS the Listeria iride" on rhe polymerizing actin tail, which is also fixed within the cyto_ plasm, by nucleating actin assembly.Actin turnover, and thus treadmilling, is mediated, as it is in the comet tails of Listeria, by the action of profilin and cofilin (Figure 17-39d).

Cell-Substrate Adhesions rWhenthe membrane has been extended and the cytoskeleton has been assembled,the plasma membrane becomesfirmly attached to the substratum. Time-lapsemicroscopy shows that actin bundles in the leading edge become anchored to structures known as focal adhesions(Figure 17-39c). The attachmenr servestwo purposes:it preventsthe leading lamella from retracting, and it attaches the cell to the substratum, allowing the cell to move forward. Given the importance of focal adhesionsand their regulation during cell locomotion, it is nor surprising that they have been found to be very rich in moieculei

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involved in signal-transduction pathways. Focal adhesions are discussedin more detail in Chapter 19' when we discuss c e l l - m a t r i xi n t e r a c t i o n s . The membrane proteins involved in attachments are called integrins, the cell-adhesionmoleculesthat mediate most cell-matrix interactions. These proteins have an external domain that binds to specific components of the extracellular matrix, such as fibronectin and collagen,and a cytoplasmic domain that links them to the actin cytoskeleton (Figure L7-39 and Chapter 19). The cell makes attachments at the front, and as the cell migrates forward, the adhesions eventually assumepositions toward the back. \7hen they reach the back of the cell, the integrins are internalized by endocytosisand transported, using both the microfilament and microtubule cytoskeletons(Chapter 18) to the front of the cell to make new adhesions(seeFigure 1'7-38,step 4). This cycle of adhesion molecules in a migrating cell resembles the way a tank usesits treads to move forward. Cell-Body Translocation After the forward attachments have been made. the bulk contents of the cell body are translocated forward (seeFigure 1'7-38)' It is believed that the nucleus and the other organelles embedded in the cytoskeletonare moved forward by myosin-dependentcortical contraction in the rear part of the cell, like squeezingthe lower half of a tube of toothpaste. Consistent with this model, myosin II is localized to the rear cell cortex. Breaking Cell Attachments Finally, in the last step of the focal adhesionsat the rear of movement (de-adhesion), the cell are broken, the integrins recycled,and the freed tail brought forward. In the light microscope,the tail is seento "snap" loosefrom its connections-perhapsby the contraction of stressfibers in the tail or by elastic tension-and it sometimes leaves a little bit of its membrane behind, still firmly attached to the substratum. The ability of a cell to move correspondsto a balancebetween the mechanical forces generatedby the cytoskeleton and the resistingforces generatedby cell adhesions.Cells cannot move if they are either too strongly attached or not attached to a surface.This relation can be demonstratedby measuringthe rate of movementin cellsthat expressvarying show that the fastest levelsof integrins.Suchmeasurements migration occurs at an intermediatelevel of adhesion,with the rate of movement falling off at high and low levels of adhesion.Cell locomotion thus resultsfrom traction forces exertedby the cell on the underlyingsubstratum.

The SmallGTP-BindingProteinsCdc42,Rac,and R h o C o n t r o lA c t i n O r g a n i z a t i o n A striking feature of a moving cell is its polarity: a cell has a front and a back. When a cell makes a turn' a new leading edgeforms in the new direction. If theseextensionsformed in all directions at once, the cell would be unable to pick a new direction of movement. To sustain movement in a particular direction, a cell requires signals to coordinate events at the front of the cell with eventsat the back and, indeed,signalstcr

tell the cell where its front is. Understandinghow this coordination occurs emergedfrom studieswith growth factors' Growth factors, such as epidermal growth factor (EGF) and platelet-derivedgrowth factor (PDGF), bind to specific cell-surfacereceptors (Chapter 1'6) and stimulate cells to move and then to divide. For example,in a wound' blood platelets becomeactivated by being exposedto collagen in the extracellular matrix at the wound edge,which helpsthe blood to clot'

tists knew that growth factors bind to very specific receptors on the cell surface and induce a signal-transduction pathway on the inner surfaceof the plasmamembrane(Chapter15)' but how that linked up to the actin machinery was mysterious' The scientiststhen found that the signal-transductionpathway activates Rac, a member of the small GTPase superfamily of Rasrelatedproteins(Chapter15)' Rac is one memberof a family of proteins that regulate microfilament organizations; tvvo others ire Cdc42 and Rho. Unfortunatel5 due to the history of their discovery,this family of proteins also has been collectively ';Rho proteins," of which Cdc42, Rac, and Rho are named members.To understandhow theseproteinswork' we have to first recall the way small GTP-bindingproteinsfunction' Like all small GTPasesof the Ras superfamily,Cdc42, Rac, and Rho act as molecular switches,inactive in the GDP-bound stateand active in the GTP-bound state (Figure 17-40)' In their GDP-bound state,they exist free in the cytoplasm in an inactive

E x t r a c e l l u l asri g n a l Receptor Exterior

are molecular 17-40The Rhofamilyof smallGTPases FIGURE existin the proteins.Rhoproteins switchesregulatedby accessory as GDI protein known a with boundformcomplexed Rho-GDP themin an whichretains displacemeinhibitor), (guanine nucleotide pathways signaling Me brane-bound statein thecytosol. inactive

and A Hall,2002, Nature42O6791 lAdaptedfrom S Etienne-Manneville AND CHEMOTAXIS t C E L ' ,M I G R A T I O NS: I G N A L I N G

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Control

D o m i n a n ta c t i v e Rho

D o m i n a n ta c t i v e Rac

D o m i n a n ta c t i v e Cdc42

EXPERIMENTAL FTGURE 17-41Dominant-active Rac,Rho, and Cdc42inducedifferent actin-containingstructures.Tolook at theeffectsof constitutively activeRac,Rho,andCdc42,growthfactor-starved fibroblasts weremicroinjected with plasmros ro express dominant-active versions of thethreeproteins. Thecellswerethen treatedwithfluorescent phalloidin, whichstains filamentous actin Dominant-active Racinduces theformation of peripheral membrane ruffles,whereas dominant-active Rhoinduces abundant contractiie stress fibersanddominant activeCdc42induces f ilopodia[From A

transductionpathway, so one can now assesswhat processes are blocked. Cdc42, Rac, and Rho were implicatedin regulationof microfilament organizationsbecauseintroduction of dominantactive mutants had dramatic effects on the actin cytoskeleton, even in the absenceof growth factors. It was discoveredthat dominant-activeCdc42 resultedin the appearanceof filopodia, dominant-active Rac resulted in the appearanceof -.-b.".r. ruffles, and dominant-active Rho resulted in the formation of stressfibers that then contracted (Figure 17-41). How can one tell if dominant-active Rac and growth factor stimulation, both of which stimulate membrane ruffle formation, operate in the same signal-transductionpathway? If growth factor stimulation leads to Rac activation, introduction of a dominant-negativeRac protein into a cell should block the ability of a growth factor to induce membrane ruffling. This is preciselywhat is found. Using this and many other biochemical strategies,scienristshave identified signaling pathways involvingCdc42, Rac, and Rho (Figure 17-42). Someof the pathways thar theseproteins regulatecontain proteins we are familiar with. Thus activation of Cdc42 stimulates actin assemblyby Arp2/3 through activation of 'JfASp, resulting in the formation of filopodia. Activation of Rac also induces Arp2/3 but through the I7AVE complex, leading to the assemblyof branched actin filaments in the leading edge (Figure 17-42). Activation of Rho has at leasttwo effects:it can activateassemblyof unbranched F-actin through a formin pathway or induce activation of nonmuscle myosin II by catalyzing, through a Rho-activatedprotein kinase, the phosphorylation of the myosin light chain and the phosphorylation and inhibition of the myosin light-chainphosphatase.Both actionsof Rho

Hall, 1998, Science279 509-514 l

form bound to a protein known as guanine nucleotide dis_ placementinhibitor (GDI). Growth factors can bind and acti-

Cell Migration Involvesthe Coordinate Regulationof Cdc42,Rac,and Rho How do each of these small GTp-binding prorelns con_ tribute to the regulation of cell migration? To answer this

is hvdrolyzed to GDp, which is stimulated by specific !]n GTPase-activating proteins (GAps).An important approachto unravelingthe functionsofRho proteinshas beento introduce into cells mutant proteins that areeither locked in the active_ Rho-GTP-state or in the inactive-Rho-GDp-stare. A mu_ tant small GTPasethat is locked in the activestateis said to be

a needle to remove a swath of cells to generatea ,,wound,'

Using this system,researchershave introduced dominant_ negativeRac into cells on the wound edge and asked how it 748

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to bringabout with effectors whichtheninteract proteins, asindicated changes cytoskeletal

affects the ability of the cells to migrate and fill the wound' SinceRac is neededfor activation of the Arp2l3 complex to form the lamellipodium, it is not surprisingthat the cells failed to form this structureand did not migrate' and so the wound did not close (Figure 1.7-43c).A very interestingresult is obtained when dominant-negativeCdc42 is introMove into wound Scratch C o n f l u e n tc e l l s duced into the cells at the wound edge:they can form a leading edge but do not orient in the correct direction-in fact, (c) they try to migrate in random directions. This suggeststhat D o m i n a n t n e g a t i v eR a c Cdc42 is critical for regulating the overall polarity of the o cell. Studiesfrom yeast (where Cdc42 was first described), D o m i n a n t n e g a t i v eC d c 4 2 c wounded-cellmonolayers,epithelialcells, and neurons reE D o m i n a n t n e g a t i v eR h o n veal that Cdc42 is a master regulator of polarity in many difControl ferent systems.Part of this regulationin animalsinvolvesits 25 50 75 100 binding to its effector,Par-6,a polarity protein that funcf woundclosure Percent tions in nematodes(whereit was first discovered),neurons' m and epithelialcells. Studiessuch as thesesuggesta generalmodel of how cell migration is controlled (Figure 1'7-44)'Signalsfrom the en17-43ThewoundedFIGURE a EXPERIMENTAL r,ironment are transmitted to Cdc42, which orients the cell. cellmonolayerassaycanbe usedto dissectsignaling The oriented cell has high Rac activity at the front to induce layerof cells pathwaysin directedcellmovement.(a)A confluent the formation of the leading edge;Rho activity is high in the a swathof aboutthreecellswideto generate to remove isscratched rear to assemblecontractile structures and activate the a freecellborderThecellsdetectthefreespaceandnewlyexposed to It is important machinery. contractile myosin-Il-based matrixandovera periodof hoursfillthe area(b) extracellular and3 hours levdifferent 5 minutes have cell can of the regions different that notice of actinin a woundedmonolayer Localization area(c) wounded the into conare migrated regulators have these Rho, so cells Rac, or the Cdc42, active els of afterscratching; Cdc42,Rac,andRhointo dominant-negative Effectof introducing trolled locally within the cell. Part of this spatial regulation (b)and(c)from occursbecausesomesmall G proteinscan work antagonisti- cellsat thewoundedge;allaffectwoundclosureIParts 1235-12441 144 Biol J Cell C D, Nooesand A Hall, 1999, cally. For example,activeRho can stimulatepathwaysthat : I G N A L I N GA N D C H E M O T A X I S C E L LM I G R A T I O N S

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Back: Rho activation L e a d i n gt o m y o s i nl l activation

Cdc42 activation at the front

signal-transductionpathways used in chemotaxis have been conservedbetweenDictyostelium amebasand human leukocytesdespitealmost a billion yearsof evolution.

ChemotacticGradientsInduceAltered PhosphoinositideLevelsBetweenthe Front a n d B a c ko f a C e l l

Contractionof mvosin ll

filamentsin bothstress fibersandcellcortex A FIGURE 17-rl4Contributionof Cdc42,Rac,and Rhoto cell movement.Theoverall polarity of a migrating celliscontrolled by Cdc42,whichisactivated at thefrontof a cell.Cdc42activation leadsto activeRacin thefrontof the cell,whichqenerates the leading edge,andactiveRhoat the backof thec;ll,whichcauses myosinll activation andcontraction ActiveRhoinhibits Rac activation, ensuring theasymmetry of thetwo activeG-proteins

lead to the inactivation of Rac. This might help ensure no leading edgestructuresform at the rear of the cell.

M i g r a t i n gC e l l sA r e S t e e r e db y C h e m o t a c t iM c olecules Under certain conditions, extracellular chemical cues guide the locomotion of a cell in a particular direction. In some cases,the movementis guided by insolublemoleculesin the underlying substratum, as in the wound-healing assay described above. In other cases,the cell sensessoluble molecules and follows them, along a concentration gradient, to their source-a processknown as chemotaxis.For example, leukocytes(white blood cells)are guided toward an infeciion by a tripeptide secretedby many bacterial cells. In another example, during the development of skeletal muscle, a secreted protein signal called scatter factor guides the migra_ tion of myoblasts to the proper locations in limb buds (Chapter 22). One of the best-studiedexamples of chemo_ taxis is the migration of Dictyosteliwm amebasalong an in_ creasrngconcentration of cAMp, which is an extracellular chemotacticagent in this organism (Figure 17-45a).Follow_ ing cAMP to its source,the amebasaggregateinto a slug and then differentiate inro a fruiting body. Despite the variety of different chemotacticmolecules_ sugars, peptides, cell metabolites,cell-wall or membrane lipids-they all work through a common and familiar mecha-

the front and back of the cell is sufficient to induce directed cell migration. Equally amazingis the finding that the internal 750

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To investigatehowDictyostelium amebassensea chemotactic gradient, investigatorshave studied the cell-surfacereceptors for extracellularcAMP and downstream signalingpathways in the expectationthat thesemust somehowsensethe concentration gradient. Before we discussthe details, let's consider how such a system might work. If a cell can sensea 2 percent differencein concentrationacrossits length, it is unlikely that simply activating actin assembly2 percent more at the front than at the back could lead to directed movement. Rather. there must be somemechanismthat amplifiesthis small external signal difference into a large internal biochemical difference.One way to do this would be for the cell to subtract the common signalfrom the front and back and only respond to a difference in signal. This is believed to be how it works. To try to understand this, investigatorshave looked at the concentrationof activecomponentsof the signalingpathway to seewhere the amplification occurs. Micrographs of cAMP receprorstagged with green fluorescentprotein (GFP) show that the receptorsare distributed uniformly on the surfaceof an ameba cell (Figure l7-45b): thereforean internal gradient must be establishedby another component of the signaling pathway. BecausecAMp receptors signal through trimeric G proteins (Chapter 16), a subunit of the trimeric G protein and other downstreamsignaling proteins were taggedwith GFP to look at their distributions. Fluorescencemicrographs show that the concentration of trimeric G proteinsis also rather uniform. Downstream of the trimeric G proteins is PI-3 kinase,an enzymethat phosphorylatesmembrane-boundinositol phospholipids(phosphoinositides), such as Pl4,S-biphosphate[PI(4,5)p2]to the signaling lipid Pl3,4,5-triphosphate[PI(3,4,5)p:] (seeFigure 16-29). RemarkablS the enzymePI-3 kinase is highly enrichedat the front of a migrating cell, as are its products.The phosphatase,

a bit more at the front than the back. This results in slightly higher levels of the signaling phospholipid at the front. The associationof the phosphatasePTEN with the membrane is very sensitiveto the level of the newly formed pI(3,4,5)p3_ so it is preferentially depleted from the front. Sinceit is less effective at dephosphorylating the pI(3,4,5)p3 at the front and more effective at dephosphorylating pI(3,4,5)p3 at the rear, a strong asymmetry of PI(3,4,5)p3results. Thus the phosphatasePTEN contributes to the background subtraction necessaryfor a cell to sensea shallow gradient of chemoattractant.

C E L Lo R G A N t z A T t o NA N D M o v E M E N T I M I C R O F I L A M ENTS

@' vid"o: Chemotaxisof a Single DictyosteliumCellto cAMP

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towardcAMP chemotaxis (green) cellsundergoing in Dictyostelium (red). myosin and actin of concentration the are shown Also which signal elevatedlevelsof signalingphosphoinositides, is generates Pl(3,4,5)P3, (c) which Pl3 kinase, The enzyme (a) cellsmigratetoward to the actin cytoskeleton. Dictyosteliurn the PTEN, whereas cells, of chemotaxing front the at enriched (a (/eft), pipet type of leukocyte) neutrophils human of cAMP and a the back at is enriched Pl(3,4,5)B phosphatase thathydrolyses a Met-Leu-Phe), migrate towarda pipetof fMLP(formylated at thefrontof the Pl(3,4,5)P3 resultin elevated Thesedistributions (right)ln the lowertwo peptideproducedby bacteria chemotactic (a)fromC polarity for movement lPart the signals which cells, panels andneutrophil Dictyostelium chemotaxing areindividual (c)fromM li1ima et al, Part 4-13 Biol 16 Cell Opin Curr 2004, Parent, years 800 million about similar, despite cellsthatlookremarkably 2002,Dev.Cell3.469-478 | of studies of results them (b)Summary separating of evolution pathways of signaling of components the concentration explorrng FIGURE17-45 Chemotaxis involves A EXPERIMENTAL

The differencein local PI(3,4,5)P3concentratronnow signals to the actin cytoskeleton to assemblea leading edgeat the front and contraction at the rear (Figure 1'7-45b), and the cell is on its way to the source of chemoattractant. This cell polarization is not stable in the absenceof the chemotactic gradient, so if the gradient changes,as might happen with a moving bacterium, the cell will also changeits direction and follow the sradient to its source.

In this chapter,we have seenthat cells have intricate mechanismsfor the spatial and temporal assembly,turnover' and attachment of microfilaments to membranes. Biochemical

C e l l M i g r a t i o n : S i g n a l i n ga n d C h e m o t a x i s r Cell migration involves the extension of an actin-rich leading edgeat the front of the cell, the formation of adhesive contacts that move backward with respectto the cell, and their subsequentrelease,combined with rear contraction to push the cell forward (seeFigure 17-38). r The assemblyand function of actin filamentsis controlled by signalingpathways through small GTP-binding proteins of the Rho family. Cdc42 regulatesoverall polarity and the formation of filopodia, Rac regulatesactin meshwork formation through the ArpZl3 complex, and Rho regulates both actin filament formation by formins as well as contraction through regulation of myosin II (seeFigure t7-42\' r Chemotaxis, the directed movement toward an attractant, involves signaling pathways that establishdifferences in phosphoinositidesbetween the front and rear of the cell, which in turn regulatethe actin cytoskeletonand direction of cell migration (seeFigure 17-45).

teractionsand the location of many of the key signalingpathways suggestthat rapid progresswill be made in this area' The protein inventories provided by genomic sequences havealso documentedthe largenumber of myosin families,yet the biochemical properties of many of these motors, or their biological functions, remain to be elucidated.Again, recent technical developments'including the ability to tag motors with fluorescent tracers such as GFP, or knocking down their

lease it at the destination-little is known about how these different eventsare coordinated or how thesetypes of myosinbasedmotors are returnedto pick up new cargo' P E R S P E C T I VFEO SRT H E F U T U R E

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Finally, although we have generally discussedmicrofila-

tissue-specific expressionof actin-bindingproteinsor myosins.

KeyTerms Arp2l3 complex723 capprngprotern722

microfilaments715 microtubules215

cnemotaxls,/.)(/ colrlrn / z I

mrcrovilli 716 motor protein 215

contractile bundles 741

myotlbnt /Jd

critical concentration, C,720

myosin head domain 233

cross-linking protein 728 duty ratio 737 l-actn / I / rrcpoola / 16

myosin LCkinase 742 myosin tarl domain 733

focal adhesionlfocal contact 7L5

profllin 721 sarcomere 730

Iormrn / 23 \r-actln / I /

sliding-filamentassay733 stressfibers 71 5 thick filaments 739

nucleation 719 potarlty / 16

GTPase superfamlly 747 integrin 747 intermediate filaments 71.l lamellipodium715 leading edge716

thin filaments 739 t h y m o s i n9 4 7 2 2 treadmrlling 721

Review the Concepts 7. Three systemsof cytoskeletalfilaments exist in mosr eukaryotic cells. Compare them in terms of composition, function, and structure. 2. Actin filamenrs have a defined polarity. \fhat is filament polarity?How is it generatedat the subunitlevel?How is filamenr polarity detectable? 3. In cells,actin filamentsform bundlesand./ornetworks. How do cells form such structures,and what specificallydetermines whether actin filamentswill form a bundle or a network? 4. Much of our undersrandingof acrin assemblyin the cell is derived from experiments using purified actin in vitro. \fhat techniquescan be used to study actin assemblyin vitro? Explain how each of theserechniquesworks. 5. The predominant forms of actin inside a cell are ATP-Gactin and ADP-F-actin. Explain how the interconversionof

6. Actin filaments at the leading edgeof a crawling cell are believedto undergo treadmilling. \il/hat is treadmilling, and what accountsfor this assemblybehavior? 7. Although purified actin can reversibly assemblein vitro, various actin-binding proteins regulatethe assemblyof actin filaments in the cell. Predict the effect on a cell's actin cytoskeleton if function-blocking antibodies against each of the following were independently microinjected into cells: profilin, thymosin-Ba, CapZ, and the Arp}l3 complex. 8. There are at least 20 different types of myosin. What propertiesdo all types share,and what makesthem different? 9. The ability of myosin to walk along an actin filament may be observedwith the aid of an appropriately equipped microscope.Describe how such assaysare typically performed. \Why is ATP required in theseassays?How can such assaysbe used to determine the direction of myosin movement or the force produced by myosin? 10. Contractile bundles occur in nonmuscle cells, although the structuresare lessorganizedthan the sarcomeresof muscle cells.rWhatis the purposeof nonmusclecontractile bundles? 11. How doesmyosin convert the chemicalenergyreleased by ATP hydrolysis into mechanicalwork? 12. Myosin II has a duty ratio of 10 percent,and its step size is about 5-15 nm. In contrast, myosin V has a much higher duty ratio (about 70 percent) and takes 36-nm steps as it walks down an actin filament. \fhat differences between myosin II and myosin V accountfor their different properties? How do the different srructuresand properties of myosin II and myosin V reflecttheir different functions in cells? 13. Contraction of both skeletal and smooth muscle is triggered by an increasein cytosolic Ca2*. Compare rhe mechanisms by which each type of muscle converts a rise in Ca2* rnto contraction. 14. Several types of cells utilize the actin cytoskeleton to power locomotion acrosssurfaces.How are different assemblies of actin filaments involved in locomotion? 15. To move in a specificdirection, migrating cellsmust utilize extracellular cuesto establishwhich portion of the cell will act as the front and which will act as the back. Describehow Gproteins appear to be involved in the signaling pathways used by migrating cells to determine direction of movement. 16. Cell motility has been described as like the motion of tank treads.At the leading edge,actin filaments form rapidly into bundles and networks that make protrusions and move the cell forward. At the rear,cell attachmentsare broken and the tail end of the cell is brought forward. N7hatprovides the traction for moving cells?How does cell-body translocarion happen? How are cell attachments releasedas cells move forward?

Analyze the Data Myosin V is an abundantnonmusclemyosin that is responsible for the transport of cargo such as organellesin many cell types. Structurally, it consistsof two identical polypeptide chains that dimerize to form a homodimer. The motor

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domains reside at the N-terminus of each chain and contain both ATP- and actin-binding sites.The rnotor domain is followed by a neck region containing six "IQ" motifs, each of which binds calmodulin, a Ca2*-binding protein. The neck domain is followed by a region capable of forming coiled coils, via which the two chains dimerize. The final 400 amino acid residuesform a globular tail domain (GTD), to which cargo binds. Myosin V would consumelarge amounts of ATP if its motor domain were always active, and a number of studies have been conducted to understand how this motor is regulated. a. The rate of ATP hydrolysis (i.e., ATP moleculeshydrolyzed per second per myosin V) was measuredin the presenceof increasingamounts of free Ca"'. The concentrai i o n o f c y t o s o l i cf r e eC a 2 ' i s n o r m a l l yl e s st h a n t 0 - 6 M b u t can be elevatedin localized areasof the cell and is often elevated in responseto a signaling event. \fhat do these data suggestabout myosin V regulation?

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References BraS D. 200t. Cell Mouements.Garland. Howard, J.20Ol. The Mechanicsof Motor Proteinsand the Cytoskeleton. Sinauer. Kreis, T., and R. Vale. 1'999.Guidebook to the Cytoskeletaland Motor Proteins.Oxford UniversityPress.

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Microfilamentsand ActinStructures Cooper,J. A., and D. A. Schafer.2000. Control of actin assembly and iisaisembly at filament ends.Curr- Opin. Cell Biol. 12:97-1.03. Holmes, K. C., D. Popp, W. Gebhard,and I7. Kabsch. 1990' Atomic model of the actin filament' Nature 347:4449 ' Kabsch.S7.,H. G. Mannherz, D. Suck,E. F. Pai, and K' C' Holmes. 1990. Atomic structureof the actin:DNaseI complex' Nature 347:3744. Pollard, T. D., L. Blanchoin,and R. D. Mullins. 2000' Molecular mechanismscontrolling actin filament dynamicsin nonmuscle cells.Ann. Reu.Biophys. Biomol. Struc- 29:545-576.

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Paavilainen,V. O., E. Bertling,S. Falck, and P.Lappalainen' 2004. Regulation of cytoskeletaldynamics by actin-monomer-binding proteins. TrendsCell Biol. 14:386-94. Theriot, J. A. 1'997.Acceleratingon a treadmill: ADF/cofilin promotesrapid actin filament turnover in the dynamic cytoskeleton' J. Cell Biol. \36:1t65-1168.

of ActinFilamentAssembly Mechanisms 05101520 lActinl,PM c. Next, the behavior of truncated myosin V' which lacks yust its C-terminal globular tail, was examined and compared to the behavior of intact myosin V. From this experiment, what can you deduce about the mechanism by which myosin V is regulated?

Gouin, E., M. D. Velch, and P. Cossart.2005. Actin-based motility of intiacellular pathogens.Curr. Opin. Microbiol' 8:35-45' Higgs, H. N. 2005. Formin proteins: a domain-basedapproach' TrendsB iochem. Sci.3O:342-35 3. Higgs, H. N., and T. D. Pollard. 2000. Regulationof actin fila-.nt .tii*otk formation through ARP2/3 complex: activation by a diversearray of proteins.Ann. Reu.Biochem. T0:61'9-676' Pruyne,D., et al. 2002 Role of formins in actin assembly:nucleation and barbedend association'Science29T:612-615' REFERENCES

753

Volkmann, N., et al. 2001. Structureof Arp2l3 complex in its activatedstateand in actin filament branch iunctions.Siience 293:2456-2459. \felch, M. D., and R. D. Mullins. 2002. Cellular control of actin nucleation.Annu. Reu.Cell Deu. Biol. tB:247-288. Ztgmond, S. H. 2004. Formin-inducednucleationof actin filaments.Curr. Opin. Cell Biol. 16:99-105. Organization of Actin-Based Cellular Structures . Bennett,V., and A. J. Baines.2001. Spectrinand ankyrin-based pathways:metazoaninventionsfor integratingcellsinto tissues. Physiological Reuiews 8l :1,35 3-1 392. . Bretscher,A., K. Edwards,and R. Fehon.2002. ERM proteins and merlin: integrators at the cell cortex. Nature Reu. Mot. Cett Biol.3:586-599. tr4cGough,A. 1998. F-actin-bindingproteins. Curr. Opin. Struc. Biol. 8:1,66-17 6. Stossel,T. P.,et al.2001. Filamins as inteqrarorsof cell mechanics and signalling.Nature Reu.Mol. Celt BioI 212y:l3g-145. Myosins: Actin-based Motor proteins Berg,J. S., B. C. Powell, and R. E. Cheney.2001. A millennial myosincensus.Mol. Biol. Cell 72:780-794. Mermall, V., P. L. Post,and M. S. Mooseker. 1998. Unconventional myosin in cell movement,membranetraffic, and signaltransduction. Science279:527-5 33. Rayment,l. 1996. The structural basisof the myosin ATpase a c t i v i r yJ.. B i o l . C h e n . 2 7 l : 1 5 8 5 0 - 1. 5 8 5 3 . . Tyska,M. J., and M. S. Mooseker.2003. Myosin-V motility: theseleverswere made for walking. Trends Cell Biot. 13:447451. Vale, R. D. 2003. The molecularmoror toolbox for inrracellular transport.Cell ll2:467480.

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Vale,R. D., and R. A. Milligan. 2000. The way thingsmove:looking under the hood of molecularmotor proteins.Science288:88-95. Myosin-Powered Movements BretscherA. 2003. Polarizedgrowth and organellesegregarion in yeast-the tracks, motors, and receptors. J. Cell Biol. 160:811-81.6. Clark, K. A., A. S. McElhinny M. C. Beckerle,and C. C. Gregorio.2002. Striatedmusclecytoarchitecture:an intricate web of form and function. Annu. Reu.Cell Deu. Biol. 18:637-706. Grazie4H. L., and S. Labeit. 2004.The giant protein titin: a major player in myocardialmechanics,signaling,and disease.Clrc. Res.94:284-295. Cell Migration: Signaling and Chemotaxis BorisS G. G., and T. M. Svitkina.2000. Actin machinery:pushing the envelope.Curr. Opin. Cell Biot. 12:104-112. 'Wennerberg. Burridge,K., and K. 2004. Rho and Rac take center stage.Cell lL6:167-179. S. 2004. Cdc42-the centreof polarity./. __Etienne-Manneville, Cell Sci.ll7 1291-1300. Etienne-Manneville,S., and A. Hall. 2002. Rho GTPasesin cell biology. Nature 420:629-63 5. Manahan, C. L., P.A. Iglesias,Y. Long, and P. N. Devreotes. 2004. Chemoattractantsignalingin Dictyostelium discoideum.Ann. Reu.Cell Deu. Biol.2O:223-253. _ Pollard, T. D., and G. G. Borisy.2003. Cellular motility driven by assemblyand disassemblyof actin filaments.Cell 112:453465. Ridley,A. J., et a|.2003. Cell migration: integratingsignals from the front to back. Science302:'1,704-1,709. Small, J. V., T. Strada, E. Vignal, and K. Ronner. 2002. The lamellipodium: where motility begins.Trends Cell Biol. 12:L12-120.

C E L LO R G A N T Z A T T O A N D M O V E M E N Tt : M t C R O F t L A M E N T 5

cLASStC

EXPERIMENT

17

1

LOOKING AT MUSCLE CONTRACTION H. Huxfey and J. Hanson, 1954, Nature 173:973-976

The contraction and relaxation of striated musclesallow us to perform all of our daily tasks. How does this happen? Scientistshave long looked to seehow muscle cells, containing many myofibrils, differ from other cells that cannot perform powerful movement. In 1,954, Jean Hanson and Hugh Huxley published their microscopy studies on muscle contraction, which demonstratedthe mechanismby which rt occurs.

Background The ability of musclesto perform work has long been a fascrnatrngprocess. Voluntary muscle contractron ls performed by striated muscles,which are named for their appearance when viewed under the microscope. By the 1950s, biologists studying the myofibrils in muscle cells had named many of the structures they could observe under the microscope. The myofibrils were seen to be made up of a repeatlng contractive unit, the sarcomere, that consists of two main regions calledthe A band, and the I band. The A band contains two darkly colored thick striations and one thin striation. The I band is made up primarily of light-colored striations, which are divided by a darkly colored line known as the Z disk. Although these structures had been characterized, their role in muscle contraction remained unclear. At the sametime, biochemistsalso tried to tackle this problem by looking for proteins that are more abundant in muscle cells than in non-muscle cells. They found muscles to contain Iarge amounts of the structural proteins actin and myosin in a complex with each other. Actin and myosin form polymers that can shorten when treated with adenosinetriphosphate (ATP).

With these observations in mind, Hanson and Huxley began their study of cross striations in muscle. In a few short years, they united the biochemical data with the microscopy observations and developeda model for muscle contraction that holds true today.

The Experiment Hanson and Huxley primarily used phase-contrast microscopy in their studiesof striatedmusclesthat they isoIated from rabbits. The technique alIowed them to obtain clear pictures of the sarcomereand to take careful measurementsof the A and the I bands. By treating the muscles with a variety of chemicals, then studying them under the phase-contrast microscope, they were able to successfullycombine biochemistry with microscopy to describe muscle structure as well as the mechanism of contraction. In their first set of studies,Hanson and Huxley employed chemicals that are known to specificallyextract either myosin or actin from myofibrils. First, they treated myofibrils with a chemical that specifically removes myosin from muscle.They used phase-contrastmicroscopy to compare untreated myofibrils to myosin-extracted myofibrils. In the untreated muscle, theY observedthe previously identified sarcomeric structure, including the darkly colored A band. lfhen they looked at the myosin-extracted cells, however, the darkly colored A band was not observed. Next, they extracted actin from the myosin-extracted muscle cells. When they extracted both myosin and actin from the myofibril, they could seeno identifiable structure to the cell under phase-contrastmicroscopy. From these experiments, they concludedthat myosin was located

primarily in the A band, whereas actin is found throughout the myofibril. 'Sfith a better understanding of the biochemical nature of muscle structures, Huxley and Hanson went on to study the mechanism of muscle contraction. They isolated individual myofibrils from muscle tissue and treated them with ATP, causing them to contract at a slow rate. Using this technique, they could take pictures of various stages of muscle contraction o b s e r v e d u s i n g p h a s e - c o n t r a s tm i croscopy. They could also mechanically induce stretching by manipulating the coverslip, which allowed them to also observethe relaxationprocess. With these techniques in hand, they examined how the structure of the myofibril changes during contraction and stretch. First, Huxley and Hanson treated myofibrils with AIP, then photographed the imagesthey observedunder phasecontrast microscopy. These pictures allowed them to measurethe lengthsof both the A band and the I band atvarious stagesof contraction' When they looked at myofibrils freely contracting' they noticed a consistentshortening of the lightly colored I band, whereasthe length of the A band remained constant (Figure 1). rifithin the A band, they observedthe formation of an increasingly dense area throughout the contractron. Next, the two scientists examined how the myofibril structure changes during a simulated muscle stretch. They stretched isolated myofibrils mounted on glass slides by manipulating the coverslip. They again Photographed phase-contrastmicroscopy imagesand measuredthe lengthsof the A and the I bands. During stretch the length of the I band increased,rather than shortened, as it had in contraction.

L O O K I N GA T M U S C L EC O N T R A C T I O N

755

Z disk

| band ______)(__

A band __________)(__ | band _

Stretched 120%

Relaxed 100%

Contracted 90%

s

Contracted 80%

1.Btt

andTheirBiological Significance 9:249l

A 1 . 5t t I o.3p

s

1 . 5l t 1 . 5l t I 0.0,u

Contracted 60%

Once again, the length of the A band remained unchanged. The dense zone that formed in the A band during contraction became less dense during stretch. From their observations,Hanson and Huxley developed a model for muscle contraction and stretch (Figure 1). In their model, the actin filaments in the I band are drawn up into the A during contraction, and thus the I band becomesshorter. This allows for increased interaction between the myosin located in the A band and the

< FIGURE 1 Schematic diagramof muscle contractionand stretchobservedby Hansonand (S),theA band of the sarcomere S 2.81t Huxley.Thelengths (A),andthe I band(l)weremeasured in muscle samples 4 l.stt I 1'3trt contracted 60 percent in lengthrelative to the relaxed muscle(bottom)or stretched to 120percent(top).The of the sarcomere, the I band,andtheA band S 2.31t lengths A 1.51t arenotedon the right.Noticethatfrom 120percent I 0.81t stretchto 60 percentcontraction the A banoooes notchangein length.However, the lengthof the I to 1 3 microns, andat 60 percent S 2.Ou bandcanstretch A 1.51t contraction, it disappears asthesarcomere shortens to I 0'51t the overall lengthof theA band.[Adapted fromJ Hanson andH E Huxley, 1955, SympSocExpBiolFibrous Proteins

A

actin filaments. As the muscle stretches, the actin filaments withdraw from the A band. From these data, Hanson and Huxley proposed that musclecontraction is driven by actin filaments moving in and out of a mass of stationary myosin filaments.

Discussion By combining microscopic observations with known biochemical treatments of muscle fibers, Hanson and Huxley were able to describethe bio-

chemical nature of muscle structures and outline a mechanism for muscle contraction. A large body of research continues to focus on understanding the processof muscle contraction. Scientists now know that muscles contract by ATP hydrolysis, driving a conformational change in myosin that allows it to pull on actin. Researchers are continuing to uncover the molecular details of this process,whereas the mechanismof contraction proposed by Hanson and Huxley remains in place.

CHAPTER

CELLORGANIZATION AND MOVEMENTII: AND MICROTUBULES INTERMEDIATE FILAMENTS (magenta), Newtlungcellin mitosis for centrosomes stained (green), (blue), microtubules chromosomes keratin intermediate (red)lCourtesy filaments o{A Khodjakor, fromNafure 4O8:423-424 (2ooo) l

types of filaments make up the animal-cell cyfhree toskeleton: microfilaments, microtubules, and interI I mediatefilaments.Why have thesethree distinct types of filaments evolved? It seems likely that their physical properties are suited to different functions. In Chapter 17 we described how actin filaments are often cross-linked into networks of bundles to form flexible and dynamic structures and to serve as tracks for the many different classesof myosin motors. Microtubules are stiff tubes that can exist as a single structure extending up to 20 pm in cells or as the bundled structuresas seenin cilia and flagella. A consequenceof their tubular design is the ability of microtubules to generate pulling and pushing forces without buckling, a property that allows single tubules to extend large distanceswithin a cell and bundles to slide past each other, as occurs in flagella and in the mitotic spindle. Microtubules' ability to extend long distancesin the cell, together with their intrinsic polarity, is exploited by microtubule-dependent motors, which use microtubules as tracks for long-range transport of organelles. Microtubules can be highly dynamic-being assembled and disassembledfrom their ends-providing the cell with the flexibility to reorganize microtubule organization as needed. In contrast to microfilaments and microtubules,intermediate filaments have great tensile strength and have evolved to withstand much larger stressesand strains. With properties akin to strong molecular ropes, they are ideally suited to endow both cells and tissueswith structural integrity and contribute to cellular organization. Intermediatefilaments do not have an intrinsic polaritv like

microfilaments and microtubules, so it is not surprising that there are no known motor proteins that use intermediate filaments as tracks. Although we discussmicrotubulesand intermediate filaments together in this chapter-and their localization in the cytoplasm can look superficially quite similar-we will see their dynamics and functions are very different. A summary of the similarities and differences among the three cytoskeletalsystemsis presentedin Figure 18-1.

OUTLINE 18.1

M i c r o t u b u l eS t r u c t u r ea n d O r g a n i z a t i o n

18.2 MicrotubuleDynamics 18.3

R e g u l a t i o no f M i c r o t u b u l eS t r u c t u r ea n d Dynamics

758 752

767

18.4

: icrotubule-Based K i n e s i na s n d D y n e i n sM Motor Proteins

18.5

: i c r o t u b u l e - B a s eSdu r f a c e C i l i aa n d F l a g e l l aM 777 Structures

18.6

Mitosis

18.7 lntermediateFilaments 18.8

781 791

Coordinationand Cooperationbetween CytoskeletalElements

757

(al

Microfilaments

Microtubules

IntermediateFilaments

ACtn DrndsAil-

o B - t u b u l i nb i n d G T P

l F s u b u n i t sd o n ' t bind a nucleotide

F o r m r i g i d g e l s ,n e t w o r k s , a n d l i n e a rb u n d l e s

R i g i da n d n o t e a s i l yb e n t

Great tensile strength

R e g u l a t e da s s e m b l yf r o m a l a r g en u m b e r o f l o c a t i o n s

R e g u l a t e da s s e m b l yf r o m a s m a l l n u m b e ro f l o c a t i o n s

Assembled onto pre-existingfilaments

H i g h l yd y n a m i c

H i g h l yd y n a m i c

J L e s sd y n a m i c

J Polarized

Polarized

Unpolarized

l Tracksfor myosins

Tracksfor kinesins and dyneins

C o n t r a c t i l em a c h i n e r va n d network at the cell cortex

O r g a n i z a t i o na n d l o n g - r a n g e t r a n s p o r to f o r g a n e l l e s

No motors

t

This chapter coversfour main topics. First, we discussthe structureand dynamicsof microtubulesand their motor proteins. Second, we examine how microtubules and their motors contribute to the movement of the specializedstructures cilia and flagella. Third, we discussthe role of microtubules in the mitotic spindle-a molecular machine that accurately segregatesduplicated chromosomes.Finally, we explore the roles of the different classesof intermediatefilaments that provide structure to the nuclear envelopeas well as strengthand organizationto cellsand tissues.Although we considermicrotubules,microfilaments,and intermediatefilaments individually, the three cytoskeletalsystemsdo not act independentlyof one another, and we consider some examples of this interdependence in the last sectionof the chapter.

C e l la n d t i s s u e integrity

< FIcURE18-1 Overviewof the physical propertiesand functionsof the three cytoskeletal systemsin animalcells. ( a )B i o p h y s i a ca n ldb i o c h e m i cpar o l perties (orange) (green) properties andbiological are shownfor eachfilament type Themicrographs s h o we x a m p l eosf e a c hf i l a m e ntty p ei n a p a r t i c u l ac re l l u l acro n t e x tb, u t n o t et h a t microtubules alsomakeup otherstructures, a n di n t e r m e d i af itlea m e n a t sl s ol i n et h ei n n e r (b)Well-spread surface of the nucleus, cultured c e l l s t a i n efdo r a c t i n( g r e e na)n ds i t e so f red) a c t i na t t a c h m e n t ot t h e s u b s t r a t u(m ( c )L o c a l i z a t ioofnm i c r o t u b u l(egsr e e na)n d (yellow) the Golgiapparatus Notice thecentral location of theGolgiapparatus, whichis collected therebytransport alongmicrotubules (d)Localization (red),a type of cytokeratins o f i n t e r m e d i afti el a m e n ta,n da c o m p o n e n t ) epithelia o f d e s m o s o m(egsr e e ni n l lls ce C y t o k e r a t ifnr o s mi n d i v i d ucael l l sa r ea t t a c h e d to eachotherthroughthe desmosomes lpart (b)courtesy or K Burridge Parl(c)courtesy of W BrownPart(d)courtesy of E Fuchs l

and flagella (Figure 18-2a, b). A careful examination of all the microtubules seenin transversesection indicated that they were made up of 13 longitudinal repeatingunirs (Figure 18-2c), now called protofilamezfs, suggestingthey all had a common structure. Microtubules purified from brain were then found to consist of a major protein, tubulin, and associatedproteins, microtubule-associatedproteins (MAPs). Purified tubulin alone can assembleinro a microtubule under favorable conditions, proving that it is the structural component of the microtubule wall. MAPs modify the assemblyand dynamics of the microtubules ass e m b l e df r o m t u b u l i n .

M i c r o t u b u l eW a l l sA r e P o l a r i z e dS t r u c t u r e sB u i l t from crB-Tubulin Dimers

nin

Microtubule Structure and

Organization In the early days of electron microscopy,cells biologists n o t e d l o n g t u b u l e s i n t h e c y t o p l a s mt h a t t h e y c a l l e d m i c r o t u b u l e s .M o r p h o l o g i c a l l y s i m i l a r m i c r o t u b u l e sw e r e s e e nm a k i n g u p t h e f i b e r s o f t h e m i t o t i c s p i n d l e ,a s c o m p o n e n t s o f a x o n s , a n d a s t h e s t r u c t u r a l e l e m e n t si n c i l i a

758

CHAPTER 18

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Purified soluble tubulin is a dimer consisting of two c l o s e l yr e l a t e ds u b u n i t so f a b o u t 5 5 , 0 0 0 d a l t o n s ,c t -a n d p , tubulin. Genomic analysesrevealthat genesencoding both cr- and B-tubulins are presentin all eukaryotes,with considerableexpansion in the number of genesin multicellular organisms.For example, budding yeast has two genes specifying o-tubulin and one for p-tubulin, whereas the soil nematode Caenorhabditis elegans has nine genes

C E L LO R G A N T Z A T T O A N D M O V E M E N Tt l M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

(b)

1 0n m tl

FIGURE 18-2 Microtubules arefound in manydifferent (a)Surface locations,and all havesimilarstructures. of the lininga rabbitoviductviewedin a scanning ciliated epithelium microscope Beating cilia,whichhavea coreof microtubules, electron propelan eggdownthe oviduct(b)Microtubules andintermediate in a quick-frozen frogaxonvisualized in a filaments anddeep-etched

(c) High-magnification view of a electronmicroscope. transmissron known as units repeating 13 singlemicrotubuleshowing andR H Kardon,1975,Ilssues protofilaments lPart(a)from R G Kessels 1982,,/ Part(b)from N Hirokawa, andCompany. W H Freeman and Organs, C Bouchetof N HirokawaPart(c)courtesy CellBiol 94:129;courtesy Marquis,2007, Biologyof the Cell99:451

encodingct-tubulin and six for B-tubulin. In addition to crand B-tubulin, all theseorganismsalso have genesspecifying a third tubulin, 1-tubulin, which servesa regulatory function. Additional isoforms of tubulin have also been discoveredthat are present only in organisms that have centriolesand basal bodies,suggestingthey are important for those structures. Each subunit of the tubulin dimer can bind one molecule of GTP (Figure18-3a).The GTP in the o-tubulin subunit is never hydrolyzed and is trapped by the interface betweenthe cr- and B-subunits.By contrast, the B-subunit has a GTPbinding site in the dimer that is exchangeablewith free GTP, and this bound GTP can be hydrolyzed. Microtubules consist of 13 laterally associatedprotofilaments forming a tubule whose external diameter is about 25 nm (Figure18-3b).Each protofilamentis made up of ctBtubulin dimers, so that the subunits alternate down a protofilament, with each subunit type repeating each 8 nm.

Becausethe protofilament is made up of tubulin dimers' each protofilament has an a-subunit at one end and a B-subunit at the other-so the protofilaments have an intrinsic polarity. In a microtubule, all the laterally associatedprotofilaments have the samepolarity, so the microtubule also has an overall polarity. The (+ ) end of the microtubule favored for polymerization is the end with exposed B-subunits,whereas the (-) end has exposed a-subunits. In microtubules, the heterodimers in adiacent protofilaments are staggeredslightly, forming tilted rows of o- and B-tubulin monomers in the microtubule wall. If you follow a row of B-subunits,for example, spiraling around a microtubule for one full turn, you will end up precisely three subunits up the protofilament' abutting an cr-subunit.Thus all microtubules have a single longitudinal seam, where an ct-subunitin one protofilament meetsa B-subunit in the next protofilament. Most microtubules in a cell consist of a simple tube, a singlet microtubule, built from 13 protofilaments' In rare

M I C R O T U B U LS E T R U C T U RAEN D O R G A N I Z A T I O N

759

(a)

cr-Tu bulin

bL,r

GTP (b)

Taxol

< FIGURE 18-3 Structureof tubulin dimersand their (a)Ribbondiagramof the organizationinto microtubules. t u b u l i nd i m e rT h eG T P( r e d b) o u n dt o t h ec t - t u b u l m i no n o m ei rs n o n e x c h a n g e aw bh l ee,r e atsh eG D P( b l u eb) o u n dt o t h e B - t u b u l i n monomerisexchangeable with freeGTPTaxol, a drugthatstabilizes microtubules andis usedto treatsomecancers, bindsto another p a r to f t h eB - s u b u n (i tb )T h eo r g a n i z a t i o fnt u b u l i ns u b u n i ti sn a microtubule Thedimersarealignedendto endintoprotofilaments, whichpacksideby sideto formthe wallof the microtubule. The p r o t oifl a m e n tasr es l i g h t lsyt a g g e r esdot h a tc t - t u b u l i n o n e p r o t o f i l a m einsti n c o n t a cwt i t h o - t u b u l i n i n t h en e i g h b o r i n g protofilaments, exceptat the seam,wherean ct-subunit contacts a ao l l a r i tiyn t h a t B - s u b u nT i t h em i c r o t u b udl ei s p l a yass t r u c t u r p s u b u n i tasr ea d d e dp r e f e r e n t i aaltl tyh ee n d ,d e s i g n a t et hde( + ) end,at whichB-tubulin monomers areexposed, Ipart(a)modified f r o m E N o g a l e se t a l , 19 9 8 , N a t u r e3 9 1 i 1 9 9 ;c o u r t e s yo f E N o g a l e sl

Dimer cr-Tubulin

B-Tubulin

(-) end Protofilament Seam

25nm

cases,singletmicrotubulescontain more or fewer protofilaments; for example,certain microtubulesin the neuronsof nematodeworms contain 11 or 15 protofilaments.In addition to the simple singlet structure, doublet or triplet microtubules are found in specializedstructuressuch as cilia and flagella(doubletmicrotubules)and centriolesand basal bodies (triplet microtubules),structureswe discussin more detail below. Each doublet or triplet containsone complete 13-protofilamentmicrotubule(A tubule) and one or two additional tubules (B and C) consistingof 10 protofilaments e a c h( F i g u r e1 8 - 4 ) .

M i c r o t u b u l e sA r e A s s e m b l e df r o m M T O C st o G e n e r a t eD i v e r s eO r g a n i z a t i o n s With the identification of tubulin as the maior structural component of microtubules,antibodiesto tubulin could be

Singlet

Doublet ( c i l i a f, l a g e l l a )

A F I G U R E l 8 -S 4 i n g l e t , d o u b l e t , a n d t r i p l e t m i c r o t u b ul n les. crosssectton, a typicalmicrotubule, a singlet, isa simpletubebuilt from 13 protofilaments ln a doubletmicrotubule, an additional set of 10 protofilaments formsa second tubule(B)by f usinqto thewall

760

CHAPTER 18

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generated and used in immunofluorescencemicroscopy to localize microtubules in cells (Figure 18-5a, b). This approach, coupled with the description of microtubules seen by electron microscopy, showed that microtubules are assembled from specific sitesto generatemany different types of organization(Figure18-5). The nucleation phaseof microtubule assemblyis such an unfavorable reaction that spontaneousnucleation does not play a significant role in microtubule assembly in vivo. Rather,all microtubulesare nucleatedfrom structuresknown as microtubule-organizingcenters,or MTOCs. In most cases the (- ) end of the microtubule staysanchoredin the MTOC. In non-mitotic cells, also known as interphasecells, the MTOC is known as the centrosomeand is generallylocated near the nucleus, producing a radial array of microtubules with their (+) endstoward the cell periphery(Figure18-5c). This radial display provides tracks for microtubule-based

Triplet ( b a s a lb o d i e s c, e n t r i o l e s )

o f a s i n g l e t ( Am) i c r o t u b uA l et t a c h m e n t a on f otherl0protofilaments to the (B)tubuleof a doubletmicrotubule creates a (C)tubuleanda tripletstructure

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

C e l lb o d y

S p i n d l ep o l e s

from microtubule A FIGURE 18-5 Microtubules are assembled in Thedistribution of microtubules organizingcenters(MTOCs). microscopy using cultured cellsasseenby immunofluorescence antibodie t ost u b u l i ni n a n i n t e r p h a cs e l (l a )a n da c e l li n m i t o s i(sb ) (c-f)Diagrams in cellsand of the distribution of microtubules allof whichareassembled fromdistinctMTOCsIn an structures, (thenucleus is interphase cell(c),the MTOCiscalleda centrosome are indicated by a blueoval);in a mitoticcell(d),thetwo MTOCs

poles(thechromosomes areshownin blue);in a calledspindle areassembled in bothaxonsanddendrites neuron(e),microtubules fromit; the froman MTOCin thecellbodyandthenreleased (f)are that makeup the shaftof a ciliumor flagellum microtubules of froman MTOCknownasa basalbodyThepolarity assembled (a)courtesy of A Bretscher by (+) and(-). Ipart isindicted microtubules of T.Wittmann Part(b)courtesv l

motor proteins to organize and transport membrane-bound compartments,such as thosecomprisingthe secretoryand endocytic pathways. During mitosis, cells completely reorganize their microtubules to form a bipolar spindle, assembled from two MTOCs called the spindle poles,to accuratelysegregatecopiesof the duplicatedchromosomes(Figure 18-5d). In another example, neurons have long processescalled axons, in which organellesare transported in both directions along microtubules (Figure 18-5e). The microtubules in axons, which can be as long as L meter in length, are not continuous and have beenreleasedfrom the MTOC but neverthelessare all of the same polarity. In the same cells, the microtubules in the dendrites have mixed polarity, although the functional significanceof this is not clear.In cilia and flagella (Figure 18-5f), microtubules are assembledfrom an MTOC calleda basalbody. In interphasecellsmicrotubulesare assembledfrom the centrosome.Electron microscopy shows that centrosomes in animal cells consist of a pair of orthogonally arranged cylindrical centriolessurroundedby apparentlyamorphous material called pericentriolar material (Figure 18-6a, arrowheads). Centrioles,which are about 0.5 pm long and 0.2 pm in diameter,are highly organizedand stable structures that consist of nine sets of triplet microtubules and are closelyrelatedin structureto basalbodies,found at the base of cilia and flagella. Centrosomesare critical for nu-

cleating microtubule assemblyin the cytoplasm. It is not the centriolesthemselvesthat nucleatethe cytoplasmicmicrotubule array but factors in the pericentriolar material' An important component is the y-tubwlin ring cotnplex (yT U R C ) ( F i g u r e s1 8 - 6 b a n d 1 8 - 7 ) ' T h i s p r o t e i n c o m p l e x i s located in the pericentriolarmaterial and consistsof many copiesof 1-tubulin, a form of tubulin distinct from o- and B-tubulin, associatedwith severalother proteins. It is currently believedthat 1-TURC acts like a split-washertemplate to bind crB-tubulin dimers for the formation of a new microtubule (Figure 18-7b).In addition to nucleating the assemblyof microtubules, centrosomesanchor and regulate the dynamicsof the (- ) end of the microtubules,which are locatedthere. Basal bodies have a structure similar to each centriole and arc the MTOC found at the base of cilia and flagella. The A and B tubules of their triplet microtubules provide a template for the assemblyof the microtubules that make up the core structure of cilia and flagella'

Microtubule Structure and Organization r Tubulin is the major structural component of microtubules and, together with microtubule-associatedproteins (MAPs), makesup microtubules(seeFigure 18-3).

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(a)

(b)

Mother

o

centriole

-

Pericentriolar material

1-TURC

FIGURE 18-6 Structureof centrosomes. (a)Thinsection of an a n i m a l - c ec lel n t r o s o ms e h o w i n tgh et w o c e n t r i o l east r i g h ta n g l e s to eachothersurrounded (arrows) by pericentiolar material (b)Diagram of a centrosome showingthe two centrioles, eachof w h i c hc o n s i sot sf n i n el i n k e do u t e rt r i p l em t i c r o t u b u leem s b e d d eidn pericentriolar material thatcontains yTURCnucleating structures

(c) lmmunofluorescence microscopyshowingthe microtubuledisplay ( g r e e n i)n a c u l t u r e da n i m a lc e l la n d t h e l o c a t i o no f t h e M T O C , usingan antibodyto a centrosomalprotein(yellow) [parts(a)and(b) from G Sluder, 2005,NatureRev.Mol CellBiol 6i-143ParI(c)courtesv of R Kuriyama l

r Free tubulin exists as an crB-dimer,with the a-subunit binding a trapped and nonhydrolyzable GTP and the Bsubunit binding an exchangeableand hydrolyzable GTP. r aB-tubulin assembles into microtubuleshaving 13 laterally associatedprotofilaments,with an a-subunit exposedat the (- ) end and a B-subunitat the (+ ) end of eachprotofilament. r In cilia and flagella,centriolesand basal bodies,doublet or triplet microtubules exist in which the additional microtubuleshave 10 protofilaments(seeFigure 18-4).

(b)

r All microtubules are nucleatedfrom microtubule-organizing centers (MTOCs), and many remain anchored with their (- ) end there. Thus the end away from the MTOC is alwaysthe (+) end.

y-TURC

r The centrosomeis the MTOC that nucleatesthe radial array of microtubules in nonmitotic cells, spindle poles are the MTOCs that nucleate the microtubules of the mitotic spindle, and basal bodies are the MTOCs that assemble microtubulesof cilia and flagella(seeFigure 18-5).

y

,t

o

Tubulin A FIGURE 18-7 The"y-tubulin ring complex(1-TURC), which nucleatesmicrotubuleassembly. (a)An immunofluorescence micrograph of in vitroassembled microtubules greenanda is labeled component of the1-TURC is labeled red,showingthatit is located specifically at oneendof the microtubule (b)Modelof how1-TURC maynucleate assembly of a microtubule byforminga template corresponding to the (-) endof a microtubule from lpart(a)Modified I J Keatingand G G Borisy,2000, Nature CellBiol 2:352; courtesyof T. .J K e a t i n g a n d GG B o r i s y l

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r Centrosomesconsist of two centrioles and the oericentriolar material that contains the ^y-TURC microtubule nucleatingcomplex (seeFigure 18-6).

ItllFl MicrotubuleDynamics Microtubules are dynamic structures due to assembly and disassemblyat their ends.The degreeof dynamicscan vary enormously, with an average microtubule lifetime of less than 1 minute for cells in mitosis and about 5-10 minutes

C E L LO R G A N T Z A T T O N D M O V E M E N Ti l : M I C R O T U B U L E AN AS N D I N T E R M E D I A TFEI L A M E N T S

for the microtubules that make up the radial array seen rn nonmitotic cells.Microtubule lifetime is longer in axons and much longer in cilia and flagella. To elucidatehow these differencesoccur, we first discusspolymerization properties of microtubule protein and the dynamic behavior of the ends of assembledmicrotubules.

(a)

a a o

M i c r o t u b u l e sA r e D y n a m i cS t r u c t u r e sD u e t o K i n e t i cD i f f e r e n c e as t T h e i r E n d s Microtubules assembleby the polymerization of dimeric oB-tubulin in a process that is greatly catalyzedby the presenceof microtubule-associatedproteins (MAPs). Microtubules assembledfrom a mixture of aB-tubulin and MAPs, collectively calledmicrotwbular protein, disassemble when chilled to 4 'C and then reassembleinto microtubules again on warming to 37 "C. This property allowed investigators to purify microtubular protein and to explore the mechanismof microtubule assemblyand dynamics when a solution of microtubule protein is warmed from 4 'C to 37'C. Analysis of the bulk polymerization propertiesof a solution of microtubule protein revealsthat it has severalfeatures in common with the polymerization of actin. However, since the assembly properties of microtubules are heavily influenced by the specific MAPs in the reaction mixture with crB-tubulin,only a few generalfeatureswill be highlighted here. First, a time courseof polymerization revealsa slow nucleationphase,followed by a rapid elongation phase, and then a steady state phase in which assemblyis balancedby disassemblgjust like the assembly o f a c t i n i n t o f i l a m e n t s ( s e e F i g u r e 1 7 - 7 ) . S e c o n d ,f o r assemblyto occur, the aB-tubulin concentration must be above the critical concentration (C.). Above this concentration, dimers polymerize into microtubules,whereas at concentrations below the C., microtubules depolymerize, similar to the behavior of G-actin and F-actin ( F i g u r e1 8 - 8 a ) . Third, at oB-tubulin concentrationshigher than the C. for polymerizatton, dimers add faster to one end (Figure 1 8 - 9 ) . B y a n a l o g y w i t h F - a c t i n a s s e m b l y ,t h e p r e f e r r e d e n d f o r a s s e m b l yi s d e s i g n a t e dt h e ( + ) e n d , w h i c h i s t h e e n d w i t h B - t u b u l i n e x p o s e d ( s e eF i g u r e 1 8 - 8 b ) . F o u r t h , the critical concentration is lower at the (* ) end than at t h e ( - ) e n d . A s a r e s u l to f t h i s p r o p e r t y ,a t s t e a d ys t a t et h e free crB-tubulin concentration is higher than the critical c o n c e n t r a t i o na t t h e ( + ) e n d b u t l o w e r t h a n a t t h e ( - ) e n d , s o s u b u n i t sa r e a d d e dt o t h e ( + ) e n d a n d s u b u n i t sd i s s o c i a t ef r o m t h e ( - ) e n d . T h i s r e s u l t si n n e t a d d i t i o n t o t h e ( + ) e n d a n d l o s s f r o m t h e ( - ) e n d , s o s u b u n i t sa p p e a rt o move down a microtubule in a processknown as treddmilling (seeFigure 18-8c), similar to that seen for actin ( s e eF i g u r e 1 7 - 1 0 b ) . B e c a u s et h e i n t r a c e l l u l a rc o n c e n t r a tion of tubulin (10-20 pM) is much higher than the critic a l c o n c e n t r a t i o n( C . ) f o r a s s e m b l y( 0 . 0 3 p M ) , p o l y m e r ization is highly favored in a cell. However, mechanisms e x i s t i n c e l l s t o r e g u l a t ew h e r e a n d w h e n m i c r o t u b u l e s oolvmerize.

cc T o t a lt u b u l i nc o n c e n t r a t i o(nd i m e r sa n d m i c r o t u b u l e s )

(b)

oos

=oo

dimers 18-8 Microtubuletreadmilling.(a)crB-tubulin A FIGURE a s s e m b il ne t om i c r o t u b u loens l yw h e np r e s e natb o v et h e c r i t i c a l (C.) AboveC.,microtubules at steadystate concentration a r ei n e q u i l i b r i uw mi t h f r e eo B - t u b u l idni m e r s(.b )T h ec r i t i c a l dimers(withboundGTP)at the two for ctB-tubulin concentrations i s h i g h e ar t t h e( + ) e n d . s i n c e t h e r a t e of addition e n d si sd i f f e r e n t (c)At steadystate,crp-tubulin to the dimersadd preferentially ( + ) e n da n da r el o s tf r o mt h e( - ) e n d ,s ot h a ts u b u n i tisn t h e m i c r o t u b u(l yee l l o wa)p p e atro m o v ed o w nt h e m i c r o t u b u loer, " t r e a d m i"l l .

I n d i v i d u a lM i c r o t u b u l e sE x h i b i tD y n a m i c Instability The properties of microtubule assemblyare similar to those of the assemblyof actin into filaments when one considersa population of growing microtubules. However, researchers found an additional phenomenon when they examined the behavior of individual microtubules within a population. They did a very simple experiment. Microtubules were assembled in vitro and then sheared to break them into shorter pieceswhose length could be analyzed.Under these conditions, one would expect the short microtubules to M I C R O T U B U LD EYNAMICS

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18-10Dynamicinstabilityof microtubules in vitro. A FIGURE in the lightmicroscope Individual microtubules canbe observed and plottedat different theirlengths timesduringassembly and disassembly. Assembly anddisassembly eachproceed at uniform rates,butthereisa bigdifference between the rateof assembly and thatof disassembly, asseenin the different slopes of the lines ismuchmorerapid(7 pm/min)than Shortening of a microtubule growth(1 pm/min), Noticetheabrupttransitions to the shrinkage andto theelongation stage(rescue) stage(catastrophe) [Adapted fromP M Bayley, K K Sharma, andS R Martin, 1994,inMicrotubules, W i l e y - Lpi s1s 1 , 8l

'Sfhat

A EXPERIMENTAL FIGURE 18-9 Microtubules grow preferentially at the (+) end. Fragment of a microtubule bundle froma flagellum wasusedasa nucleus for the in vitroaddition of o B - t u b u l iT n h en u c l e a t i nf lga g e l l af rra g m e ni st t h et h i c kb u n d l e seen in thiselectron micrograph, with the newlyformedmicrotubules (MT)radiating fromitsendsThegreater lengthof the microtubules a t o n ee n d ,t h e( + ) e n d ,i n d i c a t et hsa tt u b u l i ns u b u n i tasr ea d d e d preferentially to thisend [Courresy of G Borisy]

treadmill. However, the researchersfound that some of the microtubules grew in length, whereas others shortened very rapidly-thus indicating two distinct populations of microtubules. Further studies showed that individual microtubules could grow and then suddenly undergo a catastrophe to a shrinking phase. Moreover, sometimes a depolymerizingmicrotubule end could go through a rescue and begin growing again (Figure18-10).Although this phenomenon was first seen in vitro, analysisof fluorescently labeled tubulin microinjected into live cells showed that microtubules in cells also undergo periods of growth and shrinkage (Figure 18-11). This processof alternating between growing and shrinking states is known as dynamic instability. Thus dynamic instability is a feature of individual microtubule ends distinct from the ability of microtubules to treadmill by dimer addition at the (+ ) end and I o s sa t t h e ( - ) e n d .

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is the molecular basis of dynamic instability? If you look carefully by electron microscopy at the ends of growing and shrinking microtubules, you can see they are quite different. A growing microtubule has a blunt end, whereasa depolymerizingend has protofilaments peelingoff like ram's horns (Figure 18-12). Recent studies have provided a simple structural explanation for the two classesof microtubule ends. As we noted above, the B-subunit of the ctB-tubulin dimer is exposed on the (+) end of each protofilament. Using GTP and GDP analogs,researchershave found that artificially made single protofilaments-where there are no lateral interactionsmade up of repeating ctB-tubulin dimers containing GDP-Btubulin are curved, like a ram's horn. However, artificially made single protofilaments made up of crB-tubulin dimers with GTP-B-tubulin are straight. Thus growing microtubules with blunt ends terminate in GTP-B-tubulin, whereas shrinking ones with curled ends terminate in GDPB-tubulin. Therefore if the GTP in the terminal B-tubulins of a microtubule becomehydrolyzed,as will inevitablyhappenin a random manner, a formerly blunt-end growing microtubule will curl and a catastrophe ensue. These relationships are summarizedin Figure 1,8-1,2. These results have an additional implication. For this we have to considerin more detail a growing microtubule. The addition of another dimer to the protofilament (+ ) end of a growing microtubule involves an interaction between the new o.-subunit and the terminal B-subunit. This interaction stimulates the hydrolysis of the GTP to GDP in the former terminal B-subunir. However, the Btubulin in the newly added dimer contains GTP. Thus a

C E L LO R G A N T Z A T T O N D M O V E M E N Tl l : M I C R O T U B U L E AN AS N D I N T E R M E D I A TFEI L A M E N T S

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Video: CytoplasmicAssemblyof Microtubulesin CulturedCells < E X P E R I M E N TFAI L G U R1 E8 - 1 1 F l u o r e s € e n c e microscopyrevealsgrowth and shrinkageof individual y e l e tdu b u l i nw a s m i c r o t u b u l e isn v i v o . F l u o r e s c e nl tal b h u m a nf i b r o b l a s tTsh ec e l l s m i c r o i n j e c t iendt oc u l t u r e d i nst o t o d e p o l y m e r ipz ree e x i s t i nmgi c r o t u b u l e w e r ec h i l l e d t u b u l i nd i m e r sa n dw e r et h e ni n c u b a t eadt 3 7 " C t o a l l o w t hge f l u o r e s c etnutb u l i n r e p o l y m e r i z a t itohnu,si n c o r p o r a t i n i n t oa l lt h e c e l l ' sm i c r o t u b u l eAs r. e g i o no f t h e c e l lp e r i p h e r y c ei c r o s c o paet 0 s e c o n d2, 7 w a sv i e w e di n t h e f l u o r e s c e nm s e c o n dlsa t e ra, n d3 m i n u t e s5, 1 s e c o n dlsa t e r( l e f tt o r i g h t p a n e l s I)n t h i sp e r i o ds, e v e r aml i c r o t u b u l e lso n g a t a en d nf s n dw h i t ed o t sm a r kt h e p o s i t i o o s h o r t e nT, h el e t t e r a s o diief df r o mP J S a m m a kn d e n d so f t h r e em i c r o t u b u l el M 1988, Nature 332:.1241 G Borisy,

away? The lateral protofilament-protofilament interactions in the B-tubulin-GTP cap are sufficiently tight that they do not allow the microtubule to unpeel at its endand so the protofilaments behind the GTP-B-tubulin cap The enare constrainedfrom unpeeling(seeFigure 1'8-1,2). ergy releasedby GTP hydrolysisof the subunits behind the cap is stored within the lattice as structural strain waiting to be releasedwhen the GTP-B-tubulin is lost. If the GTPB-tubulin is lost, the stored energy can do work if some structure, such as a chromosome,is attachedto the disassemblingmicrotubule end. Thus we seethat the ability of B-tubulin to bind and hyGTP-B-tubulindrolyze GTP has three important consequencesfor microtubule biology:

growing microtubule protofilament has GDP-B-tubulin down its length and is capped by GTP-B'tubulin. As we mentioned above, an isolatedprotofilament containing GDP-B-tubulin is curved along its length, so when it is presentin a microtubule, why doesn'tit break out and peel

"1,

Assembly

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r Distinct critical concentrationsat the two ends give rise to treadmilling

The (+) endis subjectto dynamicinstability GTP

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Microtubulescan storetorsionalenergyto do work

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18-12Dynamicinstabilitydependson the presence < FIGURE takenin theelectron cap.lmages or absenceof a GTP-p-tubulin (upper)anda growing microtubule a samples of of f rozen microscope ep "l (lowedNotice thattheendof thegrowing microtubule shrinking G D P - B - t u b u l microtubule in onehascurlslikea theshrinking hasa bluntend,whereas withGTP-B-tubulin showsthata microtubule ram'shornsThediagram favoredto grow.However, isstrongly on the endof eachprotofilament at theendof theprotofilaments withGDP-B-tubulin a microtubule Switching rapiddisassembly andwillundergo structure formsa curved can rescues andcatastrophes, growingandshrinking, called between proteins. by associated is regulated of switching and the rate occur, Diagram etal, 1991, J CellBiol.114:.977 fromE-MMandelkow [lmages

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ReuCell Dev.Biol. 1997,Annu Desai andT,JMitchison, modifiedfromA 17 l 13:83-1

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LocalizedAssemblyand "search-and-Capture" H e l p O r g a n i z eM i c r o t u b u l e s

and capture sites, a cell can rapidly change its overall microtubule distribution.

'We

have now presented two concepts relating to microtubule organization and (+ ) end dynamics:microtubules are assembledfrom localizedsitesknown as MTOCs, and individual microtubules can undergo dynamic instability. Together these two processescontribute to the distribution of microtubulesin cells. In an interphase cell growing in culture, microtubules are constantly being nucleated from the centrosome and spreading out randomly "searching" the cytoplasmic space. The frequency of catastrophes and rescues,together with growth and shrinkage rates, determines the length of each microtubule-if the microtubule is subject to a high catastrophe frequency and low rescue,it will shrink back to the cell center and disappear,whereas if it has few catastrophes and is readily rescued,it will continue to grow. If the searching microtubule encountersa structure or organelle that stabilizes its (+) end to protect it from catastrophesthereby "capturing" it-the microtubule end will remain attached to the structure, whereas unattached microtubules will have a greaterfrequencyof being disassembled. So the dynamics of the microtubule end is a very important determinant of microtubule life cycle and function. "search and capture" is part of the mechanism determining the overall organization of microtubules in a cell. Moreover, by changing the rate of nucleation or local microtubule dynamics

A EXPERIMENTAL FIGURE 18-13Microtubules grow from the MTOC.Toinvestigate fromwheremicrotubules assemble in vivo, a cultured fibroblast wastreatedwith colchicine untilalmostall thecytoplasmic microtubules weredisassembled. Thecel was t h e ns t a i n e w d i t h a n t i b o d i et o s t u b u l i na n dv i e w e db y (panel[a])Thecolchicine immunofluorescence microscopy wasthen washedout to allowthe reassembly (b)shows of microtubules Panel thefirststages of reassembly, revealing growing microtubules froma centralregionabovethe nucleus (darkareas). Notein panel (a)the remaining primary cilium(arrowhead) associated withthe centrosome thatis not depolymerized by colchicine treatment under theseconditions Notealsothef luorescence fromthe cytoplasm, whichisfromunpolymerized oB-tubulin dimers. M Osborn and [From K Weber, 1976, ProcNatlAcad!ci.USA73:867-8711

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DrugsAffecting TubulinPolymerizationAre Useful Experimentallyand to TreatDiseases The conservednature of tubulins and their essential involvement in critical Drocessessuch as mitosis make them prime targets for both naturally occurring and synthetic drugs that affect polymerization or depolymerization. Historically, the first known such drug was colchicine, present in extracts of the meadow saffron, which binds tubulin dimers so thar they cannot polymerize into a microtubule. Sincemost microtubulesare in a dynamic state between dimers and polymers, the addition of colchicine sequestersall free dimers in the cytoplasm, resulting in loss of microtubules due to their natural turnover. Treatment of cultured cells with colchicine for a short time results in the depolymerizationof all the cytoplasmic microtubules, leaving the more stable tubulin-containing c e n t r o s o m e( F i g u r e 1 8 - 1 3 a ) . A l s o s e e n a f t e r c o l c h i c i n e treatment is the primary cilium, a solitary cilium on the surface of the cell that is assembledfrom one of the cen'Sfhen trioles acting as its basal body (discussedbelow). the colchicine is washed out to allow regrowth of the microtubules,they can be seento grow from the centrosome, revealingits ability to nucleatenew microtubule assembly ( F i g u r e1 8 - 1 3 b ) . Colchicine has beenused for hundreds of years to relieve the joint pain of acute gout-a famous patient was King Henry VIII of England, who was treated with colchicine to relieve this ailment. A low level of colchicine relieves the inflammation caused in gout by reducing the microtubule dynamics of white blood cells, rendering them unable to migrate efficiently to the site of inflammation. In addition to colchicine, a number of other drugs bind the tubulin dimer and restrain it from forming polymers. These include podophyllotoxin (from juniper) and nocodazole (a synthetic drug). Taxol, a plant alkaloid from the Pacific yew tree, binds and stabilizes microtubules against depolymerization. Becausetaxol stops cells from dividing by inhibiting mitosis, it has been used to treat some cancers, such as those of the breast and ovarS where the cells are especiallysensitiveto the drug. I

Microtubule Dynamics r Microtubules can treadmill using the energy of GTP hydrolysis(seeFigure 18-8). r Individual microtubule (+ ) ends can undergo dynamic instabilitS with alternating periods of growth or rapid shrinkage(seeFigure 18-10). r The B-tubulin in all microtubulesbinds primarily GDP. However, growing microtubules (* ) ends are capped by

C E L LO R G A N I Z A T I O N A N D M O V E M E N TI I : M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

GTP-B-tubulin and have blunt ends, whereas shrinking m i c r o t u b u l e sh a v e l o s t t h e G T P - B - t u b u l i nc a p a n d t h e protofilaments peel outward and disassemble(seeFigure 18-1,2).

(a)

r Growing microtubules store the energy from GTP hydrolysisin the microtubulelattice,so they have the potential to do work when disassembling. r Microtubules assembledfrom the centrosomeand exhibiting dynamic instability can "search" the cytoplasm and become "captured" by structuresor organellesthat stabilizetheir (+) end. In this way assemblycoupledwith "search and capture" can contribute to the overall distribution of microtubulesin a cell.

3 0 0n m Microtubule

fl!|F Regulationof Microtubule Structure and Dynamics

MAP

Although the wall of microtubulesis built from ctB-tubulin dimers,highly purified crB-tubulinwill assemblein vitro into microtubulesonly under specialunphysiologicalconditions. Assemblyof microtubulesin vitro under physiologicalconditions requiresthe presenceof stabilizingmicrotubuleassociated proteins, or MAPs. StabilizingMAPs representjust one class of protein that interacts with tubulin in microtubules; other classesmodify the growth propertiesof mi'Sfe crotubules or destabilize them. discuss the various classesseparately.

M i c r o t u b u l e sA r e S t a b i l i z e db y S i d e -a n d E n d B i n d i n gP r o t e i n s Severaldifferent classesof proteins stabilizemicrotubules, many of them showing cell-type-specificexpression.Among the beststudiedare the tau family of proteins,which includes tau itself, MAP2, and MAP4. Theseproteins have a modular design, with 18-residuepositively charged sequences,repeated three to four times, that binds to the negatively chargedtubulin surfaceand a domain that projects from the microtubulewall (Figure18-14).Tau proteinsare believedto stabilizemicrotubules and also to act as spacersbetween them (Figure18-14).MAP2 is found only in dendrites,where it forms fibrous cross-bridgesbetweenmicrotubulesand links microtubules to intermediatefilaments. Tau, which is much smaller than most other MAPs, is presentin both axons and dendrites.The basisfor this selectivityis still a mystery. Vhen stabilizingMAPs coat the outer wall of a microtubule, they can increasethe growth rate of microtubules or suppressthe catastrophefrequency.In many cases,the activity of the MAPs is regulated by the reversiblephosphorylation of their projection domain. PhosphorylatedMAPs are unable to bind to microtubules;thus phosphorylationpromotes microtubule disassembly. For example,microtubuleaffinity-regulatingkinase(MARK/Par-1)is a key modulator of tau proteins. Some MAPs are also phosphorylatedby a

25nm

Tau

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a E X P E R I M E N TFAI G L U R1E8 - 1 4S p a c i n go f m i c r o t u b u l e s dependson the length of the projectiondomain of proteins.Insectcellstransfected to microtubule-associated e x p r e sM s A P 2w , h i c hh a sa l o n ga r m ,o r t o e x p r e sTsa up r o t e i n , s )E l e c t r o n w h i c hh a sa s h o r ta r m ,g r o wl o n ga x o n l i kper o c e s s e( a dy m i c r o g r a po h fsc r o s s e c t i o nt sh r o u g ht h e p r o c e s s iensd u c e b cells of MAP2(/eft)or fau @ght)in transfected the expression m i c r o t u b u l(eM s T si)n M A P 2 N o t et h a tt h es p a c i n bg e t w e e n c o n t a i n i ncge l l si s l a r g etrh a ni n T a u - c o n t a i n ci negl l sB o t hc e l l a p p r o x i m a t et hl ye s a m en u m b eor f m i c r o t u b u l e s , t y p e sc o n t a i n t f M A P 2i st o e n l a r gteh e c a l i b eor f t h e a x o n l i k e b u t t h ee f f e c o m i c r o t u b u laens d bn etween p r o c e s (sb )D i a g r a mosf a s s o c i a t i o e t h e l e n g t h os f t h e p r o j e c t i oanr m si n M A P sN o t et h e d i f f e r e n ci n J hen e ta l, 1 9 9 2N M A P 2a n dT a u l P a r(ta )f r o m . C , ature360t674)

cyclin-dependentkinase (CDK) that plays a maior role in controlling the activities of proteins in the course of the cell cycle (Chapter 20). Some MAPs have recently been found to have the surprising ability to associatewith the (+) ends of microtubulesand in somecasesonly the (+ ) endsof growing, not shrinking ones (Figure 18-15). This classof proteins is known as *TIPs, and they perform varied functions when presentat the microtubule tip. Some +TIPs selectivelystabilize the (+) end againsta catastropheor enhancethe frequency of rescues,thus promoting the continued growth of the microtubule. Other +TIPs are attachmentproteins, so that when the growing microtubule encounters a structure or organelle,it can becomeattachedto it. For example,microtubulesextending into the leading edge of a migrating

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Video: In Vivo Visualizationof MicrotubuleEnds

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< E X P E R I M E N TFA IG L U R1E8 - 1 5T h e + T l Pp r o t e i nE B 1 (a)A cultured associates with the (+) endsof microtubules. cell stained with antibodies to tubulin(green) andthe + TIPproteinEB1 (red)EB1isenriched (+) end.(b) in the regionof the microtubule l n v i t r os t u d i ehsa v es h o w nt h a t E B 1b i n d ss p e c i f i c atlol yt h e microtubule seam,asseenin the enhanced electron micrograph and m o d e lb, u th o wt h i sb i n d i n lge a d tso e n r i c h m eanttt h e( + ) e n di s part(b)modified not yet known [Part(a)courtesy of T Wittmann, from Sandbladet al 2006 Cell 121:1415I

cell can becomeattachedand stabilizedby binding components there.

M i c r o t u b u l e sA r e D i s a s s e m b l ebdy E n d B i n d i n g and SeveringProteins

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A FIGURE 18-16 Proteinsthat destabilize the endsof (a)A memberof the kinesin-13 microtubules. familyenriched at microtubule endscanenhance the disassembly of thatend These proteins areATPases, andATPenhances theiractivity by dissociating themfromtheoB-tubulin dimer(b)Stathmin bindsselectively to curvedprotofilaments andenhances theirdissociation froma microtubule end Stathmin's activity isinhibited by phosphorylation 768

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Just as for microfilaments, mechanismsexist for enhancing the disassemblyof microtubules.Although most of the regulation of microtubule dynamics appearsto happen at the (+ ) end, in some situations,such as in mitosis, it can occur at both ends. Three mechanisms for microtubule destabilization arc known. One of these involves the kinesin-13 family of proteins (Figure 18-16). The kinesin-13 proteins bind and curve the end of the tubulin protofilaments into the GDPB-tubulin conformation. They then facilitate the removal of terminal tubulin dimers, thereby greatly enhancing the frequency of catastrophes.They act catalytically in the sense that they need to hydrolyze ATP to sequentiallyremove terminal tubulin dimers (Figure 18-16). Another protein, known as Op18/stathmin, was originally identified as a protein highly overexpressedin cert a i n c a n c e r s ;h e n c ep a r t o f i t s n a m e ( O n c o p r o t e i n1 8 ) . I t is a small protein that binds two tubulin dimers in a curved, GDP-B-tubulin-like conformation, which also enhancesthe rate of catastrophes.It may work by enhancing the hydrolysis of the GTP in the terminal tubulin dimer and aiding in its dissociation from the end of the microtubule. As might be expectedfor a regulator of microtubule ends, it is subject to negativeregulation by phosphorylation by a large variety of kinases. In fact, it has been found that Op1S/stathmin is inactivated by phosphorylation near the leading edge of motile cells, which contributes to preferential growth of microtubules toward the front of the cell. A third mechanism for destabilizing microtubules is through the action of a protein known as katanin (from the Japanesefor "sword"). Katanin plays a role in MTOCs, where it seversand releasesanchored microtubules.

Regulationof MicrotubuleStructureand Dynamics r Microtubules can be stabilized by by side-bindins micro(seeFigure 18-14). tubule-associated proteins(MAPs) (se

c E L L o R G A N t z A T t o NA N D M o v E M E N T l : M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

r Some MAPs, called +TIPs, bind selectivelyto the (+) end of microtubulesand can alter the dynamic properties of the microtubule or localize componentsto the searching ( + ) e n d o f t h e m i c r o t u b u l e( s e eF i g u r e1 8 - 1 5 ) . r Microtubule ends can be destabilizedby some proteins, such as the kinesin-13family of proteins and stathmin,to enhancethe frequencyof catastrophes(seeFigure 18-16).

D o r s a lg a n g l i o ny Dorsalroot

radiolabeled a m i n oa c i d s

Kinesins and Dyneins: I[[ M icrotubule-Based Motor Proteins Organellesin cells are frequently transported distancesof many micrometersalong well-definedroutes in the cytosol and delivered to particular intracellular locations. Diffusion alone cannot account for the rate, directionality,and destinations of such transport processes.Findings from early experirnentswith fish-scalepigment cells and nerve cells first demonstrated that microtubules function as tracks in the intracellular transport of various types of " c a r g o ." As already discussed,polymerization and depolymerization of microtubulescan do work using the energyprovided by GTP hydrolysis.In addition, motor proteins move along microtubulespowered by ATP hydrolysis.Two main families of motor proteins-kinesins and dyneins-are known to mediate transport along microtubules. In this sectionwe discusshow thesemotor proteins work and the functions they perform in interphasecells. In subsequent sections,we discusstheir functions in cilia and flagella and ln mltosls.

O r g a n e l l e si n A x o n sA r e T r a n s p o r t e d Along M i c r o t u b u l e si n B o t h D i r e c t i o n s A neuron must constantly supply new materials-proteins and membranes-to an axon terminal to replenish those lost in the exocytosis of neurotransmitters at the junction (synapse) with another cell (Chapter 23). B e c a u s ep r o t e i n s a n d m e m b r a n e sa r e p r i m a r i l y s y n t h e sized in the cell body, thesematerials must be transported down the axon, which can be as much as a meter in length, to the synaptic region. This movement of materia l s i s a c c o m p l i s h e do n m i c r o t u b u l e s ,w h i c h a r e a l l o r i entedwith their (+) endstoward the axon terminal (see F i g u r e1 8 - 5 e ) . The resultsof classicpulse-chase experimentsin which radioactiveprecursorswere microinjectedinto the dorsalroot ganglia near the spinal cord and then tracked along their nerve axons showed that axonal transport occurs in both directions. Anterograde transport proceeds from the cell body to the synapticterminalsand is associated with axonal growth and the deliveryof synapticvesicles.In the opposite, retrograde, direction, "old" membranesfrom the synaptic terminals move along the axon rapidly toward the cell body, where they will be degradedin lysosomes.Findings from such experiments also revealed that different

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FIGURE 18-17The rate of axonaltransport EXPERIMENTAL and gel in vivo can be determinedby radiolabeling Thecellbodiesof neuronsin the sciaticnerveare electrophoresis. l o c a t e idn d o r s a l - r ogoat n g l i (an e a trh e s p i n acl o r d )R a d i o a c t i v e i n e x p e r i m e n taanl i m a lasr e a m i n oa c i d si n j e c t eidn t ot h e s eg a n g l i a e rdo t e i n sw,h i c ha r et h e n i n c o r p o r a t ei ndt on e w l ys y n t h e s i z p e n i m a las r es a c r i f i c e d t r a n s p o r t eddo w nt h ea x o nt o t h e s y n a p s A a t v a r i o utsi m e sa f t e ri n j e c t i oann dt h e d i s s e c t esdc i a t inc e r v ei s c u t i n t os m a lsl e g m e n t so s e eh o wf a r r a d i o a c t i v el al yb e l e d canbe identified theseproteins proteinshavebeentransported; , nd a f t e rg e le l e c t r o p h o r easni sda u t o r a d i o g r a pThhyer e d ,b l u e a p u r p l ed o t sr e p r e s e g n rt o u p so f p r o t e i ntsh a ta r et r a n s p o r t e d purpleleast downthe axonat differentrates,redmostrapidly, rapid ly

materials move at different speeds (Figure 18-17). The fastest-movingmaterial, consisting of membrane-limited vesicles,has a velocity of about 3 pm/s, or 250 mm/dayrequiring about four days to travel from a cell body in your back down an axon that terminatesin your big toe. The slowest-moving material, comprising tubulin subunits and neurofilaments(the intermediate filaments found in neurons), moves only a fraction of a millimeter per day. Organelles such as mitochondria move down the axon at an intermediaterate. Axonal transport can be directly observed by video microscopy of cytoplasm extruded from a squid giant axon. The movementof vesiclesalong microtubulesin this cell-free systemrequires ATP, its rate is similar to that of fast axonal transport in intact cells, and it can proceed in

: I C R O T U B U L E - B A SMEODT O RP R O T E I N S K I N E S I NASN D D Y N E I N SM

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both the anterogradeand the retrogradedirections (Figure 1 8 - 1 8 a ) . E l e c t r o n m i c r o s c o p yo f t h e s a m e r e g i o n o f t h e axon cytoplasm reveals organellesattached to individual m i c r o t u b u l e s( F i g u r e 1 8 - 1 8 b ) . T h e s e p i o n e e r i n gi n v i t r o experimentsestablisheddefinitively that organellesmove along individual microtubulesand that their movemenrrequires ATP. Findings from experiments in which neurofilaments tagged with green fluorescent protein (GFP) were injected into cultured cells suggestthat neurofilamentspause frequently as they move down an axon. Although the peak velocity of neurofilamentsis similar to that of fast-moving vesicles,their numerous pauseslower the averagerate of transport. Thesefindings suggestthat there is no fundamen-

tal difference between fast and slow axonal transport, although why neurofilament transport stops periodically is unknown.

Kinesin-1PowersAnterogradeTransportof VesiclesDown Axons Towardthe (+) End of Microtubules The protein responsiblefor anterogradeorganelletransport was first purified from axonal extracts. Researchersfound that by mixing three components-purified organellesfrom squid axons, an organelle-freecytoplasmicaxonal extract, and taxol-stabilizedmicrotubules-organellescould be seen moving on the microtubules in an MP-dependent manner.

z/^\ Video: OrganelleMovement Along Microtubulesin a Squid Axon \\'///

Organelles

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ui A EXPERIMENTAL FIGURE18-18 DIC microscopydemonstrates microtubule-basedvesicletransport in vitro. (a)The cytoplasm was squeezedfrom a squidgiant axonwith a rolleronto a glass coverslipAfter buffercontainingATPwas addedto the preparation, it was viewedby differentialinterference contrast(DlC)mrcroscopy, and the imageswere recordedon videotapeIn the sequential imagesshown,the two organellesindicatedby open and solid trianglesmove in oppositedirections(indicatedby coloredarrows) alongthe samefilament,passeachother,and continuein their

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originaldirectionsElapsedtime in secondsappearsat the upper-right cornerof eachvideoframe (b) A regionof cytoplasmsimilarto that shown in part (a)was freeze-dried, rotaryshadowedwith platinum, and viewedin the electronmicroscope. Two largestructures attached to one microtubulearevisible;thesestructurespresumably are small vesicles that were movingalongthe microtubulewhen the p r e p a r a t i o n w a s f r o z e[ nS e e BJ S c h n a p p e t,a1l9 8 5 ,C e l4l O : 4 5 5 ; c o u r t e s y o f BS. Jc h n a p p ,DRV a l e , MP S h e e t z , a n d TR. e Se s e l

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

18-19Structureof kinesin-l.(a)Representation < FIGURE eachwith a motor with itstwo heavychains of kinesin-1, to lightchainsEachheadisattached domainandassociated linkerdomain.(b)X-ray stalkby a flexible thecoiled-coil and headswith the microtubule of the kinesin structure (a)modified fromR D Vale, bindingsitesindicated. nucleotide [Part andE.M. of E Mandelkow Part(b)courtesy 2003,Cell112:467 fromM Thormahlen etal,1998,J StrucBiol adapted Mandelkow 122:301

MT-bindingsites

However, if they omitted the axonal extract, the organelles neither bound nor moved along the microtubules, suggesting that the extract contributes a motor protein. The strategy for purifying the motor protein was basedon additional observations of organellesmoving on microtubules. It was known that if ATP was hydrolyzed to ADP, the organelles fell off the microtubules. However, if the nonhydrolyzable ATP analog AMPPNP was added, organelles remained associatedwith microtubules but did not move. This suggested that the motor linked the organelles to the microtubules yery tightly in AMPPNP but then was releasedfrom the microtubule when the AMPPNP was replaced by ATP and its subsequenthydrolysis to ADP. Researchersused this clue to purify the motor. Kinesin-1 isolatedfrom squid giant axons is a dimer of two heavy chains, each associatedwith a light chain, with a total molecular weight of about 380,000. The molecule comprisesa pair of globular head domains connectedby a short flexible linker domain to a long central stalk and terminating in a pair of small globular tail domains, which associatewith the light chains (Figure 18-19). Each domain carries out a particular function: the head domain, which binds microtubules and ATP, is responsible for the motor activity of kinesin; the linker domain is critical for forward motility; the stalk domain is involved in dimerization of the two heavy chains; and the tail domain is responsible for binding to receptors on the membrane of cargoes. Kinesin-1-dependent movement of vesicles can be tracked by in vitro motility assayssimilar to those used to study myosin-dependentmovements(seeFigure 1.7-21).In one type of assay,a vesicleor a plastic bead coated with kinesin-1is added to a glassslide along with a preparation of stabilized microtubules. In the presenceof ATP, the beads can be observed microscopically to move along a microtubule in one direction. Researchersfound that the beads coated with kinesin-1 always moved from the (-) to the (+) end of a microtubule (Figure 18-20). Thus kinesin-1 is

a (+ ) end-directed microtubule motor protein, and additional evidenceshows that it mediates anterograde axonal transport.

KinesinsForma LargeProteinFamilywith DiverseFunctions Following the discovery of kinesin-1, a number of proteins with kinesin-relatedmotor domains were identified both in genetic screens and using molecular biology approaches. There are now 14 known classesof kinesins in animals, defined as sharing amino acid sequencehomology with the motor domain of kinesin-1. The family of kinesin-related proteins is encodedby about 45 genesin the human genome. Although the function of all of these proteins has not yet

-DrivenTransportof Video:Kinesin-1 Along Microtubulesin Vitro Vesicles Vesicle

Kinesin receptor Kinesin (+) end

(-) end

le microtubu Stationary 18-20 Model of kinesin-l-catalyzedvesicle A FIGURE on the molecules, attachedto receptors transport.Kinesin-1 fromthe (-) endto the(+) thevesicles transport surface, vesicle for movement. ATPis required microtubule. endof a stationary lAdapted from R D. Valeet al ,1985, Cell 4O:559,and I Schroeret al , 1988. J Cell Biol 1O7:1185 I

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been elucidated,some of the best-studiedkinesins are involved in processes such as organelle,mRNA, and chromo, some transport and microtubule sliding and microtubule depolymerization. As with the different classesof myosin motors, the kinesin-relatedmotor domain is fused to a variety of classspecificnonmotor domains (Figure t8-21). Whereaskinesin-1 has two heavy and two light chains,membersof the kinesin2 famrly, also involved in organelletransport, have two different related heavy-chain motor domains and a third polypeptide that associateswith the tail and binds cargo. Members of the bipolar kinesin-Sfamily have four heavy chains,forming bipolar morors that can pull on antiparallel microtubulestoward the (+ ) ends.The kinesin-14motors are the only known classto move toward the (-) end of a microtubule;this classfunctions in mitosis.Yet another type, the kinesin-13family havetwo subunitsbut with the kinesinrelateddomain in the middle of the polypeptide.Kinesin-13 proteins do not have motor activity, but recall that theseare

special ATP-hydrolyzing proteins that can enhance the depolymerizationof microtubuleends(seeFigure 18-16).

K i n e s i n - 1l s a H i g h l yP r o c e s s i vM e otor How doeskinesin-1move down a microtubule?Optical trap and fluorescent-labeling techniquessimilar to those usedto characterizemyosin (seeFigures 17-26,77-27, and 17-28) have been used to study how kinesin-1moves down a microtubule and how ATP hydrolysis is converted into mechanical work. Such experimentsdemonstratethat it is a very processivemotor-taking hundreds of steps walking hand over hand down a microtubule without dissociating. During this process,the double-headed moleculetakes8-nm stepsfrom one B-tubulin subunit to the next, tracking down the sameprotofilament in the microtubule. This entails each indiuidwal head taking 16-nm steps.The two headswork in a highly coordinatedmanner so rhat one is always attached to the microtubule.

K i n e s i n - 2( h e t e r o t r i m e r i c )

K i n e s i n - 5( b i p o l a r )

- 1 3( K i n l ) Kinesin

A FIGURE18-21 Structure and function of selectedmembers o f t h e k i n e s i ns u p e r f a m i l y K . i n e s i n - 1w,h i c hi n c l u d etsh e o r i g i n a l k i n e s i ni s o l a t e fdr o m s q u i da x o n s i, s a ( + ) e n d - d i r e c t em d icrotubule motor involvedin organelletransport The kinesin-2familyhavetwo different,but closelyrelated,heavychains,and a third cargobinding subunit;this classalsotransportsorganelles in a (+) end-directed m a n n e rT h e k i n e s i n -f5a m i l yh a v ef o u r h e a v yc h a i n sa s s e m b l eidn a bipolarmannerto interactwith two antiparallel microtubules and

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a l s om o v et o w a r dt h e ( + ) e n d K i n e s i n - 1f3a m i l ym e m b e r sh a v et h e m o t o rd o m a i ni n t h e m i d d l eo f t h e i rh e a v yc h a i n sa n d d o n o t h a v e motor activitybut destabilize microtubuleends Additionalkinesin familymembersare mentionedin the text Differentkinesinshave beengivenmanydifferentnames,someof which are shown in parenthesesWe use herethe unifiednomenclature describedin C J Lawrenceet al , 2004,J CellBiol 167.19-22 [Diagrams modified from R D Vale,2003,Ceil112:467I

C E L LO R G A N I Z A T I OANN D M O V E M E N TI I M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

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KinesinMovementAlong a Microtubule ffi Podcast: u le teppingCycle illl* a n i r n a t i o nK: i n e si n -Mi cro tu b S 18-22Kinesin-1usesATPto "walk" down a < FIGURE (a)Thecycleisshownwith ADPboundto eachof microtubule. t h et w o k i n e s i n -h1e a d sB i n d i nogf o n eh e a dt o a B - t u b u l i n induces the lossof ADBgivingriseto a subunitin the microtubule stateandstrongbindingof thatheadto the nucleotide-free (steptr) Theleading headthenbindsATP(stepZ), microtubule the linkerreglonto changecausing a conformattonal whichinduces pointforward,dockintothe headdomain,andsothrustthe headbindsthe trailingheadforward(stepg;. Theleading thetrailingheadto ADBwhichinduces andreleases microtubule andthetrailing ATPto ADPandP (step4) Piisreleased, hydrolyze andthe cyclerepeat. fromthe microtuble, headcannowdissociate (b)Structural boundto Bheads(purple) modelof two kinesin Thetrailinghead,at in a microtubule of a protofilament subunits left,hasboundATPandhasthrustthe otherheadintothe leading isdockedintothe positionNoticehowthe linkerdomain(yellow) headis the linkerdomain(red)of the leading trailinghead,whereas (a)modified 2000, andR A Milligan, fromR D Vale stillfree [Part

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The ATP cycle of kinesin-1 movement is most easily viewed by starting with the leading head in a nucleotide-free state,under which conditions it is strongly bound to a B-subunit of a protofilament, and with the trailing head in a weakly bound state containing ADP (Figure 18-221.The energy associatedwith binding ATP to the leading head inducesa forward motion of the linker domain of that head that then physically docks into the core head domain. This movement resultsin the linker domain pointing forward and physically swinging the trailing head-like throwing a ballet dancer-

into a position where it becomesthe leading head. The new leading head finds the next B-tubulin subunit, which induces dissociationof the ADP and tight binding. Importantly, this step also inducesthe now trailing head to hydrolyze ATP to ADP and releaseP1and be converted into a weakly bound state.The leading head is now ready to bind AIP and swing the trailing head in front of it to repeatthe cycle.Becausethis cycle requires one head to always be firmly attached to a B-tubulin subunit in a protofilament, kinesin-1 is exceptionallv orocessiveas it moves down a microtubule.

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Myosin

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< FIGURE 18.23 Convergentstructural evolution of the ATPbinding coreof myosin headand kinesin.The commoncatalytic coresof myosin andkinesin are shownin yellow,the nucleotide in red,andthe leverarm(formyosin-ll) and ( f o rk i n e s i n - 1 ) l i n k edr o m a i n in lightpurpleIModified from R D V a l ea n d R .A M i l l i g a n , 2000, Sclence288:88 l

Nucleotide

When the x-ray structure of the kinesin head was determined, it revealeda major surprise-the catalytic core has the same overall structure as myosin's (Figure 18-23)l This occurs despiteno amino acid sequenceconservation, arguing strongly for convergentevolution to make a fold that can utilize the hydrolysis of ATP ro generate work. Moreover, the same type of three-dimensionalstructure is seenin small GTP-binding proteins, such as Ras, that undergo a conformational change on GTP hydrolysis (see Figure15-8).

Dynein Motors TransportOrganellesTowardthe ( - ) E n do f M i c r o t u b u l e s In addition to kinesin motors, which primarily mediate anterograde(+) end directedtransport of organelles,cellsuse another motor, cytoplasmic dynein, to transport organelles in a retrograde fashion toward the (- ) end of microtubules.This motor protein is very large, consistingof two Iarge (>500 kDa), two intermediate.and two small subunits. It is responsible for the ATP-dependent retrograde transport of organelles toward the (-) ends of microtubules in axons, as well as many other functions we consider in the next sections.Compared to myosins and kinesins,the family of dynein-relatedproteins is not very diverse. Like kinesin-1, cytoplasmic dynein is a two-headed molecule, built around two identical or nearly identical heavy chains. However, becauseof the enormous size of the motor domain, dynein has been lesswell characterizedin terms of its mechanochemicalacivity. A single dynein heavy chain consistsof a stem and a round head domain containing the ATPase activity, from which protrudes a stalk (Figure 1824). At the end of the stalk is a microtubule-binding site. Electron microscopy suggeststhat the power stroke of 774

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dynein involves the rotation of the round head domain (Figure18-25). Unlike kinesin-1, dynein cannot mediate cargo transport by itself. Rather, dynein-related transport requires dynactin, a large protein complex that both links dynein to its cargo and regulatesits activity (Figure 1,8-26).Dynactin consists of 11 subunits, functionally organized into two domains. One domain is built around eight copies of the actin-relatedprotein Arp1, which assemblesinto a short filament. The end correspondingto the (+ ) end of an actin filament is capped by capping protein; a number of subunits are associatedwith the (-) end. This Arp-related domain is responsiblefor binding cargo. The seconddomain of dynactin consists of a long protein called p150cl"'d,

Microtubule binding domain

Stem

Head

Stalk

FIGURE 18-24Thedomainstructureof cytoplasmic dynein. EachATPase headdomainof dyneinconsists of sevenrepeated motifslikepetalson a flower.Emerglng fromthisisa coiled-coil domainwith a microtubule bindingsiteat the end At leftisshowna numberof additional subunits thatareassociated with the heavy chains andlinkdyneinto cargothroughdynactin. fromR D [Modified Vale, 2003,Cell112:467 I

C E L LO R G A N I Z A T I O N A N D M O V E M E N TI I : M I C R O T U B U L EASN D I N T E R M E D I A TFEI L A M E N T S

Pre-stroke

which contains the dynein binding site and has a microtubule binding site at one end. Holding the two dynactin domains together is a protein called dynamitin-so named it dissociates(or "blows becausewhen it is overexpressed, apart") the two domains, making a nonfunctional complex. This feature has been very useful experimentallybecauseit has allowed researchersto identify processesthat are dependenton dynein-dynactin,which do not occur in cells. dynamitin-overexpressing

Post-stroke

K i n e s i n sa n d D y n e i n sC o o p e r a t ei n t h e T r a n s p o r ot f O r g a n e l l e sT h r o u g h o u t h e C e l l

FIGURE18-25 The power stroke of dynein. (a) Multiple dyneinmoleculesin their prestroke imagesof purifiedsingle-headed and and poststrokestateswere recordedin an electronmicroscope then averagedThe left imageshowsdyneinin the ADP-P;state, the prestrokestate,and the right imagein a which represents poststrokestate (b) A comparisonof the images nucleotide-free mechanisminvolvesa changein showsthat the force-generation orientationof the headrelativeto the stem,causinga movementof et al, modified fromS A Burgess stalk Ipart(a) the mrcrotubule-binding 421:715; courtesy of S A Burgess 2003,Nature l

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Both dynein and kinesin family members play important roles in the microtubule-dependent organization of organellesin cells (Figure 1'8-27).Becausethe orientation of microtubules is fixed by the MTOC, the direction of transport-toward or away from the cell center-depends on the motor protein. For example' the Golgi apparatus collects in the vicinity of the centrosome,where the (-) ends of microtubules lie, and is driven there by dyneindynactin. In addition, secretorycargo emerging from the endoplasmic reticulum is transported to the Golgi by dynein-dynactin.Conversely,the endoplasmicreticulum is spreadthroughout the cytoplasm and is transported there by kinesin-1,which moves toward the peripheral (+) ends of microtubules.Someorganellesof the endocyticpathway are also associatedwith dynein-dynactin,including late endosomesand lysosomes.Kinesinshave also been shown to transport mitochondria, as well as nonmembranouscargo such as specificmRNAs encodingproteins that need to be localizedduring development. organellesin an We have seenhow kinesin-1transports '$7hat to the happens axons. down fashion anterograde motor when it gets to the end of the axon? The answer is that it is carried back in a retrograde fashion on organelles

18-26Thedynactincomplex < FIGURE linkingdyneinto cargo.(a)Onedomarnof t h e c o m p l ebx i n d sc a r g oa n di s b u i l ta r o u n da t i g h st u b u n i t s s h o rfti l a m e nmt a d eu p o f a b o u e by protein Arpl capped of the actrn-related C a p ZA. n o t h edr o m a i nc o n s i s tosf t h e p r o t e i n p 15 O s t u "woh, i c hh a sa m i c r o t u b ubl ei n d i n g in attaching siteon itsdistalendandisinvolved c y t o p l a s mdi yc n e i nt o t h e c o m p l e xD y n a m i t i n h o l d st h et w o p a r t so f t h e d y n a c t icno m p l e x n i c r o g r a pohf a m e t a l t o g e t h e (r b )E l e c t r om from replicaof the dynactincomplexisolated b r a i nT h eA r p l m i n i f i l a m e(npt u r p l ea)n dt h e s i d ea r m( b l u ea) r e d y n a m i t i n /5p01s l ' " d h i g h l i q h t eIdP a r(ta )m o diief df r o mT A S c h r o e r , 2004, Annu Rev.Cell Dev. Biol 20:759 Part (b) f r o m D M E c k l e ye t a l , 1 9 9 9 ,J C e l lB i o l 1 4 7 : 3 0 7l : I C R O T U B U L E - B A SMEODT O RP R O T E I N S K I N E S I NA SN D D Y N E I N SM

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Video:Transportof SecretoryVesiclesDown Microtubules Video:Transportof VesiclesDown Microtubules from the Endoplasmic Reticulum to the Golgi cynein $ C y t o p l a s m id S K i n e s i nf a m i l y m e m b e r

A FIGURE 18-27Organelletransportby microtubule motors.Cytoplasmic (red)mediate dyneins retrograde transport of organelles towardthe(-) endof microtubules (cellcenter); (purple) kinesins mediate anterograde transport towardthe (+) end(cellperiphery) Mostorganelles haveoneor more

transported by cytoplasmic dynein. Thus kinesin-1 and dynein can associatewith the sameorganelleand a mechanism must exist that turns one motor off while activating the other, although such mechanisms are not yet fully understood. Much of what we know about the regulation of microtubule-basedorganelletransport comes from studiesusing fish (e.g., angelfish)or frog melanophores.Melanophores are cells of the vertebrate skin that conrain hundreds of dark melanin-filledpigment granulescalled melanosomes. Melanophoreseither have their pigment granulesdispersed, in which case they make the skin darker, or aggregatedat the center,which makesthe skin paler (Figure18-28).These changesin skin color, mediatedby neurotransmittersin the fish and regulated by hormones in the frog, serve in either camouflagein the caseof the fish or enhancesocialinteractions in the frog. The movementof the granulesis mediated by changes in intracellular cAMp and is dependent on microtubules. Studies investigating which motors are involved have shown that pigment granule dispersionrequires kinesin-2,whereasaggregationrequirescytoplasmic dynein/dynactin. The first hints of how these activities

CHAPTER 18

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microtubule-based motorsassociated withthem lt shouldbe noted thatthe association of motorswith organelles varies by celltype,so someof theseassociations maynotexistin allcells, whereas others notshownherealsoexistERGIC:ER-to-Golqi intermeotare comoartment

might be coordinated came from the finding that overexpressionof dynamitin inhibited granule transporr in both directions.This surprisingresult was explainedwhen it was found that dynactin binds not only to cyroplasmicdynein but also to kinesin-2-and may coordinate the activity of the two motors. The association of dynein and kinesin-2 with the same organelleis not limited to melanosomes;it has recently been suggestedthat these motors may cooperate to localize late endosomes/lysosomes and mitochondria appropriately in somecells.Thus the concept that organellescan have a number of distinct motors associatedwith them is not the exception but an emergingtheme.

Kinesinsand Dyneins: Microtubule-BasedMotor Proteins r Kinesin-1 is a microtubule (+) end-directed ATPdependent motor that transports membrane-bounil organelles(seeFigure 18-20).

C E L LO R G A N T Z A T T O N D M O V E M E N Tt l M I C R O T U B U L E AN AS N D I N T E R M E D I A TFEI L A M E N T S

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in FishMelanophores and Dispersion of PigmentGranules ViU"o:Aggregation 18-28Movementof pigmentgranulesin frog < FIGURE (a)Diagram of the microtubule-based melanophores. to the levelof cAMP according of melanosomes reorganization dyneinand by cytoplasmic areaggregated Melanosomes in the (b) of melanosomes Visualization kinesin-2. by dispersed for microscopy stateasseenby immunofluorescence dispersed (blue), andpigment (green), the DNAin the nucleus microtubules andS Rogers, (red)Ipart(a)modified fromV Gelfand granules

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a very similar structure, can propel a cell, such as sperm' through liquid. Cilia and flagella contain many different microtubule-basedmotors: axonemal dyneins are responsible for the beating of flagella and cilia, whereas kinesin-2 and cytoplasmic dynein are responsiblefor flagella and cilia assemblyand turnover.

r Kinesin-1 consistsof two heavy chains, each with an Nterminal motor domain and two light chains that associate with cargo (seeFigure 18-1,9). r The kinesin superfamily includesmotors that function in interphase and mitotic cells, transporting organelles,and sliding antiparallel microtubules and even includes one class that is not motile but destabilizesmicrotubule ends (seeFigure 18-21). r Kinesin-1 is a highly processivemotor becauseit coordinatesATP hydrolysisbetweenits two headsso that one head is always firmly bound to a microtubule (seeFigure L8-22). r Cytoplasmic dynein is a microtubule (-) end-directed ATP-dependent motor that associateswith the dynactin complex to transport cargo (seeFigure 1'8-25).

E u k a r y o t i cC i l i aa n d F l a g e l l aC o n t a i nL o n g D o u b l e tM i c r o t u b u l e sB r i d g e db y D y n e i n Motors Cilia and flagellarange in length from a few micrometers to more than 2 mm for someinsectsperm flagella.They possess a central bundle of microtubules' called the axoneme,which consistsof a so-called9 r 2 arcangementof nine doublet microtubules surrounding a central pair of singlet, yet ultrastructurally distinct, microtubules (Figure 18-29a, b). Each of the nine outer doublets consistsof an A microtubule with 13 protofilaments and a B microtubule with 10 protofilaments. All the microtubules in cilia and flagella have the samepolarity: the (+ ) ends are located at the tip. At its point of attachment in the cell, the axoneme connects with the basal body, a complicated structure containing nine triplet microtubules(seeFigure 1'8-29a). The structure of the axoneme is held together by three

r Kinesins and dyneins associatewith many different organellesto organizetheir location in cells (seeFigure 18-27).

lTlE{ Ciliaand Flagella:MicrotubuleBasedSurfaceStructures Cilia and flagella are relatedmicrotubule-basedand membranebound extensionsthat proiect from many protozoa and most animal cells. Abundant motile cilia are found on the surface of specific epithelia, such as those that line the trachea,where they beat in an orchestratedwavelike fashion to move fluids. Animal-cell flagella, which are longer but have

blets toward the central Pair. The major motor protein present in cilia and flagella is axonemal dynein, alarge, multisubunit protein related to cytoplasmic dynein. Two rows of dynein motors are attached peiiodically down the length of each A tubule of the outer doublet microtubules; these are called the inner-arm and outer-arm dyneins (seeFigure 1'8-29b).It is thesedynein motors interacting with the adjacent B tubule that bring about c i l i a a n d f l a g e l l ab e n d i n g .

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FIGURE18-29 Structural organization of cilia and flagella. (a)Ciliaand flagellaare assembled from a basalbody,a srructure b u i l ta r o u n dn i n el i n k e dt r i p l e tm i c r o t u b u l eC s o n t i n u o uw s iththeA and B microtubules of the basalbody are the A and B tubulesof the axoneme-the membrane-bound coreof the ciliumor flagellum Betweenthe basalbody and axonemeis the transitional zone The diagramand accompanying transverse sectionsof the basalbody, transitional zone,and axonemeshow their intricatesrrucrures (b) Thin sectionof a transverse sectionof a cilium(with plasma membraneremoved)with a diagramto show the identityof the structures.IPart(a)modifiedfrom S K Dutcher, 2OO1 , Curr.Opin CeltBiot 13:49-54;courtesy of 5 Dutcherpart(b)courtesy of L Tilneyl

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C i l i a r ya n d F l a g e l l a B r e a t i n gA r e p r o d u c e db y C o n t r o l l e dS l i d i n go f O u t e r D o u b l e t Microtubules Cilia and flagella are motile structuresbecauseactivation o{ the axonemaldynein motors inducesbendingin them. A close examination of this motility using video microscopy reveals

778

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that a bend startsat the baseof a cilium or flagellum and then propagatesalong the structure (Figure 18-30). A clue to how this occurscame from studiesof isolatedaxonemes.In classic experlments, axonemes were treated with a protease that cleavesthe nexin links. rWhenATP was added to the treated axonemes,the doublet microtubules slid past one another as dynein, attached to the A tubule of one doublet, ,,walked"

C E L LO R G A N T Z A T T OANN D M O V E M E N Ti l : M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

shows 18-30Videomicroscopy FIGURE < EXPERIMENTAL Chlamydomonas propel and sperm ffageffar movementsthat the cellsaremovingto the left.(a)In the forward. In bothcases, o r i g i n a taet e v e so f b e n d i n g t y p i c asl p e r mf l a g e l l u ms,u c c e s s iwv a out towardthe tip; thesewavespush the baseandarepropagated againstthe waterand propelthe cellforward.Capturedin this a bendat the baseof the spermin sequence, multiple-exposure distallyhalfwayalongthe (top) moved has frame first the b y t h e l a s tf r a m eA p a i ro f g o l db e a d so n t h ef l a g e l l u m flagellum a r es e e nt o s l i d ea p a r ta st h e b e n dm o v e st h r o u g ht h e i rr e g i o n ' occursin two (b)Beatingof the two flagellaon Chlamydomonas (top and the frames) three stroke the effective stages,called strokepullsthe Theeffective f rames). stroke(remarning recovery stroke,a throughtlrewater.Duringthe recovery organism differentwaveof bendingmovesoutwardfromthe basesof the f l a g e l l ap,u s h i n tgh ef l a g e l la l o n gt h e s u r f a coef t h e c e l lu n t i lt h e y strokeBeating reachthe positionto initiateanothereffective (a)fromC Brokaw, occurs5-10 timespersecond.IPart commonly Part(b) of C Brokaw photograph; courtesy cover 1991, J CettBiol114(6): of S Goldstein l courtesy

Careful examination of flagella on the biflagellate green

microscopy revealedthat the particles moved betweenthe outer doublet microtubules and the plasma membrane (Figure 1.8-32).Analysis of algal mutants demonstrated thai the anterogrademovement is powered by kinesin-2 and that retrogrademovement is powered by cytoplasmic dynein. \fhat is the function of IFT? Becauseall the microtubules have their growing (+ ) ends at the flagella tip, this is the site at which new tubulin subunits are added' In cells defective

down the B tubule of the adiacentdoublet (Figure 18-31b, c). In an axoneme with intact nexin links, the action of dynein inducesflagellar bendingas the microtubule doubletsare connectedto one another (Figure 18-31a). How specificsubsets of dynein are activatedand how a wave of activation is propagateddown the axoneme are not yet understood.

t o v e sM a t e r i a lU p a n d I n t r a f l a g e l l aTr r a n s p o r M D o w n C i l i aa n d F l a g e l l a Although axonemal dynein is involved in bending flagella, another type of motility has been more recently observed.

cific to IFT.

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< FIGURE 18-31Ciliaand flagellabendingmediatedby axonemaldynein.(a)Axonemal dyneinattached to an A tubule o f a n o u t e rd o u b l ept u l l so n t h e B t u b u l eo f t h e a d j a c e nt ut b u l e t r y i n gt o m o v et o t h e ( - ) e n d B e c a u st e h ea d j a c e nt ut b u l e a sre tethereb d y n e x i nt,h ef o r c eg e n e r a t ebdy d y n e i nb e n d st h ec i l i u m o r f l a g e l l u m( b, )E x p e r i m e net avli d e n cfeo r t h e m o d e il n ( a ) .W h e n t h e n e x i nl i n k sa r ec l e a v ew d i t h a p r o t e a saen dA T pa d d e dt o i n d u c ed y n e i na c t i v i t yt h, e m i c r o t u b udl eo u b l e tssl i d ep a s to n e a n o t h e r( c )E l e c t r om n i c r o g r a pohf t w o d o u b l em t i c r o t u b u l iensa protease-treated axoneme incubated with ATp In the absence of c r o s s - l i n k ipnrgo t e i n sd,o u b l em t i c r o t u b u lselsi d ee x c e s s i v eTlhy e d y n e i na r m sc a nb e s e e np r o j e c t i nf g r o mA t u b u l e a s n di n t e r a c t i n g with B tubulesof the left microtubule doublet.Ipart(c)courtesv of P S a t i rl

Figure 18-13), which lacks both the central pair of microtubules and the dynein side arms. The primary cilium is assembledfrom the centrosome,with one of the centriolesfunctioning as its basal body, Clues to the function of the primary cilium came from the discoverythat loss of a mammalian homolog of a ChlamydomonasIFT protein resultsin defectsin the primary cilium and causesautosomalrecessivepolycystic kidney disease(ADPKD). It is believedthat the primarv cilia on the epithelial cells of the kidney collecting rubule act as mechanochemicalsensorsto measurethe rate of fluid flow by the degreeto which they are bent. Primary cilia are also involved in other sensoryroles. The senseof smell is due to reception of odorants by the odorant receptorslocated in the primary cilium of olfactory sensory neurons in the nose. In another example, the rod and cone

this transport causeretinal degeneration.Given these roles for primary cilia in sensorydetection,it is not surprising that defectsin primary cilia can have wide,ranging consequences. For example, patienrs with Bardet-Biedl syndrome, which affects basal bodies and cilia, have retinal degenerationand cannot sme as well as several other disorders, suggesting that primar cilia are involved in many processesyet to be uncovered.

Ciliaand Flagella:Microtubule-Based Surface Structures

D e f e c t si n I n t r a f l a g e l l aTr r a n s p o r C t ause D i s e a s eb y A f f e c t i n gS e n s o r yp r i m a r yC i l i a \'lost vertebrate cells contain a solitary nonmotile cil_ ri ''r, known as the plimary cilium (Frgurelg-33; see 780

CHAPTER 18

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and flagella are microtubule-basedcellsurface structures with a characteristiccentral pair of singlet microtubules and nine sets of ourer doublet microtubules (seeFigure 18-29). r All cilia and flagella grow from basal bodies, srrucrures with nine setsof outer triplet microtubules and closely related to centrioles.

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APC/Cactivatedand c o h e s i n sd e g r a d e d AnaphaseA: Chromosome movementto poles A n a o h a s eB : S p i n d l ep o l e s e p a r a t i o n

Nuclearenvelopereassembly, Assemblyof contractilering

attachedby cohesins(Figure 18-34b). As discussedin more detail in Chapter 20, all theseeventsare coordinated by a rapid increasein the activity of the mitotic cyclin-CDK complex, which is a kinase that phosphorylatesmultiple protelns. The next stageof mitosis,prometaphase,is initiated by the breakdown of the nuclear envelope and the nuclear pores and disassemblyof the lamin-basednuclear lamina. Microtubules assembledfrom the spindle poles searchand "capture" chromosome pairs at specialized structures called kinetochores.Each chromatid has a kinetochore,so sister chromatids have two kinetochores, each of which becomes attached to opposite spindle poles during prometaphase. lfhen they are attached to both spindle poles, sister chromatids align equidistant from the two Prometaphase spindlepolesin a processknown as congression. continuesuntil all chromosomeshave congressed,at which point the cell entersthe next stage,metaphase,defined as the stage when all the chromosomes are aligned at the metaphaseplate. The next stage, anaphase,is induced by activation of t h e a n a p h a s e - p r o m o t i n gc o m p l e x / c y c l o s o m e( A P C / C ) . The activated APC/C leads to the destruction of the cohesins,proteins holding the sister chromatids together,so that now each separatedchromosome can be pulled to its respectivepole by the microtubules attachedto its kinetochore. This movement is known as anaphaseA. A separate

Reformationof interphasemicrotubulearray, Contractilering forms cleavagefurrow

and distinct movement also occurs: the movement of the spindle poles farther apart in a process known as inapbase B. Now that the chromosomeshave separated' the cell enters telophase,when the nuclear envelope reforms, the chromosomes decondense, and the cell is pinched into two daughter cells by the contractile ring during cytokinesis.

C e n t r o s o m eD s u p l i c a t eE a r l yi n t h e C e l lC y c l ei n Preparationfor Mitosis In order to separatethe chromosomesat mitosis, cells duplicate their MTOCs-their centrosomes-coordinately with the duplication of their chromosomesin S phase(Figure 18-35). The duplicated centrosomesseparateand become the two MTOCs-the spindle poles-of the mitotic spindle.The number of centrosomesin animal cells has to bi .'..y carefully controlled. In fact, many tumor cells have more than two centrosomes,which contribute to genetic instability resulting in missegregationof chromosomes a.td hence aneuploidy (unequal numbers of chromosomes). As cells enter mitosis, the activity of the two MTOCstheir ability to nucleate microtubules-increases greatly as they accumulate more pericentriolar material. Becausethe microtubules radiating from thesetwo MTOCs now resemble stars, they are often called mitotic asters.

783

FIGURE 18-35Relationof centrosome duplicationto the cell cycle.Afterthe pairof parentcentrioles (green) separates slightly, a d a u g h t ecre n t r i o (l eb l u eb) u d sf r o me a c ha n de l o n g a t eBsyG r , growthof the daughter centrioles iscomplete, but thetwo pairs r e m a iw n i t h i na s i n g l cee n t r o s o mcaolm p l e xE a r l iyn m i t o s i st h, e centrosome splits, andeachcentriole pairmigrates to opposite sides of the nucleusTheamountof pericentriolar material andthe activity to nucleate microtubule assembly increases greatlyin mitosisIn m i t o s i st h, e s eM T O Casr ec a l l e sd p i n d loeo l e s

astral microtubules, which extend from the spindle poles to the cell cortex (Figure 18-36). By interactingwith the cortex, the astral microtubulesperform the critical function of orienting the spindle with the axis of cell division. The secondset link the spindlepolesto the kinetochoreson the chromosomesand are therefore called kinetochore microtubules. This set of microtubules first finds the chromosomes, then attaches them through the two kinetochoresto both spindle poles and at anaphaseA transportsthem to the poles.The third set of microtubulesextendfrom eachspindlepole body toward the oppositeone and interacttogetherin an antiparallelmanner;these are calledpolar miuotubules.Thesemicrotubulesare responsible initially for pushing the duplicatedcentrosomesapart during prophase,then for maintaining the srructure of the spindle, and then for pushingthe spindlepolesapart in anaphaseB. Note that all the microtubules in each half of the symmetrical spindle have the same orientation except for some polar microtubules, which extend beyond the midpoint and interdigitate with polar microtubules from the opposite pole.

M i c r o t u b u l eD y n a m i c sI n c r e a s eD s r a m a t i c a l l yi n Mitosis

Beforewe discussthe mechanismsinvolved in this remarkable process,it is important to understandthe three distinct classes of microtubulesthat emanarefrom the spindlepoles,which is where all their (-) ends are embedded.The first classis the

Although we have drawn static imagesof the stagesof mitosis, microtubules in all stagesof mitosis are highly dynamic. As we saw above,as cellsentermitosis,the ability of their centrosomesto nucleate assembly of microtubules i n c r e a s e ss i g n i f i c a n t l y ( s e e F i g u r e 1 8 - 3 5 ) . I n a d d i t i o n , microtubulesbecomemuch more dynamic. How was this determined? In principle, you could watch microtubules and

(a)

(b)

T h e M i t o t i c S p i n d l eC o n t a i n sT h r e eC l a s s e os f Microtubules

Zone of interdigitation 'l rKinetochoreMT Kinetochore

Pole (centrosome) Polar MTs

A FIGURE18-36 Mitotic spindles have three distinct classesof microtubules.(a) In this high-voltage electronmicrograph, m i c r o t u b u l ewse r es t a i n e dw i t h b i o t i n - t a g g eadn t i - t u b u l iann t i b o d i e s to increase their size The largecylindrical objectsare chromosomes, (b) Schematic diagramcorresponding to the metaphasecellin (a) Threesetsof microtubules (MTs)make up the mitoticapparatusAll

784

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Chromosome

the microtubules havetheir(-) endsat the poles. Astralmicrotubules projecttowardthe cortexandarelinkedto it Kinetochore polarmicrotubules microtubules areconnected to chromosomes prolect towardthe cellcenterwith theirdistal(+) endsoverlapping Thespindle poleandassociated microtubules isalsoknownasa (a)courtesy mrtotrc aster[Part ofJ R Mclntosh I

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

follow their individual behaviors.However, in general,there are too many microtubules in a mitotic spindle to do this. To get an averagevalue for how dynamic microtubules are, researchershave introduced fluorescent labeled tubulin into cells, which becomesincorporated randomly into all microtubules. They then bleach the fluorescentlabel in a small region of the mitotic spindle and measurethe rate at which fluorescencecomes back in a technique known as fluorescence recouery after photobleaching (FRAP) (see Figure 1,0-1,2). Sincethe recovery of fluorescenceis due to assemblyof new microtubules from soluble fluorescent tubulin dimers. this representsthe averagerate at which microtubules turn over. In a mitotic spindle,their half-life is about 15 seconds,whereas in an interphasecell, it is about 5 minutes.It should be noted that theseare bulk measurementsand individual populations of microtubulescan be more stable.as we will see. What makes microtubulesmore dynamic in mitosis?Dynamic instability is a measure of relative contributions of growth rates, shrinkage rates, catastrophes, and rescues. Analysis of microtubule dynamicsin vivo shows that the enhanced dynamics of individual microtubules in mitosis is mostly generatedby increasedcatastrophesand fewer rescues, with little change in rates of growth (i.e., lengthening) or shrinkage (i.e., shortening).Studieswith extracts from frog oocytes have suggestedthat the main factor enhancing catastrophes in both interphaseand mitotic extractsis depolymerizing by kinesin-13 proteins. This can be seenin an in vitro

T u b u l i n+ k in e s i n - 31

T u b u l i na l o n e

assaywhere microtubule assemblyfrom pure tubulin is nucleated from purified centrosomes(Figure 1'8-37a).If kinesin-13 is added into the assay,many fewer microtubules are formed. However, if the stabilizing microtubule-associatedprotein called XMAP215 is added with the kinesin-13,many microtubules are formed due to a dramatic reduction in catastrophe frequency.It turns out that the activity of kinesin-13 does not change significantly during the cell cycle, whereas the activity of XMAP215 is inhibited by its phosphorylationduring mitosis (Figure 1,8-37b).This results in much more unstable microtubules as the cell enters mitosis (Figure 1'8-37c).

l u r i n gM i t o s i s M i c r o t u b u l e sT r e a d m i lD In addition to being highly dynamic in terms of assemblyand disassembly microtubules in the mitotic spindle are treadmilling-that is, constantly adding dimers at the microtubule (+ ) end and losingthem at the (- ) end.Treadmillingcan be revealed by expressingsmall amounts of GFP-tubulin in mitotic cells, which is incorporated randomly into microtubules, giving rise to fluorescent speckleswhere the concentration of incorporated GFP-tubulin happens to be higher. These speckles orovide a marker on the microtubules, so by following them in liuing cell, one can determine if the microtubules are station" with respectto spindlepolesor moving (Figure18-38).Exary periments such as theseshow that microtubules are constantly treadmilling in prometaphase,metaphase'and anaphase.

T u b u l i n+ k i n e sni - 13 + XMAP215

(b)

.2 o^

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ro

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N (L

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18-37 FIGURE < EXPERIMENTAT in mitosis Microtubuledynamicsincreases due to lossof a stabilizingMAP.(a)These theabilityof centrosomes reveal threepanels frompuretubulin microtubules to assemble protein (/eft);tubulinandthe destabilizing ( m i d d l e ) : k i n o r t u b u l i n , e s i n - 1a3n, d kinesin-13 protein 15 (Xenopus XMAP2 the stabilizing shows analysis Further MAPof 215kD)(ngrht). 5 isto that the majoreffectof XMAP21 3 induced by kinesin-1 catastrophes suppress in (b)Theincreased of microtubules dynamics 5 of XMAP21 isdueto the inactivation mitosis (c)Diagram the relating by phosphorylation in of microtubules stabilities dfferent Notethatin addition andmitosis. interphase and betweeninterphase stability to differential lo nucleate the abilityof MTOCs mitosis, in dramatically alsoincreases microtubules (a)fromKinoshita (seeFigure18-35). mitosis [Part Part(b)from et al, 2001,Science2g4:]340-1343 CellBiol12t267-2731 et al. 2002,Trends Kinoshita

Time (c) 0)

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1.2

E X P E R I M E N TFA TLG U R1E 8 - 3 8M i c r o t u b u l ei sn m i t o s i s treadmilltoward the spindlepoles.(a)A smallamountof GFpptKl cellsto visualize tubulinwasexpressed in cultured microtubules "speckles" andgenerate alongthemdueto randomuneven incorporation (b)Byfollowingspeckles of GFP-tubulin in time,the

direction andrateof movement of microtubules canbe determined, color-coded according to the rateshownin (c) Thisanalysis shows t h a tm i c r o t u b u lter es a d m i(lol r" f l u x " )i n t h e s ec e l l sa t a b o u 0 t 7 pm/mintowardthe poles[From L A Cameron, 2006, J Cett Biol 173: 173-179 l

T h e K i n e t o c h o r eC a p t u r e sa n d H e l p sT r a n s p o r t Chromosomes

kinetochore layers,with the (+ ) ends of the kinetochore microtubules terminating in the outer layer (Figure 18-39). Yeast kinetochores are attached by a single microtubule to their pole, human kinetochores are attached by about 30, and plant chromosomesby hundreds. How doesa kinetochorebecomeattachedto microtubules in prometaphase?Microtubules nucleated from the spindle poles are very dynamic, and when they contact the kinetochore, either laterally or at their end, this can lead to chromosomal attachment (Figure 1.8-40a, steps IE and IIil). Microtubules "captured" by kinetochores are selectively

To attach to microtubules, each chromosome has a specialized structure called a kinetochore. It is located at the centromere,a constricted region of the condensedchromosome defined by centromeric DNA. Cenrromeric DNA can vary enormouslyin size;in budding yeastit is about 125 bp, whereasin humans it is on the order of 1 Mbp. Kinetochoris contarn many protein complexesto link the centromeric DNA eventually to microtubules. In animal cells,the kinetochore consists of an inner centromere and inner and outer

J o q)

-a) u

Microtubules

FIGURE 18-39Thestructureof a mammaliankinetochore. Diagram andelectron micrograph of a mammalian kinetochore 786

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[Modified from B McEwenet al , i 998, Chromosoma107i366:courtesvof B M c E w e nl

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

stabilizedby reducingthe levelof catastrophes, therebypromoting the chancethat the attachment will persist. Recent studies have uncovered a mechanism involving the small Ran GTPasethat enhancesthe chancethat microtubules will encounter kinetochores.Recall that the Ran GTPasecycle is involved in transport of proteins in and out of the nucleusthrough nuclearpores (Chapter13; seeFigure 1.3-36).During mitosis, when the nuclear membrane and pores have disassembled,an exchangefactor for the Ran GTPase is bound to chromosomes,thereby generating a higher local concentration of Ran-GTP. Becausethe enzyme that stimulates GTP hydrolysis on Ran-Ran GAP-is evenly distributed in the cytosol, this generatesa gradient of Ran-GTP centeredon the chromosomes.Ran-GTP induces

the release of factors that promote the growth of microtubules, in this way biasing growth of microtubules nucleated from spindle poles toward chromosomes. Once attached to microtubules, the motor protein dynein/dynactin located at the kinetochore moves the chromosome pair down the microtubule toward the spindle pole (Figure 18-40a, step Z). In this orientation, the unoccupied kinetochore on the opposite side is pointing toward the distal spindle pole, and eventually a microtubule from the distal pole will capture the free kinetochore. The chromosome pair is now said to be bi-oriented (Figwe 78-40a, step B).\7ith the two kinetochores attached to opposite poles, the duplicated chromosome is now under tension, being pulled in both directions.

Kinesin-4

5& Tethereddynactin-dynein complex Attachmentby kinesin-7; m i c r o t u b u l ea s s e m b l y

Kinesin-13

+

-> Chromosome movement

FIGURE18-40 Chromosome Gaptureand congression in prometaphase. (a) In the first stageof prometaphase, chromosomes becomeattached,eitherto the end of a microtubule(IE) or to the sideof a microtubule(IE) The duplicatedchromosomeisthen drawn as dynein/dynactin toward the spindlepole by kinetochore-associated (E). Eventually, this motor movestowardthe (-) end of a microtubule a microtubulefrom the opoositeoolefindsand becomesattached is now saidto be biand the chromosome to the free kinetochore. then moveto a central oriented(!) The bi-orientedchromosomes point betweenthe spindlepolesin a processknown as chromosome congressionNote that duringthesesteps,chromosomearmspoint

Forcefrom dynein and m i c r o t u b u l e ds e p o l y m e r i z a t i o n b y k i n e s i n - 1 3a,n d b Y k i n e s i n - 4 o n c n r o m o s o m ea r m s

pole:thisisdueto chromokinesin/kinesinspindle awayfromtheclosest towardthe(+) endsof the armsmoving on thechromosome 4 motors of (b)Congressron oscillations bidirectional involves polarmicrotubules on shortening microtubules with onesetof kinetochore chromosomes, on theother.Ontheshortening onesideandtheothersetlengthening anda protein disassembly microtubule stimulates 3 side.a kinesin-'1 towardthe pole On movesthechromosome complex dynein/dynactin protein a CENP-E/kinesin-7 microtubules, thesidewithlengthening alsocontains Thekinetochore holdson to thegrowingmicrotubule from notshownherelModified proteincomplexes manyadditional Clevelandet al , Ce//112:407-421 l MtTOSTS

787

This tensionis a key part of the mechanismwhereby a cell properly segregates its chromosomes.'Whenthe chromosome is correctly bi-oriented, the cell sensesthe tension produced, and the attachments are stabilized. What happens if both kinetochoresaccidentallybecomeattachedto the samepole? In this situation, there is very little tensionon the kinetochore microtubules, and turnover of kinetochore microtubules is enhanced.Exactly how the cell sensestension is unclear.

D u p l i c a t e dC h r o m o s o m eAs r e A l i g n e db y M o t o r s a n d T r e a d m i l l i n gM i c r o t u b u l e s During prometaphase,the chromosomescome to lie at the midpoint betweenthe two spindlepoles,in a processknown as chromosome congression.During this process,chromosome pairs often oscillatebackward and forward before arriving at the midpoint. Chromosomecongressioninvolves the coordinated activity of severalmicrotubule-basedmotors together with microtubule treadmilling (Figure18-40b).The oscillating

behavior involves lengthening of microtubules attached to one kinetochoreand shorteningmicrotubulesattachedto the other kinetochore, all without losing their attachments.In metazoans, several microtubule-basedmotors contribute to this process.First, dynein/dynactin provides the strongest force pulling the chromosome pair toward the more distant pole. This movementrequiressimultaneousshorteningof the microtubule, which is enhancedby kinetochore-localized kinesin-13. The microtubules associatedwith the other kinetochore have to grow as the chromosomemoves.Anchored at this kinetochore is a kinesin-relatedmotor. kinesin-7.that holds on to the growing (+ ) end of the lengtheningmicrotubule.Contributing to congressionis also another kinesin, kinesin-4, associated with the chromosomearms.Kinesin-4,a (+l end-directedmotor, interactswith the polar microtubulesto pull the chromosomes toward the center of the spindle. rWhen the chromosomes have congressed to the metaphase plate, dynein/ dynactin is releasedfrom the kinetochores and streams down the kinetochore microtubules to the poles. These different

';Hn"nnun"

(-)

+-q, Sliding

--+

A FIGURE 18-41Chromosome movementand spindlepole separationin anaphase. Anaphase A movement ispowered by microtubule-shortening kinesin-13 proteins (E[) at the kinetochore a n da t t h es p i n d l p e o l e( E E ) N o t et h a tt h ec h r o m o s o maer m ss t i l l pointawayfromthespindle polesdueto associated chromokinesin/ kinesin-4 members, sothe depolymerization forcehasto be able 788

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to overcome theforcepullingthe armstowardthecenterof the s p i n d l eA n a p h a sBea l s oh a st w o c o m p o n e n tssl i:d i n o gf a n t i p a r a l l e l polarmicrotubules poweredby a kinesin-5 (+) end-directed motor (E[) andby dynein/dynactin located at thecellcortexpullingon (EE). [Modif astralmicrotubules iedfromCleveland erat, Cett112:40j421 I

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

activities and opposing forces work together to bring all the chromosomes to the metaphaseplate, and when successfully there,the cell is ready for anaphase.As we discussin Chapter 20, the cell has a mechanism-the spindle checkpoint-to ensurethat anaphasedoesnot proceeduntil all the chromosomes have arrived at the metaphaseplate.

AnaphaseA Moves Chromosomes to Polesby M i c r o t u b ul e S h o r t e n i n g The onset of anaphaseA is one of the most dramatic movements that can be observedin the light microscope.SuddenlS the two sister chromatids separatefrom each other and are drawn to their respectivepoles.This appearsto work so fast becausethe kinetochore microtubules are under tension, and as soon as the cohesin attachments between chromosomes are released,the chromosomesare free to move. Experimentswith isolatedmetaphasechromosomeshave shown that anaphaseA movement can be powered by microtubule shortening,utilizing the stored structural strain releasedby removing the GTP cap. This can be nicely demonstrated in vitro. lfhen metaphasechromosomesare added to purified microtubules,they bind preferentiallyto the (+) ends of the microtubules. Dilution of the mixture to reduce the concentration of free tubulin dimers results in the movement of the chromosomestoward the (-) ends by microtubule depolymerizationat the chromosome-bound(+ ) end. Recent experiments have shown that in Drosophila two kinesin-13proteins, a classof microtubule depolymerizing proteins (see Figure 1,8-1,6),contribute to chromosome movementin anaphaseA. One of the kinesin-13proteinsis localized at the kinetochore and enhancesdisassemblythere (Figure 1.8-41,E[), and the other is localizedat the spindle pole, enhancingdepolymerizationthere (Figure18-41, Eg). Thus, at least in the fly, anaphaseA is powered in part by kinesin-13proteins specificallylocalizedat the kinetochore and spindle pole to shorten the kinetochore microtubules at both their (+ ) and (- ) endsto draw the chromosomesto the poles.

AnaphaseB SeparatesPolesby the Combined A c t i o n o f K i n e s i n sa n d D y n e i n The second part of anaphaseinvolves separationof the spindle poles in a processknown as anaphaseB' A maior contributor to this movement is the involvement of the bipolar kinesin-5proteins(Figure1.8-41,EE). Thesemotors associate with the overlapping polar microtubules, and since they are (+) end-directed motors, they push the poles apart. While this is happening, the polar microtubules have to grow to accommodate the increased distance between the spindle poles-at the same time as the kinetochore microtubules are shortening for anaphaseA! Another motor-the microtubule (- ) end-directed motor cytoplasmic dynein, localized and anchored on the cell cortex-pulls on the astral microtubules and thus helps separatethe spindle poles (Figure 18-41.,8f|).

s o n t r i b u t et o S p i n d l e A d d i t i o n a lM e c h a n i s m C Formation There are a number of casesin vivo where spindles form in the absenceof centrosomes.This implies that nucleationof microtubulesfrom centrosomesis not the only way a spindle can form. Studies exploiting mitotic extracts from frog eggs-extracts that do not contain centrosomes-show that the addition of beadscovered with DNA is sufficient to assemblea relativelynormal mitotic spindle (Figure 1842\. In this system, the beads recruit preformed microtubules, and factors in the extract cooperate to make a spindle. One of the factors necessaryfor this reaction is cytoplasmic dynein, which is proposedto bind to two microtubules and migrate to their (-) ends and thereby draw them together.

CytokinesisSplitsthe DuplicatedCell in Two During late anaphaseand telophasein animal cells, the cell assemblesa microfilament-basedcontractile ring attachedto

Add fluorescent t u b u l i na n d DNA-covered beads

Xenopus egg extracts

FIGURE 18-42Mitoticspindlescanform in EXPERIMENTAL canbe extracts the absenceof centrosomes.Centrosome-free eggsto in mitosis by centrifuging isolated fromfrogoocytes arrested material fromtheorganelles andyolk When separate a soluble together labeled tubulin(green) isaddedto extracts fluorescently

spontaneously with DNA(red),mitoticspindles with beadscovered microtubules nucleated formaroundthe beadsfromrandomly lModified from Kinoshitaet al , 2002, TrendsCellBiol 12:267-273, and Antonio et al , 2000, Cell 1O2:425l

MtTOSlS

789

the plasma membrirne that will eventually contract and pinch the cell into two, a processknown as cytokinesis(see Figure 18-34).The contractilering is a thin band of actin filamentsof mixed polarity interspersedwith myosin-II bipolar filaments (seeFigure 17-34). On receivinga signal, the ring contracts first to generatea cleauagefurrow and then to pinch the cell into two. Two aspectsof the contractilering are essentialto its function. First, it has to be appropriately placed in the cell. It is known that this placementis determined by signalsprovided by the spindle,so that the ring forms equidistant between rhe two spindie poles. Recent work with Drosophila mutants has suggestedthat componentsthat accumulateat the centralpoint of the spindleduring anaphasedirect the formation of the contractile ring. However, the molecular signals involved in coordinating spindleposition and the position of the contractilering are still being unraveled. The secondimportant aspectof the contractilering is the timing of its contraction-if it contractedbeforeall chromosomes have moved to their respectivepclles, disastrous genetlcconsequences would ensue.Again, the mechanism wherebythis timing is determinedis being unraveied. In t e r p h a s e

Prophase

P l a n tC e l l sR e o r g a n i z T e h e i r M i c r o t u b u l e sa n d B u i l da N e w C e l lW a l l i n M i t o s i s lnterphaseplant cells lack a central MTOC that organizes microtubulesinto the radiating interphasearray typical of animal cells.Instead,numerous MTOCs line the cortex of plant cellsand nucleatethe assemblyof transversebands of microtubulesbelow the cell wall (Figure18-43,left).The mtcrotubules,which are of mixed polarity, are releasedfrom the cortical MTOCs by the action of katanin, a microtubule, severingprotein; loss of katanin givesrise to very long microtubulesand misshapencells.The reasonfor this is that t h e s e c o r t i c a l m i c r o t u b u l e s ,w h i c h a r e c r o s s - l i n k e db y plant-specificMAPs, aid in laying down extracellularceliulose microfibrils, the main component of the rigid cell wall (seeFigure 19-37). Although mitotic eventsin plant cellsare generallysimilar to those in animal cells, formation of the spindle and c y t o k i n e s i sh a v e u n i q u e f e a t u r e si n p l a n t s ( F i g u r e1 8 - 4 3 ) . Plant cells bundle their cortical microtubulesinto the preprophase band and reorganize them into a spindle at prophase without the aid of centrosomes.At metaphase.

Metaphase

Telophase

Preprophase band

\" /

P hr a gm o p l a s t

C e l lo l a t e F I G U R E1 8 - 4 3 M i t o s i s i n a h i g h e r p l a n t c e l l . l m m u n olfu o r e s c e n cmei c r o g r a p h( st o p )a n d c o r r e s p o n d i ndgi a g r a m s (botton) showingarrangementof microtubules in interphase and mitoticplantcells A cortjcalarrayof mtcrotubules girdlesa cell d u r i n gi n t e r p h a s eW e b so f m i c r o t u b u l ecsa pt h e g r o w i n ge n d so f p l a n tc e l l sa n d r e m a i ni n t a c td u r i n gc e l ld i v i s i o nA s a c e l le n t e r s p r o p h a s et h , e m i c r o t u b u l easr e b u n d l e da r o u n dt h e n u c l e u sa n d r e o r g a n i z ei dn t o a s p i n d l et h a t a p p e a r s i m i l atro t h a t i n m e t a p h a s e 790

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a n i m a cl e l l s B y l a t et e l o p h a s et h, e n u c l e a m r e m b r a n eh a sr e - f o r m e d a r o u n dt h e d a u g h t e n r u c l e ai n d t h e G o l g i - d e r i v epdh r a g m o p l a shta s a s s e m b l eadt t h e e q u a t o r i apl l a t e A d d i t i o n asl m a l vl e s i c l edse r i v e d f r o m t h e G o l g ic o m p l e xa c c u m u l a taet t h e e q u a t o r i apl l a t ea n d f u s e with the phragmoplast to form the new cellplate lAdapted from R H Goddard eIal, 1994,PlantPhysiol. 104:1;mcrographs courtesy of S u s aM n W i c kl

c E L Lo R G A N r z A T r o N A N D M o v E M E N Tr : M T c R o T U B U L EASN D T N T E R M E D T AFTTEL A M E N T S

the mitotic apparatusappearsmuch the same in plant and animal cells. However, the division of the cell into two is quite different from animal cells. Golgi-derived vesicles, which appear at metaphase,are transported into the mitotic apparatusalong microtubules that radiate from each end of the spindle.At telophase,thesevesiclesline up near the center of the dividing cell and then fuse to form the phragmoplast,a membranestructurethat replacesthe animal-cell contractile ring. The membranes of the vesicles forming the phragmoplastbecomethe plasma membranes of the daughtercells.The contentsof thesevesicles,such as polysaccharideprecursorsof celluloseand pectin, form the early cell plate, which developsinto the new cell wall between the dauehter cells.

Mitosis r Mitosis-the accurate separation of duplicated chromosomes-involves a molecular machine comprising dynamic treadmilling microtubules and molecular motors. r The mitotic spindlehas three classesof microtubules,all emanating from the spindle poles-kinetochore microtubules,which attach to chromosomes;polar microtubules from each spindle pole, which overlap in the middle of the spindle; and astral microtubules, which extend to the cell cortex (seeFigure 18-36).

Wl

Intermediate Filaments

The third maior filament system of eukaryotesis collectively called intermediate filaments. This name reflects their diameter of about 10 nm, which is intermediate betweenthe 6-8 nm of microfilaments and myosin thick filaments of skeletal muscle. Intermediate filaments extend throughout the cytoplasm as well as line the inner nuclear envelope of interphaseanimal cells (Figure L8-44). Intermediate filaments have severalunique properties that distinguish them from microfilaments and microtubules. First, they are biochemically much more heterogeneous-that is' many differenr, but evolutionarily related, intermediate filament subunits exist that are often expressedin a tissue-dependent manner. Second,they have great tensile strength' which is most clearly demonstrated by hair and nail that consist primarily of the intermediate filaments of dead cells' Third, they do not have an intrinsic polarity like microfilaments and microtubules, and their constituent subunits do not bind a nucleotide. Fourth, becausethey have no intrinsic polarity, it is not surprising that there are no known motors that use them as tracks. Fifth, although they are dynamic in terms of subunit exchange,they are much more stable than microfilaments and microtubules becausethe exchangerate

r In the first stage of mitosis, prophase, the nuclear chromosomes condense and the spindle poles move to either side of the nucleus (seeFigure 18-34). r At prometaphase,the nuclear envelope breaks down and microtubules emanating from the spindle poles capture chromosomepairs at their kinetochores.Each of the two kinetochores on the duplicated chromosomes becomesattachedto the two spindle poles,which allows the chromosomes to congress to the middle of the spindle. r At metaphase, chromosomes are aligned on the metaphaseplate. The spindlecheckpointsystemmonitors unattached kinetochores and delays anaphaseuntil all chromosomesare attached. r At anaphase,duplicated chromosomes separate and move toward the spindle poles by shortening of the kinetochore microtubules at both the kinetochore and spindle pole (anaphaseA). The spindle poles also move apart, pushedby bipolar kinesin-Smoving toward the (+ ) ends of the polar microtubules(anaphaseB). Spindleseparationis also facilitated by cortically located dynein pulling on ast r a l m i c r o t u b u l e s( s e eF i g u r e1 , 8 - 4 0 , 1 8 - 4 1 ) . r Redundant mechanismscontribute to the fidelity of mitosis since the mitotic spindle has the ability to selfassemblein the absenceof MTOCs. r The position of the actin-myosin basedcleavagefurrow is determined by the position of the spindle and at cytokinesiscontracts to pinch the cell in two.

of two typesof 18-44Localization FIGURE a EXPERIMENTAL lmmunofluorescence cell' in an epithelial filaments intermediate with keratin(red)andlamin of a PtK2celldoublystained micrograph can filaments (blue)antibodies of laminintermediate A meshwork the keratin whereas membrane, the nuclear be seenunderlying membrane to the plasma extendfromthe nucleus filaments [Courteso y f R D G o l d m a n] I N T E R M E D I A TFEI L A M E N T S

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is much slower. Indeed, a standard way to purify intermediate filaments is to subject cells to harsh extraction conditions in a detergent so that all membranes,microfilaments, and microtubules are solubilized,leaving a residuethat is almost exclusively intermediate filaments. Finally, intermediate filaments are not found in all eukaryotes. Fungi and plants do not have intermediate filaments, and insectsonly have one class,representedby two genesthat expresslamin A/C and B. These properties make intermediate filaments unique and important structuresof metazoans.The importance of intermediatefilaments is underscoredby the identification of more than 40 clinical disorders,some of which are discussedbelow, associatedwith defectsin genesencodingintermediatefilament proteins.To understandtheir contributions to cell and tissue structure, we first examine the structure of intermediatefilament proteins and how they assemble into a filament. We then describe the different classesof intermediate filaments and the functions they perform.

IntermediateFilamentsAre Assembledfrom S u b u n i tD i m e r s Intermediate filaments (IFs) are encoded in the human genome by 70 different genesin at least five subfamilies. The defining feature of IF proteins is the presenceof a c o n s e r v e do - h e l i c a l r o d d o m a i n o f a b o u t 3 1 0 r e s i d u e s (a) Head

- - - - - -R- o- -d- |

N-terminus

that has the sequencefeatures of a coiled-coil motif (see Figure 3-9a). Flanking the rod domain are nonhelical Nand C-terminal domains of different sizes,characteristic o f e a c hI F c l a s s . The primary building block of intermediatefilaments is a dimer (Figure 18-45a) held together through the rod domains that associateas a coiled-coil. Thesedimers then associate in an offset fashion to make tetramers, where the two dimers are in opposite orientations (Figure 18-45b). Tetramers are assembledend to end and interlocked into long protofilaments. Four protofilaments associateinto a protofibril, and four protofibrils associateside to side to generatethe 10-nm filament. Thus an intermediate filament has 15 protofilaments in it. Becausethe tetramer is symmetric, intermediatefilaments have no polarity. This description of the filament is basedon its structure rather than its mechanism of assembly:at presentit is not yet clear how intermediate filaments are assembledin vivo. Unlike microfilaments and microtubules, there are no known intermediate filament nucleating, sequestering,capping, or filament-severing protelns.

IntermediateFilamentsProteinsAre Expressed i n a T i s s u e - S p e c i fM i ca n n e r Sequenceanalysisof IF proteins revealsthat they fall into at least five distinct homology classes,with four classesshowing a strong correspondencebetweenthe sequenceclassand

(b) , Tail C-terminus

T^+-^-^-

(c)

Protofilament Protofibril

A FIGURE 18-45 Structureand assemblyof intermediate filaments.Electron micrographs anddrawings of lFproteindimers, tetramers, andmatureintermediate filaments fromAscan'an parasitic intestinal worm.(a)lFproteins formparallel dimersthrough a highlyconserved coiled-coil coredomain. Theglobular headsand tailsarequitevariable in lengthandsequence betweenintermediate

792

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(b)A tetramer filamentclasses. isformedby antiparallel, staggered side-by-side aggregation of two identical dimers(c)Tetramers aggregate endto endandlaterally intoa protofibril. In a mature filament, consisting of fourprotofibrils, theglobular domains form beaded clusters on thesurface[Adapted fromN.Geisler etal, 1998,,/, Mol Biol282:601; courtesy of UeliAebil

C E L LO R G A N I Z A T I O N A N D M O V E M E N Tl l : M I C R O T U B U L EASN D T N T E R M E D T A F TT EL A M E N T S

FUNCTION DISTRIBUTION PROPOSED Acidic keratins

Epithelial cells

Basickeratins

Epithelial cells

Tissuestrength and integrity

E p i t h e l i a cl e l l

Muscle, glial cells, mesenchymal cells

Sarcomere organlzatlon, lntegrlty

Dense bodies

S m o o t hm u s c l e

ry

Neurofilaments (NFL, NFM, and NFH)

Neurons

S k e l e t a lm u s c l e

Axon organization Axon

Nuclear structure and organization Nucleus

the developmentalorigin of the cell type in which the IF protein is expressed(Table18-1). Keratins that make up classesI and II are found in epithelia, classIII intermediatefilaments are generallyfound in cells of mesodermalorigin, and classIV make up the neurofilaments found in neurons. The lamins make up class V, 'We which are found lining the nucleus of all animal tissues. briefly summarize the five different homology classesand discusstheir roles in specifictissues. Keratins Keratins provide strength to epithelial cells. The first two homology classesare the so-calledacidic and basic keratins. There are about 50 genesin the human genomeencoding keratins, about evenly split between the acidic and basic classes.These keratin subunits assembleinto an obligate dimer, so that each dimer consists of one basic chain and one acidic chain; these are then assembledinto a filament as describedabove. The keratins are by far the most diverseof the IF protein families, with keratin pairs showing different expression patterns between distinct epithelia and also showing differentiation-specific regulation. Among these are the so-calledhard keratins that make up hair and nails. These keratins are rich in cysteineresiduesthat becomeoxidized to form disulfide bridges, thereby strengthening them. This property is exploited by hair stylists: if you do not

like the shapeof your hair, the disulfide bonds in your hair keratin can be reduced, the hair reshaped' and the as a disulfide bonds reformed by oxidation-known "permanent," The so-calledsoft, or cyto-keratins, are found in epithelial cells. The epidermal-cell layers that make up the skin provide a good example of the function of keratins. The lowest layer of cells, the basal layer which is in contact with the basal lamina, proliferates constantly' giving rise to cells called keratinocytes. After they leave the basal layer, the keratinocytes differentiate and expressabundant cytokeratins. The cytokeratins associatewith desmosomes,specializedattachment sites between cells, thereby providing sheets of cells that can withstand abrasion' The cells eventually die, leaving dead cells from which all cell organelleshave disappeared. This dead cell layer provides an essentialbarrier to water evaporation, without which we could not survive.The life of a skin cell, from birth to its loss from the animal as a skin flake, is about one month. In all epithelia, keratin filaments associatewith desmosomes,which link adjacent cells together, and hemidesmosomes.which link cells to the extracellular matrix, thereby giving cells and tissuestheir strength. These structures are describedin more detail in Chapter 19. In addition to simply providing structural support, there is increasing evidencethat keratin filaments provide some

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organization to organelles and participate in signaltransductionpathways. For example,in responseto tissue injury, rapid cell growth is induced. In epithelial cells it has been shown that the growth signal requires an interaction between a cell-growth-signalingmolecule and a specific keratin. Desmin The classIII intermediatefilamentproteinsinclude vimentin, found in mesenchymalcells; GFAP (glial fibrillary acidic protein), found in glial cells; and desmin, found in musclecells.Desmin providesstrengthand organizationto musclecells. In smooth muscle, desmin filaments link cytoplasmic dense bodies, into which rhe conrractile myofibrils are also attached,to the plasma membraneto ensurethat cells r e s i s t o v e r s t r e t c h i n g .I n s k e l e t a l m u s c l e , a l a t t i c e c o m posed of a band of desmin filaments surrounds the sarcomere. The desmin filaments encirclethe Z disk and are cross-linked to the plasma membrane. Longitudinal desmin filaments cross to neighboring Z disks within the myofibril, and connections between desmin filaments around Z disks in adjacent myofibrils serve to cross-link myofibrils into bundles within a muscle cell. The lattice is also attached to the sarcomerethrough interactions with myosin thick filaments. Becausethe desmin filaments lie outside the sarcomere,they do not actively parricipate in generatingcontractile forces. Rather, desmin plays an essential structural role in maintaining muscle integrity. In transgenicmice lacking desmin,for example,this supporting architecture is disrupted and Z disks are misaligned. The location and morphology of mitochondria in these mice are also abnormal, suggestingthat theseintermediate

(a) 20 minutes after injection

(b) 4 hours after iniection

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filaments may also contribute to organization of organelles. Neurofilaments Type IV intermediate filaments consist of the three related subunits-NF-L, NF-M, NF-H (for NF light, medium, and heavy)-that make up the neurofilaments found in the axons of nerve cells (seeFigure 18-2). The three subunitsdiffer mainly in the sizeof their C-terminal domains, and all form obligate heterodimers.Experiments with transgenic mice reveal that neurofilaments are necessary to establishthe correct diameter of axons, which deter, mines the rate at which nerve impulsesare propagateddown axons. Lamins The most widespreadintermediatefilaments are the classV lamins, which provide strength and support to the inner surfaceof the nuclearmembrane(seeFigure L8-44). Lamins are the progenitor of all IF proteins, with the cytoplasmic IFs arising by geneduplication and mutation. The lamins provide a two-dimensional network lying between the nuclear envelopeand the chromatin in the nucleus.In humans, three genes encode lamins: one encodesA-type Iamin and two encodeB type. The B-type lamin appearsto be the primordial gene and is expressed in all cells, whereas A-type lamins are developmentallyregulated. Blamins are post-translationallyisoprenylated,which presumably helps them associatewith the inner nuclear envelope membrane. In addition, they bind inner nuclear membrane proteins such as emerin and lamin-associated polypeptides (LAP2). Lamins bind multiple proteins and have been proposed to play roles in large-scalechromatin organization and the spacingof nuclear pores. As cells en-

< EXPERIMENTAL FIGURE 18-46Keratin intermediatefilamentsare dynamicas solublekeratinis incorporatedinto filaments.Monomeric typeI keratinwas p u r i f i e dc ,h e m i c a l a l yb e l ewd i t hb i o t i na, n d microinjected intolivingepithelial cellsThecells werethenfixedat different timesafterinjection andstained with an antibody to biotinandwith antibodies to keratin(a)At 20 minutes after injection, the injected biotin-labeled keratinis concentrated in smallfociscattered throughthe (/eft)andhasnot beenintegrated cytoplasm intotheendogenous keratincytoskeleton (right).(b)By4 hours,the biotin-labeled (/eff) (right)display andthe keratinfilamenls identical patterns, indicating thatthe mrcroinjected proteinhasbecomeincorporated intothe existing cytoskeleton. R K MillelK Vistrom, [From andR D Goldman, 1991 ,J CellBiol113:843; courtesy of R D Goldman l

C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E AS N D T N T E R M E D T AFTt E LAMENTs

ter mitosis, lamins become hyperphosphorylatedand disassemble;in telophasethey reassemblewith the reassembling nuclear membrane.

I n t e r m e d i a t eF i l a m e n t sA r e D y n a m i c Although intermediate filaments are much more stable than microtubules and microfilaments, IF protein subunits have been shown to be in dynamic equilibrium with the existing IF cytoskeleton. In one experiment, a biotin-labeled type I keratin was injected into fibroblasts; within 2 hours, the labeled protein had been incorporated into the already existing keratin cytoskeleton (Figure 18-46). The results of this experiment and others demonstratethat IF subunits in a soluble pool are able to add themselvesto preexistingfilaments and that subunits are able to dissociate from intacr filaments. The relative stability of intermediatefilamentspresents special challengesin mitotic cells, which must reorganize all three cytoskeletalnetworks in the course of the cell cycle. In particular, breakdown of the nuclear envelopeearly in mitosis depends on the disassemblyof the lamin filaments that form a meshwork supporting the membrane.As discussedin Chapter 20, the phosphorylation of nuclear lamins by a cyclin-dependentkinase that becomesacrive early in mitosis (prophase)inducesthe disassemblyof intact filaments and preventstheir reassembly.Later in mitos i s ( t e l o p h a s e ) r, e m o v a l o f t h e s e p h o s p h a t e sb y s p e c i f i c phosphatasespromotes lamin reassembly,which is critical to re-formation of a nuclear envelopearound the daughter chromosomes.The opposing actions of kinasesand phosphatasesthus provide a rapid mechanismfor controlling the assemblystate of lamin intermediatefilaments. Other intermediatefilamentsundergo similar disassemblyand reassemblyin the cell cycle.

K 1 4 k e r a t i n i s o f o r m s f o r m h e t e r o d i m e r st h a t a s s e m b l e into protofilaments. A mutant K14 with deletions in either the N- or the C-terminal domain can form heterodimers in vitro but does not assembleinto protofilaments. The expressionof such mutant keratin proteins in cells causesIF networks to break down into aggregates. Transgenicmice that expressa mutant K14 protein in the basal stem cells of the epidermis display gross skin abnormalities, primarily blistering of the epidermis,that resemble the human skin diseaseepidermolysisbullosa simplex (EBS).Histologicalexaminationof the blisteredareareveals a high incidenceof dead basalcells.Death of thesecellsappears to be caused by mechanical trauma from rubbing of the skin during movement of the limbs. Sfithout their normal bundlesof keratin filaments,the mutant basal cells become fragile and easilydamaged,causingthe overlyingepidermal layers to delaminate and blister (Figure 18-47lr.

Epidermis

Dermis

D e f e c t si n L a m i n sa n d K e r a t i n sC a u s eM a n y Diseases T h e r e a r e a b o u t 5 0 k n o w n m u t a t i o n si n t h e h u m a n gene for type-A lamin that are known to cause disease,many of which cause forms of Emery-Dreifuss muscular dystrophy (EDMD). Other mutations in the lamin-A gene cause dilated cardiomyopathy. It is not yet c l e a r w h y t h e s e t y p e - A l a m i n m u t a t i o n s c a u s eE D M D , but perhaps in muscle tissues the fragile nuclei cannot stand the stressand strains of the tissue, so they are the f i r s t t o s h o w s y m p t o m s . I n t e r e s t i n g l y ,o t h e r f o r m s o f EDMD have been traced to mutations in emerin, the l a m i n - b i n d i n gm e m b r a n ep r o t e i n o f t h e i n n e r n u c l e a re n velope. Yet other mutations in type-A lamin causeprogeria-accelerated aging. Thus the Hutchison-Gilford proge r i a ( " p r e m a t u r e l yo l d " ) s y n d r o m ei s c a u s e db y a s p l i c i n g error that results in a lamin A with a defectiveC-terminal oomaln. The structural integrity of the skin is essentialin order t o w i t h s t a n d a b r a s i o n .I n h u m a n s a n d m i c e , t h e K 4 a n d

Epidermis

Mutated

micecarryinga FIGURE 18-47Transgenic a EXPERIMENTAL mutant keratingeneexhibit blisteringsimilarto that in the sections bullosasimplex.Histological humandiseaseepidermolysis a mouse carrying anda transgenic through theskinof a normalmouse geneareshownInthenormalmouse, theskin mutantK14keratin layercovering in contactwiththe soft of a hardouterepidermal consists mouse, thetwo layers Intheskinfromthetransgenic innerdermallayer. (arrow)dueto weakening of thecellsat the baseof the areseparated of E Fuchs PCoulombe etal, 1991,Cell66:1301 epidermis. ] ; courtesy [From I N T E R M E D I A TFEI L A M E N T S

'

795

Like the role of desmin filaments in supporting muscle tissue, the general role of keratin filaments appearsto be to maintain the structural integrity of epithelial tissues by mechanically reinforcing the connectionsbetween cells. I

Intermediate Filaments r Intermediate filaments are the only nonpolarized fibrous component of the cytoskeletonand do not have associated motor proteins. Intermediate filaments are built from coiled-coil dimers that associatein an antiparallel fashion into tetramers and then into protofilaments, 16 of which make up the filament (seeFigure 18-45). r There are five major classesof intermediateproteins, with the nuclear lamins (classV) being the most ancient and ubiquitous in animal cells.The other four classesshow tissue-specific expression(seeTable 18-1). r Keratins (classesI and II) are found in animal hair and nails, as well as in cytokeratin filaments that associatewith desmosomesin epithelial cells to provide the cells and tissue with strength. r The class III filaments include vimentin, GFAP, and desmin, which provide structure and order to muscle Z disks and restrain smooth muscle from overextension. r The neurofilaments make up classIV and are important for the structure of axons. r Many diseasesare associatedwith defectsin intermediate filaments, especiallylaminopathies,which include a variety of conditions, and mutations in keratin genes,which can causeseveredefectsin skin (seeFigure 18-47).

mediate filaments to other structures.Some of these associate with keratin filaments to link them to desmosomes, which are junctions between epithelial cells that provides stability to a tissue,and hemidesmosomes,which are located at regions of the plasma membrane where intermediate filaments are linked to the extra-cellularmatrix (thesetopics are covered in detail in Chapter 19). Other plakins are found along intermediate filaments and have binding sites for microfilaments and microtubules. One of theseproteins, called plectin, can be seenby immunoelectron microscopy to provide connectionsbetweenmicrotubules and intermediatefilaments(Figure18-48).

Microfilamentsand MicrotubulesCooperateto TransportMelanosomes Studiesof mutant mice with light-coloredcoatshas uncovered a pathway in which microtubules and microfilaments cooperate to transport pigment granules. The color pigment in the hair is produced in cellscalled melanocytes,cellsvery similar to the fish and frog melanophoresdiscussedearlier (seeFigure 1,8-28).Melanocytes are found in the hair follicle at the base of the hair shaft and contain pigment-ladengranules called melanosomes.Melanosomesare transported to the dendritic extensions of melanocytes for subsequent exocytosis to the surrounding epithelial cells. Transport to the cell periphery is mediated, iust as in frog melanophores,by a kinesin family member. At the periphery, they are then handed off to myosin V and deliveredfor exocytosis.If the myosin V systemis defective,the melanosomesare not capturedand stay in the cell body. Thus microtubules are responsiblefor the long-range transport of melanosomes, whereas microfilament-based myosin V is responsible for capture and delivery at the cell

fftli| Coordinationand Cooperation betweenCytoskeletalElements So far, we have generally discussedthe three cytoskeletal filaments classes-microfilaments, microtubules, and intermediate filaments-as though they function independently of one another. However, the example that the microtubule-basedmitotic spindle determinesthe site of formation of the microfilament-basedcontractile ring is just one example of how these two cytoskeletal systems are coordinated.Here we mention some other examplesof linkages, physical and regulatory, between cytoskeletal elementsand their integration into other aspectsof celluIar organization.

IntermediateFilament-Associated Proteins C o n t r i b u t et o C e l l u l a rO r g a n i z a t i o n A group of proteins collectively called intermediate filament-associated proteins (IFAPs) have been identified that co-purify with intermediate filaments. Among these are the family of plakins, which are involved in attaching inter796

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FIGURE 18-48Gold-labeled A EXPERIMENTAL antibody identifiesplectincross-linksbetween intermediatefilaments and microtubules. Inthisimmunoelectron micrograph of a fibroblast cell,microtubules arehighlighted in red;intermediate filaments, in fibersbetween blue;andtheshortconnecting them,in green with gold-labeled antibodies to plectin(yellow) reveals Staining that thesefiberscontainplectin.[From T.M Svitkina, A B Verkhovsky, andG 1996, G Borisy, L CellBiol.135:991; courtesy of T.M Svitkina l

C E L LO R G A N I Z A T I OANN D M O V E M E N TI I : M I C R O T U B U L EASN D I N T E R M E D I A TFEI L A M E N T S

M i c r o t u b u l e( + ) e n d c a p t u r e\

.t, Actin assembly

Direction of oolarization cortex. This type of division of labor-long-range transport by microtubules and short range by microfilaments-has been found in many different systems,from transport in filamentous fungi to transport along axons.

< FfGURE 18-49 Cdc42regulates microfilamentsand microtubules independentlyto polarizea migrating cell.ActiveCdc42-GTP at the frontof the cell whichresults in the leadsto Racactivation. assembly of a microfilament-based leading edge(step[). Independently, Cdc42-GTP also (+) ends leadsto thecapture of microtubule andthe activation of dynein(step2; Together theseoullon microtubules to orientthe (stepB) towardthe frontof the centrosome polarizes the secretory cell.Thisreorientation pathway for the delivery alongmicrotubules molecules carriedin secretory of adhesion (stepZl). [Based inS Etiennevesicles onstudies Manneville et al, 2005,J.CellBiol170:895-901 l

r In animal cells,microtubules are generallyutilized for the Iong-range delivery of organelles,whereas microfilaments handle their local delivery. r The signalingmolecule Cdc42 coordinately regulatesmicrofilaments and microtubules during cell migration.

Cdc42CoordinatesMicrotubulesand M i c r o f i l a m e n t sD u r i n gC e l lM i g r a t i o n In Chapter 17, we discussedhow the polarity of a migrating cell is regulated by Cdc42, which resultsin the formation of an actin-basedleading edge at the front of the cell and contraction at the back (seeFigures17-44 and18-49, step [). It turns out that Cdc42 activation at the cell front also leadsto polarization of the microtubule cytoskeleton.This was originally studied in wound-healing assays(seeFigure 17-43) where it was noticed that when the cells at the edge are induced to polarize and move to fill up the scratch, the Golgi complex is moved to the front of the nucleustoward the cell front. SinceGolgi localization is dependenton the location of the MTOC (seeFigures 18-1c, 18-27), this was becausethe centrosomecame to lie in front of the nucleus.Recentstudies have suggestedhow this happens. Cdc42 activation at the front of the cell binds the polarity factor Par6, which results in the recruitment of the dynein/dynactin complex (Figure 1.8-49,step Z). Cortically localizeddynein/dynactinthen interacts with microtubules pulling on them to orient the centrosome and hence the whole radial array of microtubules (Figure 18-49, step B). This reorientation of the microtubule systemleads to the reorganization of the secretorypathway to deliver secretoryproducts, especiallyintegrins to bind the extracellular matrix, to the front of the cell for attachment to the substratum for cell migration (Figure 1,8-49,step 4).

Coordination and Cooperation between Cytoskeletal Elements r Intermediate filaments are linked both to specific attachment sites, called desmosomesand hemidesmosomes,on the plasma membrane, as well to microfilaments and microtubules(seeFigure 18-48).

In Chapters 17 and 18 we have seenhow microfilaments, microtubules, and intermediate filaments provide structure and organization to cells.'$fithout this elaboratesystem,cellswould lack all order and hence all possibility of function or division. The name "cytoskeleton" suggestsa relatively static structure on which the cell organization is hung. However, the cytoskeleton is actually a dynamic framework responding to signaltransduction pathways and operating both locally and globally to provide cellswith order to undertake their functions. In outline, we have elucidatedmany of the distinct and common functions of the three filament systems. We know many of the componentsand probably all the motors. However,in many ways this is just an exciting beginning. \fith the availablesequencedgenomesand at least in principle a complete inventory of the cytoskeletalcomponents, we have a parts list. However, a parts list is just that; what we needis to understandhow the parts come togetherin specificprocesses. A very active areaof researchtoday is to use the parts list to systematically identify the localization (through GFP fusions), functions (through RNAi knockdown), and associated partners (through isolation of protein complexes) of all cytoskeletal components. Consider that there are about 45 genes in animals that encode members of the kinesin family, yet we only know what a small subsetof them do or what cargo they carry and for what purpose. In each case it is reasonableto assumethe motors are regulated,about which very little is currently known. As we begin to put all the piecesin place, it will be increasingly possible to reconstitute specific processesin vitro. Someaspectsof the mitotic spindle have already beenreconstituted, which is an encouraging beginning, but it will be sometime before it is possibleto reconstitutethe whole process. P E R S P E C T I VFEO SRT H E F U T U R E

797

Structural biology is going play a major role becauseit will allow us to see in detail how different components work. Consider the large number of proteins that associ'We ate with the microtubule (+ ) end, the so-called*TIPs. do not know structurally how they maintain their association at the tip, and recent work has suggestedthat associations can changein different parts of the cell-again, we are only just beginning to see how these processesare regulated. Perhapsthe biggest-and most exciting-challenge is to uncover how signal-transductionpathways coordinate func'We tions between all the different cytoskeletalelements. are beginning to seeglimpsesof what is in store from the signaltransduction pathways that regulate cell polarity and allow cell migration. Although all thesestudiesare likely to be aimed at basic cell biology, as we can seefrom the studies of intraflagellar transport, such studiesoften open a window into the underlying basis of disease,from which strategiesfor treatments can be developed.The interplay between basic cell biology and medicine contributes immensely to the excitement and social value of working in this area.

KeyTerms anaphase783

kinesins 259

asters 782

kinetochores 783

axonal transportT69

lamins 794

axonerne 777

cytokinesis 783

metaphase783 microtubule-associated proteins (MAPs) 758 microtubule- o r ganizing center (MTOC) 760

desmin 794

mitosis 781

dynamic instability 764

mitotic spindle 782 plakins 796 prophase 782

basalbody 761 centromere 786 centrosome750

dyneins 759 intermediatefilamentassociatedproteins (IFAPs)795

telophase783

keratins 793

tubulin 758

protofilament 758

Review the Concepts l. Microtubules are polar filaments; that is, one end is different from the other.\(hat is the basisfor this polarity, how is polarity related to microtubule organization within the cell, and how is polarity related to the intracellular movements powered by microtubule-dependentmotors? 2. Microtubules both in vitro and in vivo undergo dynamic instability, and this type of assemblyis thought to be intrinsic to the microtubule. What is the current model that accounts for dynamic instability? 798

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3. In cells, microtubule assembly depends on other proteins as well as tubulin concentration and temperature. What types of proteins influence microtubule assembly in vivo, and how does each type affect assembly? 4. Microtubules within a cell appear to be arranged in 'S7hat specificarrays. cellular structure is responsiblefor determining the arrangement of microtubules within a cell? How many of these structures are found in a typical cell? Describe how such structures serveto nucleate microtubule assembly. 5. Many drugs that inhibit mitosis bind specifically to tubulin, microtubules, or both. What diseasesare such drugs used to treat? Functionally speaking,these drugs can be divided into two groups basedon their effect on microtubule assembly. What are the two mechanisms by which such drugs alter microtubule structure? 6. Kinesin-1 was the first member of the kinesin motor family to be identified and therefore is perhaps the bestcharacterizedfamily member. What fundamental property of kinesin was used to purify it? 7. Certain cellular components appeat to move bidirectionally on microtubules. Describehow this is possiblegiven that microtubule orientation is fixed by the MTOC. 8. The motile properties of kinesin motor proteins involve both the motor domain and the linker domain. Describethe role of eachdomain in kinesin movement,direction of movement, or both. 9. Cell swimming depends on appendages containing microtubules. What is the underlying structure of these appendages,and how do these structuresgeneratethe force required to produce swimming? 10. The mitotic spindle is often describedas a microtubulebasedcellular machine. The microtubules that constitute the mitotic spindle can be classified into three distinct types. What are the three types of spindle microtubules, and what is the function of each? 11. Mitotic spindle function relies heavily on microtubule motors. For each of the following motor proteins, predict the effect on spindle formation, function, or both of adding a drug that specifically inhibits only that motor: kinesin-5, kinesin-13,and kinesin-4. 12. The poleward movement of kinetochores, and hence chromatids, during anaphaseA requires that kinetochores maintain a hold on the shortening microtubules. How does a kinetochore hold on to shortening microtubules? 13. Anaphase B involves the separationof spindle poles. \7hat forces have been proposed to drive this separation? $7hat underlying molecular mechanismsare thought to provide theseforces? 14. Cytokinesis,the processof cytoplasmic division, occurs shortly after the separatedsisterchromatids have neared the opposite spindle poles. How is the plane of cytokinesis determined?I(hat are the respectiveroles of microtubules and actin filaments in cytokinesis? 15. Explain why there are no known motors that use intermediate filaments as tracks.

C E L LO R G A N I Z A T I O N A N D M O V E M E N TI I M I C R O T U B U L E AS N D I N T E R M E D I A TFEI L A M E N T S

Analyze the Data a. Kinesin-l contains two identical heavy chains and therefore has two identical motor domains. In contrast, kinesin-Scontains four identical heavy chains. Electron microscopic analysisof metal-shadowedkinesinsresultsin the imagesshown in the top panel.Pretreatmentof thesekinesinwith an antibody that binds specifically to the kinesin motor domain resultsin the imagesshown in the lower panel. All four images are at the same approximate magnification. What can you deduceabout the structureof kinesin-Sfrom thesedata? Kinesin-1

c. Kinesin-S can cross-bridge adjacent microtubules. Polarity marked microtubules are assembledin which tubulin attached to a red fluorescent dye is assembledto form short red microtubules, which are then elongatedwith tubulin attachedto a greenfluorescentdye. The microtubules are mixed with kinesin-S and observed by fluorescencemicroscopyas ATP is added.The following imagesshow a time sequenceof two overlapping and cross-bridgedmicrotubules as ATP is added. The arrowhead is in a fixed position. Can you explain what happens when ATP is added to microtubulescross-bridgedby kinesin-5?

Kinesin-5

No antibody

Decoratedwith k i n e s i nm o t o r d o m a i na n t i b o d y

b. To determineif kinesin-Sis a (+) or (-) end microtubule motor, polarity-marked microtubules are generatedby assemblingshort microtubulesfrom brightly fluorescenttubulin and then elongating those short, bright microtubules using less fluorescent tubulin. As a result, the microtubules are very fluorescent at one end and lessfluorescent along most of their length. A perfusion chamber is then coated with purified kinesin-s,which becomesimmobilizedon the glasssurface.The chamber is then perfused with the polarity labeled microrubules and ATI and microtubule gliding with respectto the immobilized kinesin-S is observed.The following sequenceof 'SThich imagesis obtained. end of thesemicrotubules, the bright or the lessbright end, is the (+) end? Do thesemicrotubules glide on kinesin-Swith their (+) or (-) end leading?Basedon thesedata,is kinesin-Sa (+)or (-)end microtubulemotor?

d. Eg5 is a kinesin-S family member in Xenopus. To understand Eg5 function in vivo, cells are transfectedwith RNAi directed against this motor. The following imagesare obtained of mitotic cells. ril/hat function might Eg5 play in cells?

nK2

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o

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References Structure and Organization Microtubule Badano,J. L., T. M. Teslovich,and N. Katsanis.2005. The centrosome in human geneticdisease.Natwre Reu.Mol. Cell Biol. 6:L94-205. DoxseS S. 2001. Re-evaluatingcentrosomefunction. Natwre Reu.Mol. Cell Biol.2:688-698. Dutcher,S. K. 2001. The tubulin fraternity: alpha to eta. Curr. Opin. Cell Biol. 13:49-54. Nogales,E., and H-\7 Wang. 2006. Structuralintermediatesin microtubule assemblyand disassembly:how and why? Curr. Opin. Cell Biol. 18:179-1.84.

Dynamics Microtubule Cassimeris,L.2002. The oncoprotein 18/Stathminfamily of microtubule destabilizers.Curr. ODin. Cell Biol. 14:18-24. REFEREN CES

799

Desai,A., and T. J. Mitchison. 1997. Microtubule polymerization Dyanamics.Annu. Reu.CellDeu. Biol. 13:83-7'1.7. Howard, J., and A. A. Hyman. 2003. Dynamics and mechanics of the microtubule plus end.Natwre 422:753-7 5 8. Regulation of Microtubule Structure and Dynamics Akhmanova, A., and C. C. Hoogenraad.2005. Microtubule plus-end-trackingproteins:mechanismsand functions. Curr. Opin. Cell Biol. 17:47-54. Galjart, N. 2005. CLIPs and CLASPsand cellular dynamics. Nature Reu.Mol. Cell Biol.6:487498. Kinesins and Dyneins-Microtubule-Based

Motor Proteins

Web site:kinesin home page,http://wwwproweb.org/kinesinL/ Burgess,S. A., et a|.2003. Dynein strucrureand power stroke. Nature 421:775-71,8. Dell, K. R. 2003. Dynactin policesrwo-way organelletraffic. J. Cell B iol. 160:291,-293. Dujardin, D. L., and R. B. Vallee.2002. Dynein at the cortex. Curr. Opin. Cell Biol. 14:4449. Goldstein,L. S. 2001. Kinesin molecularmotors: transport pathways,receptors,and human disease.Proc. Nat'l. Acad. Sci.

usA986999-7003. Hirokawa, N. 1998. Kinesin and dynein superfamiiyproteins and the mechanismof organelletransport. Science279:51,9-526. Hirokawa, N., and R. Takemure.2003. Biochemicaland molecular characterizationof diseaseslinked to motor oroteins.Trends Cell Biol.28:558-565. Lawrence,C. J., et aL.2004.A standardizedkinesin nomenclature.J. Cell Biol. 167:1,9-22. Schroer,T. A. 2004. Dynactin. Ann. Reu.Cell Deu. Biol. 20:759-779. Vale. R. D. 2003. The molecular motor roolbox for intracellular transport. Cell 112:467480. Vale, R. D., and R. A. Milligan. 2000. The way things move: looking under the hood of molecularmotor proteins.Science 288:88-95. 'Wordeman, L. 2005. Microtubule-depolymerizingkinesins. Curr. Opin. Cell Biol. 17:82-88. Yildiz, A., M. Tomishige,R. D. Vale, and P. R. Selvin.2004. Kinesinwalks hand-over-han d. Science303:676-67 8. Cilia and Flagella: Microtubule-Based Surface Structures 'Witman. Rosenbaum,J. L., and G. B. 2002.lntraflagellar rranspofi. Nature Reu.Mol. Cell Biol.3:813-825. Singla,V., and J. F. Reiter.2006. The primary cilium as the cells' antenna:signalingat a sensoryorganelle.Science313:629-633.

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Mitosis Web site: http://www.proweb.org/kinesin/FxnSpindleMotility.html Cleveland,D. r07.,Y. Mao, Y., and K. F. Sullivan.2003. Centromeresand kinetochores:from epigeneticsto mitotic checkpoint signaling.Cell Ll2:407 42'1.. Gadde,S., and R. Heald.2004. Mechanismsand moleculesof the mitotic spindle.Curu Biol. 14:R797-R805. Heald, R., et aL.1997. Spindleassemblyin Xenopus egg roles of centrosomesand microtubule selfextracts:resDective organization.J . Cell Biol. 138:675-628. Kinoshita, K., B. Habermann,and A. A. Hyman. 2002. XMAP215: a key componentof the dynamic microtubule cytoskeleton. Trends Cell Biol. 12:267-273. Mitchison, T. J., and E. D. Salmon.2001. Mitosis: a history of division. Nature Cell Biol. 3 :El7 -821. Rogers,G. C., et aL.2004.Two mitotic kinesinscooperateto drive sisterchromatid separationduring anaphase.Nature 427:364-370. 'Wittmann, T., A. Hyman, and A. Desai.2001. The spindle:a dynamic assemblyof microtubulesand motors. Nature Cell Biol. 3:828-E34. lntermediate Filaments Intermediate Filaments Database: http://www.interfil.org/ index.php Goldman, R. D., et aI.2002. Nuclear lamins: building blocks of nuclear architecture.G enesD eu. 16:,533-547 . Herrmann, H., and U. Aebi. 2000. Intermediatefilamentsand their associates: multi-talentedstructural elementsspecifyingcytoarchitectureand cytodynamics.Curr. Opin. Cell Biol. L2:79-90. Mattout, A., et al. 2006. Nuclear lamins, diseaseand aging. Curr. Opin. Cell Biol. 18:335-341. Coordination and Cooperation between Cytoskeletal Elements ri7ebsite: Melanophores, http://www.proweb.orglkinesin"/ Melanophore.html Chang, L., and R. D. Goldman.2004.Intermediatefilaments mediatecytoskeletalcrosstalk.Nature Reu.Mol. Cell Biol. 5:

60r-613. Etienne-Manneville,S., et al. 2005. Cdc42 and Par6-PKC( regulate the spatially localized association of Dlgl and APC to control cell polarization.l. Cell Biol. l7O:895-901,. Kodama, A., T. Lechler,and E. Fuchs.2004. Coordinating cytoskeletaltracks to polarizecellular movements.l. Cell Biol. 167: 203-207. 'Wu, X., X. Xiang, andJ. A. Hammer 11I.2006.Motor proteins at the microtubule plus-end.Trends Cell Biol. 16:135-143.

C E L LO R G A N I Z A T I O N A N D M O V E M E N Tl l : M I C R O T U B U L EASN D I N T E R M E D I A TFEI L A M E N T S

CHAPTER

I tr

CELLS INTEGRATING INTOTISSUES False-color imageof a sectionthrougha desmosome from neonatalmouseepidermisElectron microscope tomography was junction,called usedto generate an imageof a specialized cellular a desmosome, that helpsholdcellsin the skintogetherCelladhesion molecules calledcadherins areblue,membranes from adjacent cellsareeachoutlrnedin red,and relatedintracellular plaqueand intermediate molecules, cytoplasmic filaments, are orangeand lightgreen,respectively Scalebaris 30 nm [FromW He,P Cowin,andD L Stokes, 2003,Science 3O2(5642): I O9-113 l

I n the development of complex multicellular organisms I such as plants and animals, progenitor cells differentiate I i n t o d i s t i n c t" t y p e s " t h a t h a v ec h a r a c t e r i s t ci co m p o s i t i o n s . structures,and functions. Cells of a given typ. oft.., gate into a tissueto cooperatively perform a common"ggr.function: muscle contracts; nervous tissuesconduct electrical impulses; xylem tissue in plants transports water. Different tissuescan be organizedinto an organ, again to perform one or more specificfunctions. For instance,the muscles,valves, and blood vesselsof a heart work together to pump blood. The coordinated functioning of many types of cells and tissuespermits the organism to move, metabolize, reproduce, and carry out other essentialactivities. Even simple animals exhibit complex tissue organization. The adult form of the roundworm Caenorhabditis eleganscontains a mere 959 cells, yet thesecells fall into 1,2 different general cell types and many distinct subtypes.Vertebrates have hundreds of different cell types, including leukocytes (white blood cells) and erythrocytes (red blood cells); photoreceptors in the retina; adipocytes,which store fat; fibroblasts in connectivetissue; and hundreds of different subtypes of neurons in the human brain. Despite their diverseforms and functions, all animal cells can be classified as being componentsof just five main classesof tissue: epithelial tissue, connectiuetissue,muscular tissue,neruous tissue, and blood. Various cell types are arranged in precise

patterns of staggering complexity to generate tissues and organs. The costs of such complexity include increased requirements for information, material, energS and time during the developmentof an individual organism. Although the physiological costs of complex tissues and organs are high, they confer the ability to thrive in varied and variable environments,a maior evolutionary advantage.

OUTLINE x dhesion: 1 9 . 1 C e l l - C ea l ln d C e l l - M a t r i A An Overview ' 1 9 . 2 C e l l - C ea J u n c t i o n sa n d T h e i r l ln d C e l I - E C M A d h e s i o nM o l e c u l e s

808

l amina 1 9 . 3 T h e E x t r a c e l l u l aMr a t r i x l : T h e B a s a L

820

'19.4 The ExtracellularMatrix ll: Connective and Other Tissues 1 9 . 5 A d h e s i v eI n t e r a c t i o nisn M o t i l e a n d N o n m o t i l eC e l l s 19.6

P l a n tT i s s u e s

833 839

801

The complex and diverse morphologies of plants and animals are examples of the whole being greater than the sum of the individual parts, more technically describedas the emergentproperties of a complex system.For example, the distinct mechanicalproperties of rigid bones, flexible joints, and contracting musclespermit vertebratesto move efficiently and achievesubstantial size. One of the defining characteristicsof animals with complex tissuesand organs (metazoans)such as ourselvesis that the external and internal surfacesof most of their tissuesand organs, and indeed the exterior of the entire organism, are built from tightly packed sheetlikelayers of cells known as epithelia. The formation of an epithelium and its subsequentremodeling into more complex collections of epithelial and nonepithelial tis-

suesis a hallmark of the development of metazoans.Sheets of tightly attachedepithelial cells act as regulatable,selective permeability barriers, which permit the generation of chemically and functionally distinct compartments in an organism (e.g.,stomach and bloodstream).As a result, distinct and sometimesopposite functions (e.g.,digestionand synthesis)can efficientlyproceedsimultaneouslywithin an organism. Such compartmentalizationalso permits more sophisticated regulation of diverse biological functions. In many ways, the roles of complex tissuesand organs in an organism are analogousto those of organellesand membranesin individual cells. The assembly of distinct tissues and their organization into organs are determined by molecular interactions at the

C e l la d h e s i o n molecules(CAMs)

T i g h tj u n c t i o n Apical surface

ADHESIONS CELL-CELL lntermediate Adapter

G a pj u n c t i o n

Desmosome

Hemidesmosome

Basal amina Connexon

surface

E

Extracellu lar m a t r i x( E C M )

FIGURE 19-1 Overviewof majorcell-celland cell-matrix adhesiveinteractions. Schematic cutaway drawingof a typical epithelial tissue, suchasin the intestines Theapical(upper) surface of thesecellsis packed withfingerlike microvilli n thatprojectinto the intestinal lumen,andthe basal(bottom) surface Z restson extracellular matrix(ECM)TheECMassociated with epithelial cellsis usually (eg, the basal organized intovarious interconnected layers lamina, connecting fibers,connective tissue), in whichlarge, interdigitating ECMmacromolecules bindto oneanotherandto the (CAMs) cellsE Cell-adhesion molecules bindto CAMson other cells,mediating cell-cell adhesions 4, andadhesion receptors bind to various components of the ECI\4, mediating cell-matrix adhesions E gothtypesof cell-surface adhesion molecules areusually integral proteins membrane whosecytosolic domains oftenbindto multiple intracellular adapterproteinsThese adapters, directly or indirectly, (actlnor intermediate linkthe CAMto thecytoskeleton filaments) 802

CHAPTER 19

I

I N T E G R A T I NCGE L L SI N T OT I S S U E S

pathwaysAs a consequence, andto intracellular signaling information canbetransferred by CAMsandthe macromolecules to whichtheybindfromthecellexterior intothe intracellular environment andviceversaln somecases, a complex aggregate of proteins CAMs,adapters, andassociated isassembled Specific localized aggregates of CAMsor adhesion receptors formvarious typesof celljunctions that playimportant rolesin holdrng tissues togetherandfacilitating communication between cellsandtheir e n v i r o n m eT n ti g . hjtu n c t i o n6s, l y i n gj u s tu n d etrh em i c r o v i l l i , prevent thediffusionof manysubstances throughtheextracellular spaces between thecellsGapjunctions Z allowthe movement throughconnexon channels of smallmolecules andionsbetween the cytosols of adjacent cellsTheremaining threetypesof junctions, junctions adherens EI, spotdesmosomes 9, andhemidesmosomes IE, tint thecytoskeleton of a cellto othercellsor the ECM [see V Vasioukhin andE Fuchs, 2001 OpinCellBiol13:761 , Curr.

cellular level and would not be possiblewithout the temporally and spatially regulated expression of a wide array of adhesivemolecules. Cells in tissuescan adhere directly to one another (cell-cell adhesion) through specializedmembrane proteins called cell-adhesionmolecules (CAMs) that often cluster into specializedcell junctions (Figure 19-1). In the fruit fly Drosophila melanogaster,at least 500 genes F4% of the total) are estimatedto be involved in cell adhesion. Cells in animal tissues also adhere indftecdy (cellmatrix adhesion) through the binding of adhesionreceptors in the plasma membrane to components of the surrounding extracellular matrix (ECM), a complex interdigitating meshwork of proteins and polysaccharidessecretedby cells into the spacesbetween them. These two basic types of interactions not only allow cells to aggregateinto distinct tissues but also provide a meansfor the bidirectional transfer of information betweenthe exterior and the interior of cells.Such information transfer is important to many biological processes,including cell survival, proliferation, differentiation, and migration. Therefore it is not surprising that defects that interfere with the adhesiveinteractions and the associated flow of information can cause or contribute to diseases,including a wide variety of neuromuscular and skeletaldisordersand cancer. In this chapter, we examine various types of adhesive moleculesand how they interact. Becauseof the particularly well-understood nature of the adhesivemoleculesin tissuesthat form tight epithelia, as well as their very early evolutionary development,we will initially focus on epithelial tissues,such as the walls of the intestinal tract and those that form skin. Epithelial cells are normally nonmotile (sessile);however, during development, wound healing, and in certain pathologic states(e.g.,cancer),epithelial cellscan transform into more motile cells.Changes in expression and function of adhesive molecules play a key role in this transformation, as they do in normal biological processesinvolving cell movement, such as the crawling of white blood cells into sites of infection. \7e therefore follow the discussionof epithelial tissueswith a discussionof adhesion in nonepithelial, developing, and motile tissues. The evolution of plants and animals diverged before multicellular organismsarose.Thus multicellularity and the molecular means for assemblingtissuesand organs must have arisen independentlyin animal and plant lineages.Not surprisingl5 then, animals and plants exhibit many differences in the organization and development of tissues.For this reason, we first consider the organization of tissuesin animals and then deal separatelywith plants.

Cell-Celland Cell-Matrix

Adhesion:An Overview We begin with a brief orientation to the various types of adhesive molecules, their major functions in organisms, and their evolutionary origin. In subsequentsections,we exam-

ine in detail the unique structuresand properties of the various participants in cell-celland cell-matrix interactions.

C e l l - A d h e s i oM n o l e c u l e sB i n dt o O n e A n o t h e r and to lntracellularProteans A large number of CAMs fall into four major families: the cadherins, immunoglobulin (Ig) superfamilS integrins' and selectins.As the schematicstructures in Figure 19-2 illustrate, many CAMs and other adhesion moleculesare mosaicsof multiple distinct domains, many of which can be found in more than one kind of protein. Some of these domains confer the binding specificity that characterizesa particular protein. Other membrane proteins, whose structures do not belong to any of the major classesof CAMs, also participate in cell-cell adhesion in various tlssues. CAMs mediate, through their extracellular domains' adhesiveinteractions between cells of the same type (homo' typic adhesion) or between cells of different types (heterotypic adhesion).A CAM on one cell can directly bind to the samekind of CAM on an adiacent cell (homophilicbinding) or to a different class of CAM (heteropbilic binding). CAMs can be broadly distributed along the regions of plasma membranes that contact other cells or clustered in discretepatchesor spots called cell iunctions. Cell-cell adhesions can be tight and long lasting or relatively weak and transient. The associationsbetween nerve cells in the spinal cord or the metabolic cells in the liver exhibit tight adhesion. In contrast, immune-systemcells in the blood often exhibit only weak, short-lasting interactions' allowing them to roll along and pass through a blood vesselwall on their way to fight an infectionwithin a tissue. The cytosol-facingdomains of CAMs recruit setsof multifunctional adapter proteins (see Figure 19-1). These adapters act as linkers that directly or indirectly connect CAMs to elementsof the cytoskeleton(Chapters1'7and 18); they can also recruit intracellular moleculesthat function in signaling pathways to control protein activity-both intracellular proteins and the CAMs themselves-and gene expression(Chapters15 and 16). In many cases'a complex aggregateof CAMs, adapter proteins, and other associated proteins is assembledat the inner surfaceof the plasma membrane. Becausecell-celladhesionsare intrinsically associated with the cytoskeleton and signaling pathways' a cell's surroundings influence its shape and functional properties ("outside-in" effects);likewise, cellular shape and function influence a cell's surroundings ("inside-out" effects). Thus connectiuity and commwnication are intimately related properties of cells in tissues. The formation of many cell-cell adhesions entails two types of molecular interactions(Figure 19-3). First' CAMs on one cell associate laterally through their extracellular domains, cytosolic domains, or both into homodimers or higher-order oligomers in the plane of the cell's plasma membrane; these interactions are called intracellular, lateral, or cls interactions. Second, CAM oligomers on one cell bind to the sameor different CAMs on an adjacentcell; : N OVERVIEW X D H E S I O NA C E L L - C E LALN D C E L L - M A T R I A

803

Homophilic interactions Cadherins (E-cadherin)

Heterophilic interactions

lg-superfamily CAMs (NCAM)

Selectins (P-selectin)

Integrins (cwF3)

-/ Fibronectin

A cadherin

U il;]::il"

Glycoprotein

T v o el l l

(-) rgoo-ain Q tii"o*ain O ::fl:'"

FIGURE 19-2 Major familiesof cell-adhesion molecules(CAUs) and adhesionreceptors.Dimeric E-cadherins mostcommonly form (self)cross-bridges homophilic with E-cadherins on adjacent cells. Members (lg)superfamily of theimmunoglobulin of CAMscanform (shownhere)andheterophilic bothhomophilic Iinkages (nonself) (forexample, linkages. Heterodimeric integrins ctvandp3 chains) functionasCAMsor asadhesion (shownhere)thatbindto receptors verylarge,multiadhesive matrixproteins suchasfibronectin, onlya smallpartof whichisshownhereSelectins, shownasdimers. contain

a carbohydrate-binding lectindomainthat recognizes specialized sugar (shownhere)andglycolipids structures on glycoproteins on adjacent cellsNotethatCAMsoftenformhigher-order oligomers withinthe planeof the plasma membrane. Manyadhesive molecules contain multiple distinct domains, someof whicharefoundin morethanone kindof CAM.Therytoplasmic domains of theseproteins areoften associated with adapterproteins that linkthemto thecytoskeleton or pathways[See to signaling R.O Hynes, 1999, Trends Celt Biol.9(12):M33, andR O Hynes, 2002,Cell110:673-687 l

these interactions are called intercellular or trans interactions. Trans interactions sometimes induce additional cis interactions and, as a consequence,yet even more trans interactions. Adhesive interactions between cells vary considerably, depending on the parricular CAMs participating and the tissue.Just like Velcro, very tight adhesion can be gener-

ated when many weak interactionsare combined, and this is especially the case when CAMs are concentrated in small, well-defined areas,such as cellular junctions. Some CAMs require calcium ions to form effective adhesions; others do not. Furthermore, the associationof intracellular molecules with the cytosolic domains of CAMs can dramatically influence the intermolecular interactions of

> FIGURE 19-3 Modelforthe generation of cell-celladhesions.Lateralinteractions between (CAMt cell-adhesion molecules withinthe plasma membrane of a cellform dimersandlargeroligomersThepartsof the molecules that participate in thesecis Interactions varyamongthe different CAMs Subsequent transinteractions betweendistal domains of CAMson adjacent cellsgenerate a Velcro-like strongadhesion between the cells[Adapted fromM S Steinberg andp M McNutt, 1999,CurrOpinCellBiol11:5541

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Disruptions in adhesion also are characteristicof various diseases,such as metastatic cancer, in which cancerous cells leave their normal locations and spread throughout the body. Although many CAMs and adhesionreceptorswere initially identified and characterizedbecauseof their adhesive properties, they also play a major role in signaling, using many of the pathways discussedin Chapters 15 and 16. Figure 1,9-7 illustrates how one adhesion receptor,integrin, physically and functionally interactsvia adaptersand signaling kinaseswith a broad array of intracellular signaling pathways, including those initiated by receptor tyrosine kinases, to influence cell survival, gene transcription, cytoskeletal organization, cell motility, and cell proliferation. Converseln changesin the activities of signaling pathways inside of cells can influence the structuresof CAMs and adhesion receptors and thus modulate their ability to interact with other cells and ECM.

The Evolutionof MultifacetedAdhesion M o l e c u l e sE n a b l e dt h e E v o l u t i o n of DiverseAnimal Tissues Cell-cell and cell-matrix adhesionsare responsiblefor the formation, composition, architecture,and function of animal tissues.Not surprisinglS some adhesion molecules are evolutionarily ancient and are among the most highly conserved proteins in multicellular organisms. Sponges, the most primitive multicellular organisms' express certain CAMs and multiadhesive ECM molecules whose structuresare strikingly similar to those of the corresponding human proteins. The evolution of organisms with complex tissuesand organs (metazoans)has dependedon the evolution of diverse adhesion molecules with novel propertiesand functions, whose levelsof expressiondiffer in different types of cells. Some CAMs (e.g.' cadherins), adhesion receptors (e.g., integrins and immunoglobulin

ECM Ligand Bound ligand

Integrin ( a dh e s i o n receptor) Exterior Plasmamembrane

Variousadaptorsand s i g n a l i n gk i n a s e s

Cytosol

Classic s i gn al i n g pathways

Cellular responses to adhesion recepror s i gn a l i n g

Cell proliferation(cycle) Cell survival Cytoskeletalorganization C e l lm i g r a t i o n G e n et r a n s c r i p t i o n

19-7 Integrinadhesionreceptor-mediated A FIGURE signalingpathwaysthat controldiversecellfunctions.Binding o f i n t e g r i ntso t h e i rl i g a n disn d u c ecso n f o r m a t i o ncahla n g eisn n ist h t h e i rc y t o p l a s mdiocm a i n sa,l t e r n a t i nt hge i ri n t e r a c t i ow ( r c-family p r o t e i n s k i n a s e s i n c l u d s e i g n a l i n g T h e s e cytoplasmic kinase[lLK])and kinaselFAKl,integrin-linked focaladhesion kinases, v i,n c u l i nt h) a tt r a n s m si ti g n a l s , axillin a d a p t opr r o t e i n(seg , t a l i n p

cellproliferation, pathways, therebyinfluencing signaling viadiverse andgene migration, cell organization, cytoskeletal cellsurvival, pathways shownhere of the components of the Many transcription. pathways, signaling with othercell-surface-activated areshared i ndC h a p t e r1s5a n d1 6 .[ M o d i f i ferdo mW G u oa n dF G discusse Giancotti, 2OO4,Nat Rev.Mol CellBiol 5(10):8'16-826,and R O Hynes, 2002, Cell 110:,673-687l

: N OVERVIEW X D H E S I O NA C E L L - C E LALN D C E L L - M A T R I A

807

superfamily CAMs), and ECM components(type IV collagen, laminin, nidogen/entactin,and perlecan-likeproteoglycans)are highly conserved,whereasothers are not. For example,fruit flies do not have certain types of collagenor the ECM protein fibronectin that play crucial roles in mammals. A common feature of adhesiveproteins is repeating domains forming very large proteins. The overall length of these molecules,combined with their ability to bind numerous ligands via distinct functional domains, likely played a role their evolution. The diversity of adhesivemoleculesarisesin large part from two phenomena that can generatenumerous closely related proteins, called isoforms, that constitute a protein family. In some cases,the different members of a protein family are encoded by multiple genes that ,.or. ]ro" common ancestor by gene duplication and divergent evolution (Chapter 6). In other cases,a single gene produces an RNA transcript that can undergo alternative splicing to yield multiple mRNAs, each encoding a distinct protein isoform (Chapter 8). Both phenomena contribute to the diversity of some protein families such as the cadherins.Particular isoforms of an adhesiveprotein are often expressedin some cell types and tissuesbut not others.

Cell-Celland Cell-Matrix Adhesion: An Overview r Cell-cell and cell-extracellularmatrix (ECM) interactions are critical for assembling cells into tissues, controlling cell shape and function, and determining the developmentalfate of cells and tissues.Diseasesresult from abnormalities in the srrucruresor expressronof adhesion molecules. r Cell-adhesionmolecules (CAMs) mediate direct cell-cell adhesions(homotypic and heterotypic),and cell-surface adhesion receptors mediate cell-matrix adhesions (see Figure 19-1). These interactions bind cells into tissues and facilitate communication between cells and their envlronments. The cytosolic domains of CAMs and adhesionreceprors nd adapter proteins that mediate interaction with cyskeletalfibers and intracellular signaling proteins. r The major families of cell-surface adhesion molecules are the cadherins,selectins,Ig-superfamily CAMs, and integrins(seeFigure 19-2). r Tight cell-cell adhesionsentail both cis (lateral or inrracellular) oligomerization of CAMs and trans (intercellular) interaction of like (homophilic) or different (heterophilic) CAMs (seeFigure 19-3).The combinationof cis and trans interactions produces a Velcro-like adhesionbetweencells. r The extracellular matrix (ECM) is a complex meshwork of proteins and polysaccharidesthat contributes to the structure and function of a tissue. The maior classesof ECM moleculesare proteoglycans,collagens,and multiadhesivematrix proteins (fibronectin, laminin). C H A P T E R1 9

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TNTEGRATTN CG E L L St N T O T t S s U E S

r The evolution of adhesion molecules with specialized structures and functions permits cells to assembleinto diverseclassesof tissueswith varying functions.

Cell-Cell and CelI-ECM Junctions andTheirAdhesionMolecules Cells in epithelial and nonepithelial tissuesusemany, but not all, of the samecell-cell and cell-matrix adhesionmolecules. Becauseof the relatively simple organization of epithelia, as well as their fundamental role in evolution and development, we begin our detailed discussion of adhesion with the epithelium.

E p i t h e l i aC l e l l sH a v eD i s t i n c tA p i c a l ,L a t e r a l , a n d B a s a lS u r f a c e s Cells that form epithelial tissuesare said to be polarized becausetheir plasma membranes are organized into at least two discreteregions. TypicallS the distinct surfaces of a polarized epithelial cell are called the apical (top), lateral (side),and basal (baseor bottom) surfaces(Figure 19-8). The area of the apical surface is often greatly expanded by the formation of microvilli. Adhesion molecules play essentialroles in generating and maintaining thesestructures. Epithelia in different body locations have characteristic morphologiesand functions (Figure 19-8). Stratified (multilayered)epitheliacommonly serveas barriers and protective surfaces(e.g.,the skin), whereas simple (single-layer) epithelia often selectivelymove ions and small molecules from one side of the layer to the other. For instance, the simple columnar epitheliumlining the stomachsecreteshydrochloric acid into the lumen; a similar epithelium lining the small intestine transports products of digestion from the lumen of the intestine across the basolateral surface into the blood (seeFigure 1,1,-29). In simple columnar epithelia, adhesive interactions between the lateral surfaceshold the cells together into a two-dimensional sheet,whereasthose at the basal surface connect the cells to a specializedunderlying extracellular matrix called the basal lamina. Often the basal andlateral surfaces are similar in composition and together are called the basolateral surface.The basolateralsurfacesof most simple epithelia are usually on the side of the cell closestto the blood vessels,whereas the apical surface is not in direct contact with other cells or the ECM. In animals with closed circulatory systems, blood flows through vesselswhose inner lining is composed of flattened epithelial cells called endothelial cells. The apical side of endothelial cells, which facesthe blood. is usuallv called the lwminal surfaceand the opposite basal side, thl abluminal surface. In general, epithelial cells are sessile,immobile cells, in that adhesionmoleculesfirmly and stably attach them to one another and their associatedECM. One especiallyimportant

Although hundreds of individual adhesion-moleculemediated interactions are sufficient to causecells to adhere, junctions play specialroles in imparting strength and rigidity to a tissue,transmitting information betweenthe extracellular and the intracellular space, controlling the passageof Basal ions and moleculesacrosscell layers' and serving as conduits sudace for the movement of ions and moleculesfrom the cytoplasm Basal of one cell to that of its immediate neighbor.Particularly imlamina portant to epithelial sheetsis the formation of junctions that help form tight seals between the cells and thus allow the ( b )S i m p l es q u a m o u s sheetto serveas a barrier to the flow of moleculesfrom one side of the sheetto the other. Three maior classesof animal cell junctions are prominent featuresof simple columnar epithelia (Figure 1'9-9 and Table 19-2). Anchoring iunctions and tight iunctions perform the key task of holding the tissue together. These ( c )T r a n s i t i o n a l junctions are organized into three parts: (1) adhesiveproteins in the plasma membrane that connect one cell to another cell on the lateral surfaces(CAMs) or to the extracellular matrix on the basal surfaces (adhesion receptors); (2) adapterproteins, which connect the CAMs or adhesion receptors to cytoskeletalfilaments and signaling molecules; and (3 ) the cytoskeletalfilaments themselves.Tight junctions also control the flow of solutes through the extracellular spacesbetween the cells, forming an epithelial sheet.Tight junctions are found primarily in epithelial cells' whereas (d) Stratified squamous (nonkeratinized) anchoring junctions can be seen in both epithelial and nonepithelial cells. The third class of junctions, gap iunctions, permit the rapid diffusion of small, water-solublemolecules between the cytoplasm of adjacent cells. They share with anchoring and tight junctions the role of helping a cell communicatewith its environmentsbut are structurally very different from anchoring junctions and tight junctions and do not play a key role in strengthening cell-cell and cellECM adhesions.Gap junctions, found in both epithelial and '19-8Principal typesof epithelia.Theapicaland A FIGURE cells, resemble cell-cell iunctions in plants characteristics nonepithelial cellsexhibitdistinctive surfaces of epithelial basolateral which we discussin Section19.6' calledplasmodesmata, (a)Simple cells,including of elongated epithelia consist columnar Thiee types of anchoring junctions are present in cells. tract) andcervical cells(inthe liningof thestomach mucus-secreting Two participate in cell-cell adhesion,whereasthe third par(b)Simple cells(inthe liningof thesmallintestine) andabsorptive ticipatesin cell-matrix adhesion.Adherensiwnctionsconnect composed of thincells,linethe bloodvessels epithelia, squamous (c)Transitional the lateral membranes of adiacent epithelial cells and are (endothelial andmanybodycavities cells/endothelium) shapes, layers of cellswith different of several usually located near the apical surface,just below the tight composed epithelia, (e g the (Figure 1'9-9).A circumferential belt of actin and and contraction junctions subject to expansion cavities linecertain , (nonkeratinized) line epithelia squamous bladder)(d)Stratified urinary myosin filaments in a complex with the adherensiunction resist abrasion theselinings suchasthe mouthandvagina; surfaces functions as a tension cable that can internally brace the cell of or secretion in theabsorptron do not participate andgenerally and thereby control its shape. Epithelial and some other a thinfibrous Thebasallamina, intoor out of thecavity, materials types of cells,such as smooth muscle and heart cells,are also all supports andotherECMcomponents, networkof collagen bound tightly together by desmosomes,snaplike points of tissue connective themto the underlying andconnects epithelia contact sometimescalled spot desmosomes.Hemidesmosomes,found mainly on the basal surfaceof epithelial cells, mechanismused to generatestrong, stable adhesionsis to anchor an epithelium to components of the underlying exconcentratesubsetsof these moleculesinto clusters called tracellular matrix, much like nails holding down a carpet' runctlons. Bundlesof intermediatefilaments running parallel to the cell surface or through the cell interconnect spot desmosomes T h r e eT y p e so f J u n c t i o n sM e d i a t eM a n y C e l l - C e l l and hemidesmosomes'imparting shape and rigidity to the Interactions and Cell-ECM cell. Adherensiunctions and desmosomesare found in many different types of cells; hemidesmosomesappear to be reto one another All epithelialcells in a sheetare connected strictedto epithelialcells. and the extracellular matrix bv specialized iunctions. ( a ) S i m p l ec o l u m n a r

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(b)

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Tight junction A d h e r e n sj u n c t i o n Lateral surface

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FIGURE 19-9 Principaltypesof celljunctionsconnectingthe columnarepithelialcellsliningthe smallintestine.(a)Schematic cutawaydrawingof intestinal epithelial cellsThebasalsurface of the cellsrests on a basallamina, andtheapical sudace isoacked with fingerlike microvilli thatproject intotheintestinal lumenTightjunctions, lyingjustunderthemicrovilli, prevent thediffusron of manysubstances between theintestinal lumenandthebloodthrouqh theextracellular

s p a c eb e t w e e cne l l sG a pj u n c t i o nasl l o wt h e m o v e m e notf s m a l l molecules andionsbetweenthe cytosols of adjacent cells.The nn s c t i o n s ,p o t r e m a i n i ntgh r e et y p e so f j u n c t i o n s - a d h e r ej u desmosomes, andhemidesmosomes-are critical to cell-cell andcell(b)Electron matrixadhesion andsignaling micrograph of a thinsection of intestinal epithelial cells,showingrelative locations of thedifferent junctionsIPart (b)C Jacobson etal,2001,]ournat CeilBiot.152:435-450 l

Desmosomesand hemidesmosomes help transmit shear forces from one region of a cell layer to the epithelium as a whole, providing strength and rigidity to the enrire epithelial cell layer. They are especiallyimportant in maintaining the integrity of skin epithelia.For instance,mutations that interfere with hemidesmosomal anchoring in the skin can lead to blistering in which the epithelium becomesdetached from its matrix foundation and e*tracellular fluid accumulates at the basolateral surface, forcing the skin to balloon outward.

many different types of cadherinsin vertebrates,because many differenr types of cells in widely diverse rissuesuse these CAMs to mediate adhesion and communication. The brain expressesthe largest number of different cadherins, presumably owing to the necessityof forming many very specificcell-cellcontactsto help establishits complex wiring diagram. Invertebrates,however, are able to function with fewer than 20 cadherins.

C a d h e r i n sM e d i a t eC e l l - C e lAl d h e s i o n s i n A d h e r e n sJ u n c t i o n sa n d D e s m o s o m e s The primary CAMs in adherensjunctions and desmosomes belong to the cadherin family. In vertebrates, rhis protein family of more than 100 members can be qrouped into at least six subfamilies, including classicalcadherins and desmosomal cadherins, which we will describe below, as well asprotocadherinsand others. The diversity of cadherins arisesfrom the presenceof multiple cadherin genesand alternative RNA splicing. It is not surprising that there are 810

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T N T E G R A TC I NEGL L tSN T ol s s u E s

Classical Cadherins The "classical" cadherinsinclude E-, N-, and P-cadherins.E- and N-cadherinsare the most widely expressed,particularly during early differentiation. Sheetsof polarized epithelial cells,such as those that line the small intestineor kidney tubules,contain abundant E-cadherinalong their lateral surfaces.Although E-cadherinis concentratedin adherensjunctions, it is present throughout the lateral surfaces,where it is thought to link adjacentcell membranes.The results of experimentswith L cells, a line of cultured mouse fibroblasts, demonstratedthat E-cadherinspreferentiallymediate homophilic interactions. L cells expressno cadherins and adhere poorly to themselvesor to other cells.\(hen the

ADHESI0N TYPI

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Cell-cell

Cadherins

Actin filaments

Shape,tension, signaling

2. Desmosomes

Cell-cell

Desmosomal cadherins

Intermediate filaments

Strength, durability, slgnallng

3. Hemidesmosomes

Cell-matrix

Integrin(ct694)

Intermediatefilaments

Shape,rigiditv'signaling

Cell-cell

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Actin filaments

Controllingsoluteflow, signaling

Connexins, innexins, pannexlns

Possibleindirect connectlons to cytoskeleton through adapters to other junctions

Communication; small-molecule transport between cells

Anchoring junctions

Tight junctions

Communication; molecule transport between cells

E-cadheringene was introduced into L cells, the engineered L cellswere found to adherepreferentially cadherin-expressing to other cells expressingE-cadherin (Figure 19-10). These L cells expressingE-cadherin formed epithelial-like aggregates with one another and with epithelialcellsisolatedfrom lungs. Although most E-cadherins exhibit primarily homophilic binding, somemediateheterophilicinteractions. The adhesivenessof cadherins dependson the presence of extracellular Caz*, the property that gave rise to their name (calcium adhering).For example,the adhesionof L cells expressingE-cadherin is prevented when the cells are No c a d h e r i nt r a n s g e n e

bathedin a solutionthat is low in Ca2* (Figure19-10).Some adhesion moleculesrequire some minimal amount of Ca"in the extracellular fluid to function properly, whereas others (e.g.,IgCAMs) are Ca2*-independent. The role of E-cadherin in adhesion can also be demonstrated in experiments with cultured epithelial cells called Madin-Darby canine kidney (MDCK) cells (Figure 9-34)' A green fluorescent-protein-labeledform of E-cadherin has been used in these cells to show that clusters of E-cadherin mediate the initial attachment and subsequent zippering

C a d h e r i nt r a n s g e n e

adherensiunctions. Each classicalcadherin contains a single transmembrane domain, a relatively short C-terminal cytosolic domain, and five extracellular "cadherin" domains (seeFigure 1'9-2)'The extracellular domains are necessaryfor Ca2* binding and cadherin-mediatedcell-cell adhesion. Cadherin-mediated adhesion entails both lateral (intracellular) and trans (intercellular)molecularinteractions(seeFigure 1,9-3)'TheCa"C a 2 * m e d i a t e s E c a d h e r i n F I G U R 1 E 9 1 0 E X P E R I M E N T A L a binding sites,located betweenthe cadherin repeats'serveto d e p e n d e na t d h e s i o no f L c e l l s .U n d e sr t a n d a rcde l lc u l t u r e rigidify the cadherin oligomers.The cadherin oligomers subfluid,L cells in theextracellular of calcium in the presence conditions form intercellular complexes to generatecell-cell of a genethat causes seq,retttly intosheets(/eff)Introduction do not aggregate then additional lateral contacts' resulting in a and in theiragqregation adhesion in thesecellsresults of E-cadherin the expression of cadherinsinto clusters.In this way' multiup" "zippering (center) but not of calcium clumpsin the presence intoepithelial-like interactions sum to produce a very tight pt.io*-alfittity (right)Bar,60pm [From L Adams etal, 1998, Cynthia rn itsabsence adhesion. intercellular J C e l lB i o l 1 4 2 ( 4 ) : 1 0 5 - 111 9 l 811

cELL-cELLANDcELLEcMJUNcT|oNsANDTHEIRADHESIoNMoLEcULEs

Podcast:E-cadherinZipper

not just the N-terminal domains, participate by interdigitation in trans associations. The C-terminal cytosolic domain of classicalcadherins Timeafter m i x i n gc e l l s is linked to the acin cytoskeleton by adapter proteins ( hr s) : (Figure 19-12). These linkages are essential for strong 0 adhesion, apparently owing primarily to their contributing to increasedlateral associations.For example, disruption of the interactions between classicalcadherins and a- or B-catenin-two common adapter proteins that link these cadherins to actin filaments-dramatically reduces A EXPERIMENTAL FtcURE19-11 E-cadherin mediates cadherin-mediatedcell-cell adhesion. This disruption ocadhesiveconnections in culturedMDCKepithelialcells.An curs spontaneously in tumor cells, which sometimesfail to E-cadherin genefusedto greenf luorescent protein(GFp) was express cr-catenin, and can be induced experimentally by introduced intocultured MDCKcells. Thecellswerethenmixed depleting the cytosolic pool of accessibleB-catenin.The together in a calcium-containing mediumandthedistribution of cytosolic domains of cadherinsalso interact with intracellufluorescent E-cadherin wasvisualized overtime(shownin hours). lar signalingmoleculessuch as B-cateninand p12O-catenin. Clusters of E-cadherin mediate theinitialattachment andsuosequent InterestinglS B-catenin not only mediates cytoskeletal zippering up of theepithelial cells.[From Cynthia L Adams erat, 1998. attachment but can also translocate to the nucleus and J CellBiol 142(4).1105-1j191 alter gene transcription in the ITnt signaling pathway (see Figure 16-32). Cadherinsplay a critical role during tissuedifferentiaThe resultsof domain swap experiments,in which an extion. Each classicalcadherin has a characteristictissuedistracellular domain of one kind of cadherin is replaced with tribution. In the course of differentiation, the amount or the correspondingdomain of a different cadherin, have indinature of the cell-surface cadherins changes, affecting cated that the specificity of binding resides,at least in part, many aspectsof cell-celladhesionand cell migration. For in the most distal (farthestfrom the membrane)extracellular instance,the reorganizationof tissuesduring morphogendomain, the N-terminal domain. Cadherin-mediatedadheesis is often accompaniedby the conversion of nonmotile sion was commonly thought to require only head-to-head epithelial cells into motile precursor cells for other tissues interactions between the N-terminal domains of cadherin (mesenchymal cells). Such epith elial-mesenchymal tr ansioligomers on adjacent cells, as depicted in Figure 19-12. tions are associatedwith a reduction in the expressionof However, some experimentssuggestthat under some condiE-cadherin (Figure 1,9-13a,b). The conversion of epithetions, at least three cadherin domains from each molecule. lial cells into malignant carcinoma cells, such as in certain

B-Catenin

Cell 1

Plasma membrane

A FIGURE 19-12Proteinconstituents of typicaladherens junctions.Theexoplasmic domains of E-cadherin dimersclustered junctions at adherens on adjacent cellsformCa+2-dependent homophilic interactions. Thecytosolic domains of the E_cadherins binddirectly or indirectly to multiple (eg , B_ proteins adapter catenrn) thatconnect thejunctions (F-actin) to actinfilaments of the

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Cell 2

cytoskeleton andparticipate in intracellular pathways. signaling Somewhat different setsof adapter proteins areillustrated in the two cellsto emphasize thata variety of adapters caninteract with junctionsSomeof theseadapters, adherens suchasZOj , can interact with several different CAMs[Adapted fromV Vasioukhin and E Fuchs, 2001, Curr.OpinCellBiol 13:761

( a )A d h e r e n te p i t h e l i acl e l l s ( b ) M o t i l em e s e n c h y m a l cells

The cadherin desmogleinwas identified through studies of an unusual but revealing skin diseasecalled pemphigus uulgaris, an autoimmune disease.Patients with a u t o i m m u n e d i s o r d e r s s y n t h e s i z ea n t i b o d i e s t h a t b i n d to a normal body protein. In pemphigus vulgaris the autoantibodies disrupt adhesion between epithelial cells, causing blisters of the skin and mucous membranes.The predominant autoantibody was shown to be specific for

{al Plasma

( c )C a n c e r o u cs e l l s ,n o c a d h e r i n

membrane In t e r c eIlu l ar space

N o r m a lc e l l s i n e p i t h e l i alli n i n g o f g a s t r i cg l a n d s e x p r e s sc a d h e r i n FIGURE19-13 E-cadherinactivity is lost A EXPERIMENTAL during the epithelial-mesenchymaltransition and cancer of the expression progression.A proteincalledSnailthat suppresses transitions(a) is associated with epithelial-mesenchymal E-cadherin of the NormalepithelialMDCK cellsgrown in culture (b) Expression snallgene in MDCKcellscausesthem to undergoan epithelialdetectedby of E-cadherin transition(c) Distribution mesenchymal staining(darkbrown)in thin sectionsof tissue immunohistochemical is from a patientwith hereditarydiffusegastriccancerE-cadherin boardersof normalstomachgastricgland seenat the intercellular is seenat the bordersof epithelialcells(upper right);no E-cadherin (a)and(b)fromAlfonso underlyinginvasivecarcinomacells [Panels of M A Nieto) lmagescourtesy Arias,2001,Cell105:425-431; Martinez (c)fromF.Carneiro et al , 2004,J Pathol203(2):681-687 Panel l ductal breast tumors or hereditary diffuse gastric cancer (Figure 19-13c), is also marked by a loss of E-cadherin actrvrty.

D e s m o s o m a l C a d h e r i n s D e s m o s o m e s( F i g u r e 1 9 - 1 , 4 ) contain two specializedcadherin proteins, desmoglein and desmocollin, whose cytosolic domains are distinct from those in the classical cadherins. The cytosolic domains of desmosomalcadherins interact with adapters proteins such as plakoglobin (similar in structure to B-catenin), plakophilins, and a member of the plakin family of adapters called desmoplakin. These adapters, which form the thick cytoplasmic plaques characteristic of desmosomes,in turn interact with intermediatefilaments.

D e s m o g l e i na n d desmocollin (cadherins)

lntermediatefilaments

C y t o p l a s m i cp l a q u e (Plakoglobin, desmoPlakins, plakophilins) Cytoplasmicplaques

P l a s m am e m b r a n e s

,0'2pm ,

(a)Modelof a desmosome 19-14Desmosomes. A FIGURE of intermediate to thesrdes with attachments cells epithelial between anddesmocollin CAMsdesmoglein Thetransmembrane filaments, boundto the familyAdapterproteins belongto thecadherin plakoglobin, of the CAMsinclude domains cytoplasmic (b)Electron of a thin micrograph andplakophilins desmoplakins, differentiated two cultured connecting a desmosome of section from radiate filaments of intermediate Bundles humankeratinocytes plaques that linethe inner cytoplasmic thetwo darklystaining (a),seeB M plasma membranes lPart of theadjacent surface Curr'OpinCell 1993, R Garrod, D 11:551, and 1993,Neuron Gumbiner, of R vanBuskirk l Biol5:3OPart(b)courtesy

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desmoglein;indeed,the addition of such antibodiesto normal skin inducesthe formation of blistersand disruption of cell adhesion.I 6

The firm epithelialcell-celladhesionsmediatedby cadherinsin adherensjunctions permits the formation of a secondclassof intercellularjunctionsin epithelia-tight junctions.

c

T i g h t J u n c t i o n sS e a lO f f B o d y C a v i t i e sa n d R e s t r i cD t i f f u s i o no f M e m b r a n eC o m p o n e n t s For polarizedepithelialcellsto function as barriersand mediators of selectivetransport, extracellularfluids surrounding their apical and basolateralmembranesmust be kept separate.Tight lunctions berween adjacent epirhelial ceils are usuallylocatedin a band surroundingthe cell just below the apicalsurfaceand help establishand maintain cell polarity (Figure79-I5). Thesespecialized junctionsform a barrier that sealsoff body cavitiessuch as the intestinallumen and the blood (e.g.,the blood-brainbarrier). Tight junctions prevent the diffusion of macromolecules and, to varying degrees,small water-solublemoleculesand ions acrossan epithelialsheetvia the spacesbetweencells. They also maintain the polarity of epiihelial cells by preventing the diffusion of membrane proteins and glycolipids betweenthe apicaland the basolateralregionsof the plasma membrane, ensuring that these regions contain different membrane components. As a consequence,movement of many nutrients acrossthe intestinal epithelium is in large part through the trdnscellular pdthway via specific membrane-boundrransportproteins (seeFigure lI-29). Tight junctions are composedof thin bands of plasmamembrane proteins that completely encircle the cell and are in contact with similar thin bands on adiacent cells. When t h i n s e c t i o n so f c e l l sa r e v i e w e di n a n e l e c t r o nm i c r o s c o D e . the lateral surfacesof adjacentcells appear to touch each other at intervals and even to fuse in the zone just below the apical surface(seeFigure 19 -9b). In freeze-fracrurepreparations, tight junctions appear as an interlocking network of ridgesand groovesin the plasmamembrane(Figure 19-15a).

Intercellular space L i n k a g eo f p r o t e i p p a , r t i c l e si n a d j a c e n t ceils

i.

n:i'

> FIGURE 19-15Tightjunctions.(a)Freeze-fracture preparation of tightjunctionzonebetween two intestinal epithelial cellsThe fracture planepasses throughthe plasma membrane of oneof the two adjacent cellsA honeycomb-like networkof rldgesandgrooves belowthe microvilli constitutes thetightjunctlonzone (b)Schematic drawingshowshow a tightjunctionmightbe formedby the linkage of rowsof proteinparticles in adjacent cells.In the insermrcrograpn ( c ) of an ultrathin sectional viewof a tightjunction, the adlacent cells canbe seenin closecontactwherethe rowsof proterns interact(c) Asshownin theseschematic drawings of the majorproteins in tight j u n c t i o n bs o , t ho c c l u d iann dc l a u d i n -c1o n t a ifno u rt r a n s m e m b r a n e helices, whereas thejunctionadhesron (JAM)hasa single molecule transmembrane domarn anda largeextracellular (a)courtesy region[part of L A Staehelin Drawing in part(b)adapted fromL A Staehelin andB E Hull, 1978,9ci An 238(5):140,andD proc Nat,l.Acad Goodenough,1999, Sci USA96:319Photograph in part(b)courtesy of S Tsukita et al, 2001,NatureRev. Mol.CellBiol.2:285 Drawing in part(c)adapted fromS Tsukita et al, 2001. rVature ReizMol. CellBiol 2:2851

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Rows of prorern particles

N

Occludin

Claudin-1

^ \/-: -/ N>

Very high magnification revealsthat rows of protein particles 3-4 nm in diameter form the ridges seen in freezefracturemicrographsof tight junctions.In the model shown in Figure 19-15b, the tight junction is formed by a double row of theseparticles,one row donatedby eachcell. Treatment of an epithelium with the proteasetrypsin destroysthe tight junctions,supportingthe proposalthat proteinsare essential structural components of these junctions. The two principal integral-membraneproteins found in tight iunctions are occludin and clawdin When investigators engineered mice with mutations inactivating the occludin gene, which was thought to be essentialfor tight junction formation, the mice still had morphologicallydistinct tight junctions. Further analysisled to the discoveryof claudin. Each of theseproteins has four membrane-spanningct helices(Figure 19-15c).The claudin multigenefamily encodesnumerous homologousproteinsthat exhibit distinct tissue-specific patternsof expression.A group of iunction adhesionmolecules (JAMs) have been found to contribute to homophilic adhesionand other functionsof tight junctions.Thesemolecules,which contain a singletransmembranect helix, belong to the Ig superfamily of CAMs. The extracellular domains of rows of occludin,claudin, and JAM proteins in the plasma membraneof one cell apparentlyform extremelytight links with similar rows of the same proteins in an adiacentcell, creating a tight seal. Ca2*-dependentcadherin-mediated adhesionalso plays an important role in tight junction formation, stability,and functton. The long C-terminalcytosolicsegmentof occludin binds to PDZ domains in certain large cytosolicadapterproteins' Thesedomains are found in various cytosolicproteins and mediate binding to the C-termini of particular plasmamembrane proteins or to each other. PDZ-containing adapter proteins associatedwith occludin are bound, in turn, to other cytoskeletaland signaling proteins and to actin fibers.Theseinteractionsappear to stabilizethe linkage betweenoccludin and claudin moleculesthat is essential for maintaining the integrity of tight iunctions. The Ctermini of claudins also bind to the intracellular,multiplePDZ-domain-containing adaptor protein ZO-1, which is also found in adherensjunctions(seeFigure 1'9-72).Thus, as cytosolic is the casefor adherensjunctionsand desmosomes, adaptor proteins and their connectionsto the cytoskeleton are critical componentsof tight junctions. Plasma-membraneproteins cannot diffuse in the plane of the membranepast tight lunctions. Theseiunctions also restrict the lateral movement of lipids in the exoplasmic leaflet of the plasma membrane in the apical and basolate r a l r e g i o n so f e p i t h e l i a lc e l l s .I n d e e d ,t h e l i p i d c o m p o s i tions of the exoplasmic leaflet in these two regions are distinct. Essentiallyall glycolipids are present in the exop l a s m i c f a c e o f t h e a p i c a l m e m b r a n e ,a s a r e a l l p r o t e i n s linked to the membrane by a glycosylphosphatidylinositol ( G P I ) a n c h o r ( s e eF i g u r e 1 0 - 1 9 ) . I n c o n t r a s t ,l i p i d s i n t h e cytosolic leaflet in the apical and basolateral regions of epithelial cells have the same composition and can apparently diffuse laterally from one region of the membraneto the other.

A simple experiment demonstratesthe impermeability of certain tight junctionsto many water-solublesubstances. In this experiment, lanthanum hydroxide (an electron-dense colloid of high molecularweight) is injectedinto the pancreatic blood vesselof an experimental animal; a few minutes later' the pancreatic epithelial acinar cells are fixed and prepared for microscopy. As shown in Figure 19-76, the lanthanum hydroxide diffuses from the blood into the spacethat separatesthe lateral surfacesof adjacentacinar cellsbut cannot p e n e t r a t ep a s tt h e t i g h t i u n c t i o n . The barrier to diffusion provided by tight junctions is not absolute.Owing at leastin part to the varying propertiesof the different types of claudin molecules located in different tight junctions, their permeability to ions, small molecules, and water varies enormously among different epithelial tissues.In epitheliawith "leaky" tight junctions, small molecules can move from one side of the cell layer to the other through the paracellularpathway in addition to the transcellular pathway (Figure19-17). The leakinessof tight junctions can be altered by intracellular signaling pathways, especiallyG protein and cyclic AMP-coupled pathways (Chapter 15). The regulation of tight junction permeability is often studied by measuringion flux (electricalresistance)or the movement of radioactive or fluorescentmoleculesacrossmonolayersof MDCK cells. p1fll The importance of paracellular transport is illustrated in severalhuman diseases.In hereditary hypoIl magnesemia,defectsin the cldudinlS geneprevent the normaL paracellular flow of magnesium in the kidney. This results in an abnormally low blood level of magnesium, which can lead to convulsions.Furthermore' a mutation in

Apical surface of left cell

Apical surface o f r i g h tc e l l

T i g h tj u n c t i o n

LateraI surface

Lateral surface o f r i g h tc e l l

of left cell

L a n t h a n u mh y d r o x i d e (betweencells)

19-16Tightjunctionsprevent FIGURE a EXPERIMENTAL space throughextracellular passageof largemolecules are in the pancreas betweenepithelialcells.Tightjunctions hydroxide lanthanum colloid to the largewater-soluble impermeable sideof the epithelium (darkstain)administered fromthe basolateral of D Friend lCourtesv l

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815

P a r a c e l l u l aTrr a n s c e l l u l a r pathway pathway

memorane

A FIGURE 19-17Transcellular and paracellular pathwaysof transepithelial transport.Transcellu lartransport requires the cellular uptakeof molecules on onesideandsubsequent release o n t h eo p p o s i tsei d eb y m e c h a n i s m d is c u s s iendC h a p t e1r 1 I n paracellular transport, molecules moveextracellularly throughparts of tightjunctions, whosepermeability to smallmolecules andions depends on thecomposition of thejunctional components andthe physiologic stateof the epithelial cells[Adapted fromS Tsukita etal, 2001, NatureReuMol CellBiol2:2851

the claudinl4 genecauseshereditary deafness,apparently by altering transport around hair-cell epithelia in the cochlea of the inner ear. Toxins produced by Vibrio cholerae, which causes cholera, and several other enteric (gastrointestinaltract) bacteria alter the permeability barrier of the intestinal epithelium by altering the composirion or activiry of tight junctions. Other bacterial toxins can affectthe ion-pumping activity of membrane transport proteins in intestinal epithelial cells. Toxin-induced changes in tight junction permeability (increasedparacellulartransport) and in protein-mediatedion pumping (increasedtranscellulartransport) can result in massiveloss of internal body ions and water into the gastrointestinaltract, which in turn leadsto diarrhea and potentially lethal dehydration. I

I n t e g r i n sM e d i a t eC e l l - E C M Adhesions i n E p i t h e l i aC l ells

of anchoring junctions called hemidesmosomes(seeFigure 19-9a). Hemidesmosomescomprise several integral membrane proteins linked via cytoplasmic adaptor proteins (e.g.,plakins) to keratin-basedintermediatefilaments.The principal ECM adhesionreceptorin hemidesmosomes is integrin a684,. a member of the integrin family of proteins ( s e eF i g u r e1 9 - 2 ) .

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combinations, are known. A single B chain can interact with any one of multiple o chains,forming integrins that bind different ligands. This phenomenon of combinatorial diuersity allows a relatively small number of components to serve a large number of distinct functions. Although most cells expressseveraldistinct integrins that bind the sameor different ligands, many integrins are expressedpredominantly in certain types of cells. Not only do many integrins bind more than one ligand but severalof their ligands bind to multiple integrins. All integrins appear to have evolved from two ancient general subgroups: those that bind proteins containing the tripeptide sequenceArg-Gly-Asp, usually called the RGD sequence(e.g., fibronectin) and those that bind laminin. Severalintegrin o subunits contain a distinctive inserted domain, the l-domain, which can mediate binding of certain integrins (e.g.,cr1B1and e2B1) to various collagensin the ECM. Some integrins with I-domains are expressedexclusively on leukocytesand red and white blood cell precursor (hematopoietic)cells.Thesedomains recognizecell-adhesion moleculeson other cells, including members of the Ig superfamily (e.g., ICAMs, VCAMs), and thus participate in cellcell adhesion. Integrins typically exhibit low affinities for their ligands, with dissociation constants Kp between 10 6 and 10 7 mollL. However, the multiple weak interacrions generated by the binding of hundredsor thousandsof integrin molecules to their ligands on cells or in the extracellular matrix allow a cell to remain firmly anchored to its ligandexpresslngtarget. Parts of both the c and the B subunits of an integrin molecule contribute to the primary extracellular ligand-binding site (seeFigure 19-2). Ligand binding to integrins also requires the simultaneous binding of divalent cations. Like other cell-surfaceadhesivemolecules,the cytosolic region of integrins interacts with adapter proteins that in turn bind to the cytoskeletonand intracellular signalingmolecules.Most integrins are linked to the actin cytoskeleton, such as the ct6B1 and cr3B1 integrins that connect the basal surface of epithelial cells to the basal lamina via laminin. However, the cytosolic domain of the B4 chain in the a6B4 integrin in hemidesmosomes,which is much longer than those of other B integrins, binds to specializedadapter proteins that in turn interact with keratin-basedintermediatefilamenrs. As we will see,the diversity of integrins and their ECM ligands enablesintegrins to participate in a wide array of key biological processes,including the migration of cells to their correct locations in the formation of the body plan of an embryo (morphogenesis)and in the inflammatory response. The importance of integrins in diverse processesis highIighted by the defectsexhibited by knockout mice engineered to have mutations in each of almost all of the integrin subunit genes.These defectsinclude maior abnormalities in development,blood vesselformation, Ieukocytefunction. inflammation,bone remodeling,and hemosrasis. Despitetireir differences,all theseprocessesdepend on integrin-mediated regulated interactions between the cytoskeleton and either the ECM or CAMs on other cells.

ct1B1

Many types

Mainly

a291.

Many types

Mainly collagens; also laminins

(l381

Many types

Laminins

o"4pl

Hematopoietic cells

Fibronectin; VCAM-1

Ct581

Fibroblasts

Fibronectin

CI691

Many types

Laminins

ctl92

T lymphocytes

ctMp2

Monocytes

Platelets

a6g4

Epithelialcells

ICAM-2 Serumproteins(e.g.,C3b, fibrinogen,factor X); ICAM-1 S e r u mp r o t e i n s( e . g .f,i b r i n o gen,von'Willebrandfactor, vitronectin);fibronectin Laminin

'The

proteins' Some integrins are grouped into subfamilies having a common B subunit. Ligands shown in red are CAMs; all others are ECM or serum subunits can have multiple spliced isoforms w i r h d i f f e r e n t c y t o s o l i cd o m a i n s . souRCE:R. O. Hynes, L992, Cell 69:L1.

In addition to their adhesionfunction, integrins can mediate outside-inand inside-outsignaling(seeFigure 19-7). The engagementof integrins by their extracellular ligands can, through adapter proteins bound to the integrin cytosolic region, influence the cytoskeleton and intracellular signaling pathways (outside-in signaling). Conversely,intracellular signaling pathways can alter, from the cytoplasm, the structure of integrins and consequentlytheir abilities to adhereto their extracellular ligands and mediatecell-celland cell-matrix interactions (inside-out signaling). Integrinmediated signaling pathways influence processesas diverse as cell survival, cell proliferation, and programmed cell death (Chapter 27).

G a p J u n c t i o n sC o m p o s e do f C o n n e x i n sA l l o w S m a l lM o l e c u l e st o P a s sD i r e c t l yB e t w e e n A d j a c e n tC e l l s Early electron micrographs of virtually all animal cells that were in contact revealedsitesof cell-cellcontact with a characteristic intercellular gap (Figure 19-1,8a1.This feature prompted early morphologiststo call theseregionsgap junctions. In retrospect,the most important feature of thesejunctions is not the gap itself but a well-defined set of cylindrical particlesthat cross the gap and composepores connecting the cytoplasmsof adjacentcells.

In many tissues,large numbers of gap junctional particles cluster together in patches(e.g', along the lateral surfacesof epithelialcells;seeFigure 19-9). Vhen the plasma membrane is purified and then shearedinto small fragments' some piecesmainly containing patches of gap junctions are

The effectivepore size of gap junctions can be measured by inyectinga cell with a fluorescentdye covalently linked to moleculesof various sizesand observingwith a fluorescence microscope whether the dye passesinto neighboring cells' Gap junctions between mammalian cells permit the passage of moleculesas large as 1'2 nm in diameter.In insects,these junctions are permeableto moleculesas large as 2 nm in diameter.Generally speaking,moleculessmaller than 1200 Da passfreely and those larger than 2000 Da do not pass;the purr"g. of intermediate-sizedmoleculesis variable and limit.d. thus ions, many low-molecular-weight precursors of cellular macromolecules'products of intermediary metabolism, and small intracellular signaling moleculescan pass from cell to cell through gap junctions.

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(a)

Gap j un c t i o n 50 nm ,,"13 ,i1.;,,tl-,, 50 nm (c)

< F I G U R E1 9 - 1 8 G a p j u n c t i o n s . ( a )I n t h i s t h i n s e c t i o nt h r o u g ha g a p l u n c t i o nc o n n e c t i n g t w o m o u s el i v e rc e l l s t, h e t w o p l a s m a m e m b r a n e as r e c l o s e l ya s s o c i a t efdo r a d i s t a n c eo f s e v e r ahl u n d r e d n a n o m e t e r ss,e p a r a t e d b y a " g a p " o f 2 - 3 n m ( b )N u m e r o u s r o u g h l yh e x a g o n apl a r t i c l eas r ev i s i b l ei n t h i s p e r p e n d i c u l aorr, e n face,view of the cytosolicface of a regionof plasmamembrane e n r i c h e di n g a p j u n c t i o n sE a c hp a r t i c l ea l i g n sw i t h a s i m i l a rp a r t i c l e o n a n a d j a c e nct e l l ,f o r m i n ga c h a n n e cl o n n e c t i n g the two cells ( c ) S c h e m a t im c o d e lo f a g a p l u n c t i o nc o n n e c t i n g two plasma m e m b r a n e sB o t h m e m b r a n e cs o n t a i nc o n n e x o nh e m i c h a n n e l s , c y l i n d e ros f s i xd u m b b e l l - s h a p ecdo n n e x i nm o l e c u l e sT w o c o n n e x o njso i n i n t h e g a p b e t w e e nt h e c e l l st o f o r m a g a p - l u n c t i o n channel,1 5-2 O nm in diameter,that connectsthe cytosolsof the t w o c e l l s ( d ) E l e c t r o nd e n s i t yo f a r e c o m b i n a ngt a p - j u n c t i o n channeldeterminedby electroncrystallography (Left)Sideview of the completestructureorientedas in part (c) N4: membrane bilayer;E : extracellular gap; C : cytosol (Rrgrht) View looking d o w n o n t h e c o n n e x o nf r o m t h e c y t o s o pl e r p e n d i c u l at or t h e m e m b r a n eb i l a y e r sS, u p e r i m p o s eodn t h e e l e c t r o nd e n s i t ym a p a r e m o d e l so f t h e t r a n s m e m b r a ncer h e l i c e s( g o l d ) f, o u r p e r s u b u n i t ,2 4 p e r c o n n e x o nh e m i c h a n n e[lP a r(ta )c o u r t e soyf D G o o d e n o u gpha r t( b ) courtesy of N GilulaPart(d)adapted fromS j Fleishman et al ,2004,Mol Ceil 15(6):879-888 l

Connexon hemichannel

+=*4

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I n t e r c e l l u l agr a p

In nervoustissue,someneuronsare connectedby gap junctions through which ions pass rapidly, thereby allowing very rapid transmission of electric signals. Impulse transmission through theseconnections,calledelectricalsynapses,is almost a thousandfoldas rapid as ar chemicalsynapses(Chapter23). Gap junctions are also presentln many non-neuronaltissues, where they help to integratethe electricaland metabolicactivities of many cells.In the heart, for instance,gap junctionsrapidly pass ionic signalsamong muscle cells, which are tightly interconnectedvia desmosomes,and thus contribute to the electricallystimulatedcoordinatecontraction of cardiac muscle cellsduring a beat.As discussedin Chapter 15, someextracellular hormonal signalsinduce the production or releaseof small intracellular signalingmoleculescalled secondmessengers (e.g., cyclic AMP, IP3, and Ca2*) that regulate cellular 818

.

c H A p r E R1 9 |

T N T E G R A T cTENLGL st N r o l s s u E s

metabolism. Becausesecond messengerscan be transferred betweencellsthrough gap junctions,hormonal stimulation of one cell can trigger a coordinatedresponseby that samecell and many of its neighbors.Suchgap junction-mediatedsignaling plays an important role, for example,in the secretionof digestiveenzymesby the pancreasand in the coordinatedmuscular contractile waves (peristalsis)in the intestine.Another vivid example of gap junction-mediatedtransport is the phenomenon of metabolic coupling,or metabolic cooperation,in which a cell transfersnutrients or intermediarymetabolitesto a neighboringcell that is itself unable to synrhesizethem. Gap junctions play critical roles in the developmentof egg cellsin the ovary by mediatingthe movementof both metabolitesand signaling moleculesbetween an oocyte and its surrounding granulosacellsas well as betweenneighboringgranulosacells.

A current model of the structure of the gap junction is shown in Figure 19-1,8c,d. Vertebrategap junctions are composed of connexins, a family of structurally related transmembrane proteins with molecular weights between 26,000 and 60,000.A completelydifferentfamily of proteins,the innexins, forms the gap junctions in invertebrates.A third family of innexinJike proteins, called pannexins,was recently discovered in both vertebratesand invertebrates.Each vertebrate hexagonal particle consists of 12 noncovalently associatedconnexin molecules: 6 form a cylindrical connexin hemichannel in one plasma membrane that is joined to a connexin hemichannelin the adjacent cell membrane, forming the continuous aqueous channel betrveenthe cells. Each individual connexin molecule spansthe plasma membrane four times with a topology similar Pannexinsare capableof to that of occludin (seeFigure1,9-1,5). forming intercellular channels as well; however, pannexin hemichannelsmay also function to permit direct exchange betweenthe intracellular and extracellular spaces. There are 21 differentconnexingenesin humans,with different setsof connexinsexpressedin different cell types.This diversity togetherwith the generationof mutant mice with inactivating mutations in connexin genes,has highlighted the importanceof connexinsin a wide variety of cellular systems. Some cells expressa single connexin that forms homotypic channels.Most cells,however,expressat leasttwo connexinsl thesedifferent proteins assembleinto heteromericconnexins, which in turn form heterotypicgap-junctionchannels.Diversity in channel composition leads to differencesin channel permeability. For example, channels made from a 43-kDa connexin isoform, Cx43-the most ubiquitously expressed connexin-are more than 1OO-foldas permeableto ADP and ATP as those made from Cx32 (32 kDa). The permeability of gap junctions^can be regulated by changesin the intracellular pH and Ca'* concentration and phosphorylation of connexin. One example of the physioIogical regulation of gap junctions is mammalian childbirth. The muscle cells in the mammalian uterus must contract strongly and synchronously during labor to expel the fetus. To facilitate this coordinate activity, immediately prior to and during labor there is an approximately five- to tenfold increasein the amount of the major myometrial connexin, Cx43, and an increasein the number and size of gap junctions, which decreaserapidly postpartum. Assemblyof connexins,their trafficking within cells,and formation of functional gap junctions apparently depend on N-cadherin and its associatedjunctional proteins (e.g., a- and B-cateninsZO-1,, ZO-2) as well as desmosomalproteins (plakoglobin, desmoplakin, and plakophrlin-2). PDZ domainsinZO-l andZO-2 bind to the C-terminusof Cx43 and apparently mediate its interaction with catenins and N-cadherin. The relevanceof these relationships is particularly evident in the heart, which depends on adjacent gap junctions (for rapid coordinated electrical coupling) and adherensjunctions and desmosomes(for mechanicalcoupling between cardiomyocytes)for the intercellular integration of electricalactivity and movement required for normal cardiac function. It is noteworthy that ZO-1 servesas an adaptor for adherens (see Figure 1,9-12),tight, and gap junctions,

suggestingthis and other adapters can help integrate the formation and functions of thesediverseiunctions. Mutations in connexin genescauseat least eight huincluding neurosensorydeafness(Cx26 man diseases, and Cx31). cataractor heart malformations (Cx43, Cx45, and CxS0). and the X-linked form of Charcot-Marie-Tooth disease(Cx32), which is marked by progressive degeneration of peripheral nerves.I

Cell-Celland CelI-ECMJunctions and Their Adhesion Molecules r Polarized epithelial cells have distinct apical, basal' and lateral surfaces.Microvilli projecting from the apical surfacesof many epithelial cellsconsiderablyexpand the cells' surfaceareas. r Three maior classesof cell junctions-anchoring junctions, tight junctions, and gap junctions-assemble epithelial cells into sheetsand mediate communication between them (seeFigures19-'l' and 19-9).Anchoring junctionscan be further subdivided into adherens iunctions' desmosomes,and hemidesmosomes. r Adherens junctions and desmosomes are cadherincontaining anchoring junctions that bind the membranesof adjacentcells,giving strengthand rigidity to the entire tissue. r Cadherinsare cell-adhesionmolecules(CAMs) responsible for Ca2*-dependent interactions between cells in epithelial and other tissues.They promote strong cell-cell adhesion by mediating both lateral intracellular and intercellular interactions. r Adapter proteins that bind to the cytosolic domain of cadherinsand other CAMs and adhesionreceptorsmediate the associationof cytoskeletaland signalingmoleculeswith the plasma membrane (seeFigure 1,9-12).Strong cell-cell adhesion dependson the linkage of the interacting CAMs to the cytoskeleton. r Tight junctions block the diffusion of proteins and some lipids in the plane of the plasma membrane'contributing to the polarity of epithelialcells.They also limit and regulatethe extracellular (paracellular)flow of water and solutesfrom one sideof the epitheliumto the other (seeFigure 19-1'7). r Hemidesmosomesare integrin-containing anchoring junctions that attach cellsto elementsof the underlying extracellular matrix. r Integrins are alarge family of crBheterodimeric cell-surface proteins that mediate both cell-cell and cell-matrix adhesions and inside-outand outside-insignalingin numeroustissues. ap junctions are constructed of multiple copiesof conn proteins, assembledinto a transmembrane channel interconnectsthe cytoplasmsof two adjacentcells (see Figure 19-18). Small moleculesand ions can passthrough gap junctions, permitting metabolic and electricalcoupling of adiacentcells.

J U N C T I O NASN D T H E I RA D H E S I O NM O L E C U L E S C E L L - C E LALN D C E L L . E C M

819

The Extracellular Matrixl: TheBasalLamina In animals, the extracellular matrix helps organize cells into tissuesand coordinatestheir cellular functions by activating intracellular signaling pathways that control cell growth, proliferation, and gene expression. Many functions of the matrix require transmembraneadhesionreceptorsthat bind directly to ECM components and that also interact, through adapter proteins, with the cytoskeleton.A principal class of adhesion receptors that mediate cell-matrix adhesion is integrins (Section 19.2). However, other types of molecules also function as important adhesionreceptors. Adhesion receprors bind to three types of molecules abundantin the extracellularmatrix of all tissues: r Proteoglycans,a group of glycoproteins that cushion cells and bind a wide variety of extracellular molecules r Collagen fibers, which provide structural integrity and mechanicalstrength and resilience r Solublemultiadhesivematrix proteins, such as laminin and fibronectin, which bind to and cross-link cell-surface adhesionreceptorsand other ECM components. We begin our description of the structuresand functions of thesemajor ECM components in the context of the basal lamina-the specializedextracellular matrix sheetthat plays (a)

a particularly important role in determining the overall architecture and function of epithelial tissue. In the next section, we discuss the ECM molecules commonly found in nonepithelial tissues,including connectivetissue.

T h e B a s a lL a m i n aP r o v i d e sa F o u n d a t i o n f o r A s s e m b l yo f C e l l si n t o T i s s u e s In animals, most organized groups of cells in epithelial and nonepithelial tissuesare underlain or surrounded by the basal lamina, a sheetlikemeshwork of ECM components usually no more than 60-120 nm thick (Figure 19-191. The basal lamina is structured differently in different tissues.In columnar and other epithelia (e.g., intestinal lining, skin), it is a foundation on which only one surface of the cellsrests.In other tissues,such as muscle or fat, the basal lamina surrounds each cell. Basal laminae play important roles in regeneration after tissue damage and in embryonic development. For insrance, the basal lamina helps four- and eight-celledembryos adhere together in a ball. In the development of the nervous system, neurons migrate along ECM pathways that contain basal lamina components.In higher animals, two distinct basal laminae are employed to form a tight barrier that limits diffusion of molecules between the blood and the brain (bloodbrain barrier), and in the kidney a specializedbasallamina serves as a selectivepermeability blood filter. Thus the basal lamina is important for organizing cells into tissues and distinct compartmenrs, tissue repair, and guiding (b)

Cytosol

Basalsurface

,e:

Connective tissue

Basal amina

A FIGURE19-19 The basal lamina separating epithelial cells and some other cells from connective tissue. (a)Transmission electronmicrographof a thin sectionof cells(top)and underlying connectivetissue(bottom) The electron-dense layerof the basal laminacan be seento follow the undulationof the basalsurfaceof the cells.(b) Electronmicrographof a quick-freeze deep-etch preparationof skeletalmuscleshowingthe relationof the plasma

820

c H A P T E R1 9

|

T N T E G R A T TC NE GL L St N T OT t S S U E S

Cell-surface receptorproteins

C o l l a g e nf i b e r s

membrane,basallamina,and surroundingconnectivetissue.In this preparation, the basallaminais revealedas a meshworkof filamentousproteinsthat associates with the plasmamembraneand the thickercollagenfibersof the connectivetissue [part(a)courtesy of P FitzGerald Part(b)from D W Fawcett,1981,TheCell,2ded, SaunderV PhotoResearchers; courtesy of JohnHeuser l

migrating cells during development.It is thereforenot surprising that basal lamina components have been highly conservedthroughout evolution. Most of the ECM components in the basal lamina are synthesizedby the cells that rest on it. Four ubiquitous protein components are found in basal laminae (Figure1,9-20):

L a m i n i n ,a M u l t i a d h e s i v eM a t r i x P r o t e i n ,H e l p s C r o s s - l i nC k o m p o n e n t so f t h e B a s a lL a m i n a

t Perlecan, alarge multidomain proteoglycan that binds to and cross-linksmany ECM componentsand cell-surface molecules

Laminin, the principal multiadhesivematrix protein in basal laminae, is a heterotrimeric, cross-shapedprotein with a total molecularweight of 820,000 (Figure1'9-21).At least 15 laminin isoforms, eachcontaining slightly different polypeptide chains, have been identified. Globular LG domains at the C-terminus of the laminin a subunit mediate Ca2*dependentbinding to specificcarbohydrateson certain cellsurfacemoleculessuch as syndecanand dystroglycan,which will be described further in Section 1,9.4.LG domains are found in a wide variety of proteins and can mediate binding to steroids and proteins as well as carbohydrates. For example, LG domains in the a chain of laminin can mediate binding to certain integrins, including a6B4 integrin in hemidesmosomeson the basal surfaces of epithelial cells. Laminin is the principal basal laminal ligand of a5B4 and other integrins(Table19-3).

t Nidogen (also called entactin), a rodlike molecule that cross-linkstype IV collagen,perlecan,and laminin and helps incorporate other componentsinto the ECM

TypelV Collagenls a Major Sheet-Forming StructuralComponentof the BasalLamina

Other ECM molecules are incorporated into various basal laminae, depending on the tissue and particular functional requirementsof the basal lamina. As depictedin Figure 19-1, one side of the basallamina is linked to cells by adhesionreceptors,including cr6B4integrin in hemidesmosomes,which binds to laminin in the basal lamina. The other side of the basal lamina is anchoredto the adjacentconnectivetissueby a layer of fibers of collagenembeddedin a proteoglycan-richmatrix. In stratified squamous epithelia (e.g.,skin), this linkage is mediatedby anchoring fibrils of type VII collagen. Together,the basal lamina and collagen-anchoringfibrils form the structure calledthe basement membrane.

Type IV collagen is a principal structural component of all basallaminaeand can bind to certain integrin adhesionreceptors. CollagenIV is one of more than2} typesof collagenthat participate in the formation of distinct extracellularmatrices in various tissues(Table 1'9-4).Although they differ in certain structural features and tissue distribution, all collagensare trimeric proteinsmade from three polypeptides,usually called collagen cr chains. All three ct chains can be identical (homotrimeric) or different (heterotrimeric).A trimeric collagen moleculecontains one or more three-strandedsegments,each with a similar triple-helical structure (Figure 19-22a1.Each strand contributed by one of the a chainsis twisted into a lefthanded helix, and three such strands from the three ct chains wrap around each other to form a right-handed triple helix.

. Type IV collagen, trimeric moleculeswith both rodlike and globular domains that form a two-dimensional network t Laminins, a family of multiadhesive,cross-shapedproteins that form a fibrous two-dimensional network with type IV collagen and that also bind to integrins and other adhesionreceptors

19-20 Major protein componentsof the basal A FIGURE andlaminin eachformtwo-dimensional lamina.TypelV collagen molecules whicharecross-linked by entactin andperlecan networks,

[Adaptedf rom B Albertset al , 1994, MolecularBiologyof the Cell,3d ed , G a r l a n dp, 9 9 1 l

T H E E X T R A C E L L U L AMRA T R I X l : T H E B A S A LL A M I N A

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A FIGURE 19-22The collagentriple helix.(a)(teft)Sideviewof thecrystal structure of a polypeptide f ragment whosesequence is basedon repeating setsof threeaminoacids,Gly-X-Y characteristic q.chains(Center) of collagen Eachchainistwistedintoa left-handed helix,andthreechains wraparoundeachotherto forma right-handed triplehelix.Theschematic model(nglht) clearly illustrates thetriple (b)Viewdowntheaxisof thetriple helical natureof thestructure. (orange) helixTheprotonsidechains of theglycine residues pointinto theverynarrowspacebetweenthe polypeptide chainsin thecenterof thetriplehelix.In mutations in collagen in whichotheraminoacids glycine, replace theprotonin glycine isreplaced by largergroups that disrupt the packing of thechains anddestabilize thetriple-helical structure fromR Z Kramer etal, 2001, J MolBiol311(1):131 lAdapted ] FIGURE 19-21Laminin,a heterotrimeric multiadhesive matrixproteinfound in all basallaminae.(a)Schematic model showingthegeneral shape,location of globular domains, andcoiledcoilregionin whichlaminin's threechains arecovalently linkedby several disulfide bonds.Different regions of lamininbindto cellsurface receptors andvarious (b)Electron matrixcomponents. micrographs of intactlamininmolecule, showingitscharacteristic (/eft)andthe carbohydrate-binding crossappearance LGdomains (right).leart(a) nearthe C-terminus adapted fromG R Martin andR Timpl, 1987, Ann Rev. Cell Biol3:57,andK yamada, 199j,J BiolChem 256:12809 Part(b)fromR Timplet al , 2000,MatrixBiol 19:309; photograph at rjghtcourtesy of JUrgen Engel l

The collagen triple helix can form becauseof an unusual abundanceof three amino acids:glycine,proline, and a modified form of proline called hydroxyproline (seeFigure 2-15). They make up the characteristicrepeatingmotif Gly-X-l where X and Y can be any amino acid but are often proline and hydroxyproline and less often lysine and hydroxylysine. Glycine is essentialbecauseits small side chain, a hydrogen atom, is the only one that can fit into the crowded centerof the three-strandedhelix (Figure19-22b). Hydrogen bonds help hold the three chains together. Although the rigid peptidyl-proline and peptidyl-hydroxyproline linkages are not compatible with formation of a classic single-strandedo. helix, they stabilize the distinctive threestrandedcollagen helix. The hydroxyl group in hydroxypro-

822

CHAPTER 19

I

I N T E G R A T I NCGE L L SI N T OT I S S U E S

Iine helps hold its ring in a conformation that stabilizesthe three-strandedhelix. The unique properties of each type of collagen are due mainly to differencesin (1) the number and lengths of the collagenous, triple-helical segments;(2) the segmentsthat flank or interrupt the triple-helical segmentsand that fold into other kinds of three-dimensionalstructures;and (3) the covalent modification of the cr chains (e.g., hydroxylation, glycosylation,oxidation, cross-linking).For example, the chains in type IV collagen,which is unique to basal laminae, are designatedIVo chains.Mammals expresssix homologous IVa chains,which assembleinto a seriesof type IV collagens with distinct properties. All subtypes of type IV collagen, however, form a 400-nm-long triple helix (Figure 19-23) that is interrupted about 24 times with nonhelical segmentsand flanked by large globular domains at the C-termini of the chains and smaller globular domains at the N,termini. The nonhelical regions introduce flexibility into the molecule. Through both lateral associarionsand interactions entailing the globular N- and C-termini, type IV collagen molecules assembleinto a branching, irregular two-dimensional fibrous network that forms the lattice on which the basal lamina is built (Figure 19-23). In the kidnen a double basal lamina, the glomerular basement membrane, separatesthe epithelium that Iines the urinary spacefrom the endothelium that lines the

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collagen triple-helical structure (seeFigure 1,9-22).Different collagens are distinguished by the length and chemicalmodifications of their crchainsand by the presence of segmentsthat interrupt or flank their triple-helical reglons.

their own distinct fibrils in the matrix of most connectivetissues.Although severaltypes of cells are found in connective tissues,the various ECM components are produced largely by cells called fibroblasts.

r Perlecan,a large secretedproteoglycanpresentprimarily in the basal lamina, binds many ECM componentsand adhesionreceptors.Proteoglycansconsist of membraneassociatedor secretedcore proteins covalently linked to one or more specializedpolysaccharidechains called gly(GAGs). cosaminoglycans

F i b r i l l a rC o l l a g e n sA r e t h e M a j o r F i b r o u s Proteinsin the ECMof ConnectiveTissues

TheExtracellular Matrixll: and OtherTissues Connective Connectivetissue,such as tendon and cartilage,differs from other solid tissuesin that most of its volume is made up of extracellular matrix rather than cells. This matrix is packed with insoluble protein fibers and contains proteoglycans, various multiadhesive proteins, and a specialized glycosaminoglycancalled hyaluronan.The most abundant fibrous protein in connectivetissue is collagen.Rubberlike elastin fibers, which can be stretchedand relaxed,also are presentin deformablesites (e.g.,skin, tendons,heart). The fibronectins. a familv of multiadhesivematrix proteins. form

About 80-90 percent of the collagen in the body consistsof fibrillar collagens(types I, II, and III), located primarily in connectivetissues(seeTable L9-4).Becauseof its abundance in tendon-rich tissuesuch as rat tail, type I collagenis easyto isolate and was the first collagento be charactetized.Itsfundamental structural unit is a long (300-nm)' thin (1.5-nmdiameter)triple helix (seeFigure1,9-22)consistingof two ct1(I) chains and one cr2(I)chain, each 1050 amino acids in length. The triple-strandedmoleculesassociateinto higher-orderpolymers called collagen fibrils, which in turn often aggregateinto larger bundlescalledcollagenfibers (Figure1'9-24). Quantitatively minor classesof collagen include fibrilassociatedcollagens,which link the fibrillar collagensto one another or to other ECM compoflentsi sheet-forming and anchoring collagens, which form two-dimensional networks in basal laminae (type IV) and connect the basal lamina in skin to the underlying connective tissue (type VII); transmembrane collagens, which function as adhesion reand host defense ceptors(e.g.,BP180 in hemidesmosomes);

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of fibrillarcollagens. < FIGURE 19-24Biosynthesis on aresynthesized ctchains Step[: Procollagen (ER) reticulum withtheendoplasmic associated ribosomes are oligosaccharides andasparagine-linked membrane, propeptide StepZ: Propeptides addedto theC-terminal linkedby to formtrimersandarecovalently associate in the Gly-X-Y residues and selected bonds, disulfide (certain prolines modified arecovalently tripletrepeats galactose arehydroxylated, andlysines lGallor galactoseto somehydroxylysines, isattached glucose lhexagonsl StepE: The prolines arecis-+ transisomerized). stabilization formatron, zipperlike facilitate modifications protein andbindingbythe chaperone of triplehelices, or the helices 13),whichmaystabilize Hsp47(Chapter of thetrimersor both premature prevent aggregation aretransported Steps4 andEl: Thefoldedprocollagens wheresomelateral to andthroughthe Golgiapparatus, a s s o c r a t ironnt os m a lbl u n d l etsa k e sp l a c eT h ec h a i n s (step6), the N- andC-terminal arethensecreted (stepZ), andthe trimers propeptrdes areremoved a s s e m bilnet of i b r i l sa n da r ec o v a l e n tcl yr o s s - l i n k e d givesthe of thetrrmers (stepts). The67-nmstaggering micrographs in electron appearance fibrilsa striated 2002, andB Brodsky, (inset)[Adapted fromA V Persikov i SA99(3):1101-11031 l c a dS c U P r o cN a t ' A

f i n r i t a s s e m b l ya n d c r o s s - l i n k i n g

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collagens, which help the body recognize and eliminate pathogens.Interestingln severalcollagens (e.g.,types XVIII and XV) are also proteoglycans with covalently attached GAGs (seeTable l9-4).

Historically, British sailors were provided with limes to prevent scurvy, Ieading to their being called "limeys." Mutations in lysyl hydroxylasegenesalso can causeconnective-tissuedefects.I

F i b r i l l a rC o l l a g e nl s S e c r e t e da n d A s s e m b l e d i n t o F i b r i l sO u t s i d eo f t h e C e l l

TypeI and ll CollagensAssociatewith N o n f i b r i l l a rC o l l a g e n st o F o r mD i v e r s e Structures

Fibrillar collagensare secretedproteins, produced primarily by fibroblasts in the ECM. Collagen biosynthesisand secretion follow the normal pathway for a secretedprotein, described in detail in Chapters 13 and 14. The collagen ct chains are synthesizedas longer precursors,called pro-c chains, by ribosomes attached to the endoplasmicreticulum (ER). The pro-a chainsundergo a seriesof covalentmodifications and fold into triple-helical procollagen moleculesbefore their releasefrom cells (seeFigure 19-24). After the secretionof procollagen from the cell, extracellular peptidasesremove the N-terminal and C-terminal propeptides. In fibrillar collagens, the resulting molecules, which consist almost entirely of a triple-strandedhelix, associatelaterallyto generatefibrils with a diameterof 50-200 nm. In fibrils, adjacent collagen moleculesare displacedfrom one another by 67 nm, about one-quarterof their length. This staggeredarray produces a striated effect that can be seenin both light and electronmicroscopicimagesof collagenfibrils (Figure 19-24, inset). The unique properties of the fibrous collagens(e.g.,types I, II, ilI) are mainly due to the formation of fibrils. Short non-triple-helical segmentsat either end of the fibrillar collagen a chains are of particular importance in the formation of collagen fibrils. Lysine and hydroxylysine side chains in thesesegmentsare covalently modified by extracellular lysyl oxidasesto form aldehydesin place of the amine group at the end of the side chain. These reactive aldehyde groups form covalent cross-linkswith lysine, hydroxylysine, and histidine residuesin adjacent molecules.These crosslinks stabilizethe side-by,side packing of collagenmolecules and generatea very strong fibril. The removal of the propeptides and covalent cross-linking take place in the extracellular spaceto prevent the potentially catastrophic assemblyof fibrils within the cell. The post-translationalmodifications of pro-o chains are crucial for the formation of mature collagenmoleculesand their assemblyinto fibrils. Defectsin thesemodifications have serious consequences, as ancient mariners frequently experienced.For example, ascorbic acid (vitamin C) is an essentialcofactor for the hydroxylases responsiblefor adding hydroxyl groups to proline and lysine residuesin pro-ct chains. In cells deprived of ascorbate,as in the diseasescuruy,the pro-o.chainsare not hydroxylated sufficientlyto form stabletriple-helicalprocollagenat normal body temperature,and the procollagenthat forms can'sfithout not assembleinto normal fibrils. the structural support of collagen, blood vessels,tendons, and skin become fragile. Fresh fruit in the diet can suDDly sufficient vitamin C to support the formation of noi-rl collagen. 826

.

cHAprER 19 |

T N T E G R A TcTEN LG L tsN T ol s s u E s

Collagensdiffer in the structuresof the fibers they form and how these fibers are organized into networks. Of the predominant types of collagen found in connectivetissues,type I collagen forms long fibers, whereasnetworks of type II collagen are more meshlike. In tendons, for instance,the long type I collagen fibers connect muscles to bones and must withstand enormous forces. Becausetype I collagen fibers have great tensile strength,tendons can be stretchedwithout being broken. Indeed, gram for gram, type I collagen is stronger than steel.Two quantitatively minor fibrillar collagens,type V and type XI, coassembleinto fibers with type I collagen, thereby regulating the structuresand properties of the fibers. Incorporation of type V collagen,for example,results in smaller-diameterfibers. Type I collagen fibrils are also used as the reinforcing rods in the construction of bone. Bones and teeth are hard and strong becausethey contain large amounts of dahllite, a crystalline calcium- and phosphate-containingmineral. Most bones are about 70 percent mineral and 30 percent protein, the vast majority of which is type I collagen. Bones form when certain cells (chondrocytesand osteoblasts)secrete collagen fibrils that are then mineralizedby deposition of small dahllite crystals. In many connectivetissues,particularly skeletal muscle, type VI collagenand proteoglycansare noncovalently bound to the sidesof type I fibrils and may bind the fibrils together to form thicker collagen fibers (Figure 19-25a).Type VI collagen is unusual in that the molecule consistsof a relatively short triple helix with globular domains at both ends. The Iateral association of two type VI monomers generatesan "antiparallel" dimer. The end-to-end associationof these dimers through their globular domains forms type VI "microfibrils." These microfibrils have a beads-on-a-string appearance,with about 50-nm-long triple-helical regions separatedby 40-nm-long globular domains. The fibrils of type II collagen, the major collagen in cartilage, are smaller in diameter than type I fibrils and are oriented randomly in a viscousproteoglycan matrix. The rigid collagen fibrils impart strength to the matrix and allow it to resist large deformations in shape. Type II fibrils are crosslinked to matrix proteoglycansby type IX collagen,another fibril-associatedcollagen.Type IX collagenand severalrelated types have two or three triple-helical segmentsconnected by flexible kinks and an N-terminal globular segment (Figure 19-25b). The globular N-terminal segment of type IX collagen extends from the fibrils at the end of one of its helical segments,as does a GAG chain that is sometimes Iinked to one of the type IX chains. These protruding nonhelical structures are thought to anchor the type II fibril to

(a)

(b)

Type I collagenfibrils

Type ll collagenfibril

abnormalities. Skin abnormalities have also been reported with type VI collagen disease.I

and Their ConstituentGAGsPlay Proteoglycans DiverseRolesin the ECM

Chondroitin su lfate

TypeVl collagen

Proteoglycan

A FIGURE 19-25 Interactions of fibrouscollagenswith (a)In tendons, nonfibrousfibril-associated collagens. typeI fibrils arealloriented in the direction of thestress aoplied to thetendon. Proteoglycans andtypeVl collagen bindnoncovalently to fibrils, coatingthesurfaceThemicrofibrils of typeVl collagen, which containglobular andtriple-helical segments, bindto typeI fibrilsand linkthemtogetherintothickerfibers(b)In cartilage, typelX collagen molecules arecovalently boundat regular intervals alongtypell fibrils A chondroitin sulfate chain,covalently linkedto thea2(lX)chainat theflexible kink,projects outwardfromthefibril,asdoestheglobular N-terminal regionlPart(a),seeR R Bruns etal, 1986, I CellBiol103:393 Part(b),seeL M Shaw andB Olson, 1991,Trends Biochem Sci18:1 91.1 proteoglycansand other components of the matrix. The interrupted triple-helical structure of type IX and related collagensprevents them from assemblinginto fibrils, although they can associatewith fibrils formed from other collagen types and form covalent cross-linksto them. Mutations affecting type I collagen and its associated proteinscausea variety of human diseases.Certain mutations in the genesencodingthe type I collagena1(I) or o2(I) chains lead to osteogenesisimperfecta, or brittle-bone disease.Becauseevery third position in a collagen ct chain must be a glycine for the triple helix to form (seeFigure 1,9-22), mutations of glycine to almost any other amino acid are deleterious, resulting in poorly formed and unstablehelices.Only one defectivea chain of the three in a collagenmoleculecan disrupt the whole molecule'striple-helicalstructureand function. A mutation in a single copy (allele) of either the cr1(I) geneor the c2(I) gene,which are located on nonsex chromosomes(autosomes),can causethis disorder.Thus it normally shows autosomal dominant inheritance. Absence or malfunctioning of collagen-fibril-associated microfibrils in muscle tissuedue to mutations in the type VI collagen genescausedominant or recessivecongenital muscular dystrophieswith generalizedmuscleweakness,respiratory insufficiency,muscle wasting, and muscle-relatedjoint

As we saw with perlecan in the basal lamina, proteoglycans play an important role in cell-ECM adhesion.Proteoglycans are a subset of secretedor cell-surface-attachedglycoproteins containing covalently linked specializedpolysaccharide chains called glycosaminoglycans(GAGs). GAGs are long linear polymers of specific repeating disaccharides.Usually one sugaris either a uronic acid (o-glucuronic acid or L-iduronic acid) or o-galactose; the other sugar is N-acetylglucosamine or N-acetylgalactosamine(Figure 1'9-26).One or both of the sugars contain at least one anionic group (carboxylate or sulfate).Thus each GAG chain bearsmany negativecharges. GAGs are classified into several major types based on the nature of the repeating disaccharideunit: heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. A hypersulfatedform of heparan sulfate called heparin, produced mostly by mast cells, plays a key role in allergic reactions.It is also used medically as an anticlotting drug becauseof its ability to activate a natural clotting inhibitor called antithrombin III. '!(ith the exception of hyaluronan, which is discussed below, all the major GAGs occur naturally as components of proteoglycans. Like other secretedand transmembrane glycoproteins, proteoglycan core proteins are synthesized on the endoplasmic reticulum (Chapter 13). The GAG chains are assembledon these cores in the Golgi complex. To generate heparan or chondroitin sulfate chains, a three-sugar"linker" is first attached to the hydroxyl side chains of certain serine residues in a core protein; thus the linker is an Olinked oligosaccharide(Figure L9-27a). In contrast, the linkers for the addition of keratan sulfate chains are oligosaccharidechains attached to asparagine residues; such Nlinked oligosaccharides are presentin many glycoproteins (Chapter 14), although only a subsetcarry GAG chains. All GAG chains are elongated by the alternating addition of sugar monomers to form the disacchariderepeatscharacteristicof a particular GAG; the chains are often modified subsequently by the covalent linkage of small molecules such as sulfate. The mechanismsresponsible for determining which proteins are modified with GAGs, the sequenceof disaccharidesto be added, the sitesto be sulfated, and the lengths of the GAG chains are unknown. The ratio of polysaccharideto protein in all proteoglycans is much higher than that in most other glycoproteins. Function of GAG Chain Modifications As is the casewith the sequenceof amino acids in proteins, the arrangement of the sugar residuesin GAG chains and the modification of specific sugars(e.g.,addition of sulfate) in the chains can determine their function and that of the proteoglycans containing them. For example, groupings of certain modified sugars in the GAG chains of heparin sulfate proteoglycans can control the binding of growth factors to certain receptors or the activities of proteins in the blood-clotting cascade.

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( a ) H y a l u r o n a n( n < 2 5 , 0 0 0 )

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lV-AcetylD-glucosamine

(b) Chondroitin(or dermatan)sulfate (n s 250\ t>u3

For years, the chemical and structural complexity of proteoglycansposed a daunting barrier to an analysis of their structures and an understanding of their many diverse functions. In recent years, investigatorsemploying classicaland state-of-the-artbiochemical techniques(e.g., capillary highpressureliquid chromatography), mass spectrometry, and genetics have begun to elucidate the detailed structures and functions of theseubiquitous ECM molecules.The resultsof ongoing studies suggest that sets of sugar-residuesequences containing some modifications in common, rather than single unique sequences,are responsiblefor specifying distinct GAG functions.A casein point is a set of five-residue(pentasaccharide) sequencesfound in a subsetof heparin GAGs that control the activity of antithrombin III (ATIII), an inhibitor of the

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M a n= m a n n o s e GlcNAc= N-acetylglucosamine SA= sialicacid '19-27Hydroxyl(O-)linked polysaccharides. (a) A FIGURE (GAG), Synthesis of a glycosaminoglycan in thiscasechondroitin is initiated sulfate, by transfer of a xylose residue to a serineresidue FIGURE 19-26The repeatingdisaccharides of glycosaminogly- in thecoreprotein, mostlikelyin the Golgicomplex, followedby cans(GAGs),the polysaccharide componentsof proteoglycans. sequential addition of two galactose residues. Glucuronic acidandNEachof thefourclasses of GAGsisformedby polymerization of acetylgalactosamine residues arethenaddedsequentially to these monomerunitsintorepeats of a particular disaccharide andsubsequent linkingsugars, formingthechondroitin sulfatechainHeparan sulfate modifications, including additionof sulfategroupsandinversion chainsareconnected to coreproteins bythesamethree-sugar linker. (epimerization) groupon carbon5 of o-glucuronic of the carboxyl (b)Mucin-type O-linked chains arecovalently boundto glycoproteins acidto yieldr-iduronic acid Heparin isgenerated (GalNAc) by hypersulfation viaan N-acetylgalactosamine monosaccharide to whichare of heparan sulfate, whereas hyaluronan isunsulfated. Thenumber covalently attached a variety of othersugars(c)Certain specialized (n)of disaccharides typically foundin eachglycosaminoglycan chain O-linked oligosaccharides, suchasthosefoundin the protein isgiven.Thesquiggly linesrepresent covalent bondsthatareoriented dystroglycan, (Man) areboundto proteins viamannose eitherabove(o-glucuronic acid)or below(r-iduronic acid)the rinq. monosaccharides. 828

C H A P T E R1 9

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T N T E G R A T T NCG E L L St N T O T T S S U E S

A FIGURE 19-28Pentasaccharide GAGsequence that regulatesthe activity of antithrombin lll (ATlll).Setsof modified five-residue sequences in the muchlongerGAGcalledheparin with thecomposition shownherebindto ATlllandactivate it, thereby inhibiting bloodclottingThesulfategroupsin redtypeareessential for thisheparin function; the modifications in bluetypemaybe present but arenot essential Othersetsof modified GAGsequences arethoughtto regulate the activity of othertargetproteins.

key blood-clotting proteasethrombin. When thesepentasacin heparin are sulfatedat two specificposicharide sequences tions, heparin can activateATIII, thereby inhibiting clot formation (Figure 19-28). Severalother sulfatescan be presentin the active pentasaccharidein various combinations, but they are not essentialfor the anticlotting activity of heparin. The rationale for generatingsetsof similar activesequences rather than a singleunique sequenceand the mechanismsthat control GAG biosynthetic pathways, permitting the generation of such activesequences, are not well understood, Diversity of Proteoglycans The proteoglycansconstitute a remarkably diversegroup of moleculesthat are abundant in the extracellularmatrix of all animal tissuesand are also expressedon the cell surface.For example, of the five major classesof heparan sulfate proteoglycans,three are located in the extracellularmatrix (perlecan,agrin, and type XVIII collagen) and two are cell-surfaceproteins. The latter include integral membrane proteins (syndecans)and GPl-anchored proteins (glypicans);the GAG chains in both types of cellsurface proteoglycans extend into the extracellular space. The sequencesand lengthsof proteoglycancore proteins vary considerably,and the number of attachedGAG chainsranges from just a few to more than 100. Moreover, a core protein is often linked to two different types of GAG chains,generating a "hybrid" proteoglycan. The basal laminal proteoglycan perlecanis primarily a heparan sulfate proteoglycan (HSPG) with three to four GAG chains, although it sometimescan have a bound chondroitin sulfate chain. Additional diversity in proteoglycansoccurs becausethe numbers of chains,compositions, and sequencesof the GAGs attachedto otherwise identical core proteins can differ considerably.Laboratory generationand analysisof mutants with defectsin proteoglycan production in Drosopbila melanogaster lfrurt fly), C. elegans(roundworm), and mice have clearly shown that proteoglycansplay critical roles in development,most likely as modulators of various signalingpathways. Syndecansare cell-surfaceproteoglycansexpressedby epithelial and nonepithelialcellsthat bind to collagensand mul-

tiadhesivematrix proteins(e.g.,fibronectin),anchoringcellsto the extracellular matrix. Like that of many integral membrane proteins, the cytosolic domain of syndecaninteracts with the actin cytoskeleton and in some caseswith intracellular regulatory molecules.In addition, cell-surfaceproteoglycans like syndecanbind many protein growth factors and other external signalingmolecules,thereby helping to regulatecellular metabolism and function. For instance,syndecansin the hypothalamic region of the brain modulate feeding behavior in response to food deprivation. They do so by participating in the binding to cell-surfacereceptors of antisatiety peptides that help control feeding behavior. In the fed state, the syndecanextracellular domain decorated with heparan sulfate chains is released from the surfaceby proteolysis,thus suppressingthe activity of the antisatiety peptides and feeding behavior. In mice engineeredto overexpressthe syndecan-l genein the hypothalamic region of the brain and other tissues,normal control of feeding by antisatietypeptidesis disrupted and the animals overeatand becomeobese.

HyaluronanResistsCompression,Facilitates C e l lM i g r a t i o n ,a n d G i v e sC a r t i l a g e Its Gel-likeProperties Hyaluronan, also called hyaluronic acid (HA) or hyaluronate, is a nonsulfated GAG (seeFigure 1,9-26a)made by a plasmamembrane-bound enzyme (HA synthase) that is directly secretedinto the extracellular space.(A similar approach is used by plant cellsto make their ECM component cellulose.) HA is a major component of the extracellular matrix that surrounds migrating and proliferating cells, particularly in embryonic tissues.In addition, hyaluronan forms the backbone of complex proteoglycan aggregatesfound in many extracellular matrices, particularly cartilage. Becauseof its remarkable physical properties,hyaluronan imparts stiffness and resilienceas well as a lubricating quality to many types of connectivetissuesuch as jolnts. Hyaluronan molecules range in length from a few disacchariderepeatsto =25,000. The typical hyaluronan in ioints such as the elbow has 10,000 repeatsfor a total mass of 4 x 106 Da and length of 10 pm (about the diameter of a small cell). Individual segmentsof a hyaluronan molecule fold into a rodlike conformation becauseof the B glycosidic linkages betweenthe sugarsand extensiveintrachain hydrogen bonding. Mutual repulsion between negatively charged carboxylate groups that protrude outward at regular intervals also contributes to these local rigid structures.Overall' however, hyaluronan is not a long, rigid rod as is fibrillar collagen; rather, in solution it is very flexible, bending and twisting into many conformations, forming a random coil. Becauseof the large number of anionic residueson its surface, the typical hyaluronan molecule binds a large amount of water and behavesas if it were a large hydrated spherewith a diameter of =500 nm. As the concentration of hyaluronan increases,the long chains begin to entangle, forming a viscousgel. Even at low concentrations,hyaluronan forms a hydrated gel; when placed in a confining space, such as in a matrix between two cells, the long hyaluronan

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moleculeswill tend to push outward. This outward pushing createsa swelling, or turgor pressure,within the extracellular space. In addition, the binding of cations by COOgroups on the surface of hyaluronan increasesthe concentration of ions and thus the osmotic pressurein the gel. As a result, large amounts of water are taken up into the matrix, contributing to the turgor pressure. These swelling forces give connectivetissuestheir ability to resist compression forces, in contrast with collagen fibers, which are able to resist stretching forces. Hyaluronan is bound to the surface of many migrating cells by a number of adhesion receptors (e.g., one called CD44) containing HA-binding domains, each with a similar three-dimensional conformation. Becauseof its loose, hydrated,porous nature,the hyaluronan"coat" bound to cells appearsto keep cellsapart from one another,giving them the freedom to move about and proliferate. The cessationof cell movement and the initiation of cell-cell attachmentsare frequently correlated with a decreasein hyaluronan, a decrease in HA-binding cell-surfacemolecules,and an increasein the extracellular enzyme hyaluronidase, which degrades hyaluronan in the matrix. Thesefunctions of hyaluronan are particularly important during the many cell migrations that facilitate differentiation and in the releaseof a mammalian egg cell (oocyte) from its surrounding cells after ovulation. The predominant proteoglycan in cartilage, calledaggrecan, assembleswith hyaluronan into very large aggregates, illustrative of the complex structures that proteoglycans sometimesform. The backbone of the cartilageproteoglycan aggregateis a long molecule of hyaluronan to which multiple aggrecanmoleculesare bound tightly but noncovalently (Figure 19-29a). A single aggrecanaggregate,one of the largestmacromolecularcomplexesknown, can be more than 4 mm long and have a volume larger than that of abacterial cell. These aggregatesgive cartilage its unique gel-like properties and its resistanceto deformation, essentialfor distributing the load in weight-bearingJornts. The aggrecan core protein (=250,000 MW) has one N-terminal globular domain that binds with high affinity to a specific disaccharide sequencewithin hyaluronan. This specific sequenceis generated by covalent modification of some of the repeatingdisaccharidesin the hyaluronan chain. The interaction between aggrecanand hyaluronan is facilitated by a link protein that binds to both the aggrecancore protein and hyaluronan (Figure 19-29b). Aggrecan and the link protein have in common a "link" domain. =100 amino acids long, that is found in numerous matrix and cell-surface hyaluronan-binding proteins in both cartilaginous and noncartilaginoustissues.Almost certainly theseproteins arosein the course of evolution from a single ancestralgenethat encodedjust this domain.

FibronectinsInterconnectCellsand Matrix, I n f l u e n c i n gC e l lS h a p e ,D i f f e r e n t i a t i o n , and Movement Many different cell types synthesizefibronectin, an abundant multiadhesive matrix protein found in all vertebrates. 830

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T N T E G R A T Tc N GL st N T ol s s u E s EL

Aggrecan (b)

H v a l u r o n am n olecule

+

Linkprotein Keratan sulfate

N-terminal Hyaluronan-binding domain

Chondroitin sulfate

Linking sugars Aggrecancore protein

FIGURE 19-29 Structureof proteoglycanaggregatefrom cartilage.(a)Electron micrograph of an aggrecan aggregate from fetalbovineepiphyseal cartilageAggrecan coreproteins arebound (b)Schematic at =40-nmintervals to a molecule of hyaluronan representation of an aggrecan monomer boundto hyaluronan In aggrecan, bothkeratan sulfateandchondroitin sulfatechains are attached to the coreprotein. TheN-terminal domainof thecore proteinbindsnoncovalently to a hyaluronan molecule. Binding is facilitated by a linkprotein, whichbindsto boththe hyaluronan molecule andthe aggrecan coreprotein. Eachaggrecan coreprotein has127Ser-Gly sequences at whichGAGchains canbe addedThe molecular weightof an aggrecan monomer averages 2 x 106.The entireaggregate, whichmaycontainupwardof 100aggrecan monomers, hasa molecular weightin excess of 2 x 108andisabout aslargeasthe bacterium E coli.lPart(a)from J A Buckwalterand L Rosenberg, 1983, CollRe/Res3:489; courtesyof L Rosenberg l The discoverythat fibronectin functions as an adhesivemolecule stemmed from observations that it is oresent on the surfacesof normal fibroblastic cells, which udh.r. tightly to petri dishesin laboratory experiments,but is absentfrom the surfacesof tumorigenic (i.e., cancerous)cells, which adhere weakly. The 20 or so isoforms of fibronectin are generated by alternative splicing of the RNA transcript produced from a single gene (see Figure 4-16). Fibronecrins are essential

for the migration and differentiation of many cell types in embryogenesis.Theseproteins are also important for wound healing becausethey promote blood clotting and facilitate the migration of macrophagesand other immune cells into the affectedarea. Fibronectins help attach cells to the extracellular matrix by binding to other ECM components, particularly fibrous collagens and heparan sulfate proteoglycans, and to cellsurface adhesion receptors such as integrins (seeFigure 192). Through their interactions with adhesionreceptors(e.g., cr5B1 integrin), fibronectins influence the shape and movement of cells and the organization of the cytoskeleton.ConverselSby regulating their receptor-mediatedattachmentsto fibronectin and other ECM components,cells can sculpt the immediate ECM environment to suit their needs. Fibronectins are dimers of two similar polypeptides linked at their C-termini by two disulfide bonds; each chain is about 50-70 nm long and 2-3 nm thick. Partial digestion of fibronectin with low amounts of proteasesand analysisof the fragments showed that each chain comprises several functional regions with different ligand-binding specificities (Figure 1.9-30a).Each region, in turn, contains multiple copies of certain sequencesthat can be classifiedinto one of three types. These classificationsare designatedfibronectin type I, II, and III repeats,on the basisof similaritiesin amino acid sequence,although the sequencesof any two repeatsof a given type are not identical. These linked repeatsgive the molecule the appearanceof beadson a string. The combination of different repeats composing the regions confers on fibronectin its ability to bind multiple ligands. One of the type III repeatsin the cell-binding region of fibronectin mediatesbinding to certain integrins. The results of studies with synthetic peptides correspondingto parts of

this repeat identified the tripeptide sequenceArg-Gly-Asp, usually called the RGD sequence,as the minimal sequence within this repeat required for recognition by those integrins. In one study, heptapeptidescontaining the RGD sequence or a variation of this sequencewere tested for their ability to mediate the adhesionof rat kidney cellsto a culture dish. The results showed that heptapeptidescontaining the RGD sequencemimicked intact fibronectin's ability to stimulate integrin-mediatedadhesion,whereasvariant heptapeptides lacking this sequencewere ineffective (Figure 19-31'). A three-dimensionalmodel of fibronectin binding to integrin based on structures of parts of both fibronectin and integrin has been assembled.In a high-resolution structure of the integrin-binding fibronectin type III repeat and its neighboring type III domain, the RGD sequenceis at the apex of a loop that protrudes outward from the molecule,in a position facilitating binding to integrins (see Figure 1930b). Although the RGD sequenceis required for binding to severalintegrins, its affinity for integrins is substantiallyless than that of intact fibronectin or of the entire cell-binding region in fibronectin. Thus structural features near the RGD sequencein fibronectins (e.g.,parts of adjacentrepeats,such as the synergyregion; seeFigure 19-30b) and in other RGDcontaining proteins enhance their binding to certain integrins. Moreover, the simple soluble dimeric forms of fibronectin produced by the liver or fibroblasts are initially in a nonfunctional conformation that binds poorly to integrins becausethe RGD sequenceis not readily accessible.The adsorption of fibronectin to a collagen matrix or the basallamina or, experimentallg to a plastic tissue-culturedish results in a conformational changethat enhancesits ability to bind to cells. Possibl5 this conformational change increasesthe accessibilityof the RGD sequencefor integrin binding'

(a)

(b) Fibrin, heparan suifate coilagen binding binding

k

C e l lb i n d i n g '-------^.E l l l B ElllA

Heparan Fibrin sulfate binding binding

fypelrcpeat Type ll repeat

'ab

lllcs

Synergy regron RGD sequence

tvpe lll repeat

Integrin

A FIGURE 19-30Organizationof fibronectinand its binding isshowndockedbytwo to integrin.(a)Scale modelof f ibronectin to theextracellular domains of integrin. Onlyoneof typelll repeats whicharelinkedby disulfided bondsnear thetwo similar chains, in the dimeric theirC-termini, fibronectin molecule isshownEach of three chaincontains about2446aminoacidsandiscomposed (typel, ll,or lll repeats). The typesof repeating aminoacidsequences ElllA,ElllB-bothtypelll repeats-andlllCSdomainarevariably intothe structure at locations indicated by arrowsCirculating spliced fibronectin lacksoneor bothof ElllAandElllB. At leastfivedifferent maybe present in the lllCSregionasa resultof alternative sequences

multirepeat(seeFigure several 4-16).Eachchaincontains splicing bindingsites someof whichcontainspecific regions, containing fibrin(a sulfate, for heparan (madeup of multiple-binding repeats) integrins. andcell-surface collagen, of bloodclots), majorconstituent the cell-binding known as is also domain Theintegrin-binding from weredetermined s domains of fibronectin domainStructures (b)A high-resolution showsthat structure of the molecule. fragments (red)extends outwardin a loopfromits the RGDbindingsequence asthe typellldomainon the samesideof fibronectin compact binding (blue), high-affinity to also contributes which region synergy Cell84:161 al, 1996, D J Leahyet from ] to integrins. [Adapted

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to disrupt fibronectin-integrin binding. Thus fibronectin moleculesremain bound to integrin while cell-generatedmechanical forces induce fibril formation. In effect, the integrins through adapter proteins transmit the intracellular forces generatedby the actin cytoskeletonto extracellular fibronectin. Gradually, the initially formed fibronectin fibrils mature into highly stable matrix components by covalent crossJinking. In some electron micrographic images, exterior fibronectin fibrils appear to be aligned in a seemingly continuous line with bundles of actin fibers within the cell (Figure 1,9-32b).These observations and the results from other studies provided the first example of a molecularly

o

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E 0.4

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o

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10 100 1000 (nmol/ml) Peptideconcentration

A EXPERIMENTAL FIGURE 19-31A specifictripeptidesequence (RGD)in the cell-bindingregion of fibronectinis requiredfor adhesionof cells.Thecell-binding regionof fibronectin contarns an integrin-binding hexapeptide sequence, GRGDSP in thesingle-letter aminoacidcode(seeFigure 2-14)Together with an additional C(C)residue terminal cysteine thisheptapeptide andseveral variants weresynthesized chemically. Differentconcentrations of each peptidewereaddedto polystyrene synthetic dishesthat hadthe (b) Fibronectin Cell Plasma Actin-containing proteinimmunoglobulin G (lgG)firmlyattached to theirsurfaces; the fibrils exterior membrane microfilaments peptides werethenchemically cross-linked to the lgG.Subsequently, cultured normalrat kidneycellswereaddedto the dishes and incubated for 30 minutes to allowadhesion. Afterthe nonbound cellswerewashedaway,the relative amountsof cellsthat had adhered firmlyweredetermined by staining the boundcellswith a dyeandmeasuring the intensity of thestaining with a spectrophotometer. Theresultsshownhereindicatethat cell adhesion increased abovethe background levelwith increasing peptideconcentration for thosepeptides containing the RGD sequence but notfor thevariants (modification lacking thissequence proc.Nat't. underlined). M D Pierschbacher andE Ruoslahti. [From 1984. A EXPERIMENTAL FIGURE 19-32Integrinsmediatelinkage Acad.Sci.USA81:5985.1 between fibronectinin the extracellularmatrix and the cytoskeleton.(a)lmmunofluorescent micrograph of a fixedcultured Microscopy and other experimental approaches (e.g., (green) fibroblast showingcolocalization of thea5p1 integrin and biochemical binding experiments) have demonstrated the actin-containing stress fibers(red).Thecellwasincubated withtwo role of integrins in crosslinking fibronectin and other ECM typesof monoclonal antibody: an integrin-specific antibody linkedto a greenfluorescing componentsto the cytoskeleton.For example,the colocalizadyeandan actin-specific antibody linkedto a red fluorescing dye.Stress fibersarelongbundles of actinmicrofilaments tion of cytoskeletalactin filaments and integrins within cells thatradiateinwardfrompointswherethecellcontacts a substratum. can be visualized by fluorescencemicroscopy (Figure I9-32a). At thedistalendof thesefibers,nearthe plasma membrane, the The binding of cell-surface integrins to fibronectin in the coincidence (green) of actin(red)andfibronectin-binding integrin matrix induces the actin cytoskeleton-dependent movement produces (b)Electron a yellowfluorescence. micrograph of the of someintegrin moleculesin the plane of the membrane.The junctionof fibronectin and actin fibers in a cultured fibroblast. ensuingmechanicaltension due to the relative movement of Individual actin-containing 7-nmmicrofilaments, components of a different integrins bound to a singlefibronectin dimer stretches stress fiber,endat the obliquely sectioned cellmembrane. The the fibronectin. This stretching promotes self-associationof microfilaments appearalignedin closeproximity to thethicker, fibronectins into multimeric fibrils. densely stained fibronectin fibrilson the outside of the cell lpart(a) The force needed to unfold and expose functional selff r o m JD u b a n d e ,t 1 a9 l 8 8J,. C eBl l i o l1. 0 7 : 1 3 8P5a r t ( b f r)o m lJ S i n g e r , associationsitesin fibronectin is much lessthan that needed 1979,Cell16:675; courtesy of l. J Singer; copyright 1979,MlT.l

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19-34 Model for integrin < FIGURE modelis activation.(teft)Themolecular of the basedon the x-raycrystalstructure regionof cvB3integrinin its extracellular ("bent")form,with thea low-affinity inactive, s u b u n i tn s h a d eosf b l u ea n dt h eB s u b u n i tn sites shades of red.Themajorligand-binding wherethe areat thetip of the molecule, propeller domainof thec subunit(darkblue) An RGD andBAdomatn(darkred)interact. peptideligandisshownin yellow.(Rrgrht) isthoughtto be due Activation of integrins thatinclude changes to conformational keymovements of the molecule; straightening which andBAdomains, nearthe propeller andseparation the affinityfor ligands; increases in altered resulting domains, of thecytoplasmic proteins. with adapter interactions lAdapted Opin.CellBiol et al, 2002,Curr. fromM Arnaout 2002,Cell11O:673]l 14:641, andR O Hynes,

basis of this diseasecame from the discovery that people with DMD carry mutations in the gene encoding a protein named dystrophin. This very large protein was found to be a cytosolic adapter protein, binding to actin filaments and to an adhesion receptor called dystroglycan. Dystroglycan is synthesizedas a large glycoprotein precursor that is proteolytically cleavedinto two subunits.The ct subunit is a peripheral membraneprotein' and the B subunit is a transmembraneprotein whose extracellulardomain associateswith the a subunit (Figure 1,9-35t.Multiple O-linked oligosaccharidesare attached covalently to side-chain hydroxyl groups of serine and threonine residues in the a subunit. Unlike the most abundant O-linked (also called mucin-like) oligosaccharides in which an N-acetylgalactosamine (GalNAc) is the first sugar in the chain linked directly to the hydroxy group of the side chain of serine or theonine or the linkage in proteoglycans' many of the Olinked chains in dystroglycan are directly linked to the hydroxyl group via a mannosesugar (seeFigure 1,9-271' These specializedO-linked oligosaccharidesbind to various basal lamina components' including the LG domains of multiadhesivematrix protein laminin and the proteoglycans ConnectionsBetweenthe ECM perlecanand agrin. The neurexins,a family of adhesionmoland CytoskeletonAre Defective ecules expressed by neurons' also are bound via these i n M u s c u l a rD y s t r o p h y oligosaccharides,whose detailed heterogeneousstructures The importance of the adhesion-receptor-mediated and mechanismsof synthesishave not been fully elucidated. The transmembrane segment of the dystroglycan B sublinkage between ECM components and the cytoskelewith a complex of integral membraneproteins; unit associates ton is highlighted by a set of hereditary muscle-wastingdisits cytosolic domain binds dystrophin and other adapter proeases,collectively called muscular dystrophies. Duchenne teins, as well as various intracellular signaling proteins (Figmuscular dystrophy (DMD), the most common type, is a ure 19-35). The resulting large, heteromericassemblage,the sex-linked disorder, affecting 1 in 3300 boys, that results in dystrophin gly coprotein complex (D GC), links the extracellucardiac or respiratory failure, usually in the late teens or lar matrix to the cytoskeleton and signaling pathways within early twenties. The first clue to understandingthe molecular

Integrin Expression The attachment of cells to ECM components can also be modulated by altering the number of integrin moleculesexposedon the cell surface.The cr4B1 integrin, which is found on many hematopoieticcells, offers an example of this regulatory mechanism. For these hematopoietic cells to proliferate and differentiate, they must be attached to fibronectin synthesizedby supportive ("stromal") cellsin the bone marrow. The o4B1 integrin on hematopoietic cells binds to a Glu-Ile-Leu-Asp-Val(EILDV) sequencein fibronectin, thereby anchoring the cells to the matrix. This integrin also binds to a sequencein a CAM called vascular CAM-1 (VCAM-1), which is presenton stromal cells of the bone marrow. Thus hematopoietic cells directly contact the stromal cells as well as attach to the matrix. Late in their differentiation, hematopoietic cells decreasetheir expressionof ct4B1 integrin; the resulting reduction in the number of ct4B1integrin moleculeson the cell surfaceis thought to allow mature blood cellsto detachfrom the matrix and stromal cells in the bone marrow and subsequently enter the circulation.

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Laminin

Perlecan

- Basallamina

o,p-Dystroglycan -

Sarcoglycancomplex

o-linked sugar -

N - l i n k e ds u g a r

Sarcospan

Cytosol

NOS

Syntrophins

Actin FIGURE 19-35 Dystrophinglycoproteincomplex(DGC)in skeletalmusclecells.Thisschematic modelshowsthatthe DGC comprises threesubcomplexes: thect,Bdystroglycan subcomplex; the sarcoglycan/sarcospan subcomplex proteins; of integral membrane andthe cytosolic adapter subcomplex comprising dystrophin, other adapterproteins, andsignaling molecules ThroughitsO-linked sugars, bindsto components of the basallamina, B-dystroglycan suchaslamininandperlecan, proteins, andcellsurface sucnas neurexin in neuronsDystrophin-the proteindefective in Duchenne muscular dystrophy-links to the actincytoskeleton, B-dystroglycan anda-dystrobrevin linksdystrophin to the sarcoglycan/sarcospan subcomplex. (NOS) Nitricoxidesynthase produces nitricoxide,a g a s e o ussi g n a l i nmgo l e c u laen, dG R B 2 i sa c o m p o n e n o tf s i g n a l i n g pathways activated (Chapter15) by certaincell-surface receptors fromS J Winder, 2001,Trends [Adapted Biochem Sci26:118, andD E Michele andK P Campbell, 2003, J BiolChem278(18):15457-154601

muscleand other types of cells.For instance,the signalingenzyme nitric oxide synthase(NOS) is associatedthrough syntrophin with the cytosolic dystrophin subcomplex in skeletal muscle.The rise in intracellular Ca2t during musclecontraction activatesNOS to produce nitric oxide (NO), a signaling molecule that diffuses into smooth muscle cells surrounding nearby blood vessels.NO promotes smooth muscle relaxation, leading to a local rise in the flow of blood supplying nutrients and oxygen to the skeletal muscle. Mutations in dystrophin, other DGC components, laminin, or enzymesthat add the O-linked sugarsto dystroglycan can all disrupt the DGC-mediated link between the exterior and the interior of muscle cells and causemuscular dystrophies.In addition, dystroglycan mutations have been shown to greatly reduce the clustering of acetylcholine receptors on muscle cells at the neuromuscular junctions, which also is dependenton the basallamina proteins laminin and agrin. These and possibly orher effects of DGC defects apparently lead to a cumulative weakening of the mechanical stability of muscle cells as they undergo contraction and relaxation, resulting in deterioration of the cells and muscuIar dystrophy. 836

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Dystroglycan provides an elegant-and medically relevant-example of the intricate networks of connectivity in cell biology. Dystroglycan was originally discoveredin the context of studying DMD. However, it was later shown to be expressedin nonmuscle cells and, through its binding to laminin, to play a key role in the assemblyand stability of at Ieastsome basementmembranes.Thus it is essentialfor normal development (Chapter 22). Additional studies led to its identification as a cell-surface receptor for the virus that causesthe frequendy fatal human diseaseLassa fever and other related viruses,all of which bind via the same specialized O-linked sugars that mediate binding to laminin. Furthermore, dystroglycan is also the receptor on specialized cells in the nervous system-Schwann cells-to which binds the pathogenic bacterium Mycobacteriwm leprae,the causative organism of leprosy.I

INTEGRATING C E L L SI N T O T I S S U E S

l g C A M sM e d i a t eC e l l - C e lAl d h e s i o ni n N e u r o n a l and Other Tissues Numerous transmembrane proteins characterized by the presenceof multiple immunoglobulin domains (repeats)in their extracellular regions constitute the immunoglobulin (Ig) superfamily of CAMs, or IgCAMs. The Ig domain is a common protein motif, containing 70-110 residues, that was first identified in antibodies, the antigen-binding immunoglobulins, but has a much older evolutionary origin in CAMs. The human, D. melanogaster,and C. elegans genomesincludeabout765,150, and 64 genes,respectively, that encodeproteins containing Ig domains. ImmunoglobuIin domains are found in a wide variety of cell-surfaceproteins, including T-cell receptors produced by lymphocytes and many proteins that take part in adhesiveinteractions. Among the IgCAMs are neural CAMs; intercellular CAMs (ICAMs), which function in the movement of leukocytes into tissues;and junction adhesionmolecules(JAMs), which are presenrin tight junctions. As their name implies, neural CAMs are of particular importance in neural tissues.One type, the NCAMs, primarily mediate homophilic interactions. First expressed during morphogenesis,NCAMs play an important role in the differentiation of muscle, glial, and nerve cells. Their role in cell adhesion has been directly demonstrated by the inhibition of adhesion with anti-NCAM antibodies. Numerous NCAM isoforms, encoded by a single gene, are generatedby alternative mRNA splicing and by differences in glycosylation.Other neural CAMs (e.g., L1-CAM) are encoded by different genes.In humans, mutations in different parts of the L1-CAM genecausevarious neuropathologies (e.g., mental retardation, congenital hydrocephalus, and spasticity). An NCAM comprisesan extracellular region with five Ig repeats and two fibronectin type III repeats,a single membrane-spanningsegment,and a cytosolic segmentthat interacts with the cytoskeleton (seeFigure 19-2).In contrast, the extracellular region of LI-CAM has six Ig repeatsand four fibronectin type III repeats.As with cadherins,cis (intracellular) interactions and trans (intercellular) interactions

probably play key roles in IgCAM-mediated adhesion (see Figure 19-3); however, adhesion mediated by IgCAMs is Ca2*-independent. The covalent attachment of multiple chains of sialic acid, a negativelycharged sugar derivative,to NCAMs alters their adhesiveproperties. In embryonic tissues such as brain, polysialic acid constitutesas much as 25 percent of the mass of NCAMs. Possibly becauseof repulsion betweenthe many negativelychargedsugarsin theseNCAMs, cell-cellcontacts are fairly transient, being made and then broken, a property necessaryfor the developmentof the nervous system.In contrast, NCAMs from adult tissuescontain only one-third as much sialic acid, permitting more stable adhesions.

LeukocyteMovement into Tissuesls Orchestratedby a PreciselyTimed Sequence of AdhesiveInteractions In adult organisms,severaltypes of white blood cells (leukocytes) participate in the defenseagainst infection causedby foreign invaders (e.g., bacteria and viruses) and tissue damage due to trauma or inflammation. To fight infection and clear away damaged tissue, these cells must move rapidly from the blood, where they circulate as unattached, relatively quiescentcells, into the underlying tissueat sitesof infection, inflammation, or damage. We know a great deal about the movement into tissue, termed extrauasation, of four types of leukocytes:neutrophils, which releaseseveral antibacterial proteins; monocytes, the precursors of macrophages,which can engulf and destroy foreign particles;and T and B lymphocytes,the antigen-recognizingcells of the immune system (Chapter 24). Extravasation requires the successiveformation and breakage of cell-cell contacts between leukocytes in the blood and endothelial cells lining the vessels.Some of thesecontactsare mediatedby selectins,a family of CAMs that mediate leukocyte-vascularcell interactions. A key player in theseinteractionsis P-selectin,which is localized to the blood-facing surface of endothelial cells. All selectins contain a Ca2*-dependentlectin domain, which is located at the distal end of the extracellular region of the molecule and recognizesoligosaccharidesin glycoproteins or glycolipids (seeFigure 1.9-2).For example,the primary ligand for P- and E-selectinsis an oligosaccharidecalled the sialyl Lewis-x antigen, a part of longer oligosaccharides presentin abundanceon leukocyteglycoproteinsand glycolipids. Figure 19-35 illustrates the basic sequenceof cell-cell interactions leading to the extravasation of leukocytes.Various inflammatory signalsreleasedin areasof infection or inflammation first cause activation of the endothelium. P-selectin exposed on the surface of activated endothelial cells mediatesthe weak adhesion of passingleukocytes.Becauseof the force of the blood flow and the rapid "on" and "off" rates of P-selectin binding to its ligands, these "trapped" leukocytes are slowed but not stopped and literally roll along the surface of the endothelium. Among the signals that promote activation of the endothelium are

chemokines,a group of small secretedproteins (8-12 kDa) produced by a wide variety of cells, including endothelial cells and leukocytes. For tight adhesion to occur between activated endothelial cells and leukocytes, B2-containing integrins on the surfaces of leukocytes also must be activated by chemokines or other local activation signals such as platelet-actiuating factor (PAF). Platelet-activatingfactor is unusual in that it is a phospholipid, rather than a protein; it is exposed on the surface of activated endothelial cells at the same time that P-selectin is exposed. The binding of PAF or other activators to their receptors on leukocytes leads to activation of the leukocyte integrins to their highaffinity form (seeFigure 1,9-341.(Most of the receptors for chemokines and PAF are members of the G proteincoupled receptorsuperfamilydiscussedin Chapter 15') Activated integrins on leukocytes then bind to distinct IgCAMs on the surface of endothelial cells. These include ICAM-2, which is expressedconstitutivelS and ICAM-1' ICAM-1, whose synthesisalong with that of E-selectinand P-selectin is induced by activation, does not usually contribute substantially to leukocyte endothelial cell adhesion immediately after activation but rather participates at later times in casesof chronic inflammation. The resulting tight adhesion mediated by the Ca2*-independentintegrin-ICAM interactions leads to the cessation of rolling and to the spreading of leukocytes on the surface of the endothelium; soon the adhered cells move between adjacent endothelial cells and into the underlying tissue. The selective adhesion of leukocytes to the endothelium near sites of infection or inflammation thus depends on the sequential appearance and activation of several different CAMs on the surfaces of the interacting cells. Different types of leukocytes express specific integrins containing the B2 subunit: for example, aLB2 by T lymphocytes and aMB2 by monocytes, the circulating precursors of tissue macrophages.Nonetheless' all leukocytes move into tissuesby the samegeneralmechanismdepicted i n F i g u r e1 . 9 - 3 6 . Many of the CAMs usedto direct leukocyteadhesionare sharedamong different types of leukocytesand target tissues. Yet often only a particular type of leukocyte is directed to a particular tissue. How is this specificity achieved?A threeitep model has been proposed to account for the cell-type specificity of such leukocyte-endothelial-cell interactions. First, endothelial activation promotes initial relatively weak, transient, and reversiblebinding (e.g., the interaction of selectins and their carbohydrate ligands).'Without additional local activation signals,the leukocyte will quickly move on. Second,cells in the immediate vicinity of the site of infection or inflammation releaseor expresson their surfaceschemical signals(e.g.,chemokines,PAF) that activateonly specialsubsets(dependingon their complementof chemokinereceptors) of the lransiently attached leukocytes. Third, additional activation-dependent CAMs (e.g., integrins) engage their binding partners, leading to strong sustainedadhesion.Only if the proper combination of CAMs' binding partners' and activation signalsare engagedtogether with the appropriate

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Animation:Cell-Cell flltt Adhesionin LeukocyteExtravasation

z

E Leukocyte (restingstate) S e l e c t i nl i g a n d (specific carbohydrate)

E Leukocyteactivation (PAFactivatesintegrin)

E n d o t h e l i aal c t i v a t i o na n d leukocyteattachmentand rolling

o"LB2 integrin

PAF receptor

lcAM-2

P-selectin

lcAM-1

-..+ Vesiclecontaining P-selectin

Extravasation

E A F I G U R1 E9 - 3 6 S e q u e n c o e f c e l l - c e liln t e r a c t i o n lse a d i n g to tight binding of leukocytesto activatedendothelialcells and subsequentextravasation.Step E: In the absence of i n f l a m m a t i oonr i n f e c t i o nl e, u k o c y t easn de n d o t h e l icael l l sl i n i n g b l o o dv e s s e a l sr ei n a r e s t i n sgt a t eS t e p Z : I n f l a m m a t osriyg n a l s r e l e a s eodn l yi n a r e a o s f i n fl a m m a t i o n i n, f e c t i o no,r b o t ha c t i v a t e restingendothelial cellsto movevesicle-sequestered selectins to t h e c e l ls u r f a c eT h ee x p o s esde l e c t i nmse d i a t e l o o s eb i n d i n go f leukocytes by interacting with carbohydrate ligandson leukocytes A c t i v a t i oonf t h e e n d o t h e l i uaml s oc a u s essy n t h e soi sf p l a t e l e t -

timing at a specificsite will a given leukocyteadherestrongly. Suchcombinatorial diversity and crosstalk allows a small set of CAMs to servediversefunctions throughout the body-a good example of biological parsimony. Leukocyte-adhesion deficiency is caused by a genetic ffi IITI . detect in the synthesisof the integrin B2 subunit. peoIil ple with this disorder are susceptibleto repeatedbacterial infections because their leukocytes cannot extravasate properly and thus fight the infection within the tissue. Some pathogenic viruses have evolved mechanisms to exploit for their own purposes cell-surfaceproteins that participate in the normal responseto inflammation. For example, many of the RNA viruses that cause the common cold (rhinoviruses)bind to and enrercellsthrough ICAM-1, and chemokine receptors can be important entry sites for human immunodeficiency virus (HIV), the cause of AIDS. Integrins appear to participate in the binding andlor inter838

c H A P T E R1 9

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T N T E G R A T T NCG E L L St N T O T T S S U E S

F i r m a d h e s i o nv i a integrin/ICAM binding

A activating factor(PAF) and ICAM-1,bothexpressed on the cell surface. PAFandotherusually secreted activators, including c h e m o k i n et sh,e ni n d u c ec h a n g eisn t h e s h a p eosf t h e l e u k o c y t e s andactivation of leukocyte integrins suchasctlp2,whichis e x p r e s s ebdy T l y m p h o c y t eBs. T h es u b s e q u etni gt h tb i n d i n g betweenactivated integrins on leukocytes and CAMson the e 9 . , I C A M - 2a n dI C A M - 1r)e s u l tisn f i r ma d h e s i o n e n d o t h e l i u(m (extravasation) 4 andsubsequent movement intothe underlying j992,Celt68:303.] tissue E [Adapted fromR O Hynes andA Lander,

nalization of a wide variety of viruses, including reoviruses (causing fever and gastroenteritis,especiallyin infants), adenoviruses(causingconjunctivitis,acute respiratory disease), and foot-and-mouth diseasevirus (causing fever in cattle and pigs). I

Adhesive lnteractions in Diverse Motile and Nonmotile Cells r Many cells have integrin-containing aggregates(e.g.,focal adhesions,3-D adhesions,podosomes)that physically and functionally connect cells to the extracellular matrix and facilitate inside-out and outside-in signaling. r Via interaction with integrins, the three-dimensional structure of the ECM surrounding a cell can profoundly influence the behavior of the cell.

r Integrins exist in two conformations that differ in the affinity for ligands and interactions with cytosolic adapter proteins (seeFigure 19-341;switching between these two conformations allows regulation of integrin activity, which is important for control of cell adhesionand movements. r Dystroglycan, an adhesion receptor,forms a large complex with dystrophin, other adapter proteins, and signaling molecules(seeFigure 19-35). This complex links the actin cytoskeletonto the surrounding matrix, providing mechanical stability to muscle.Mutations in various componentsof this complex causedifferent types of muscular dystrophy. r Neural cell-adhesionmolecules,which belong to the immunoglobulin (Ig) family of CAMs, mediate Ca2*independentcell-cell adhesionin neural and other tissues. r The combinatorial and sequentialinteraction of several types of CAMs (e.g., selectins, integrins, and ICAMs) is critical for the specific and tight adhesionof different types of leukocytes to endothelial cells in responseto local signalsinduced by infection or inflamm a t i o n ( s e eF i g u r e 1 , 9 - 3 6 ) .

PlantTissues 'We turn now to the assemblyof plant cellsinto trssues. The overall structural organization of plants is generally simpler than that of animals. For instance,plants have

only four broad types of cells, which in mature plants form four basic classesof tissue: dermal tissue interactswith the environment; uascwlartissue transportswater and dissolved substances(e.g., sugars,ions); space-fillingground tisswe constitutesthe maior sites of metabolism; and sporogenous tisswe forms the reproductive organs. Plant tissues are organized into just four main organ systems: stems have support and transport functions' rools provide anchorage and absorb and store nutrients, leauesate the sitesof photosynthesis, and flotuers enclosethe reproductive structures' Thus at the cell, tissue,and organ levels,plants are generally lesscomplex than most animals. Moreover, unlike animals' plants do not replaceor repair old or damagedcells or tissues;they simply grow new organs. Indeed, the developmentalfate of any given plant cell is primarily based on its position in the organism rather than its lineage(Chapter 21), whereasboth are important in animals. Thus in both plants and animals a cell'sdirect communication with it neighborsis important. Most importantly for this chapter and in contrast with animals, few cells in plants directly contact one another through molecules incorporated into their plasma membranes. Instead, plant cells are typically surrounded by a rigid cell wall that contacts the cell walls of adjacentcells (Figure 1.9-37a).Also in contrast with animal cells,a plant cell rarely changesits position in the organism relative to other cells.Thesefeaturesof plants and their organization have determinedthe distinctive molecular mechanismsby

(a) Nucleus

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Primary wall

Pectin Cellulose microfibril Hemicellulose Plasmodesmata

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of 19-37Structureof the plantcellwall. (a)Overview A FIGURE of a typicalplantcell,in whichthe organelle-filled theorganization by a well-defined issurrounded membrane cellwith itsplasma representation matrixcalledthe cellwall (b)Schematic extracellular . e l l u l o saen dh e m i c e l l u l oasr ea r r a n g e d l a l lo f a n o n i o nC o f t h ec e l w Thesrzeof in a matrixof pectinpolymers intoat leastthreelayers the aredrawnto scaleTosimplify andtheirseparations the polymers andothermatrix cross-links mostof the hemicellulose diagram, (e g , extensin, lignin)arenot shown(c)Fast-freeze, constituents

of the cellwallof the gardenpeatn micrograph electron deep-etch by chemical wereremoved polysaccharides pectin whichsomeof the ibrils,and microf cellulose fibers are thicker abundant The treatment (b) (arrowheads) cross-links lPart thethinnerf ibersarehemicellulose ed, Ihe 1991,in C Lloyd, andK R Roberts, fromM McCann adapted p 126 as of PtantGrowthand Form,AcademicPress, Basis Cytoskeletat T, M , Hamann G , Facette S, Brininstool C , Bauer in Somerville modified H S, Youngs Vorwerk 5, RaabT., A, Persson E, Paredez MilneJ . Osborne ,39(12)1315-1323l Part(c)fromT.FujinoandT.ltoh, 1998,PlantCellPhysiol P L A N TT I S S U E S

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which their cellsare incorporated into tissuesand communicate with one another.

T h e P l a n tC e l lW a l l l s a L a m i n a t eo f C e l l u l o s e F i b r i l si n a M a t r i x o f G l y c o p r o t e i n s The plant extracellular matrix, or cell wall, which is mainly composedof polysaccharidesand is =O.Z pm thick, completely coats the outside of the plant cell's plasma membrane. This structure servessome of the same functions as those of the ECM produced by animal cells, even though the two structures are composed of entirely different macromolecules and have a different organization. About 1000 genesin the plant Arabidopsis are devoted to the synthesis and functioning of its cell wall, including approximately 414 glycosyltransferase and more than 316 glycosyl hydrolasegenes.Like animal cell ECM, the plant cell wall connects cells into tissues,signals a plant cell to grow and divide, and controls the shape of plant organs. It is a dynamic structure that plays important roles in controlling the differentiation of plant cells during embryogenesisand growth and provides a barrier to protect against pathogen infection. Just as the extracellular matrix helps define the shapes of animal cells, the cell wall defines the shapes of plant cells. \7hen the cell wall is digestedaway from plant cells by hydrolytic enzymes, spherical cells enclosed by a plasma membrane are left. Becausea major function of a plant cell wall is to withstand the osmotic turgor pressureof the cell (between14.5 and 435 pounds per squareinch!), the cell wall is built for lateral strength. It is arranged into layers of cellulose microfibrils-bundles of 30-36 chains of long (as much as 7 pm or greater), linear, extensively hydrogen-bonded polymers of glucose in B glycosidic linkages. The cellulose microfibrils are embedded in a matrix composed of pectin, a polymer of t-galacturonic acid and other monosaccharides, and hemicellulose,a short, highly branched polymer of severalfive- and six-carbon monosaccharides.The mechanical strength of the cell wall depends on cross-linking of the microfibrils by hemicellulosechains (Figure 19-37b, c). The layers of microfibrils prevent the cell wall from stretching laterally. Cellulose microfibrils are synthesized on the exoplasmic face of the plasma membrane from UDP-glucose and ADP-glucose formed in the cytosol. The polymerizing enzyme, called cellulose synthase, moves within the plane of the plasma membrane along tracks of intracellular microtubules as cellulose is formed, providing a distinctive mechanism for intracellular/extracellular communrcatron. Unlike cellulose,pectin and hemicelluloseare synthesized in the Golgi apparatus and transported to the cell surface, where they form an interlinked network that helps bind the walls of adjacent cells to one another and cushions them. When purified, pectin binds water and forms a gel in the presenceof Ca2* and borate ionshencethe use of pectinsin many processedfoods. As much as 15 percent of the cell wall may be composed of ex-

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tensin, a glycoprotein that contains abundant hydroxyproline and serine.Most of the hydroxyproline residues are linked to short chains of arabinose (a five-carbon monosaccharide), and the serine residues are linked to galactose.Carbohydrate accountsfor about 65 percent of extensin by weight, and its protein backbone forms an extendedrodlike helix with the hydroxyl or O-linked carbohydrates protruding outward. Lignin-a complex, insoluble polymer of phenolic residues-associateswith celIulose and is a strengthening material. Like cartilage proteoglycans, lignin resists compression forces on the matrlx. The cell wall is a selectivefilter whose permeability is controlled largely by pectins in the wall matrix. $Thereas water and ions diffuse freely acrosscell walls, the diffusion of large molecules,including proteins larger than 20 kDa, is limited. This limitation may account for why many plant hormones are small, water-solublemolecules.which can diffuse across the cell wall and interact with receptors in the plasma membrane of plant cells.

Looseningof the CellWall PermitsPlant Cell Growth Becausethe cell wall surrounding a plant cell prevents it from expanding, the wall's structure must be loosenedwhen the cell grows. The amount, type, and direction of plant-cell growth are regulated by small-moleculehormones (e.g., indoleaceticacid) called auxins. The auxin-inducedweakening of the cell wall permits the expansion of the intracellular vacuole by uptake of water, Ieadingto elongation of the cell. 'We can grasp the magnitude of this phenomenon by considering that, if all cells in a redwood tree were reduced to the size of a typical liver cell, the tree would have a maximum height of only I meter. The cell wall undergoesits greatestchangesat the meristem of a root or shoot tip. Thesesitesare where cells divide and grow. Young meristematic cells are connected by thin primary cell walls, which can be loosenedand stretchedto allow subsequent cell elongation. After cell elongation ceases,the cell wall is generally thickened, either by the secretion of additional macromoleculesinto the primary wall or, more usually, by the formation of a secondarycell wall composed of severallayers. Most of the cell eventually degenerates,leaving only the cell wall in mature tissuessuch as the xylem-the tubes that conduct salts and water from the roots through the stemsto the leaves.The unique properties of wood and of plant fibers such as cotton are due to the molecular properties of the cell walls in the tissuesof origin.

Plasmodesmata DirectlyConnectthe Cytosols o f A d j a c e n tC e l l si n H i g h e rP l a n t s The presenceof a cell wall separatingcells in plants imposes barriers to cell-cell communication-and thus cell-type differentiation-not faced by animals. One distinctive mechanism used by plant cells to communicate directly is through

specializedcell-cell junctions called plasmodesmata,which extend through the cell wall. Like gap junctions, plasmodesmata ate channelsthat connect the cytosol of a cell with that of an adiacentcell. The diameter of the channel is about 30-60 nm, and its length can vary and be greater than 1 pm. The density of plasmodesmatavaries dependingon the plant and cell type, and even the smallestmeristematic cells have more than 1000 interconnectionswith their neighbors. AIthough a variety of proteins that are physically or functionally associatedwith plasmodesmatahave been identified, key structural protein components of plasmodesmataremain to be identified. Moleculessmallerthan about 1000 Da, includinga variety of metabolic and signaling compounds (ions, sugars, amino acids),generallycan diffuse through plasmodesmata. However, the size of the channel through which molecules pass is highly regulated. In some circumstances,the channel is clamped shut; in others, it is dilated sufficiently to permit the passageof moleculeslarger than 10,000 Da. Among the factors that affect the permeabilitv of plasmodesmatais the cytosolic Ca2* concentration,*iitr an increasein cytosolic Ca2* reversibly inhibiting movement of moleculesthrough thesestructures. Although plasmodesmataand gap junctions resemble each other functionally with respectto forming channelsfor small molecule diffusion, their structuresdiffer dramatically in two significantways (Figure 19-38). The plasma membranesof the adjacentplant cellsmergeto form a continuous channel, the annulus, at each plasmodesma, whereas the membranesof cellsat a gap junction are not continuous with each other. In addition, plasmodesmataexhibit many additional complex structural and functional characteristics.For example,they contain within the channel an extensionof the endoplasmic reticulum called a desmotubule that passes through the annulus,which connectsthe cytosolsof adjacent plant cells.They also have a variety of specializedproteins at the entrance of the channel and running throughout the length of the channel, including special cytoskeletal,motor' and docking proteins that regulate the sizes and types of moleculesthat can passthrough the channel. Many types of moleculesspreadfrom cell to cell through plasmodesmata, including proteins called non-cell-autonomous proteins (NCAPs, including some transcription factors), nucleic acid/protein complexes, metabolic products, and plant viruses.It appearsthat some of theserequire specialchaperones to facilitate transport. Specializedkinasesmay also phosphorylate plasmodesmalcomponents to regulate their activities(e.g.,opening of the channels).Solublemolecules passthrough the cytosolicannulus,or sleeve(about 3-4 nm in diameter), that lies between the plasma membrane and desmotubule, whereas membrane-bound molecules or certain proteins within the ER lumen can pass from cell to cell via the desmotubule.Plasmodesmata appearto play an especially important role in regulating the development of plant cells and tissues,as is suggestedby their ability to mediate intracellular movement of transcription factors and ribonuclear protein complexes.

(a)

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(a)Schematic modelof a 19-38Plasmodesmata. FIGURE of the an extension showingthedesmotubule, plasmodesma (ER), a plasma-membraneandtheannulus, reticulum endoplasmic of the cytosols that interconnects filledwith cytosol linedchannel of a thin sections (b) of micrographs Electron cells. adjacent (teft) plasmodesmata) individual indicate leaf(brackets sugarcane runningthrough viewshowingERanddesmotubule Longitudinal viewsof (Right) cross-sectional Perpendicular eachannulus. the connecting structures spoke in someof which plasmodesmata, (b) from K' seen. can be lPart the desmotubule to membrane olasma 18 l 184:307-3 1991 andR F Evert, , Planta Robrnson-Beers

O n l y a F e w A d h e s i v eM o l e c u l e sH a v eB e e n l d e n t i f i e di n P l a n t s Systematicanalysisof the Arabidopsls genomeand biochemical analysisof other plant speciesprovide no evidencefor the existenceof plant homologs of most animal CAMs, adhesion receptors,and ECM components.This finding is not surprising, given the dramatically different nature of cell-cell and cell-matrix/wall interactionsin animals and plants. Among the adhesive-typeproteins apparently unique to plants are five wall-associatedkinases(VAKs) and'WAK-like proteins expressedin the plasma membraneof Arabidopsis cells.The extracellularregionsin all theseproteins contain multiple epidermalgrowth factor (EGF) repeats'frequently

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found in animal cell-surface receptors, which may directly participate in binding to orher molecules. Some NTAKs have beenshown to bind to glycine-richproteins in the cell wall, thereby mediating membrane-wall contacts. These Arabidopsisproteins have a singletransmembranedomain and an intracellular cytosolic tyrosine kinase domain, which may participate in signaling pathways somewhat like the receptor tyrosine kinasesdiscussedin Chapter 15. The results of in vitro binding assayscombined with in vivo studiesand analysesof plant mutants have identifiedseveral macromoleculesin the ECM that are important for adhesion. For example,normal adhesionof pollen, which contains sperm cells,to the stigma or style in the female reproductive organ of the Easterlily requiresa cysteine-richprotein called stigma/stylar cysteine-richadhesin (SCA) and a specialized pectin that can bind to SCA (Figure 19-39).A small,probably ECM embedded,-10 kDa protein calledchymocyaninworks in conjunction with SCA to help direct the movement of the sperm-containingpollen tube (chemotaxis)to the ovary. Disruption of the geneencoding glucuronyltransferase1, a key enzyme in pectin biosynthesis,has provided a striking ilIustration of the imporrance of pectins in intercellular adhesion

in plant meristems.Normally, specializedpectin moleculeshelp hold the cells in meristems tightly together. Nfhen grown in culture as a cluster of relatively undifferentiated cells, called a callus, normal meristematic cells adhere tightly and can differentiate into chlorophyll-producing cells, giving the callus a green color. Eventually the callus will generateshoots. In contrast, mutant cells with an inactivated glucuronyltransferaseL gene are large, associateloosely with each other, and do not differentiate normally forming a yellow callus. The introduction of a normal glucuronyltransferase1 geneinto the mutant cells restorestheir ability to adhere and differentiate normally. The paucity of plant adhesive molecules identified to date, in contrast with the many well-defined animal adhesive molecules,may be due to the technical difficulties in working with the ECtrzUcellwall of plants. Adhesive interactions are often likely to play different roles in plant and animal biologS at least in part becauseof their differencesin development and physiology.

Plant Tissues r The integration of cells into tissuesin plants is fundamentally different from the assemblyof animal tissues,primarily becauseeach plant cell is surrounded by a relatively rigid cell wall. r The plant cell wall comprises layers of cellulose microfibrils embedded within a matrix of hemicellulose, pectin, extensin,and other lessabundant molecules. r Cellulose,alarge,linear glucosepolymer,assemblesspontaneouslyinto microfibrils stabilizedby hydrogen bonding. r The cell wall defines the shapes of plant cells and restricts their elongation. Auxin-induced looseningof the cell wall permits elongation. r Adjacent plant cells can communicate through plasmodesmata, junctions that allow molecules to pass through complex channelsconnecting the cytosols of adjacent cells (seeFigure 19-38). r Plants do not produce homologs of the common adhesive moleculesfound in animals. Only a few adhesivemoleculesunique to plants have beenwell documentedto date.

A deeper understanding of the integration of cells into tissues in complex organisms will draw on insights and techEXPERIMENTAL FTGURE 19-39An in vitro assaywas usedto niques from virtually all subdisciplines of molecular cell identify moleculesrequiredfor adherenceof pollentubesto biology-biochemistrS biophysics,microscop5 genetics,gethe stylar matrix. In thisassay, extracellular stylarmatrixcollected nomics, proteomics, and developmental biology-together (SE) fromlilystyles or an artificial matrixisdriedontonitrocellulose with bioengineering (NC).Pollen and membranes computer science.This area of cell tubescontaining spermarethenaddedand biology is undergoing explosivegrowth. theirbindingto thedriedmatrixisassessed. Inthisscanning electron micrograph, thetipsof pollentubes(arrows) An important set of questions for the future deals with canbeseenbinding to driedstylarmatrix. Thistypeof assay hasshownthatpollenadherence the mechanisms by which cells detect and respond to medepends on stigma/stylar (SCA) cysteine-rich adhesin chanical forces on them and the extracellular matrix. as well anda pectinthat bindsto SCA.[From G y Jauheral, 1997,SexplantReprod 10:1731 as the influence of their three-dimensional arrangementsand 842

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interactions.A relatedquestionis how this information is used to control cell and tissuestructure and function. Theseissues involve the fields of biomechanicsand mechanotransduction. can induce distinct patternsof geneexShearor other stresses pressionand cell growth and can greatly alter cell metabolism its binding to its ECM ligands (laminin, etc'). and responsesto extracellularstimuli. MechanosensitivenonA struitural hallmark of CAMs, adhesion receptors, and selectivecation channels(NSCws)' a least some of which apECM proteins is the presenceof multiple domains that impart pear to be membersof the transient receptor potential (TRP) diversi functions to a single polypeptide chain. It is generally cation channel family, are activated by the stretch of plasma evolutionarily by membrane and are important players in mechanotransduc- agreedthat such multidomain proteins arose the distinct encoding sequences DNA of distinct the assembly tion, such as that involved in sensingsound in the ear,which is opportuprovide domains multiple encoding Genes mediated,in part, by specializedcadherins.Most of the classes domains. diversity functional and sequence enormous generate nities to of moleculesdiscussedin this chapter-ECM, adhesionreceptors, CAMs, intracellularadapters,and the cytoskeleton-are and mechanthought to play crucial roles in mechanosensing otransduction.Future researchshould give us a far more sophisticatedunderstandingof the rolesof the three-dimensional organizationof cellsand ECM componentsand the forcesacting on them under normal and pathologicalconditionsin controlling the structuresand activitiesof tissues.Applications of suchunderstandingwill provide new methodsto explorebasic celVtissuebiology and provide improved technologiesfor the searchfor novel therapiesfor disease. Although junctions help play a key role in forming stable epithelial tissues and defining the shapesand functional properties of epithelia, they are not static. Remodeling in terms of replacementof older moleculeswith more recently synthesizedmolecules is ongoing, and the dynamic properties of junctions open the door to more substantial changes when necessary(the epithelial-mesenchymaltransition durlar basis of functional cell-cell and cell-matrix attachmentsing development, wound healing, etc.). Understanding the the "wiring"-in the nervous systemand how that wiring ultimolecular mechanismsunderlying the relationship between mately peimits complex neuronal control and, indeed, the stability and dynamic changewill provide new insights into intellect required to understandmolecular cell biology' morphogenesis,maintaining tissue integrity and function, and responseto (or induction of) pathology. Numerous questions relate to intracellular signaling KeyTerms from CAMs and adhesionreceptors.Such signaling must be integratedwith other cellular signalingpathways that are acgap junction 809 adhesionreceptor 803 tivated by various external signals (e.g., growth factors) so glycosaminoglycan adherensjunction 809 that the cell responds appropriately and in a coordinated (GAG\ 822 809 anchoring lunction fashion to many different simultaneousinternal and external hyaluronan 825 basallamina 808 stimuli. It appearsthat small GTPaseproteins participate in immunoglobulin cellat least some of the integratedpathways associatedwith sigcadherin 803 adhesionmolecule naling between cellular junctions. How are the logic circuits cell-adhesionmolecule (IgCAM)835 constructed that allow cross talk between diverse signaling (cAM) 803 integrin 803 pathways? How do these circuits integrate the information cellwall839 from thesepathways?How is the combination of outside-in laminin 821 collagen 805 and inside-out signaling mediated by CAMs and adhesion multiadhesivematrix connexin819 receptorsmerged into such circuits? protein 805 'We desmosome809 can expect ever-increasingprogress in the exploration paracellular pathwaY 815 of the influenceof glycobiology (the study of biology of oligoepithelium 802 plasmodesmata841 and polysaccharides)on cell biology. The importance of epithelial-mesench Ymal proteoglycan 805 specializedGAG sequencesin controlling cellular activities' transition 833 their RGD sequence816 especiallyinteractionsbetweensomegrowth factors and extracellular matrix receptors,is now clear.With the identification of the biosynselectin803 (ECM)833 thetic mechanismsby which thesecomplex structuresare gensyndecan 829 fibrillar collagen 825 erated and the development of tools to manipulate GAG tight junction 809 fibronectin 805 structures and test their functions in cultured systemsand K E YT E R M S .

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Reviewthe Concepts t. Using specific examples, describe the two phenomena that give rise to the diversity of adhesivemolecules. 2. Cadherins are known to mediate homophilic interactions between cells. !(/hat is a homophilic interaction. and how can it be demonstraredexperimentallyfor E-cadherins? 3. Together with their role in connecting the lateral membranes of adjacent epithelial cells, adherensjunctions play a role in controlling cell shape.What proteins and ,trrritrrr., are involved in this role? 4. IJfhat is the normal function of tight junctions? What can happen to tissueswhen tight junctions do not function properly? 'Sfhat 5. is collagen,and how is it synthesized?How do we know that collagen is required for tissueintegrity? 6. Using structural models, explain how integrins mediate outside-in and inside-out signaling. 7. Compare the functions and properties of each of three types of macromoleculesthat are abundant in the extracelluIar matrix of all tissues. 8.- Many proteoglycans have cell-signaling roles. Regulation of feeding behavior by syndecansin ttre hypothalamic region of the brain is one example.How is this reguLtion accompl-ished? You have synthesized an oligopeptide conraining an ? RGD sequencesurrounded by other amino acids. What is

the effect of this peptide when added to a fibroblast cell culture grown on a layer of fibronectin absorbedto the tissueculture dish?Vhy doesthis happen? 10. Blood clotting is a crucial function for mammalian survival. How do the multiadhesive properties of fibronectin lead to the recruitment of plateletsto blood clots? 11. How do changesin molecular connections between the extracellular matrix (ECM) and cytoskeleton give rise to Duchennemuscular dystrophy? 72, To fight infection, leukocytes move rapidly from the blood into the tissue sites of infection. lfhat is this orocess called?How are adhesionmoleculesinvolved in this process? 13. The structure of a plant cell wall needsto loosen to accommodate cell growth. What signaling molecule controls this process? L4. Compare plasmodesmatain plant cells to gap iunctions in animal cells.

Analyzethe Data Researchershave isolated two E-cadherin mutant isoforms that are hypothesizedro function differently from the isoform of the wild-type E-cadherin. An E-cadherin negative mammary carcinoma cell line was transfected with the mutant E-cadheringenesA (part a in the figure, triangles) or B (part b) (triangles)or the wild-type E-cadheringene(black circles)and

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compared to untransfectedcells (open circles)in an aggregation assay.In this assay,cells are first dissociatedby trypsin treatment and then allowed to aggregatein solution over a period of minutes. Aggregating cells from mutants A and B are presentedin panels a and b respectively.To demonstrate that the observedadhesionwas cadherin mediated,the cells were pretreatedwith a nonspecificantibody (left panel) or a functionblocking anti-E-cadherinmonoclonal antibody (right panel)' a. Vhy do cells transfected with the wild-type Ecadherin gene have greater aggregation than control, untransfectedcells? b. From these data, what can be said about the function of mutants A and B? Sfhy does the addition of the anti-E-cadherinmonc. oclonal antibodS but not the nonspecific antibody, block aggregation? 'What would happen to the aggregation ability of d. geneif the the cellstransfectedwith the wild-type E-c^adherin assaywere performed in media low in Ca'-?

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Nelson, C. M., and M. J. Bissell.2005. Modeling dynamic reciprocity: engineeringthree-dimensionalculture modeli oi b.east a.chitecture,function, and neoplastictransformation.Sem. Cancer Biol. l5(51:342-352. Reizes,O., et al. 2001. Transgenicexpressionof syndecan-1un?y9rs a physiologicalcontrol of feedingbehavior by syndecan-3. Cell 106:1,05-1,1.6. _Rougon, G., and O. Hobert. 2003. New insightsinto the diversity and function of neuronal immunoglobulin supeifamily molecules. Ann. Reu.Neurosci. 26:207J3 8. Shimaoka,M., J. Takagi, and T. A. Springer.2002. Conformational regulationof integrin structureand funttion. Ann. Reu.Biophys. Biomol. Struc. 3l:48 5-51,6. Somers,,J7.S.,J. Tang, G. D. Shaw,and R. T. Camphausen. 2000. Insightsinto the molecular basisof leukocytetetlering and rolling revealedby structuresof P- and E-selectinbound to SLe(X) and PSGL-1.Cell lO3:467479. Stein,E., and M. Tessier-Iavigne.2001,.Hierarchical organiza_ tion of.guidancereceptors:silencingof netrin attraction by Slit through a Robo/DCC receprorcomplex. Science291t192b-193g. Xiong, J. P.,et al. 2001. Crystal strucrureof the extracellular segmentof integrin aYg3. Science294:339-345.

PlantTissues Bacic,A. 2005. Breakingan impassein pectin biosynthesis. Proc. Nat'l. Acad. Sci.USA 103(15):5 $9-5640. Delmer, D. P., and C. H. Haigler. 2002.The regulationof mera_ . _ bolic flux to cellulose,a major sink for carbon in plants.Metab. Eng.4:22-28. Iwai, H., N. Masaoka, T. Ishii, and S. Satoh.2000. A pectin glu_ curonyltransferasegene is essentialfor intercellular attachment iri the plant meristem.Proc. Nat'l. Acad. Sci.IISA 99:1.631,9-16324. Jorgensen,R. A., and \W.J. Lucas.2006. Teachingresources: movementof macromoleculesin plant cellsthrough plasmodesmata. Sci. STKE (3231:tr2. pollen tube guidance: Ki-, , !., J..Dong, and E. M. Lord.2004. the role of adhesionand chemotropicmolecules.Curr. Tip. Deu. Biol.6l:67-79. Lord, E. M., and J. C. Mollet. 2002.plant cell adhesion:a bioassayfacilitates discovery of the first pectin biosynthetic gene. Proc. Nat'1.Acad. Sci.USA 99:1.5843-15845. Lord, E. M., and S. D. Russell.2002.The mechanismsof oolli_ nation and fertilization in plants.Ann. Reu.Cell Deuel. Biol. 1 8 : 81 - 10 5 . L9uql,T. J., and.!7.J. Lucas. 2006.lntegrativeplant biology: , r!le of phloem long-disrancemacromolecul"i tr"ffi.-king. Ann.neu. Plant Biol. 57:203-232. Pennell,R. 1998. Cell walls: structuresand signals.Curr. Opin. Plant Biol.1:504-510. Roberts,A. G., and K. J. Oparka. 2003. plasmodesmataand rhe control of symplastictransporr.Plant Cell Enuiron.26:103-124. 'Whetten, R.If., J.J. MacKaS and R. R. Sederoff.199g. Recent advancesin understandinglignin biosynthesis.Ann. Reu.plant Physiol. Plant Mol. Biol. 49:585-609. _ Somerville,C., et al. 2004.Toward a systemsapproachto un_ derstanding plant cell w alls. Science306(57OS\ O206-2211. Zambryski, P.,and K. Crawford. 2000. plasmodesmata:sate_ keepersfor cell-to-celltransport of developmentalsignalsin plants. Ann. Reu.Cell Deuel. Biol. 16:393421.

CHAPTER

THt REGULATING EUKARYOTICCELL CYCLE against embryostainedwith anttbodies A two-cellC e/egans protein(green) a spindlecheckpoint tubulin(red)andCeBUB-1, on the is localized CeBUB-1 with DAPI(blue). DNAisstained spindlemicrotubules and kinetochore-attached chromosomes posteriorcell(nght) lt is presumed in the smaller, duringmetaphase attachmentandtenston.Thelarger, to monitorchromosome is no and CeBUB-1 anteriorcell(/eft)hasalreadyenteredanaphase, or spindlemicrotubules on the chromosomes longerdetectable embryo of thissecondcellcyclein the C e/egans Thusasynchrony presence spindle a functional of of both the observation the allows after and itsabsence checkpointproteinduringmetaphase, anaphaseIEncanadaetal initiationof ,2005,Mol BiolCe//15:1056]

control of cell division is vital to all organisms.In l\roper Pu.i..11t1ar organisms,cell division must be balanced with cell growth so that cell size is properly maintained. I If severaldivisions occur before parental cells have reached the proper size, daughter cells eventually become too small to be viable. If cells grow too large before cell division, the cells function improperly and the number of cells increases slowly. In developing multicellular organisms, the replication of each cell must be preciselycontrolled and timed to faithfully and reproducibly completethe developmentalprogram in every individual. Each type of cell in every tissue must control its replication precisely for normal development of complex organs such as the brain or the kidney. In a normal adult, cells divide only when and where they are needed.However, loss of normal controls on cell replication is the fundamental defect in cancer,an all-too-familiar diseasethat kills one in every six people in the developedworld (Chapter 25). The molecular mechanismsregulating eukaryotic cell division discussedin this chapter have gone a long way in explaining the loss of replication control in cancer cells. Appropriateln the initial experiments that elucidated the master regulators of cell division in all eukaryoteswere awardedthe Nobel prize in 2001. The term cell cycle refers to the ordered seriesof macromolecular events that lead to cell division and the produc-

tion of two daughter cells' each containing chromosomes identical with those of the parental cell' Two main molecular processestake place during the cell cycle, with resting intervals in between:during the S phaseof the cycle, the parental

OUTLINE 849

20.1

Overview of the Cell Cycleand lts Control

20.2

Control of Mitosisby Cyclinsand MPFActivity 853

20.3

KinaseRegulationDuring Cyclin-Dependent Mitosis

20.4

MolecularMechanismsfor Regulating Mitotic Events

20.5

and Ubiquitin-ProteinLigase Cyclin-CDK Control of 5 Phase

872

20.6

C e l l - C y c lCe o n t r o li n M a m m a l i a nC e l l s

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20.7

Regulation Checkpointsin Cell-Cycle

884

20.8

Meiosis:A SpecialTypeof Cell Division

892

847

chromosomes are duplicated; in mitosis (M phase), the resulting daughter chromosomes are distributed to each daughter cell (Figure 20-1). High accuracy and fidelity are required to assurethat each daughter cell inherits the correct number of each chromosome.Further, chromosome replication and cell division must occur in the proper order in every cell division. If a cell undergoesthe eventsof mitosis before the replication of all chromosomeshas been completed, at Ieastone daughter cell will lose geneticinformation. If a second round of replication occurs in one region of a chromosome before cell division occurs, the genesencoded in that region are increasedin number out of proportion to other

OverviewAnimation:Cell-Cycle Control{tttt

Daughter cells

Chromosome decondensation, re-formationof n u c l e a re n v e l o p e , cytokinesis

Chromosome condensation, n u c l e a re n v e l o p e breakdown, chromosome segregatton

DNAsvnthesis

FIGURE 20-1 Thefate of a singleparentalchromosome throughoutthe eukaryoticcellcycle.Following (M), mitosis daughter cellscontain2n chromosomes in djploidorqanisms and 1n chromosomes in haploid organisms. In proliferating cells,G1is the periodbetween the "birth"of a cellfollowingmitosis andthe initiation of DNAsynthesis, whichmarksthe beqinnino of the S phase. At theendof theS phase, cellsenterG2containing twice the numberof chromosomes asG1cells(4nin diploidorganisms, 2n in haploid organisms). Theendof G2ismarkedbythe onsetof mitosis, duringwhichnumerous eventsleading to celldivision occur.TheG1,S,andG2phases arecollectivelv referred to as interphase, the periodbetweenone mitosis.nO tf,. next Most nonproliferating cellsin vertebrates leavethecellcvclein G,. entering the Gostate.Althoughchromosomes condense only duringmitosis, heretheyareshownin condensed formthroughout thecellcycleto emphasize the numberof chromosomes at each stageForsimplicity, the nuclear envelope is not depicted

848

C H A P T E R2 0

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genes, a phenomenon that often leads to an imbalance of gene expressionthat is incompatible with viability. To achieve the required level of accvracy and fidelity in chromosome replication and chromosome segregation to daughter cells during mitosis, and to coordinate these with cell growth and developmental programs, cell division is controlled by checkpoint surveillancemechanismsthat prevent initiation of each step in cell division until earlier steps on which it depends have been completed. Mutations that inactivate or alter the normal operation of thesecheckpoints contribute to the generation of cancer cells becausethey result in chromosomal rearrangementsand abnormal numbers of chromosomes,which lead to mutations and changesin gene expression level that cause uncontrolled cell growth (Chapter 25). Normally, such chromosomal abnormalities are prevented by multiple layers of control mechanismsthat regulatethe eukaryotic cell cycle. In the late 1980s, it became clear that the molecular processesregulating the two key events in the cell cyclechromosome replication and segregation-are fundamentally similar in all eukaryotic cells. Initially, it was surprising to many researchersthat cellsas diverseas baker'syeastand developing human neurons use nearly identical proteins to regulate their division. However, like transcription and p.ot.in synthesis,control of cell division appearsto be a fundamental cellular processthat evolved and was largely optimized early in eukaryotic evolution. Becauseof this similariry researchwith diverse organisms,each with its own particular experimental advantages,has contributed to a growing understanding of how these events are coordinated and controlled. Biochemical,genetic, imaging, and micromanipulation techniquesall have been employed in studying various aspectsof the eukaryotic cell cycle. These studies have revealedthat cell division is controlled primarily by regulating the timing of nuclear DNA replication and mitosis. The master controllers of these events are a small number of heterodimeric protein kinasesthat contain a regulatory subunit (cyclin) and catalytic subunit (cyclin-dependent kinase, or CDK). Thesekinasesregulatethe activitiesof multiple proteins involved in DNA replication and mitosis by phosphorylating them at specificregulatory sites,activating some and inhibiting others to coordinate their activities. Regulated degradation of proteins also plays a prominent role in important cell-cycletransitions. Sinceprotein degradation is irreversible,this ensuresthat the processesmove in only one direction.

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

Overviewof the CellCycleand Its Control \We begin our discussionby reviewing the stagesof the eukaryotic cell cycle, presenting a summary of the current model of how the cycle is regulated, and briefly describing key experimental systemsthat have provided revealing information about cell-cycleregulation. As mentioned earlier, since the fundamental molecules involved in cell-cyclecontrol are highly homologous in all eukaryotes,virtually everything learned about control of the cell cycle, whether it is from studies of yeast, sea urchins, or frogs, is relevant to control of the cell cycle in human cells.

The Cell Cyclels an OrderedSeriesof Events L e a d i n gt o C e l lR e p l i c a t i o n As illustrated in Figure 20-1', the cell cycle is divided into four major phases.Cycling (replicating)mammalian somatic cellsgrow in sizeand synthesizeRNAs and proteins required for DNA synthesisduring the G1 (first gap) phase. \7hen cells have reachedthe appropriate size and have synthesized the required proteins, they enter the S (synthesis)phase,the period in which they are actively replicating their chromosomes.After progressingthrough a secondgap phase,the G2 phase, cells begin the complicated process of mitosis, also called the M (mitotic) phase, which is divided into several stages(seeFigure20-2, top). In discussingmitosis, we commonly use the term chromosome for the replicated structuresthat condenseand become visible in the light microscopeduring the prophaseperiod of mitosis. Thus each chromosome is composed of the two daughter DNA moleculesresulting from DNA replication, plus the histones and other chromosomal proteins associated with them (see Figure 5-40). The two identical daughter DNA moleculesand associatedchromosomal proteins that form one chromosome are called sister chromatids. Sisterchromatids are attached to each other by protein cross-links along their lengths. In vertebrates'these cross-linksbecomeconfined to a singleregion of association called the centromere as chromosome condensation progresses. During interphase,the portion of the cell cycle between the end of one M phase and the beginning of the next, the outer nuclear membrane is continuous with the endoplasmic 'With the onset of mitosis in reticulum (seeFigure 9-L,9l). prophase,the nuclear enveloperetractsinto the endoplasmic reticulum in most cells from higher eukaryotes, and Golgi membranesbreak down into vesicles.As describedin Chapter 18. cellular microtubules form the mitotic apparatus' consisting of a football-shaped bundle of microtubules (the spindle) with a star-shapedcluster of microtubules radiating from eachend, or spindle pole. During the metaphaseperiod of mitosis, a multiprotein complex, the kinetochore' assembles at each centromere. The kinetochores of sister chromatids then associatewith microtubules coming from opposite spindle poles (seeFigure L8-36), and chromosomesalign

in a plane in the center of the cell. During the anaphaseperiod of mitosis, sisterchromatids separate.They initially are pulled by motor proteins along the spindle microtubules toward the oppositepoles and then are further separatedas the mitotic spindleelongates(seeFigure 18-41). Once chromosome separation is complete, the mitotic soindle disassemblesand chromosomes decondenseduring tilophase. The nuclear envelopere-forms around the segregated chromosomesas they decondense.The physical division of the cytoplasm, called cytokinesis, then yields two

the nuclear envelope,which then pinches off, forming two nuclei at the time of cytokinesis. In vertebratesand diploid yeasts' cells in G1 have a diploid number of chromosomes (2n), one inherited from .u.h p"..rrt. In haploid yeasts'cells in G1 have one of each chromosome (1n),the haploid number. Rapidly replicating human cells progressthrough the full cell cycle in about 24 hours: mitosis takes =30 minutes; G1, t hours; the S phase, 10 hours; and G2,4.5 hours. In contrast,the full cycletakes only =90 minutes in rapidly growing yeast cells' In multicellular organisms' most differentiated cells

thereby providing control of cell proliferation'

RegulatedProteinPhosphorylationand DegradationControl PassageThrough the Cell Cycle As mentioned in the chapter introduction, passagethrough the cell cycle is controlled by heterodimeric protein kinases' The concentrationsof the cyclins, the regulatory subunits of the heterodimers,increaseand decreaseas cells progress through the cell cycle' The concentrations of the catalytic s,rbr'r.titsof these kinases, called cyclin-dependent kinases (CDKs), do not fluctuate in such a characteristicmanner in yeast cells, but they have no kinase activity unless they are associatedwith a cyclin' Each CDK can associatewith a small number of different cyclins that determine the sub-

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Metaphase H i g h m i t o t i cc y c l i n High MPF activity

Late anaphase Prophase

cdhl

Polyubiquitination

4g!uq APC/C

Synthesisof mitotic cyclin

Interphase

f"[?iiT:T[:lf Telophase FIGURE 20-10 Regulationof mitoticcyclinlevelsin cycling Xenopusearlyembryoniccells.In lateanaphase, the anaphase(APC/C) promoting polyubiquitinylates complex mitoticcyclinsAsthe cyclins aredegraded by proteasomes, MPFkinase activity declines precipitously, triggering the onsetof telophase. APC/Cactivity is directed towardmitoticcyclins by a specificity factor,calledCdh7,

Control of Mitosis by Cyclinsand MPF Activity r MPF is a protein kinase that requiresa mitotic cyclin for activity. The protein kinase activity of MPF stimulates the onset of mitosis by phosphorylating multiple specific protein substrates,most of which remain to be identified. r In the synchronously dividing cells of early Xenopus and seaurchin embryos,the concentrationof mitotic cyclins(e.g., cyclin B) and MPF activiry increaseas cells enter mitosis and then fall as cells exit mitosis (seeFigures 20-7 and20-8). r The rise and fall in MPF activity during the cell cycle result from concomitant synthesisand degradationof mitotic cyclin protein (seeFigure20-9). r The multisubunit anaphase-promoting complex (APC/C) is a ubiquitin ligase that recognizesa conserved destruction box sequencein mitotic cyclins and promotes their polyubiquitination, marking the proteins for rapid degradation by proteasomes.The resulting decreasein MPF activity leads to completion of mitosis. r The ubiquitin ligase activity of APC/C is controlled so that mitotic cyclins are polyubiquitinylated only during

by G1cyclin-CDK andthereby inactivated whichis phosphorylated the calledCdcl4 removes A specificphosphatase complexes phosphate factorlatein anaphase. fromthespecificity regulatory of in G1,theconcentration factoris inhibited Oncethespecificity reaching a highenoughlevelto eventually mitoticcyclinincreases, mitosis. entryintothesubsequent stimulate

late anaphase(seeFigure 20-1'0).Deactivation of APC/C in G1 permits accumulation of mitotic cyclins during the next cell cycle. This results in the cyclical increases and decreasesin MPF activity that cause the entry into and exit from mitosis.

Kinase Cyclin-Dependent DuringMitosis Regulation The studies with Xenopws egg extracts describedin the previous section showed that continuous synthesisof a mitotic cyclin followed by its periodic degradation at late anaphase is required for the rapid cycles of mitosis observedin early Xenopus embryos. Identification of the catalytic protein kinase subunit of MPF and insight into its regulation initially came from genetic analysis of the cell cycle in the fission yeastS. pombe. An advantageof geneticstudiesis that genes involved in a processcan be identified (and readily cloned from yeasts)without any prior knowledge of the biochemical activities of the proteins they encode. S.pombe grows as a rod-shapedcell that increasesin length as it grows and then divides in the middle during mitosis to NU R I N GM I T O S I S C Y C L I N - D E P E N D EKNI N T A S ER E G U L A T I OD

859

Video: Mitosis and Cell Divisionin S. pombe Cytokinesis

,)

, (,

N u c l e a rd i v i s i o n Chromosome segregation

DNA replication Spindle formation

A FIGURE 20-11ThefissionyeastS. pombe.(a)Scanning electron micrograph of S.pombecellsat variousstagesof the cellcycle.Longcellsareaboutto enter mitosis; shortcellshavejustpassed through (b)Maineventsin the 5. pombe cytokinesis cellcycle.Notethatthe nuclear envelope doesnot disassemble duringmitosis in 5 pombeandotheryeasts[Part(a)courtesy of N Hajibagheri l

Chromosome condensation

/---l---\ (a)) \______

S p i n d l ep o l e body duplication

producetwo daughtercellsof equalsize(Figure20-1,1,1. Unlike most mammalian cells that grow primarily during G1, this yeastdoesmost of its growing during the G2 phaseof the cell cycle. Entry into mitosis is carefully regulatedin responsero cell sizein order to properly coordinate cell division with cell growth. Consequently,this organism is ideal for isolating mutants in genesthat regulateentry into mitosis sincemutations that alter the timing of mitosis yield cells of abnormal size,a readily observedphenotype. Temperature-sensitive mutants of S. pombe with conditional defectsin the ability to progressthrough the cell cycle are easily recognized becausethey causecharacteristicchangesin cell length at the nonpermissivetemperature. Many such mutants have been isolated, and fall into two groups. In the first group are cdc mvtantq which fail to progressthrough one of the phasesof the cell cycle at the nonpermissivetemperature; they form extremely long cellsbecausethey continue to grow in length, but fail to divide. In contrast, uee mutantsform smallerthan-normal cells becausethey are defectivein the proteins that normally prevent cells from dividing when they are too small. In S. pombe wild-type genesare indicated in italics with a superscriptplus sign (e.g.,cdc2* ); geneswith a recessive mutation, in italics with a superscriptminus sign (e.g.,cdc2 l.The protein encoded by a particular gene is designatedby the gene symbol in roman type with an initial capital letter (e.g., Cdc2). In this section we seehow geneticanalysesas well as structural studiesof the proteins involved allowed elucidation of the basic mechanismscontrolling entry into mitosis. First we discusshow mitotic regulatory geneswere identified in S. pombe, 850

C H A P T E R2 0

I

Cell growth

and how they were shown to be analogovs to Xenopu.sMPF. Next we explore the mechanismsused by S. pombe to regulate mitotic cyclin-CDK activity. Mammalian cells regulate mitotic cyclin-CDK in a similar manner,and we end the sectionwith an analysisof the structure of a human CDK and how its activity dependson phosphorylation-inducedconformational changes.

MPFComponentsAre ConservedBetween Lower and Higher Eukaryotes Mutations in cdc2, one of several different cdc genesin S. pombe, produce opposite phenotypesdependingon whether the mutation is recessiveor dominant (Figure 20-1,2).Recessive mutations (cdc2-) give rise to abnormally long cells, whereas dominant mutations (cdc2D) give rise to abnormally small cells, the wee phenotype. As discussedin Chapter 5, recessivemutations generally causea /oss of the wildtype protein function; in diploid cells, both allelesmust be mutant in order for the mutant phenotypeto be observed.In contrast, dominant mutations generally result in a gain in protein function, either becauseof overproduction or lack of regulation; in this case,the presenceof only one mutant allele confers the mutant phenotype in diploid cells. The finding that a loss of Cdc2 activity (cdc2- mutants) preventsentry into mitosis and a gain of Cdc2 activity (cdc2Dmutants) brings on mitosis earlier than normal identified Cdc2 as a key regulator of entry into mitosis in S. pombe. The wild-type cdc2+ gene contained in a S. pombe plasmid library was identified and isolated by its ability to

REGULATING T H E E U K A R Y O T I C E L LC Y C L E

cdc2* (wild type)

mutant

cdc2(recessive)

wild type

cdc2D (dominant)

A EXPERIMENTAL FIGURE 20-12 Recessive and dominant5. pombecdc2mutants have oppositephenotypes.Thewild-type cell(cdc2*)is depictedjust beforecytokinesis with two normal-size daughter cells.A recessive cdc2 mutantcannotentermitosis at the nonpermissive temperature andappears asan elongated cellwith a singlenucleus, whichcontains duplicated chromosomes. A dominant prematurelv cdc2D mutantentersmitosis beforereachino normalsize

fromcytokinesis aresmaller cellsresulting in Gz;thus,thetwo daughter on thannormal-theyhavetheweephenotypeUppermicrographs a cdc2-ts mutantandwt cells5 h aftershiftto the the rightcompare a cdc2D Lowermicrographs compare temperature non-permissive photos: cellfixationmethod.[Top mutantto wt usingan alternative MolecGenGenet.146t167; 1976, P Nurse, PThuriaux, andK Nasmyth, photos. P Nurse, 2002,ChemBio Chem.3:596]1 Bottom

complement cdc2- mutants (see Figure 20-4). Sequencing showed that cdc2* encodesa 34-kDa protein with homology to eukaryotic protein kinases.In subsequentstudies,researchersidentified cDNA clones from other organismsthat could complement S. pombe cdc2- mutants. Remarkably, they isolated a human cDNA encoding a protein identical with S. pombe Cdc2 in 63 percent of its residues.At the time of this experiment it was a tremendoussurprise to scientists that a human protein could perform the essentialfunctions of a protein from so distantly related an organism as S. pombe. The complementation of S. pombe cdc2- mutants by human Cdc2 was one of the first demonstrations that proteins performing fundamental cellular processes are highly conservedbetween all eukaryotic organisms. Isolation and sequencingof another S. pombe cdc gene (cdc13*), which also is required for entry into mitosis, revealedthat it encodesa protein with homology to seaurchin and Xenopws cyclin B. Further studies showed that a heterodimer of Cdc13 and CdcL forms the S. pombe MPF. Like Xenopus MPF, this heterodimer has protein kinase activity that phosphorylateshistone H1. Moreover, the H1 protein kinase activity rises as S. pombe cells enter mitosis and falls as they exit mitosis in parallel with the rise and fall in the Cdc13 protein level. These findings, which are completely analogousto the resultsobtained with Xenopus egg extracts (seeFigure 20-9a), identified Cdc13 as the mitotic cyclin in S. pombe. Further studies showed that the isolated Cdc2 protein and its homologs in other eukaryoteshave little protein kinase activity until they are bound by a cyclin. Hence, this family of protein kinases became known as cyclindependentkinases,or CDKs.

Researcherssoon found that antibodies raised against a highly conservedregion of Cdc2 recognizea polypeptide that co-purifies with MPF purified from Xenopzs eggs. Thus Xenopus MPF is also composed of a CDK (called CDKI\ plus a mitotic cyclin, cyclin B. This convergenceof findings from biochemicalstudiesin an invertebrate(seaurchin) and a vertebrate(Xenopus)and from geneticstudiesin a yeastindicatedthat entry into mitosis is controlled by analogousmitotic cyclin-CDK complexesin all eukaryotes(Figure20-2).

P h o s p h o r y l a t i oonf t h e C D KS u b u n i tR e g u l a t e s the KinaseActivity of MPF As we saw from the studiesin Xenopws egg extracts and comparable biochemicalstudiesin S. pombe, one way of regulating MPF activity is to control the stability of mitotic cyclins.Mitotic cyclins are suddenlydegradedin late anaphasebecausethey are polyubiquitinylated by the activated APC/C. A similar APC/C complex operatesin S. pombe and all eukaryotes.Sincea cyclin must be bound to a CDK for it to have significant kinase activity the degradation of mitotic cyclin causesa drop in MPF activity. However, in S. pombe and all other eukaryotes,additional layers of regulation are used by the cell to ensure that cyclin-CDK complexesare activeonly at the appropriate time in the cell cycle. These additional layers of control were first revealed by studying mutations in S. pombe genesother than cdc2* (encodingthe S. pombe CDK) or cdc13* (encodingthe S. pombe mitotic cyclin) that also affect cell size at the nonpercdc25missivetemperature.For example,temperature-sensitive nonpermissive temperature, mutants cannot enter mitosis at the producing elongatedcells.On the other hand, overexpressionof NU R I N GM I T O S I S C Y C L I N - D E P E N D EKNI N T A S ER E G U L A T I OD

861

(a)

encoded proteins with suitable expressionvectors. The deduced sequencesof Cdc25 and Weel and biochemicalstudies Deficitof Cdc25 of the proteins demonstratedthat they regulatethe kinase acor tivity of S. pombe MPF by phosphorylating and dephosphoExcessof Weel E l o n g a t e dc e l l s rylating specificregulatory sitesin the CDK subunit of MPF. ( i n c r e a s eG d 2) Figure 20-14 illustrates the functions of four proteins that regulate the protein kinase activity of the S. pombe Deficitof Weel CDK. First is the mitotic cyclin of S. pombe, which associor ateswith the CDK to form MPF with extremely low activity. Excess of Cdc25 S m a l lc e l l s Secondis the Weel protein-tyrosine kinase,which phospho(decreasedG2) rylatesan inhibitory tyrosineresidue(Y15) in the CDK subunit. Third is another kinase, designatedCDK-actiuating kinase (CAK), which phosphorylates an activating threonine (b) residue (T161). \fhen both residuesare phosphorylated, S. pombe MPF MPF is inactive. FinallS the Cdc25 phosphataseremovesthe phosphate from Y15, yielding highly active MPF. Sitespecificmutagenesisthat changedthe Y15 in S. pombe CDK to a phenylalanine, which cannot be phosphorylated, proWeel duced mutants with the wee phenotype, similar to that of E X P E R I M E N TFA L U R2E0 - 1 3C d c 2 5a n d W e e l h a v e IG wee-1, mutants. Both mutations prevent the inhibitory phosopposingeffectson S. pombe MPFactivity.(a)Cellsthat lack phorylation at Y15, resulting in the inability to properly regCdc25or Weel activity, asa resultof recessive temperature-sensitive ulate MPF activity, leading to premature entry into mitosis. genes,havetheopposite mutations in the corresponding phenotype As discussedfurther in Section20.7, the checkpoint surLikewise, cellswith multiple copies of plasmids wild-type containing * veillance systemsthat ensurethat chromosome replication is cdc25*or weel , andwhichthusproduce an excess of the encoded complete and that there is no unrepaired damage to chroproteins, (b) haveopposite phenotypes Thesephenotypes implythat mosomesor DNA before initiating mitosis function by regu(-+)by Cdc25and the mitoticcyclin-CDK complex isactivated (-l) bVWeel Seetextfor furtherdiscussion lating the activities of the inhibitory Weel kinase and the acinhibited tivating Cdc25 phosphatase.Weel and Cdc25 homologs exist in higher eukaryotes,and very similar checkpoint conCdc25 from a plasmid presentin multiple copiesper cell detrol systemsoperate in human cells. creases the lengthof G2, causingprematureentry into mitosis and small (wee) cells (Figure 20-13a). Conversely,loss-offunction mutations in the weel* genecausesprematureentry C o n f o r m a t i o n aCl h a n g e sl n d u c e db y C y c l i n into mitosis resultingin small cells,whereasoverproductionof B i n d i n ga n d P h o s p h o r y l a t i oInn c r e a s eM P F Weel protein increasesthe length of G2 and resuhs in elonActivity gated cells. A logical interpretation of these findings is that Cdc25 protein stimulatesthe kinaseactiviry of S. pombe MPF, Unlike both fission and budding yeasts,each of which prowhereas\7ee1 protein inhibits MPF activity (Figure 20-13b). duce just one CDK, vertebratesproduce several CDKs (see In subsequentstudies,the wild-type cdc25* and weel+ Table 20-1). The three-dimensional structureof one human geneswere isolated, sequenced,and used to produce the cyclin-dependentkinase (CDKZ) has been determinedand

a)

Inactive MPF

lnactive MPF

Inactive MPF

Weel

CAK

+ Y15

T161

Y 1 5 T161

Y15

@

@ @

FIGURE 20-14 Regulationof the kinaseactivityol S. pombe mitosis-promoting factor(MPF).Interaction of mitoticcyclin ( C d c 1 3w)i t hc y c l i n - d e p e n dkei nnat s(eC d c 2f )o r m sM P FT. h eC D K subunitcanbe phosphorylated at two regulatory sites:byWee'1 at tyrosine (CAK)at threonine 15 (Y15)andby CDK-activating kinase 1 6 1( T 1 6 1 )R e m o v oa fl t h ep h o s p h a ot en Y 15 b y C d c 2 5 CHAPTER 20

I

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+ T161

Substrate-bindino surface

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phosphatase yieldsactiveMPFin whichthe CDKsubunitis p h o s p h o r y l aat et Td16 1 ,a n di su n p h o s p h o r y l aatteYd15 . T h e mitoticcyclinsubunitcontributes to the specificity of substrate bindingby MPF,probably byformingpartof thesubstrate-binding (crosshatch), surface whichalsoincludes the inhibitorv Y15residue

R E G U L A T I NTGH E E U K A R Y O T IC CE L LC Y C L E

(a) FreeCDK2

(b) Low-activitycyclinA-CDK2

A FIGURE 20-15Structuralmodelsof humanCDK2,which is homofogousto the 5. pombecyclin-dependent kinase(COf1.16; Free,inactive CDK2unbound to cyclinA. InfreeCDK2,theT-loop blocksaccess of proteinsubstrates to the 1-phosphate of the bound ATP,shownasa ball-and-stick model.Theconformations of the regions highlighted in yellowarealtered whenCDKisboundto cyclin A. (b)Unphosphorylated, low-activity cyclinA-CDK2complex. Conformational changes induced by binding of a domainof cyclin A (green) causethe T-loopto pullawayfromthe activesiteof CDK2,so proteins thatsubstrate canbrnd.Thect1helixin CDK2,which

intothe with cyclinA, movesseveral angstroms interacts extensively for the keycatalytic sidechainsrequired catalytic cleft,repositioning phosphotransfer equivalent reactionTheredballmarksthe position high-activity 161in 5.pombeCdc2.(c)Phosphorylated, to threonine changes induced by Theconformational cyclin A-CDK2complex. (redball)altertheshape phosphorylation of theactivating threonine greatly increasing theaffinityfor surface, of thesubstrate-binding proteinsubstrates et al.,1996, of P D Jeffrey. SeeA A Russo [Courtesy Biol3:696,1 Nature Struct.

provides insight into how cyclin binding and phosphorylation of CDKs regulatetheir protein kinase activity. Although the three-dimensionalstructures of the S. pombe CDK and most other CDKs have not been determined,their extensive sequencehomology with human CDK2 suggeststhat all these CDKs have a similar structure and are regulated by a similar mechanism. Unphosphorylated, inactive CDK2 contains a flexible region, called the T-loop, that blocks accessof protein substratesto the active site where ATP is bound (Figure 20-15a). Steric blocking by the T-loop largely explains why free CDK2, unbound to cyclin, has little protein kinase activity. Unphosphorylated CDK2 bound to one of its cyclin partners has minimal but detectableprotein kinase activity in vitro, although it may be essentiallyinactive in vivo. Extensiveinteractions betweenthe cyclin and the T-loop causea dramatic shift in the position of the Tloop, thereby exposing the CDK2 active site (Figure 2015b). Binding of the cyclin also shifts the position of the a1 helix in CDK2, modifying its substrate-binding surface. High activity of the cyclin-CDK complex requires phosphorylation of the activating threonine, located in the T-loop, causing additional conformational changesin the cyclin-CDK2 complex that gready increaseits affinity for protein substrates (Figure 20-15c). As a result, the kinase activity of the phosphorylated complex is a hundredfold greater than that of the unphosphorylated complex. The inhibitory tyrosine residue (Y15) in the S. pombe CDK is in the region of the protein that binds the ATP phosphates.Vertebrate CDK2 proteins contain an additional inhibitory residue,threonine-14(T14), that is located in the

same region of the protein. Phosphorylationof Y15 and T14 in these proteins prevents binding of ATP becauseof electrostaticrepulsion between the phosphateslinked to the protein and the phosphatesof ATP. Thus these phosphorylations inhibit protein kinase activity even when the CDK protein is bound by a cyclin and the activating residue is phosphorylated. So far we have discussedtwo mechanisms for controlling cyclin-CDK activity: (1) regulation of the concentration of mitotic cyclins as outlined in Figure 20-1.0 and (2) regulation of the kinase activity of MPF as outlined in Figure 20-L4.In Section 20.5 we shall see that the protein kinase activities of cyclin-CDK complexes can also be regulated by CDK inhibitory proteins that bind to CDKs or cyclin-CDK complexes, blocking their ability to interact with substrates.

Cyclin-DependentKinase Regulation During Mitosis r In the fission yeast S. pombe, the cdc2t gene encodesa cyclin-dependentprotein kinase (CDK) that associates with a mitotic cyclin encoded by the cdc13+ gene.The resulting mitotic cyclin-CDK heterodimer is equivalent to Xenopus MPF. Mutants that lack either the mitotic cyclin or the CDK fail to enter mitosis and, therefore' form elongated cells. r The protein kinase activity of the mitotic cyclin-CDK complex (MPF) depends on the phosphorylation state of two residues in the catalytic CDK subunit (see Figure 20-1,4). The activity is greatest when threonine 161 is D U R I N GM I T O 5 I S CYCLIN-DEPENDEK N ITN A S ER E G U L A T I O N

863

phosphorylated and is inhibited by'Wee1-catalyzedphosphorylation of tyrosine 15, which interferes with correct binding of ATP. This inhibitory phosphate is removed by the Cdc25 protein phosphatase. r The human cyclin-CDK2 complex is similar to MPF from Xenopus and S. pombe. Structural studies with the human proteins reveal that cyclin binding to CDK2 and phosphorylation of the activating threonine (equivalent to threonine 161 in the S. pombe CDK) causeconformational changes that expose the active site and modify the substrate-binding surface so that it has high activity and affinity for protein substrates(seeFigure 20-1,5}.

,

1l't

,

MolecularMechanisms for RegulatingMitotic Events In the previous sections,we have seen that a regulated increasein MPF activity inducesentry into mitosis.Presumably the entry into mitosis is a consequenceof the phosphorylation of specific proteins by the protein kinase activity of MPF. However, until recently, the vast majority of proteins phosphorylated by MPF were not determined;consequently, precisely how MPF induces enrry into mitosis is not well understood. Although many of the recently identified substratesof MPF remain to be studied,analysisof a small number of substrateshas provided examplesthat show how their phosphorylation by MPF mediatesmany of the early events of mitosis: chromosome condensation,formation of the mitotic spindle, and disassemblyof the nuclear envelope(see F i g u r e1 8 - 3 4 ) . Recall that a decreasein mitotic cyclins and the associated inactivation of MPF coincides with the later stagesof mitosis (see Figure 20-9a). Just before this, in early anaphase,sister chromatids separateand move to opposite spindle poles. During telophase, microtubule dynamics return to interphase conditions, the chromosomes decondense,the nuclear envelope re-forms, the Golgi complex is remodeled,and cytokinesisoccurs.Someof theseprocesses are triggered by dephosphorylation; others, by protein degradation. In this section, we discuss the molecular mechanisms and specificproteins associatedwith some of the eventsthat characterizeearly and late mitosis. These mechanismsillustrate how cyclin-CDK complexes together with ubiquitinprotein ligasescontrol passagethrough the mitotic phase of the cell cycle.

P h o s p h o r y l a t i oonf N u c l e a rL a m i n sa n d O t h e r ProteinsPromotesEarly Mitotic Events The nuclear envelopeis a double-membraneextensionof the rough endoplasmicreticulum containing many nuclear pore complexes (seeFigure 9-1 and Figure 13-32). The lipid bilayer of the inner nuclear membrane is supported by the

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Lamin tetramer

Phosphorylated l a m i nd i m e r s

A FIGURE 20-16 The nuclearlaminaand its depolymerization. (a)Electron micrograph of the nuclearlaminafrom a Xenopus oocyte Notethe regularmeshlike networkof laminintermediate f i l a m e n t sT.h i ss t r u c t u rlei e sa d j a c e nt o t t h e i n n e rn u c l e a r m e m b r a n(es e eF i g u r e1 8 - 4 4 )( b )S c h e m a tdi ci a g r a mosf t h e s t r u c t u roef t h e n u c l e alra m i n aT. w op e r p e n d i c u sl aert so f 1 0 - n m d i a m e t efri l a m e n tbsu i l to f l a m i n A s , B ,a n dC f o r mt h e n u c l e a r l a m i n a( t o p ) I. n d i v i d u a l al m i nf i l a m e n tasr ef o r m e db y e n d - t o - e n d p o l y m e r i z a t ioofnl a m i nt e t r a m e r w s ,h i c hc o n s i sot f t w o l a m i n coiled-coil dimers(middle). Theredand greencirclesrepresent r - t e r m i n a ln dC - t e r m i n ad lo m a i n sr ,e s p e c t i v e l y t h e g l o b u l aN P h o s p h o r y l a toi of n s p e c i f isce r i n er e s i d u ense a rt h e e n d so f t h e r o d l i k ec e n t r asl e c t i o n o f l a m i nd i m e r sc a u s etsh e t e t r a m e rtso (bottom)As a result, depolymerize the nuclear laminadisintegrates. Nature 323:560; courtesy of U Aebi; [Part(a)fromU Aebiet al , 1986, part(b)adapted fromA Murrayand T Hunt,1993,TheCellCycle: An lntroduction, W H Freeman andCompany l

REGULATING THE EUKARYOTIC C E L LC Y C L E

nuclear lamina, a meshwork of lamin filaments located adjacentto the inside face of the nuclear envelope(Figure 20-1,6a).The three nuclear lamins (A, B, and C) presentin vertebratecells belong to the classof cytoskeletalproteins, the intermediate filaments, that are critical in supporting c e l l u l a rm e m b r a n e s( C h a p t e r1 8 ) . Lamins A and C, which are encoded by the same transcription unit and produced by alternative splicing of a single pre-mRNA, are identical except for a 133-residueregion at the C-terminus of lamin A, which is absent in lamin C. Lamin B, encoded by a different transcription unit, is modified post-transcriptionally by the addition of a hydrophobic isoprenyl group near its carboxyl terminus. This fatty acid becomesembeddedin the inner nuclear membrane, thereby anchoring the nuclear lamina to the membrane (seeFigure 10-19). All three nuclear lamins form dimers containing a rodlike cr-helicalcoiled-coil central section and globular

head and tail domains; polymerization of these dimers through head-to-headand tail-to-tail associationsgenerates the intermediate filaments that compose the nuclear lamina ( s e eF i g u r e1 8 - 4 5 ) . Once MPF is activated at the end of G2 through eventsdescribedin the last section,MPF phosphorylatesspecificserine residuesin all three nuclearlamins. This causesdepolymerization of the lamin intermediatefilaments (Figure20-16b). The phosphorylatedlamin A and C dimers are releasedinto solution, whereas the phosphorylated lamin B dimers remain associatedwith the nuclear membrane via their isoprenyl anchor.Depolymerizationof the nuclearlamins leadsto disintegration of the nuclear lamina meshwork and contributesto disassemblyof the nuclearenvelope.The experimentsummarized in Figure 20-17 shows that disassemblyof the nuclear envelope,which normally occursearly in mitosis, dependson phosphorylation of lamin A.

(a) Interphase

(b) Prophase

(c) Metaphase

L a m i nA s t a i n

L a m i nA s t a i n

DNA stain

DNA stain

Cellswith mutanthumanlaminA

Cellswith wild-typehumanlaminA EXPERIMENTAL FIGURE 20-17 Phosphorylation of human laminA causeslamindepolymerization. Site-directed mutagenesis wasusedto prepare a mutanthuman/amrnA gene encoding a proteinin whichalanines replace the serines that normally arephosphorylated in wild-type laminA (seeFigure 2016b).As a result, the mutantlaminA cannotbe phosphorylated Expression vectors carrying thewild-type or mutanthumangene wereseparately transfected intocultured hamster cellsBecause the transfected /amlngenesareexpressed at muchhigherlevels thanthe endogenous hamster lamingene,mostof the laminA produced in transfected cellsis humanlaminA Transfected cellsat various stages in the cellcyclethenwerestained with a fluorescent-labeled

specific for humanlaminA andwith a antibody monoclonal dyethat bindsto DNA.Thebrightbandof fluorescence fluorescent for in interphase cellsstained of the nucleus aroundthe perimeter (unphosphorylated) polymerized laminA humanlaminA represents (a).In cellsexpressing humanlaminA, the diffuselamin thewild-type (b andmetaphase in prophase the cytoplasm staining throughout bandin metaphase of the brightperipheral andc)andthe absence (c)indicate littlelamin of laminA In contrast, depolymerization the mutantlaminA in cellsexpressing occurred depolymerization werefullycondensed DNAstaining showedthatthe chromosomes or mutantlaminA. eitherwild-type in cellsexpressing by metaphase lFrom R Healdand F.McKeon, 1990, Cell 61:5791

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CuriouslS spontaneousdominant mutations in the lamin A/C gene ILMNA/ cause the rare syndrome Hutchinson-Guilford progeria. Patientsexpressingone of these mutant lamins A and C undergo a gready accelerated rate of aging. Other LMNA mutations causestriated muscular diseases,abnormal fat cell function, and peripheral 'While nerve cell diseases. the molecular mechanismsunderlying thesesymptoms are not understood,the observation that different mutations in the LMNA gene produce distinct syndromes suggeststhat lamin A and C perform several different functions in normal cells. If that were the case, mutations that affect one or another of these functions might produce the distinct group of symptoms that constitute the different svndromes associated with lamin A/C mutations. I MPF-catalyzed phosphorylation of specific nucleoporins (Figure20-18n) causesnuclear pore complexesto dissociate into subcomplexesduring prophase. Phosphorylation of integral membrane proteins of the inner nuclear membrane (Figure 20-1,8f|lr is thought to decreasetheir affinity for chromatin and further contribute to disassemblyof the nuclear envelope.The weakening of the associationsbetween

I

N u c l e apr o r e proteins

Cytoplasm

50nm

FIGURE 20-18 Nuclearenvelopeproteinsphosphorylated by MPF.(n) Components (NPC) porecomplex of the nuclear are phosphorylated by MPFin prophase, causing NPCs to dissociate into soluble andmembrane-associated (Z) MpF NPCsubcomplexes. phosphorylation (lNM)proteins of innernuclear membrane inhibits theirinteractions with the nuclear lamrna andchromatin(E) MPF phosphorylation of nuclear laminscauses theirdepolymerization and dissolution of the nuclear lamina(4) MPFphosphorylation of proteins chromatin induces chromatin condensation andinhibits interactions between chromatin andthe nuclear envelope[Adapted fromB Burke andJ Ellenberg, 2002,NatRev. Mol.CellBiol3:487l

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the inner nuclear membrane proteins and the nuclear lamina (Figure 20-18E|) and chromatin (Figure 20-1.881)allows sheetsof inner nuclear membrane to retract into the endoplasmic reticulum, which is continuous with the outer nuclear membrane. Several lines of evidence indicate that MPF-catalyzed phosphorylation also plays a role in chromosome condensation and formation of the mitotic spindle apparatus.For instance,geneticexperimentsin the budding yeastS. cereuisiae identified a family of structural maintenance of chromosomesproteins, or SMC proteins, that are required for normal chromosome segregation.These large proteins (=1200 amino acids) contain characteristicATPasedomains at their N- and C-termini and long regionsthat participate in coiledcoil structures(seeFigure6-38a). Immunoprecipitation studieswith antibodies specificfor Xenopus SMC proteins revealedthat in cycling egg extracts some SMC proteins are part of a multiprotein complex calledcondensin,which becomesphosphorylated as cells enter mitosis. $fhen the anti-SMC antibodies were used to deplete condensinfrom an egg extract, the extract lost its ability to condenseadded sperm chromatin following the initial decondensationphase. Other in vitro experiments showed that phosphorylated purified condensin binds to DNA and winds it into supercoils (seeFigure 4-8), whereas unphosphorylated condensindoes not. Theseresults and the observation that a condensin subunit is phosphorylated by MPF in vitro have led to the model that condensincomplexesare activated by phosphorylation catalyzedby MPF. Once activated, condensincomplexesare proposed to bind to DNA at intervals along the chromosome, forming successively smaller loops that result in chromosome condensation (see Figure 6-38c). Phosphorylationof microtubule-associated proteins by MPF probably is required for the dramatic changes in microtubule dynamics that result in the formation of the mitotic spindle and asters (Chapter 18). In addition, phosphorylation of proteins associatedwith the endoplasmic reticulum (ER) and Golgi complex, by MPF or other protein kinasesactivated by MPF-catalyzedphosphorylation, is thought to alter the trafficking of vesiclesbetween the ER and Golgi to favor trafficking in the direction of the ER during prophase. As a result, the Golgi complex membranes are transferred to the ER, and vesicular traffic from the ER through the Golgi to the cell surface(Chapter 14), seen in interphase cells, does not occur during mrtosrs. Many of the direct substratesof MPF have beenidentified in S. cereuisiaeby engineering a CDK mutant that can utilize an analog of ATP that is not bound by other kinases (Figure 20-1,9).This ATP analog has a bulky benzyl group attached to N5 of the adenine.This makes the analog too large to fit into the ATP-binding pocket of wild-type protein kinases. However, the ATP-binding pocket of the mutant CDK was modified to accommodate this large ATP analog. Consequentl5 only the mutant CDK can utilize this

REGULATING THE EUKARYOTIC C E L LC Y C L E

A FIGURE 20-19 ATPanalog-dependent CDKmutant. (a)Representation of the ATP-binding andcatalytic sitesof wild-type S.cerevisiae CDK(Cdc28). BoundATPanda phenylalanine sidechain (pink)ln thevicinity of thebindingpocketareshownin stickformat. (b)BulkyATPanalogs groupbound suchasthosecontaining a benzyl to the N6amrnonitrogen aretoo largeto fit intotheATP-binding pocketof wild-typeproteinkinases andthuscannotbe utilizedby

at position CDKmutant,the phenylalanine them.In the 5. cerevrslae whichlacksa largesidechain.Themutant to glycine, 88 ischanged These exhibitshighproteinkinaseactivityusingN6-(benzyl)ATP of the PKA CDKarebasedon crystalstructures modelsof 5. cerevisiae extensivehomologywith the kinase kinasedomain,whichshares et al.,2003,Nature CDK [SeeJ A. Ubersax domainof S. cerevisiae K Shahet al , 1997,Proc.Nat'lAcad.Sci.USA94:3565.1 425:859:

ATP analog as a substratefor transferring its ^y-phosphate to a protein side chain. !(hen the N5-benzyl ATP analog with a labeled "y-phosphatewas incubated with yeast cell extracts and recombinant yeast MPF containing the mutant CDK, multiple proteins were labeled. True yeast MPF in vivo substratescould be verified among these potential substratesby treatment of cells expressingthe mutant CDK in place of the wild-type protein with a similar derivative of another ATP analog that inhibits protein kinases. This derivative of the kinase inhibitor also contains a bulky substitution at the adenine N5 position so that it can bind to and inhibit only the mutant CDK. It is sterically blocked from binding to all other kinasesand consequentlyinhibits only the mutant CDK engineeredin these cells. Treatment of cells with this specific mutant CDK inhibitor resulted in the dephosphorylation of most of the putative MPF targets identified initiallS indicating that theseproteins are indeed phosphorylated by the CDK in vivo as well as in vitro. This procedure identified most of the known CDK substrates plus more than 150 additional yeast proteins. These are currently being analyzed for their functions in cell cycle processes.

(APC/C) leads to the proteasomal destruction of thesecyclins (see Figure 20-1'0). Additional experiments with Xenopus egg extracts provided evidence that degradation of cyclin B, the Xenopzs mitotic cyclin, and the resulting decreasein MPF activity are required for chromosome decondensation but not for chromosome segregation (Figure

Unlinkingof SisterChromatidsInitiates Anaphase We saw earlier that in late anaphase,polyubiquitination of mitotic cyclins by the anaphase-promoting complex

20-20a.b\. To determine if ubiquitin-dependent degradation of another protein is required for chromosome segregation, researchers prepared a peptide containing the cyclin destruction-box sequenceand the site of polyubiquitination. When this peptide was added to a reaction mixture containing untreated egg extract and sperm nuclei, decondensation of the chromosomes and, more interestingly, movement of chromosomes toward the spindle poles were greatly delayed at peptide concentrations of 20-40 pg/ml and blocked altogether at higher concentrations (Figure 20-20c). The added excess destruction-box peptide is thought to act as a substrate for the APC/C-directed polyubiquitination system, competing with the normal endogenous target proteins and thereby delaying or preventing their degradation by proteasomes. Competition with cyclin B delays cyclin B degradation, accounting for the observed inhibition of chromosome decondensation. The observationthat chromosome segregationalso was inhibited in this experiment but not in the experiment with mutant nondegradable cyclin B (see Figure 20-20b) lndicated that segregationdepends on polyubiquitination of a

M I T O T I CE V E N T S FORREGULATING M O L E C U L A RM E C H A N I S M S

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(a) RNasetreatedextract+ mRNA encodingwild-typecyclin B

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< E X P E R I M E N TFAI L G U R2E0 - 2 0 O n s e to f a n a p h a s e dependson polyubiquitinationof proteinsother than n i x t u r ecso n t a i n eadn u n t r e a t eodr R N a s e c y c l i nB . T h er e a c t i o m treatedXenopusegg extractand isolated Xenopusspermnuclei, p l u so t h e rc o m p o n e n itns d i c a t ebde l o wC . h r o m o s o mw e se r e v i s u a l i z ewdi t h a f l u o r e s c e D n tN A - b i n d i ndgy e .F l u o r e s c e n t r h o d a m i n e - l a b et luebdu l i ni n t h e r e a c t i o nwsa si n c o r p o r a t e d i n t om i c r o t u b u l epse, r m i t t i nogb s e r v a t i oonf t h e m i t o t i cs p i n d l e (a, b) Afterthe eggextractwastreatedwith RNase apparatus. t o d e s t r oey n d o g e n o umsR N A sa,n R N a sien h i b i t owr a sa d d e d . T h e nm R N Ae n c o d i negi t h e w r i l d - t y p cey c l i nB o r a m u t a n t n o n d e g r a d a bclyec l i nB w a sa d d e dT h et i m ea t w h i c ht h e c o n d e n s ecdh r o m o s o m ae ns da s s e m b l esdp i n d l e apparatus b e c a m vei s i b l a e f t e ra d d i t i o n o f s p e r mn u c l eits d e s i g n a t e d 0 m i n u t e sI .n t h e p r e s e n coef w i l d - t y p cey c l i nB ( a ) ,c o n d e n s e d c h r o m o s o m ae tst a c h etdo t h e s o i n d l e m i c r o t u b u la en sd s e g r e g a t et odw a r dt h e p o l e so f t h e s p i n d l eB. y4 0 m i n u t e s , h u si s n o tv i s i b l ea) ,n dt h e t h es p i n d l e h a dd e p o l y m e r i z(et d e N As t a i n i n ga)sc y c l i n c h r o m o s o mh ea s dd e c o n d e n s (eddi f f u s D B w a sd e g r a d e dI n t h e p r e s e n coef n o n d e g r a d a bclyec l i nB ( b ) , p o l e sb y 15 m i n u t e sa,s c h r o m o s o m sees g r e g a t et od t h e s p i n d l e i n ( a ) ,b u t t h e s p i n d l e microtubule d si d n o t d e p o l y m e r iaz ne dt h e c h r o m o s o m de isd n o t d e c o n d e n seev e na f t e r8 0 m i n u t e sT. h e s e o b s e r v a t i oinnsd i c a tteh a td e g r a d a t i oonf c y c l i nB i s n o t r e q u i r e d f o r c h r o m o s o msee g r e g a t i odnu r i n ga n a p h a s a e l,t h o u g h it is r e q u i r efdo r d e p o l y m e r i z a toi of ns p i n d l e m i c r o t u b u la en sd ( c )V a r i o u s c h r o m o s o mdee c o n d e n s a t idounr i n gt e l o p h a s e c o n c e n t r a t i oonfsa s h o r tp e p t i d ec o n t a i n i ntgh e c y c l i nB destruction boxwereaddedto extracts that had not beentreated w i t h R N a s et h; e s a m p l ews e r es t a i n e fdo r D N Aa t 1 5 o r 3 5 m i n u t ea s f t e rf o r m a t i o n o f t h es p i n d l e a p p a r a t uT s h et w o l o w e s t p e p t i d ec o n c e n t r a t i odnesl a y e cdh r o m o s o msee g r e g a t i oann, d t h e h i g h e cr o n c e n t r a t i ocnosm p l e t e il n y h i b i t ecdh r o m o s o m e s e g r e g a t i oInn.t h i se x p e r i m e nt th,e a d d e dd e s t r u c t i obno x p e p t i d ei st h o u g h t o c o m p e t i t i v ei n t PC/C-mediated l yh i b iA p o l y u b i q u i t i n a t oi of nc y c l i nB a sw e l la sa n o t h etra r g e tp r o t e i n is required whosedegradation for chromosome segregation lFrom et al , 1993,Cell73:1393; S L Holloway courtesy of A, W Murray J

different target protein by the same ubiquitin-protein ligasethat binds the cyclin B destructionbox and the isolated destruction-boxpeptide. As mentioned earlier, each sister chromatid of a metaphasechromosome is attached to microtubules via its kinetochore, a complex of proteins assembledat the centromere. The opposite ends of these kinetochore microtubules associatewith one of the spindle poles (seeFigure 1,8-36).At metaphase,the spindle is in a state of tension, with forces pulling the two kinetochores toward the opposite spindle poles balanced by forces pushing the spindle poles apart. Sisterchromatids do not separate,becausethey are held together at their centromeresby multiprotein complexes called coheslas.Among the proteins composing the cohesin complexes are members of the SMC protein family discussedin the previous section (seeFigure 6-38). Vhen Xenopus egg extracts were depletedof cohesin by treatment with antibodies specific for the cohesin SMC proteins, the depleted extracts were able to replicate the DNA in added

sperm nuclei, but the resulting sister chromatids did not associateproperly with each other. Furthermore, in S. cereuisiae with temperature-sensitivemutations in cohesin subunits, incubation at the nonpermissive temperature causes errors in chromosome segregationduring mitosis. Since attachment of sister chromatids to spindle fibers from opposite spindle poles requires linkage between them, this is the expectedresult if sister chromatids of thesemutant cells are not associatedduring mitosis. These findings demonstrate that cohesin is necessaryfor the cohesion between sister chromatids. Cohesin moleculesassociatewith chromosomesin late G1. Figure 20-21 presentsone model for how the circular cohesin complexes link daughter chromosomes as they replicate in S phase. According to this model, either the DNA replication fork passesthrough the cohesin circles, or cohesincircles open to let the replication fork passand then close around both daughter chromatids. This leaves cohesin links along the full length of the daughter chromatids. In some organisms, such as C. elegans,protein links persist in the chromosome arms until they are broken in anaphase. However, in S. cereuisiae and vertebrates, phosphorylation of cohesins by protein kinases that are activated by MPF causescohesin complexes to dissociatefrom the chromatid arms in late prophase. As opposed to cohesin complexes in the chromosome arms' the same type of cohesin moleculesin the vicinity of the centromeredo not dissociate,and continue to hold sister chromatids in the region of the centromere. Analysis of S. cereuisiaemutants defective in mitotic chromosomal segregation revealed that a specific isoform of a protein phosphatase called PP2A normally associateswith centromeres(observedin human chromosomesin Figure 20-22). This phosphataserapidly dephosphorylatescohesin complexes phosphorylated in late prophase by the kinase mentioned above. This occurs only in the vicinity of the centromere where the phosphatase is bound, so that centromere-associatedcohesin complexes do not dissociate during late prophase like cohesin complexesin the chromosome arms, but rather continue to link chromatids at the centromere. Further studies of yeast mutants have led to the model depicted in Figure 20-23 for how the APC/C regulates sister chromatid separation to initiate anaphase. Cohesin SMC proteins link sister chromatids at the centromere. The cross-linking activity of cohesin dependson secwrin,which is found in all eukaryotes. Prior to anaphase,securin binds to and inhibits separase,a ubiquitous protease. Once all chromosome kinetochores have attached to spindle microtubules, the APC/C is directed by a specificity factor called Cdc20 to polyubiquitinylate securin (note that this specificity factor is distinct from Cdh1, which directs the APC/C to polyubiquitinylate B-type cyclins). Polyubiquitinylated securin is rapidly degraded by proteasomes, thereby releasingseparase.Free from its inhibitor, separase cleaves a small subunit of cohesin called kleisin, breaking the protein circles linking sister chromatids. Once this link is broken, anaphasebegins,as the poleward

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A FIGURE 20-21 Modelfor cohesinlinkageof daughter chromosomes. Thereisstrongevidence thatthe cohesin complex is circular, likeotherSMCproteincomplexes (seeFigure 6-38),but it is not knownwhethera singlecohesin ringlinksdaughter chromatids, or whethertwo rings,oneeacharoundtheseparate sister chromatids, arelinkedto eachotherlikelinksin a chain,possibly

force exerted on kinetochoresmoves sisterchromatids toward the opposite spindle poles. Because Cdc20-the specificity factor that directs APC/C to securin-is activated before Cdhl-the specificity factor that directs APC/C to mitotic cyclins-MPF activity does not decreaseuntil after the chromosomes have segregated.As a result of this temporal order in the activation of the two APC/C specificity factors, the chromosomesremain in the condensedstate and reassemblyof the nuclear envelope does not occur until chromosomes

phase G2 phase G2

Metaphase involving several linkedcohesin circles between sisterchromatids. Passage of a replication forkthrougha cohesin ringresults in linking of sisterchromatids Invertebrate cells,cohesins arereleased from chromosome armsduringprophase andearlymetaphase, andby the endof metaphase areretained onlyin the regionof the centromere. K Nasmyth andC H Haering, 2005, Ann Rev. Biochem 74:595] [From

are moved to the proper position. As we shall seein Section 20.7, Cdc20 and Cdhl are regulated by checkpoint surveillancemechanisms.Cdc20 is inhibited until every kinetochore has attached to a spindle fiber and tension is applied to the kinetochores of all sister chromatids, pulling them toward opposite spindle poles. Cdh1, on the other hand, is inhibited until daughter chromosomeshave been separatedby a sufficient distancein anaphaseto ensure that the separatedchromosomesare included in separate nuclei as the nuclear envelopesre-form and the cell divides.

Chromosome D e c o n d e n s a t i oann d R e a s s e m b l y o f t h e N u c l e a rE n v e l o p eD e p e n do n Dephosphorylationof MPFSubstrates

A F|GURE 2O-22Localizationof PP2Asubtypeat the centromereof a humanmetaphasechromosome. DNAisstained blue.A markerproteinfor centromeres wasdetected with a specific (red),aswasPP2Asubtype antibody B56a(green)Bar: 1 pm [From S Tomoyaet al , 2006, NatureM1i46l

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Earlier we discussedhow MPF-mediatedphosphorylation of nuclear lamins, nucleoporins,and proteins in the inner nuclear membrane contributes to the dissociation of nuclear pore complexesand retraction of the nuclear membrane into the ER. When chromosomes have separated sufficiently during anaphase, the chromosome segregation checkpoint surveillance mechanism activates the

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Securin

a A FIGURE 20-23 Regulationof cohesincleavage.Separase, protease the smallkleisin subunitof cohesin thatcancleave isinhibited complexes, beforeanaphase bythe bindingof securin microtubules and haveattached to spindle Whenallthe kinetochores isproperly andoriented, the Cdc20 the spindle apparatus assembled with theAPC/Canddirects it to specificity factorassociates

by degradation securin securinFollowing polyubiquitinylate subunit, the kleisin cleaves separase proteasomes, the released to be pulled allowingsisterchromatids circles, thecohesin breaking that ispullingthemtowardopposite apparatus apartby thespindle 2005,Ann Rev. andC H Haering, fromK,Nasmyth spindlepoles.lAdapted 74:595.1 Biochem

protein phosphatase Cdc14. Cdc14 removes phosphate groups that were added to proteins by MPF and, consequently, is an active antagonist of MPF function. Importantly, Cdc1,4 also dephosphorylates and consequently activatesthe Cdhl specificity factor. This allows Cdhl to bind to the APCiC complex, directing it to polyubiquitinylate mitotic cyclins, inducing their degradation (see F i g u r e2 0 - 1 , 0 ) . Reversal of MPF phosphorylation changesthe activities of many proteins back to their usual state in interphasecells. Dephosphorylation of condensins,histone H1 and other chromatin-associatedproteins leads to the decondensation of mitotic chromosomes in telophase. Dephosphorylated inner nuclear membrane proteins are thought to bind to chromatin once again. As a result, multiple projections of regions of the ER membrane containing these proteins are thought to associatewith the surface of the decondensingchromosomes and then fuse with one another to form a continuous double membrane around each chromosome (Figure 20-24). Dephosphorylation of nuclear pore subcomplexesallows them to reassembleinto complete NPCs traversing the inner and outer membranessoon after fusion of the ER proiections. Ran'GTR required for driving most nuclear import and e x p o r t ( C h a p t e r 1 3 ) , s t i m u l a t e sb o t h f u s i o n o f t h e E R projections to form daughter nuclear envelopesand assembly of NPCs from the nuclear pore subcomplexesthat were generatedby MPF phosphorylation of nucleoporins in prophase (Figure 20-24). The Ran'GTP concentration is highest in the microvicinity of the decondensingchromosomes becausethe Ran-guanine nucleotide-exchange factor (Ran-GEF) is bound to chromatin. Consequentln membrane fusion is stimulated at the surfacesof decondensing chromosomes, forming sheets of nuclear membrane with inserted NPCs. The reassemblyof nuclear envelopescontaining NPCs around each chromosome forms individual mini-nuclei

called karyomeres. Subsequent fusion of the karyomeres associated with each spindle pole generates the two daughter-cellnuclei, each containing a full set of chromosomes. Dephosphorylated lamins A and C appear to be imported through the reassembledNPCs during this period and reassembleinto a new nuclear lamina. Reassembly of the nuclear lamina in the daughter nuclei probably is initiated on lamin B molecules,which remain associated with the ER membrane via their isoprenyl anchors throughout mitosis and become localized to the inner membrane of the reassembled nuclear envelopes of karyomeres.

Molecular Mechanismsfor Regulating Mitotic Events r Early in mitosis, MPF-catalyzed phosphorylation of lamins A, B, and C, and of nucleoporins and inner nuclear envelope proteins causes depolymerization of lamin filaments (see Figure 20-1,6\ and dissociation of nuclear pores into pore subcomplexes,leading to disassembly of the nuclear envelope and its retraction into the ER. r Phosphorylation of condensin complexes by MPF or a kinase regulatedby MPF promotes chromosomecondensation early in mitosis. r Sister chromatids formed by DNA replication in S phase are linked at the centromere by cohesin complexes that contain DNA-binding SMC proteins and other protelns. r At the onset of anaphase, the APC/C is directed by Cdc20 to polyubiquitinylate securin,which subsequentlyis degraded by proteasomes.This activates separase,which cleaveskleisin, a subunit of cohesin,thereby unlinking sister chromatids (seeFigure 20-23).

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Video: NuclearEnvelopeDynamics DuringMitosis

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Chromatin Membrane recruitment

FIGURE20-24 Model for reassemblyof the nuclear envelope during telophase. Extensions of the endoplasmic reticulum(ER)associate with eachdecondensing chromosomeand t h e n f u s ew i t h o n e a n o t h e rf,o r m i n ga d o u b l em e m b r a n ea r o u n d the chromosomeDephosphorylated nuclearpore subcomplexes r e a s s e m b il ne t o n u c l e apr o r e s f, o r m i n gi n d i v i d u aml i n i - n u c l ec ia l l e d karyomeresThe enclosedchromosomefurtherdecondenses, and subsequent fusionof the nuclearenvelopes of all the karyomeres at e a c hs p i n d l ep o l ef o r m sa s i n g l en u c l e u sc o n t a i n i n a g f u l l s e to f chromosomesNPC: nuclearpore complex lAdapted fromB Burke andJ Ellenberg,2002, NatureRev. Mol.CellBiol 3:487I

After sisterchromatidshave moved to the spindlepoles, e APC/C is directed by Cdhl to polyubiquitinylate mitotic cyclins, leading to rheir destruction and causing the decreasein MPF activity that marks the onset of telophase. r The fall in MPF activity in telophase allows phosphatases such as Cdc14 to remove the regulatory phosphatesfrom condensin, lamins, nucleoporins, and other nuclear membrane proteins, permitting the decondensation of chromosomesand the reassemblyof the nuclear membrane, nuclear lamina, and nuclear pore complexes.

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r The associationof Ran-GEF with chromatin results in a high local concentrationof Ran.GTP near the decondensing chromosomes,promoting the fusion of nuclear envelope extensions from the ER around each chromosome. This forms karyomeresthat then fuse to form daughter cell nuclei (seeFigure 20-24).

Cyclin-CDK and Ubiquitin-Protein LigaseControlof S phase In most vertebrate cells, the key decision determining whether or not a cell will divide is the decisionto enter the S phase. In most cases,once a vertebrate cell has become committed to entering the S phase, it does so a few hours later and progressesthrough the remainder of the cell cycle until it ccrmpletesmitosis. The budding yeastSaccharomyces cereuisideregulatesits proliferation similarly, and much of our current understanding of the molecular mechanisms controlling entry into the S phase and the control of DNA replication originated with geneticstudiesof S. cereuisiae. S. cereuisiaecells replicate by budding (Figure 20-ZS). Both mother and daughtercells remain in the G1 period of the cell cycle while growing, although it takes the initially larger mother cells a shorter time to reacha size compatible with cell division. \Vhen S. cereuisiaecells in G1 have grown sufficiently, they begin a program of gene expression that leadsto entry into the S phase.If G1 cells are shifted from a rich medium to a medium low in nutrients before they reach a critical size,they remain in G1 and grow slowly until they are large enough to enter the S phase.However, once G1 cells reach the critical size,they becomecommitted to completing the cell cycle, enrering the S phase and proceeding through G2 and mitosis, even if they are shifted to a medium low in nutrients. The point in late G1 of growing S. cereuisiaecells when they become irrevocably committed to entering the S phaseand traversingthe entire cell cycle is called S7l4RT. As we shall seein Section20.6, a comparablephenomenonoccurs in replicatingmammaliancells. In this section, our focus is on the G1 -+ S transition as we explore the molecular eventsthat constitute START. Just as in mitosis, entry into S phase is controlled by the activity of cyclin-CDKs. However, the regulatory mechanismsgoverning the activity of these cyclin-CDKs differ from those used by mitotic cyclin-CDK complexes.We discussthe roles of G1 cyclin-CDKs and S-phasecyclin-CDKs in initiating DNA synthesisand ensuring that DNA replication occurs only onceper cell cycle,as well as how the cell cycle"resets" after mitosis, in preparation for the next cell division.

A Cyclin-Dependen Kti n a s e( C D K )l s C r i t i c a fl o r S-PhaseEntry in S. cerevisiae All S. cereuisiae cells carrying a mutation in a particular cdc gene arrest with the same size bud at the nonpermissive

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Daughter cell Chromosome segregation; nuclear division

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S p i n d l ep o l e bodY duplication

Q r - ) / a r oemergence DNA replication

(a)Scanning 20-25 The buddingyeast5. cerevisiae. A FfGURE stagesof the of 5. cerevisiae cellsat various electronmicrograph c e l lc y c l eT. h el a r g etrh eb u d ,w h i c he m e r g east t h ee n do f t h e G t phase,the furtheralongin the cyclethe cellis (b)Maineventsin than cellcycle.Daughter cellsarebornsmaller the 5. cerevrslae mothercellsand mustgrowto a greaterextentin G1beforethey

arelargeenoughto enterthe S phase.As in 5. pombe,tne Unlike5. doesnot breakdownduringmitosis. envelope nuclear do chromosomes the small5 cerevrslae pombechromosomes, (a) [Part to be visibleby lightmicroscopy. sufficiently not condense andL Herskowitz l of E Schachtbach courtesy

temperature(seeFigure 5-6b). Each type of mutant has a terminal phenotype with a particular bud size: no bud, intermediate-sizedbuds, or large buds. Note that in S. cereuisiae wild-type genes are indicated in italic capital letters (e.g., CDC28) and recessivemutant genes in italic lowercase letters (e.g., cdc28); the corresponding wild-type protein is written in roman letters with an initial capital (e.g.,Cdc28), similar to S. pombe proteins. Temperature-sensitivemutants in the cdc28 gene, now known to encodethe S. cereuisiaeCDK, do not form buds at the nonpermissive temperature. This phenotype indicates that Cdc28 function is required for entry into the S phase. 'When these mutants are shifted to the nonpermissivetemperature, they behavelike wild-type cells suddenly deprived of nutrients; that is, cdc28 mttant cells that have grown large enough to pass START at the time of the temperature shift continue through the cell cycle normally and undergo mitosis, whereas those that are too small to have passed

START when shifted to the nonpermissivetemperature do not enter the S phase even though nutrients are plentiful. Even though cdc28 cellsblocked in G1 continue to grow in size at the nonpermissive temperature' they cannot pass START and enter the S phase.Thus they appear as large cells with no bud. The wild-type CDC28 gene was isolated by its ability to complement mutant cdc28 cells at the nonpermissive temperature (see Figure 20-4\' Sequencing of CDC28 showed that the encoded protein is homologous to known protein kinases,and when Cdc28 protein was expressedin E. coli, it exhibited low protein kinase activity. Like S. pombe, S. cereuisiaecontains only a single cyclin-dependent protein kinase (CDK) that functions directly in cell-cycle control. Sequencecomparisons have shown that the CDKs in the two speciesare highly homologous. The difference in the phenotypes of S. pombe and S' cereuisiaecells with temperature-sensitivemutations in

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their CDK genes can be explained by the physiology of the two yeasts.In S. pombe cells growing in rich media, cell-cyclecontrol is exerted primarily at the G2 -+ M trans i t i o n ( i . e . ,e n r r y t o m i t o s i s ) .I n m a n y S . p o m b e C D K r e cessivemutants, including those initially isolated which gave the phenotype depicted in Figure 20-12, enough CDK activity is maintained at the nonpermissivetemperature to permit cells to enter the S phase, but not enough to permit entry into mitosis. Such mutant cells are observedto be elongatedcells arrestedin G2. At the nonpermissive temperature, cultures of completely defective CDK mutants include some cells arrestedin G1 and some arrestedin G2, depending on their location in the cell cycle at the time of the temperature shift. Conversely,cellcycle regulation in S. cereuisiaeis exerted primarily at the G 1 - + S t r a n s i t i o n ( i . e . ,e n t r y t o r h e S p h a s e ) .T h e r e f o r e , partially defectivemutants of CDK are arrestedin G1, but completely defective CDK mutanrs are arrested in either G1 or G2, depending on their location in the cell cycle at the time of the temperature shift. These observations demonstratethat both the S. pombe and the S. cereuisiae CDKs are required for entry into both the S phase and mrtosrs.

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ThreeG1CyclinsAssociatewith S. cerevisiae CDKto Form S-Phase-Promoting Factors By the late 1980s,it was clearthat mitosis-promotingfactor (MPF) is composedof two subunits:a CDK and a mitotic Btype cyclin required to activate the catalytic subunit. By analogy, it seemed likely that S. cereuisiaecontains an Sphase-promoting factor (SPF)that phosphorylates and regulates proteins required for DNA synthesis.Similar to MpE, SPFwas proposedto be a heterodimercomposedof the S. cereuisiaeCDK and a cyclin, in this caseone that acts in G1 (seeFigure20-2). To identify this putative G 1 cyclin, researcherslooked for genesthat, when expressedat high concentration,could suppress certain temperature-sensitivemutations jn the S. cereuisiae CDK. The rationale of this approach is illustrated in Figure 20-26. Researchersisolated two such genes, designated CLN1 and CLN2. Using a different approach, researchersidentified a dominant mutation in a third sene c a l l e dC L N J Sequencingof the three CLN genesshowed that they encodedrelated proteins, each of which includesan =100residue region exhibiting significant homology with Btype cyclins from sea urchin, Xenopus, human, and S. pombe. This region encodesthe cyclin domain that interacts with CDKs and is included in the domain of the human cyclin shown in Figure 20-15b, c. The finding that the three CIn proteins contain this region of homology with mitotic cyclins suggestedthat they were the sought-after S. cereuisiaeG1 cyclins. (Note that the homologous CDKbinding domain found in various cyclins differs from the destruction box mentioned earlier,which is found only in B-type cyclins.)

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A E X P E R I M E N TFAI L G U R2E0 - 2 6 G e n e se n c o d i n gt w o S. cerevisiaeG.,cyclinswere identified by their ability to suppressa temperature-sensitive mutant CDK.Thisgenetic screen i s b a s e do n d i f f e r e n c ei nst h e i n t e r a c t r o bn es t w e e n G1 cyclinsandwild-typeandtemperature-sensitive (ts)S. cerevrsiae C D K s( a )W i l d - t y pcee l l sp r o d u c a e n o r m aC l D Kt h a ta s s o c i a t e s w i t h G r c y c l i n sf o, r m i n gt h e a c t i v eS - p h a s e - p r o m o ft a i ncgt o r ( S P Fr)e, s u l t i nign c o l o n yf o r m a t i o a n t b o t ht h e p e r m i s s i va en d t h e n o n p e r m i s stievm e p e r a t u(rie , 2 5 ' a n d 3 6 ' C ) ( b )S o m e cdc29"mutantsexpress a mutantCDKwith low affinityfor G., c y c l i na t 3 6 " C .T h e s em u t a n t sp r o d u c e n o u g hG 1c y c l i n - C D K ( S P Ft o ) s u p p o rgt r o w t ha n dc o l o n yd e v e l o p m eantt 2 5 " C ,b u t n o t a t 3 6 ' C ( c )W h e nc d c 2 8 t ' c e lw l se r et r a n s f o r m ewdi t h a S c e r e v i s i age n o m i lci b r a r cy l o n e di n a h i g h - c o ppyl a s m i dt h , ree t y p e so f c o l o n i efso r m e da t 3 6 ' C : o n ec o n t a i n ead p l a s m i d carryingthe wild-typeCDC2B gene;the othertwo contained p l a s m i dcsa r r y i negi t h e tr h e C L N Io r C L N 2g e n e I n t r a n s f o r m e d c e l l sc a r r y i ntgh e C l N To r C L N 2g e n e t, h e c o n c e n t r a t i o n f the encoded G 1c y c l i ni s h i g he n o u g ht o o f f s e t h e l o w a f f i n i t yo f t h e m u t a nC t D Kf o r a G , c y c l i a n t 3 6 ' C , s ot h a te n o u g h S P Ff o r m st o s u p p o ret n t r yi n t ot h e S p h a s ea n ds u b s e q u e n t m i t o s i sU. n t r a n s f o r mceddc 2 ? tc' e l l sa n dc e l l st r a n s f o r m e d w i t h p l a s m i dcsa r r y i nogt h e rg e n e sa r ea r r e s t eidn G 1a n dd o n o t f o r m c o l o n i e s [ S e eJ A H a d w i g e re t a l , 1 9 8 9 , p r o c N a t ' l A c a d S c i U S A8 6 : 6 2 5 5 l

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the mitotic cyclin caused a shortened Gz and premature entry into mitosis, whereas inhibition of the mitotic cyclin by mutation resulted in a lengthenedG2 (seeFigure 20-1'2)' Thus these results confirmed that the S. cereuisiaeCIn proteins are G1 cyclins that regulate passagethrough the G1

Gene-knockout experiments showed that S. cereuisiae cells can grow in rich medium if they carry any one of the three G1 cyclin genes.As the data presentedinFigure20-27 indicate, overproduction of one G1 cyclin decreasesthe fraction of cells in G1, demonstrating that high levels of the G1 cyclin-CDK complex drive cells through START prematurely. Moreover, in the absenceof all three of the G1 cyclins, cells becomearrestedin G1, indicating that a G1 cyclin-CDK heterodimer, or SPF, is required for S. cereuisiae cells to enter the S phase. These findings are reminiscent of the results for the S. pombe mitotic cyclin with regard to passagethrough G2 and entry into mitosis. Overproduction of

Podcast:G1-cyclinControl of Entry into S-phase High-levelexpressionof G1cyclin

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20-27Gl cyclinis requiredfor 5. FIGURE A EXPERIMENTAL cerevisiaecellsto enter 5 phase,and overexpressionof G1cyclin prematurelydrivesthem into the 5 phase.Theyeastexpression (top)carriedoneof the three5. vectorusedin theseexperiments whichis Gr cyclingeneslinkedto the strongGAI7 promoter, cerevisiae the Todetermine ispresent in the medium. turnedoff whenglucose to a fluorescent proportion of cellsin Gr andGz,cellswereexposed througha fluorescencedyethat bindsto DNAandthenwerepassed (see DNAcontentof G2 the Figure 9-28) Since cell sorter activated cellsin the candistinguish thisprocedure cellsistwicethatof G1cells, with an empty phases(a)Wildtypecellstransformed two cell-cycle of cellsin Gr and the normaldistribution vectordisplayed expression (b)In (Glc) of glucose. glucose addition and after of G2in the absence

G2 G1 Fluorescence -->

with the G1cyclin wild-typecellstransformed of glucose, the absence percentage of cells a higher-than-normal vectordisplayed expression of the G1cyclin overexpression in the S phaseandG2because of the Gr the Gr period(topcurve)'Whenexpression decreased glucose, the cell of by addition off shut was vector cyclinfromthe to normal(bottomcurve)'(c)Cellswith returned distribution with the Gr in allthreeG1cyclingenesandtransformed mutations

Cell591127 I etal, 1989, fromH E Richardson phase. [Adapted

I G A S EC O N T R O LO F S P H A S E A N D U B I Q U I T I N . P R O T E LI N CYCLIN-CDK

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open-readingframe that inhibits translation initiation at the Cln3 open reading frame. This inhibition is diminished when nutrients are in abundance,leading to activation of the TOR pathway and the subsequentincreasein translation initiation factor activity (seeFigure g-30). Since Cln3 is a highly unstable protein, its concenrratron fluctuates with the translation rate of its mRNA. Consequently, the amount and activity of mid-G1 cyclin-CDK complexes, which depend on the concentration of the mid-G1 cyclin protein, are largely regulated by the nutrient level. Once sufficient mid-G1 cyclin is synthesizedfrom its mRNA, the mid-G1 cyclin-CDK complex phosphorylates and activates two related transcription factors, SBF and MBF. These induce transcription of the late-G1 cyclin genes,CLNI and CLN2, whose encodedproteins accelerate entry into the S phase. Thus regulation of CLN3 mRNA translation in responseto the concentrationof nutrients in the medium is thought to be primarily responsible for controlling the length of G1 in S. cereuisiae.In addition to the late-G1 cyclins, SBF and MBF also stimulare tran-

called early S-phasecyclins.Inactivation of Cdhl allows the S-phasecyclin-CDK complexesto accumulate in late G1. The specificity factor Cdhl is phosphorylated and inactivated by both late-G1 and B-type cyclin-CDK complexes, and thus remains inhibited throughout S, G2, and M phaseuntil late anaphasewhen the Cdc14 phosphatase is activated and removes the inhibitory phosphate from

cdh1. D e g r a d a t i o no f t h e S - P h a s Ien h i b i t o rT r i g g e r s DNAReplication As the S-phasecyclin-CDK heterodimersaccumulatein late G1, they are immediately inactivated by binding of an inhibitor, called Sic1, that is expressedlate in mitosis and in early G1. BecauseSicl specificallyinhibits B-type cyclinCDK complexes, but has no effect on the G1 cyclin-CDK complexes,it functions as an S-phaseinhibitor. Entry into the S phaseis defined by the initiation of DNA replication. ln S. cereuisiaecells this occurs when the Sicl inhibitor is precipitouslydegradedfollowing its polyubiquitination by the distinct ubiquitin-protein ligasecalled SCF mentioned earlier (Figure 20-28; seealso Figure20-2). Once Sicl is degraded,the S-phasecyclin-CDK complexesinduce DNA replication by phosphorylatingseveralproteins in prereplication complexesbound to replication origins. This mechanism for activating the S-phasecyclin-CDK complexes-that is, inhibiting them as the cyclinsare synthesizedand then precipitously degradingthe inhibitor-permits the suddenacrivation of large numbers of complexes,as opposedto the gradual increasein kinaseactivity that would result if no inhibitor were presentduring synthesisof the S-phasecyclins. We can now seethat regulated proteasomal degradation directed by two ubiquitin-protein ligase complexes, SCF and APC/C, controls three major transitions in the cell cycle: onset of the S phase through degradation of Sicl by SCF, the beginning of anaphasethrough degradation of securin by the APC/C, and exit from mitosis through degradation of B-type cyclins by the APC/C. The ApC/C is

are required for initiation of DNA synthesis, they are

Polyubiquitinationof p h o s p h o r y l a t eS di c l ; proteasomal degradation

-_____+

z

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FIGURE 20-28 Controlof S phaseonset in S. cerevisiaeby regulatedproteolysisof the S-phaseinhibitor,Sic1.TheS_phase cyclin-CDK (Clb5-CDK complexes andClb6-CDK) beqinto accumutate in G1,butareinhibited bySlc1. Thisinhibition pr.u.nt,initiation of DNA replication untilthe cellisfullyprepared. G1cyclin-CDK complexes assembled in lateG1(Cln1-CDK andCln2-CDK) phosphorylate Siclat 876

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multiple sites(step[), marking it for polyubiquitination bytheSCF ubiquitinligase, andsubsequent proteasomal (stepZ). degradation TheactiveS-phase cyclin-CDK complexes thentriggerinitiation of DNA (stepB) by phosphorylating synthesis components of pre-initiation complexes assembled on DNAreplication originsin earlyG1 lAdapted fromR W Kingetal,1996,Science2T4:1652l

R E G U L A T T NTGH E E U K A R y o l c C E L Lc y c l E

20-29 ActivitYof 5. < FIGURE ae cycli n-CDKcomPlexes cerevisi through the courseof the cell cycle. bandsis Thewidthof the colored proportional to the approximately proteinkinase or proposed demonstrated cyclin-CDK of the indicated activity produces Mid-G1cyclin-CDK complexes. a single S cerevisiae cln3-cDK whose kinase cyclin-dependent (CDK) ,'z cyclins, by thevarious iscontrolled actrvity duringdifferent whichareexpressed portions of the cellcycle.

Mitotic cyclin-GDKs

Late S-phase/ early M-phase cyclin-CDKs ctb3,4CDK

Late-G1cyclin-CDKs Cln1.2-CDK

clb5,6-cDK EarlyS-phasecyclin-CDKs

directed to polyubiquitinylate the anaphaseinhibitor securin by the Cdc20 specificity factor (seeFigure20-23). The APC/CCdc20 complex also directs the degradation of S-phasecyclins and much of the mitotic cyclin, but sufficient mitotic cyclin remains to maintain chromosome condensation until late anaphase.Then, another specificity factor, Cdh1, targets the APC/C to the remainingB-typecyclins(seeFigure20-10). In contrast to the APC/C, the SCF ubiquitin-protein ligaseis not regulatedby phosphorylation of specificityfactors, but rather by phosphorylationof its substrate,Sic1. Sicl is phosphorylated by G1 cyclin-CDKs (see Figure 20-28). It must be phosphorylated at at least six sites,which are relatively poor substratesfor the G1 cyclin-CDKs, before it is bound sufficiently well by SCF to be polyubiquitinylated. This difference in strategy for regulating the ubiquitinprotein ligase activities of SCF and APC/C probably occurs becausethe APC/C has severalsubstrates,including securin and B-type cyclins, which must be degraded at different times in the cycle. In contrast, entry into the S phaserequires the degradation of only a single protein, the Sicl inhibitor. Also, the requirement for phosphorylating multiple weak sitesin Sicl delaysthe onset of S phaseuntil G1 cyclin-CDK activity has reachedits peak and virtually all other G1 cyclinCDK substrateshave been phosphorylated. An obvious advantage of proteolysis for controlling passagethrough these critical points in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed irreversibly in one direction through the cycle.

Multiple CyclinsRegulatethe KinaseActivity of 5. cerevisraeCDKDuring Different Cell-CyclePhases theybethroughtheSphase, Asbuddingyeastcellsprogress gin transcribing genes encoding two additional B-type

tosis, with the help of two other mitotic cyclins. Theseadditional mitotic cyclins are expressedwhen S. cereuisiaecells complete chromosome replication and enter G2. They function as late mitotic cyclins,associatingwith the CDK to form

R e p l i c a t i o na t E a c hO r i g i n l s l n i t i a t e dO n l y O n c e D u r i n gt h e C e l lC Y c l e As discussed in Chapter 4, eukaryotic chromosomes are replicated from multiple replication origins' Initiation of replication from these origins occurs throughout S phase'

I G A 5 EC O N T R O LO F S P H A 5 E C Y C LNI - C D KA N D U B I Q U I T I N . P R O T E LI N

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877

However, no eukaryotic origin initiates more than once per S phase. Moreover, the S phase continues until replication from multiple origins along the length of each chromosome results in complete replication of the entire chromosome. Thesetwo factors ensurethat the correct genecopy number is maintained as cells proliferate. Yeast replication origins contain an 11-base-pairconservedcore sequenceto which is bound a hexamericprotein, the origin-recognition complex (ORC), required for initiation of DNA synthesis.DNase I footprinting analysis(Figure 7-17) and immunoprecipitation of chromatin protelns crosslinked to specificDNA sequences (Figure7-31\ duringvarious phasesof the cell cycle indicate that the ORC remains associatedwith origins during all phasesof the cycle. Several additional replication initiation factors required to initiate

DNA synthesisat origins were identified in geneticstudiesin S. cereuisiae.These DNA replication initiation factors associate with the ORC at origins during G1, but not during G2 or M. During G1 the various initiation factors assemblewith the ORC into a prereplication complex ar each origin (Figure20-30). The restriction of origin "firing" to once and only once per cell cycle in S. cereuisiaeis enforcedby the alternating cycle of B-type cyclin-CDK activity levels through the cell cycle: Iow in telophasethrough G1 and high in S, G2, and M through anaphase(seeFigure 20-29). As we just discussed, S-phasecyclin-CDK complexes become active at the beginning of S phase when their specific inhibitor, Sic1, is degraded. The prereplication complexes assembledat origins early in G1 (Figure 20-30, step [) initiate DNA synthesisin

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< FIGURE 20-30 Assemblyand regulationof p r e r e p l i c a t i ocno m p l e x e sS. t e p[ : D u r i n ge a r l yG 1 , u n p h o s p h o r y l arteepdl i c a t i oi n i t i a t i ofna c t o r sa s s e m ool en a n o r i g i n - r e c o g n i tci o n m p l e(xO R Cb) o u n dt o a r e p l i c a t i o n o r i g i nt o g e n e r a tae p r e r e p l i c a t icoonm p l e xS t e p[ : I n t h e S p h a s eS, - p h a scey c l i n - C DcKo m p l e x easn dD D K phosphorylate components of the prereplication complex S t e pS : T h i s l e a d st o b i n d i n go f C d c 4 5a, c t i v a t i oonf t h e h e x a m e rM i cC M h e l i c a s ewsh, i c hu n w i n dt h e p a r e n t a l D N As t r a n d sa,n dr e l e a soef t h e p h o s p h o r y l a tCeddc 6a n d C d t l i n i t i a t i ofna c t o r sR P Ab i n d st o t h e r e s u l t i nsgi n g r e s t r a n d eD d N A .S t e p4 : l n i t i a t i oonf s y n t h e sbi sy t h e D N A p o l y m e r a cs re- p r i m a s( see eF i g u r 4e - 3 1 ) S . t e pE : O t h e r components necessary for replication fork movement are recruited, and bidirectional synthesis awayfromthe o r i g i nc o n t i n u east e a c hf o r k O R Cb i n d st o t h e o r i g i n s e q u e n ci n e t h e d a u g h t edro u b l e - s t r a n dDeN d A ,b u t t h e p h o s p h o r y l a ti e nd i t i a t i ofna c t o r cs a n n oat s s e m b al e p r e r e p l i c a t icoonm p l eox n i t . B - t y p e c y c l i n - C DcK omplexes maintainthe initiationfactorsin a phosphorylated state t h r o u g h o ut ht e r e m a i n d eorf S ,G 2 ,a n de a r l ya n a p h a s e (fop) Thesefactorscannotassemble into new p r e r e p l i c a t icoonm p l e x eusn t i lt h e ya r ed e p h o s p h o r y l a t e d b y C d c 1 4p h o s p h a t aasnedB - t y p e c y c l i nasr ed e g r a d e d f o l l o w i n tgh e i rp o l y u b i q u i t i n a tbi oynt h e A p C / Ci n I a t e a n a p h a sS e e v e r a ld d i t i o n a f al c t o r sr e q u i r efdo r replication arenot shown

S phasewhen they are phosphorylatedby the S-phasecyclinCDKs and a secondheterodimericprotein kinase, DDK, expressedin G1 along with other proteins involved in DNA replication (step E). Although the complete set of proteins that must be phosphorylated to activate initiation of DNA synthesishas not yet been determined,there is evidencethat phosphorylation of at least one subunit of the hexameric and of another initiation factor called Cdc6 is MCM belica.se required. Following their phosphorylation, the helicaseunwinds the DNA, and the resulting single-strandedDNA is bound by the single-strandedbinding protein RPA and other replication factors (Figure 20-30 steps B, 4, and 5; see also Figure4-31). As the replication forks progressaway from each origin' the phosphorylated initiation factors are displacedfrom the chromatin. However, ORC complexes immediately bind to the origin sequencein the replicated daughter duplex DNAs and remain bound throughout the cell cycle (seeFigure 2030, step [). Origins can fire only once during the S phase becausethe phosphorylated initiation factors cannot reassembleinto a prereplication complex. Consequentlgphosphorylation of componentsof the prereplication complex by S-phasecyclin-CDK complexes and the DDK complex simultaneously activates initiation of DNA replication at an origin and inhibits re-initiation of replication at that origin. As we have noted, B-type cyclin-CDK complexesremain active throughout the S phase, G2, and early anaphase'maintaining the phosphorylated state of the replication initiation factors that prevents the assembly of new prereplication complexes(step[). When the Cdc14 phosphatase is activated in late anaphaseand the APC/C-Cdh1 complex triggers degradation of all B-type cyclins in telophase,phosphateson the initiation factors are removed by the unopposed Cdc14 phosphatase. This allows the reassembly of prereplication complexesduring early G1. As discussedpreviously, the inhibition of APC/C activity in G1 setsthe stagefor accumulation of the S-phasecyclins needed for onset of the next S (1 ) phase.This regulatory mechanismhas two consequences: prereplication complexes are assembledonly during G1, when the activity of B-type cyclin-CDK complexes is low, and (2) each origin initiates replication one time only during the S phase, when S phase cyclin-CDK complex activity is high. As a result, chromosomal DNA is replicated only one time each cell cycle.

r Once active mid-G1 cyclin-CDK complexes accumulate in mid-late G1, they phosphorylate and activate two transcription factors that stimulate expression of the late-G1 cyclins, as well as enzymesand other proteins required for DNA replication, and the early S-phaseB-type cyclins. r The late-G1 cyclin-CDK complexes phosphorylate and inhibit Cdh1, the specificity factor that directs the anaphase-promotingcomplex (APC/C) to B-type cyclins, thus permitting accumulation of S-phaseB-type cyclins in late G1. r S-phasecyclin-CDK complexesinitially are inhibited by Sic1. Polyubiquitination of Sicl by the SCF ubiquitinprotein ligasemarks Sicl for proteasomal degradation' reieasing activated S-phasecyclin-CDK complexesthat trigger onset of the S phase (seeFigure 20-28). Late S-phase/earlyM-phase B-type cyclins, expressed ter in the S phase,form heterodimerswith the CDK that also promote DNA replication and initiate spindle formation early in mitosis. t Late M-phase B-type cyclins, expressedin G2' form heterodimers with the CDK that stimulate mitotic events. r In late anaphase,the specificity factor Cdhl is activated by dephosphorylationand then directsAPC/C to polyubiquitinylate all the B-type cyclins' Their subsequentproteasomal degradation inactivates MPF activity, permitting exit from mitosis (seeFigure 20-1'0). r DNA replication is initiated from prereplication complexesassembledat origins during early G1. S-phasecyclinCDK complexessimultaneouslytrigger initiation from prereplication complexes and inhibit assembly of new prereplication complexes by phosphorylating components of the prereplication complex (seeFigure 20-30). r Initiation of DNA replication occurs at each origin, but only once, until a cell proceedsthrough anaphase,when activation of APC/C leads to the degradation of B-type cyclins. The block on re-initiation of DNA replication until replicated chromosomes have segregated assures that daughter cells contain the proper number of chromosomes per cell.

Controlin Mammalian Cell-Cycle Cells In multicellular organisms, precise control of the cell cycle

Cyclin-CDKand Ubiquitin-Protein LigaseControl of S phase t S. cereuisiaeexpressesa single cyclin-dependentprotein kinase (CDK), which interactswith severaldifferent cyclins during different phasesof the cell cycle (seeFigure 20-29). r Three G1 cyclins are active in G1. The concentration of the mid-G1 cyclin mRNA does not vary significantly through the cell cycle, but its translation is regulatedby the availability of nutrients.

ing G1, entering the G6 state (seeFigure 20-1)' Some differentiatedcells (e.g.,fibroblasts and lymphocytes)can be stimulated to reenter the cycle and replicate. Many postmitotic C E L L - C Y C LCEO N T R O LI N M A M M A L I A N C E L L S

879

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A EXPERIMENTAL FtcURE20-31 Microinjection experiments with anti-cyclinD antibody demonstratethat cyclinD is requiredfor passagethrough the restrictionpoint. TheGsarrested mammalian cellsusedin theseexperiments passthe restriction point14-16hoursafteraddition of growthfactorsand enterthe S phase6-8 hourslater.(a)Outlineof experimental protocol. At various times1O-16hoursafteradditionof growth factors(Il), somecellsweremicroinjected with rabbitantibodies against cyclinD (Z). Bromodeoxyuridine (BrdU), a thymidine analog, wasthenaddedto the medium(E), andthe uninjected controlcells (/eft)and microinjected experimental cells(nghf)wereincubated for an additional 16 hours.Eachsample wasthenanalvzed to determine the percentage of cellsthathadincorporated BrdUintonewly synthesized DNA(4), indicating thattheyhadentered the S phase. (b)Analysis of controlcellsandexperimental cellsiniected with anticyclinD antibody 8 hoursafteradditionof growthfactors. Thethree micrographs showthesamefieldof cellsstained16 hoursafter

addition of BrdUto the medium. Cellswerestained with different fluorescent agentsto visualize DNA(top),BrdU(middle), andanti(bottom). cyclinD antibody Notethatthetwo cellsin thisfield injected (theredcellsin the bottom with anti-cyclin D antibody micrograph) did not incorporate BrdUintonuclear DNA,asindicated bytheirlackof staining in the middlemicrograph (c)percentage of controlcells(bluebars)andexpenmental cells(redbars)that incorporated BrdU.Mostcellsinjected with anti-cyclin D antibodies 10or 12 hoursafteraddition of growthfactorsfailedto enterthe S phase, indicated by the low levelof BrdUincorporation In contrast, anti-cyclin D antibodies hadlittleeffecton entryintothe S phaseand DNAsynthesis wheninjected at 14or 16 hours,thatis,aftercells hadpassed the restriction point.Theseresults indicate thatcyclinD is required to passthe restriction point,butoncecellshavepassed the restrictjon point,theydo not require cyclinD to enterthe S phase (b)and(c)adapted 6-8 hourslater.IParts fromV Baldin et al..1993,Genes & Devel. T:812.1

M a m m a l i a nR e s t r i c t i o nP o i n t l s A n a l o g o u st o STARTin Yeast Cells Most studies of mammalian cell-cycle control have been done with cultured cells that require certain polypeptide growth factors (mitogens) to stimulate cell proliferation. 880

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Binding of thesegrowth factors to specificreceptor proteins that span the plasma membrane initiates a cascadeof signal transduction that ultimately influences transcription and cell-cyclecontrol (Chapters15 and 16). Mammalian cells cultured in the absenceof growth factors are arrestedwith a diploid complement of chromosomes in the Ge period of the cell cycle. If growth factors are added to the culture medium, thesequiescentcellspassthrough the restriction point 14-15 hours later, enter the S phase 6-8 hours after that, and traversethe remainder of the cell cycle (seeFigure 20-2).The restriction point is the time after addition of growth factors when cells no longer require the presenceof growth factors to enter S phase.Like START in yeast cells, the restriction point is the point in the cell cycle at which mammalian cells becomecommitted to entering the S phase and completing the cell cycle, which takes about 24 hours for most cultured mammalian cells.

Multiple CDKsand CyclinsRegulatePassageof M a m m a l i a nC e l l sT h r o u g ht h e C e l lC y c l e Unlike S. pombe and S. cereuisiae,whicheachproduce a single cyclin-dependentkinase (CDK) to regulate the cell cycle, mammalian cells use a small family of related CDKs to regulate progressionthrough the cell cycle. Four CDKs are expressed at significant levels in most mammalian cells and play a role in regulating the cell cycle. Named CDK1, 2, 4, and 6, these proteins were identified by the ability of their cDNA clonesto complement certain cdc yeastmutants or by their homology to other CDKs. Like S. cereuisiae,mammalian cells express multiple cyclins. Cyclin A and cyclin B, which function in the S phase,G2, and early mitosis, initially were detectedas proteins whose concentration oscillatesin experimentswith synchronously cycling early sea urchin and clam embryos (seeFigure 20-8). Homologous cyclin A and cyclin B proteins have been found in all multicellular animals examined.The cDNAs encoding three related human D-type cyclins and cyclin E were isolated basedon their ability to complement S. cereuisiaecells mutant in all three genesencodingG1 cyclins.The relativeamounts of the three D-ryp. cyclins expressedin various cell types differ' Here we refer to them collectivelyas cyclin D. Cyclins D and E are the mammalian mid- and late-G1cyclins, respectively. Experiments in which cultured mammalian cells were microinjected with anti-cyclin D antibody at various times after addition of growth factors demonstrated that cyclin D is essentialfor passagethrough the restriction point (Figure20-31'). Figure 20-32 presentsa current model for the periods of the cell cycle in which different cyclin-CDK complexesact in Gs-arresredmammalian cells stimulated to divide by the addition of growth factors. In the absenceof growth factors, cultured Ge cells expressneither cyclins nor CDKs; the absenceof these critical proteins explains why Ge cells do not progressthrough the cell cycle and replicate. Table 20-1, presentedearly in this chapter, summarizesthe various cyclins and CDKs that we have mentioned and the portions of the cell cycle in which they are active. The cyclins fall

into two major groups' G1 cyclins and B-type cyclins, which function in S, G2, and M. Although it is not possibleto draw a simple one-to-onecorrespondencebetweenthe functions of the severalcyclins and CDKs in S. pombe, S. cereuisiae'and vertebrates, the various cyclin-CDK complexes they form can be broadly consideredin terms of their functions in mid-G1, IateG1, S, and M phases.All B-type cyclins contain a conserveddestruction box sequencethat is recognizedby the APC/C-Cdh1 ubiquitin-protein ligase,whereas G1 cyclins lack this sequence. Thus the APC/C regulates only the activity of those cyclinCDK complexesthat include B-type cyclins.

RegulatedExpressionof Two Classesof Genes ReturnsGs MammalianCellsto the Cell Cycle Addition of growth factors to Gg-arrestedmammalian cells induces transcription of multiple genes,most of which fall into one of two classes-early-responseot delayed-response genes-depending on how soon their encoded mRNAs appear.Transcription of early-responsegenesis induced within a few minutes after addition of growth factors by signaltransduction cascadesthat activate preexisting transcription factors in the cytosol or nucleus (Chapter 16). Many of the early-responsegenesencodetranscription factors' such as cFos and c-Jun, that stimulate transcription of the delayedresponsegenes. Mutant, unregulated forms of both c-Fos anJ c-Jun are expressedby oncogenicretroviruses (Chapter 25); the discoverythat the activatedviral forms of theseproteins (v-Fos and v-Jun) can transform normal cells into cancer cells led to identification of the normal, regulated cellular forms of thesetranscription factors. After peaking at about 30 minutes following addition of growth factors, the concentrations of the early-response mRNAs fall to a lower level that is maintained as long as growth factors are present in the medium. This decreasein early-responsemRNA levels is mediated by early-response protelns. Expression of delayed-responsegenes depends on proteins encoded by early-response genes. Some delayedresponsegenesencode additional transcription factors (see below); others encode mid- and late-G1 cyclins and CDKs' The mid-G1 cyclins and their associating CDKs are ex-

centrations fall precipitously.As a consequence'the cells do not passthe restriction point and do not replicate' In addition to being controlled by transcription of the

ter l6,leading to activation of the mTOR pathway and the resulting activation of translation initiation factors (see cELLs c E L l - c y c L Ec o N T R o Ll N M A M M A L I A N

t

881

Mitotic cyclin-CDKs CyclinA-CDK1 CyclinB-CDK1

< FIGURE 20-32Activityof mammaliancyclin-CDK complexesthrough the courseof the cell cycle. Go Cultured G6cellsareinduced to divideby treatment with growthfactorsThewidthof thecolored bandsis proportional to the proteinkinase \ actrvity Mid-Gl cyclin-CDKs approximately "CyclinD" refers of the indicated complexes to allthree Cyclin D-CDK4 D-typecyclins Cyclin D-CDK6

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Late-G1cyclin-CDK CyclinE-CDK2

Figure 8-30).As a resuk, translationof cyclin D mRNA and other mRNAs is stimulated. Agents that inhibit activation of translation initiation factors, such as TGF-8, inhibit translation of cyclin D mRNA and thus inhibit cell proliferation.

PassageThrough the Restrictionpoint Depends on Phosphorylationof the Tumor-Suppressor Rb Protein Somemembersof a small family of related transcription factors, referred to collectively as E2F factors, are encoded by delayed-response genes.These transcription factors activate genesencoding many of the proteins involved in DNA synthesis. They also stimulate transcription of genesencoding the late-G1cyclin, the S-phasecyclin, and the S-phaseCDK. Thus the E2Fs function in late G1 similarly to the S. cereuisiae tanscription factors SBF and MBF. In addition, E2Fs autostimulate transcription of their own genes.E2Fs function as transcriptional repressorswhen bound to Rb protein, which in turn binds histone deacetylaseand methylasecomplexes.As discussedin Chapter 7, histone deacetylationand methylation of specific histone lysines causeschromatin to assumea condensed,transcriptionally inactive form. Rb protein was initially identified as the produc of the prototype tumor-suppressorgene,RB. The products of tumor-suppressor genesfunction in various ways to inhibit progression through the cell cycle (Chapter 25). Loss-of-function mutations in RB are associatedwith the diseaseh ereditary retinoblastoma. A childwith this disease inherits one normal RB+ allele from one parent and one mutant RB- allele from the other. If rhe Rt+ allele in any of the trillions of cells that make up the human body becomesmutated to a RB- allele,then no functional Rb pro_ tein is expressedand the cell or one of its descendanisis likely to become cancerous. For reasons that are not 882

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understood,this generallyhappensin a retinal cell leading to the retinal tumors that characterizethis disease.Subsequently it was discoveredthat Rb function is inactiyated in almost all cancer cells, either by mutations in both alleles of RB, or by abnormal regulation of Rb phosphorylation.I Rb protein is one of the most significant substratesof mammalian G1 cyclin-CDK complexes. Phosphorylation of Rb protein at multiple sites prevents its associationwith E2Fs, thereby permitting E2Fs to activatetranscription of genesrequired for entry into S phase. As shown in Figure ZO-33, phosphorylation of Rb protein is initiated by the mid-G1 cyclin-CDK complexesin mid G1. Once the late-G1cyclin and CDK are induced by phosphorylation of some Rb, the resulting late-G1 cyclin-CDK complex further phosphorylates Rb in late G1. Sfhen late-G1 cyclin-CDK accumulatesto a Mid Gl

Late G1

FIGURE 20-33 Regulationof Rband E2Factivitiesin mid-lateGr. Stimulation of Gocellswith mitogens inouces expression of CDK4,CDK6,D-typecyclins, andthe E2Ftranscription factors, allencoded by delayed-response genes.Rbproteininitially inhibits E2FactivityWhensignaling frommitogens issustained, the resulting cyclinD-CDK4/6 complexes beginphosphorylating Rb, releasing someE2F, whichstimulates transcription of the genes encoding cyclinE,CDK2,andE2Fitself(autostimulation) Thecyclin E-CDK2 complexes furtherphosphorylate Rb,resulting in positive feedback loops(bluearrows) thatleadto a rapidrisernrne expression andactrvity of bothE2FandcyclinE-CDK2 asthecell approaches the G1-+ Stransition

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

critical threshold level, further phosphorylation of Rb by the late-G1 complex continues even when mid-G1 cyclin-CDK activity is removed. This is one of the principal biochemical eventsresponsiblefor passagethrough the restriction point. At this point, further phosphorylation of Rb by the late-G1 cyclin-CDK occurs even when mitogens are withdrawn and mid-G1 cyclin-CDK levels fall. SinceE2F stimulates its own expressionand that of the late-G1 cyclin and CDK, positive cross-regulationof E2F and late-G1 cyclin-CDK produces a rapid rise of both activities in late G1. As they accumulate,S-phasecyclin-CDK and mitotic cyclin-CDK complexesmaintain Rb protein in the phosphoryIated state throughout the S, G2, and early M phases.After cells complete anaphaseand enter early G1 or Ge, the fall in cyclin-CDK levels leads to dephosphorylation of Rb. As a consequence,hypophosphorylated Rb is available to inhibit E2F activity during early G1 of the next cycle and in Gearrestedcells.

C y c l i nA l s R e q u i r e df o r D N A S y n t h e s i sa n d C D K 1f o r E n t r yi n t o M i t o s i s High levels of E2Fs activate transcription of the cyclin A gene as mammalian cells approach the G1 -+ S transition. (Despite its name, cyclin A is a B-type cyclin, seeTable 201.) Disruption of cyclin A function inhibits DNA synthesis in mammalian cells,suggestingthat cyclin A is the S-phase cyclin and that, along with CDK2, it may function like S. cereuisiaeS-phasecyclin-CDK complexes to trigger initiation of DNA synthesis.There is also evidencethat the mammalian late-G1cyclin-CDK complexesalso contribute to activation of prereplication complexes. Note that CDK2 complexes with both the late-G1 and the S-phasecyclins (seeFigure 20-32). Three related CDK lnhibitory proteins, or CKls (p27*'ot,p57*t", and p21cIP),appearto sharethe function of the S. cereuisiaeS-phaseinhibitor Sicl (seeFigure 20-28). Phosphorylation of p27*tnt by late-G1cyclin-CDK targets it for polyubiquitination by the mammalian SCF complex (see Figure 20-28). The mechanisms for degrading p2lcrP and pSZrtPzare lesswell understood. The activity of mammalian cyclin-CDK2 complexes is also regulated by phosphorylation and dephosphorylation mechanisms similar to those controlling the S. pombe mitosis-promoting factor, MPF (see Figure 20-1.4). The Cdc25A phosphatase,which removes the inhibitory phosphate from CDK2, is a mammalian equivalent of S. pombe Cdc25 except that it functions at the G1 -+ S transition rather than the G2 -+ M transition. The mammalian phosphatase normally is activated late in G1, but is degradedin the responseof mammalian cellsto DNA damageto prevent the cellsfrom enteringS phase(seeSection20.7). Once late-G1cyclin-CDK and S-phasecyclin-CDK are activated by Cdc25A and the S-phaseinhibitors have been degraded, DNA replication is initiated at prereplication complexes. The general mechanism is thought to parallel that in S. cereuisiae(seeFigure 20-30), although small differencesare found in vertebrates.Phosohorvlation of DNA

replication preinitiation complexes at replication origins by late-G1cyclin-CDK and S-phasecyclin-CDK likely promotes initiation of DNA replication. As in yeast, phosphorylation of these initiation factors likely prevents reassemblyof prereplication complexes until the cell passesthrough mitosis, thereby assuring that replication from each origin occurs only once during each cell cycle. In metazoans, a second small protein, geminin, contributes to the inhibition of reinitiation at origins until cells complete a full cell cycle. Geminin is expressedin late G1; it binds and inhibits replication initiation factors as they are releasedfrom preinitiation complexes once DNA replication is initiated during S phase (Figure 20-30, step B), contributing to the inhibition of re-initiation at an origin. Geminin contains a destruction box at its N-terminus that is recognizedby the APC/C-Cdh1, causingit to be polyubiquitinylated in late anaphaseand degraded by proteasomes.This frees the replication initiation factors, which are dephosphorylatedby Cdc14 phosphatase, to bind to ORC on replication origins forming preinitiation complexesduring the following G1 phase. The principal mammalian CDK in G2 and mitosis is CDK1 (seeFigure 20-32). This CDK, which is highly homologous with S. pombe CDK, associateswith cyclins A and B. The mRNAs encoding either of thesemammalian cyclins can promote meiotic maturation when injected into Xenopus oocytesarrestedin G2 (seeFigure 20-6), demonstrating that they function as mitotic cyclins. In somatic vertebrate cells, cyclin A-CDK1 and cyclin B-CDK1 function together as the equivalentof the S. pombe MPF (mitotic cyclin-CDK). The kinase activity of these mammalian complexes also is regulatedby proteins analogousto those that control the activity of the S. pombe MPF (see Figure 20-141. The inhibitory phosphate on CDK1 is removed by Cdc2SC phosphatase, which is analogous to S. pombe Cdc25 phosphatase. In cycling mammalian cells, cyclin B is first synthesized Iate in the S phaseand increasesin concentrationas cellsproceed through G2, peaking during metaphaseand dropping after late anaphase.This parallels the time course of cyclin B expressionin Xenopwscycling egg extracts (seeFigure 20-9). In human cells, cyclin B first accumulatesin the cytosol and then enters the nucleus iust before the nuclear enveloperetracts into the ER early in mitosis. Thus MPF activity is controlled not only by phosphorylation and dephosphorylation but also by regulation of its import into the nucleus.In fact, cyclin B shuttles between the nucleus and cytosol, and the changein its localization during the cell cycle results from a changein the relative ratesof import and export. As rn Xenopus eggsand S. cereuisiae,cyclins A and B are polyubiquitinylated by the APC/C-Cdh1 complex during late anaphase and then are degradedby proteasomes(seeFigure 20-1'0).

Two Typesof Cyclin'CDKlnhibitors Contribute to Cell-CycleControl in Mammals As noted above, three related CKIs-p21crP, p27KrPr,and p57KlP2-inhibit late-G1 cyclin-CDK and S-phasecyclinCDK activity and must be degradedbefore DNA replication C E L L - C Y C LCEO N T R O LI N M A M M A L I A N C E L L S

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can begin. These same CDK inhibitory proteins also can bind to and inhibit the other mammalian cyclin-CDK complexes involved in cell-cycle control. As we discuss later, p21tt' plays a role in the responseof mammalian cells to DNA__damage.Experiments with knockout mice lacking p27*t" have shown that this CKI is particularly importani in controlling generalizedcell proliferation soon after birth. Although p27KrP1knockouts are larger than normal, most develop normally otherwise. In contrast, p57'tnt knockouts exhibit defectsin cell differenriation, and most die shortly after birth owing to defectivedevelopmentof various organs. A second class of cyclin-CDK inhibitors called lNK4s (lzhibitors of Ainase4) includes severalsmall, closelyrelated proteins that interact only with the mid-G1 CDKs, CDK4 and CDK5, and thus function specificallyin controlling the mid-G1 phase.Binding of INK4s to CDK4 and CDK5 blocks their interaction with cyclin D and hencetheir protein kinase activity. The resulting decreasedphosphorylation of Rb protein prevents transcriptional activation by E2Fs and entry into the S phase.One INK4 calledp16 is a tumor suppressor, like Rb protein discussedearlier.The presenceof two mutant p16 allelesin a large fraction of human cancersis evidence for the important role of p1.6 in controlling the cell cycle (Chapter25).

r The activity of S-phasecyclin-CDK, induced by high E2F activity, initially is held in check by CKIs, which function like an S-phaseinhibitor, and by the presenceof an inhibitory phosphate on CDK2, the S-phaseCDK. Proteasomal degradation of the inhibitors and activation of the Cdc25A phosphatase,as cells approach the G1 -+ S transition, generateactive S-phasecyclin-CDK. Along with the late-G1 cyclin-CDK, this complex activatesprereplication complexesto initiate DNA synthesisby a mechanismsimilar to that in S. cereuisiae(seeFigure 20-30). r Cyclin A-CDK1 and cyclin B-CDK1 induce the events of mitosis through early anaphase. Cyclins A and B are polyubiquitinylated by the anaphase-promotingcomplex (APC/C) during late anaphaseand then are degraded by proteasomes, r The activity of mammalian mitotic cyclin-CDK complexesis regulatedby phosphorylation and dephosphorylation similarly to the mechanism in S. pombe, with the Cdc25C phosphataseremoving inhibitory phosphates(see Figure20-1,4). r The activities of mammalian cyclin-CDK complexesalso are regulated by CDK inhibitors (CKIs), which bind to and inhibit each of the mammalian cyclin-CDK complexes,and INK4 proteins, which block passagethrough G1 by specifically inhibiting G1 CDKs (CDK4 and CDK5).

Cell-CycleControl in Mammalian Cells r Various polypeptide growth facrors called mitogens stimulate cultured mammalian cells to proliferate by inducing expressionof early-responsegenes.Many of these encode transcription factors that stimulare expressionof delayed-responsegenes encoding the G1 CDKs, G1 cyclins, and E2F transcription factors. r Once cells passthe restriction point, they can enter the S phase and complete S, G2, and mitosis in the absenceof growth factors. r Mammalian cells use severalCDKs and cyclins to regulate passagethrough the cell cycle. Cyclin D-CDK4 and cyclin D-CDK6 function in mid to late G1; cyclin E-CDK2, in late G1 and early S; cyclin A-CDK2, in S; and cyclin A-CDK1 and cyclin B-CDK1 in G2 and M through anaphase(seeFigure 20-32). r Unphosphorylated Rb protein binds to E2Fs, converting them into transcriptional repressors.Phosphorylation of Rb by the mid-G1 cyclin-CDK liberates E2Fs to activare transcription of genes encoding the late-G1 cyclin and CDK, as well as other proteins required for the S phase. E2Fs also autostimulate transcription of their own genes. r The late-G1cyclin-CDK further phosphorylatesRb, further activating E2Fs. Once a critical level of late-G1cyclinCDK has been expressed,a positive feedback loop with E2F results in a rapid rise of both activities that drives passagethrough the restriction point (seeFigure 20-33).

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Checkpoints in Cell-Cycle Regulation Before proceeding, let's review the major steps in the eukaryotic cell cycle summarized in Figure 20-34.In continuously cycling cells,cyclin-CDK complexesare absentin early G1. Hypophosphorylated DNA replication initiation factors are free to bind to ORC complexesat DNA replication origins, generating prereplication complexes that are inactive until they are phosphorylated by an S-phase cyclin-CDK (step E). In mid-G1, mid-G1 cyclin-CDKs are expressedand phosphorylate the APC/C specificity factor Cdh1, inactivating it and allowing newly synthesizedB-type cyclins (and geminin in vertebrates) to accumulate when they are expressed(stepZ).The mid-G1 cyclin-CDKsalso phosphorylate specific transcription facrors, activating expression of late-G1 and S-phasecyclins (and CDK in vertebrate somatic cells) (step S). However, as B-type cyclins are expressed, they are immediately bound by inhibitors. \fhen G1 cyclinCDK activities reach peak levels, they phosphorylate these inhibitors at multiple sites (step @), marking them for polyubiquitination by the SCF ubiquitin-protein ligase, and subsequentdegradation by proteasomes(step g). This rapid degradation of S-phasecyclin-CDK inhibitors releasesS-phasecyclin-CDK activities,which phosphorylate key regulatory sitesin prereplication complexes,stimulating initiation of DNA replication at multiple origins (step 6).

REGULATING THE EUKARYOTIC C E L LC Y C L E

CdcA phosphatase activatesCdhl and APC/C-Cdh1/proteasome degradesmitotic cyclins

r

APCIC-Cdc2Ol proteasome

,a--\ /

\

\*/

Telophaseand cytokinesis

333.1ii" s

E

Anaphase Early G1

DNA prereplication complexesassemble at origins

z

Mid-late Gl

z Metaphase

G. cyclin-CDKactivates E eipression of S-phasecyclin-

Restriction point

CDK comoonents

q

Cdc25phosphatase activatesmitotic cyclinCDKs,which activate early mitotic events

tr--:----g

G, cyclin-CDKinactivatesCdhl

'{

tr wdtl!trp S-phase cycrin-cDK w activatesprereplication comprexes

d[

G. cyclin-CDKphosphorylates 5-'phaseinhibitor

SCFrproreasome

g:3;:::..fl,e,l_fo?r'".0 inhibitor

DNA replication A FIGURE20-34 Fundamental processesin the eukaryotic cell cycle.Seethe text for discussion

Mitotic cyclin-CDKs are expressedin late S phase and G2. When DNA replication has been completed, they are activated by Cdc25 phosphatase,and either theS or other protein kinasesthat they activate,phosphorylatespecificregulatory sitesin more than a hundred proteins including histone H1, condensins and cohesins, additional chromatinassociatedproteins, microtubule-associatedproteins, nuclear lamins, inner nuclear membrane proteins, and nuclear pore complex proteins. These multiple, specific phosphorylations induce the early eventsof mitosis including chromosome condensation,remodeling of microtubules into the mitotic spindle apparatus, and, in animals and plants, retraction of the nuclear envelopeinto the ER (step Z). Once every kinetochore of each sister chromatid has attached to spindle microtubule fibers during metaphase,inhibition of the Cdc20 specificity factor is lifted. This results in

active APC/C-Cdc20 and polyubiquitination and proteasomal degradationof securin (step E ). Securindegradationreleasesthe proteolytic activity of separase,which then cleaves the cohesin rings at centromeresthat hold sister chromatids together.The forces exerted by the mitotic spindle apparatus then pull the releasedsister chromatids toward opposite spindle poles. The resulting sudden separation of all sister chromatids marks the beginning of anaphase. Once the daughter chromosomes have separated sufficiently to ensure equal segregationof all chromosomes to daughter cells during cytokinesis,the Cdc14 phosphataseis activated. Cdc1,4dephosphorylatesand activatesthe Cdhl APC/C specificity factor, resulting in the polyubiquitination and proteasomal degradation of all B-type cyclins (and geminin in vertebrates),and consequently,the loss of MPF activity (step p). Sites on the multiple proteins that were

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phosphorylated by cyclin-CDKs are dephosphorylated by Cdc1.4.This returns the proteins to their interphase functions, resulting in decondensationof chromosomes,formation of an interphasemicrotubule cytoskeletonwith a single microtubule organiztngcenter,and reassemblyof the nuclear envelopeduring telophase,followed by cytokinesis.The dephosphorylated DNA replication-initiation factors (released by geminin degradation in vertebrates)then reassemble preinitiation complexeson ORC complexesbound to repli, cation origins in daughter cells, in preparation for the next cell cycle (step[).

Successfulcompletion of the cell cycle has severalgeneral requirements.Each processsummarizedin Figure 20-34 must go to completion beforesubsequentstepsare undertaken,and the stepsmust occur in the correct order.Catastrophicgenetic damagecan occur if cellsprogressto the next phaseof the cell cycle beforethe previousphaseis properly completed.For example, when S-phasecellsare induced to enter mitosis by fusion to a cell in mitosis, the MPF presentin the mitotic cell forcesthe chromosomesof the S-phasecell to condense.This premature entry into mitosis results in fragmentation of the Sphasechromosomes,a disastrousconsequence for a cell.

KINASESAND PHOSPHMASES CAK kinase

Activatescyclin-CDKs

Veel kinase

Inhibitscyclin-CDKs

Cdc25phosphatase

Activates cyclin-CDKs

cdc14 phosphatase

Activatescdhl to inhibit mitotic cyclin-cDK

Cdc25Aphosphatase

Acrivares verrebrare S-phase cyclin-CDK

Cdc25Cphosphatase

Acrivatesverrebraremitotic cyclin-CDK

ATM/MR kinases

Checkpointcontrols,activateChkl/Chk2 kinases

Chkl/Chk2 kinases

Checkpointcontrols,inactivateCdc25Cand Cdc25Aphosphatases to inducecell-cyclearrest

INHIBITORY PROTEINS Bindsand inhibits S-phase cyclin-CDKs CKIs p27KIP1,p57*"t, and p21cIP

Bind and inhibit cyclin-CDKs

INK4

Binds and inhibits mid-G1 CDKs

Mad2

Spindle-assemblycheckpoint control, binds Cdc20 and prevents onset of anaphaseand inactivation of B-type cyclin-CDKs

Rb

Binds E2Fs, preventing transcription of multiple cell cycle genes

UBIQUITIN-PROTEINLIGASES SCF

Degradation of phosphorylated Sicl or p27KrP1to activare S-phasecyclin-CDKs

APC/C + Cdc20

Inducesdegradationof Securin,initiating anaphase.Inducespartial degradation of B-type cyclins

APC/C + Cdhl

Induces complete degradation of B-type cyclins to initiate teiophase,and geminin in metazoans to allow formation of prereplication complexes on DNA replication origins

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'We have seenhow progression through the cell cycle is governed by precise regulation of the activities of multiple cyclin-CDK complexes. TabIe 20-2 summarizes the various types of regulators of cyclin-CDK activity. The key cellcycle events of DNA replication and chromosome segregation must be accomplishedwith extraordinary accuracy and fidelity. To ensure that these processesoccur correctly and in the proper order, cells have evolved multiple additional levels of regulation controlling these fundamental cell-cycle events. Collectively, these additional regulatory mechanismsare known as checkpoints(Figure20-35). Several examplesof cell-cyclecheckpointshave beendiscussed earlier in the chapter. In this section' we consider these and additional checkpoints in terms of the major cell-cycle processessummarizedabove.Control mechanismsthat operateat thesecheckpoints ensure that chromosomes are intact and that each stageof the cell cycle is completed before

Another example of the importance of order of events in the cell cycle concernsattachmentof kinetochoresto microtubules of the mitotic spindle during metaphase.If anaphaseis initiated before both kinetochoresof a replicated chromosome become attached to microtubules from opposite spindle poles, daughter cells are produced that have missing or extra chromosomes,an outcome called nondisjwnction. Nflhennondisjunction occurs in mitotic cells, it can lead to the misregulation of genes,and contribute to the development of cancer.When nondisjunction occurs during the meiotic division that generatesa human egg, Down syndrome can occur from trisomy of chromosome 21, resulting in developmental abnormalities and mental retardation. Other mechanismscan also generatetrisomy. (Trisomy of any of the human chromosomes can occur, but for every other chromosome except chromosome 21, trisomy results in embryonic lethality or death shortly after birth.) I

E

a

Spindle'Position checkPoint

Spindle-assembly checkpoint Mad2 I

I

I

APc/c-cdh1

C d c 1 4+

p o l y u b i q u i t i n a t i o<nof B-typecyclins

Sicl

I

APC/C-Cdc20 polyubiquitination of securin

ATM/R

v

Ia

Telophase

o?3

\ Anaphase

I Cyclin D-CDK4I6 |

CyclintuB-CDK1+ ,/ Cdc25C

E lntra-S-phase checkpoint

DNA-damase checkpoint

P21ctP

M-phase entrY S-phase <entry

TI chkl .t

cyctinE/A_CDK2

TI 1

p21ctP

I

CyclinA-CDK2

T1

ATR

p21ctP

@ DNA-damage checkpoint

IT

ps3

1l

Cdc2bA

p53 Cdc2bA

t

ATM/R +

Chkl/2

|

ATM/R --+ Chkl/2

FIGURE 20-35 Overviewof checkpointcontrolsin the cell (0) prevents activation of cyclin checkpoint cycle.Theintra-S-phase (i e , mitosis-promoting factor,MPF)by A-CDK1andcyclinB-CDK1 proteinkinase cascade thatphosphorylates activation of an ATR-Chk1 Cdc25C, thereby inhibiting entryintomitosisInthe andinactivates (E), Mad2andotherproteins inhibit checkpoint spindle-assembly for factorCdc20required of theAPC/Cspecificity activation preventing polyubiquitination of securin, thereby entryintoanaphase (B) prevents release of theCdc14 Thespindle-position checkpoint phosphatase activation of theAPC/C f romnucleoli, thereby blocking

of Bfor APC/Cpolyubiquitination factor(Cdh1)required specificity in decrease result, the As a of Sicl induction well as as cyclins type doesnot occur'In for the eventsof telophase MPFactivityrequired (4), theATMor ATR checkpoint the initialphaseof theDNA-damage thentrigqer (ATM/R) kinases Theactive isactivated. proteinkinase (llil blocking pathway and pathways: Chk-Cdc25A lE), the two pathway, andthep53-p21crP through5 phase, entryintoor passage leading to arrestin G1,S,andGz(!E-!E|). Seethetextfor further pathways thatinhibitprogression indicate Redsymbols discussion throughthecellcycle

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Intra-S phase checkpoint

Ensures all DNA repiicatron is complete before entering M-phase

Spindle-assembly checkpoint Ensuresall chromosome kinetochores are attached to spindle microtubules before anaphase

ATR detectsreplicationforks

Inhibition of Cdc25C ro prevent activation of mitotic cyclin-CDKs, blocking early mitotic events

Mad2 detectskinetochores unattached to microtubules

Inhibition of Cdc20 to prevent activation of separaseand onset of anaphase

Spindle-position checkpoint

Ensuresall chromosomes are properly segregatedto daughter cells before telophase and cytokinesis

(5. cereuisiae) Tem-1detects properpositionof spindle pole body in bud

Prevention of Cdc14 activation and degradation of mitotrc cyclins, blocking late mitotic events

DNA-damage checkpoinr

Detects damage to DNA throughout the cell cycle

ATM, ATR detectDNA damage

Inhibition of Cdc25A to prevent entry into S phase; pzltto inhibition of all cyclin-CDK complexes to induce cell cycle arrest

the following stageis initiated. Our understandingof these control mechanismsat the molecular level has advanced considerably in recent years. Table 20-3 lists four maior cell cycle checkpointsand summarizesthe control mechanisms used at each checkooint.

for orderly progressionof the fundamentalprocessesof the cell cycle (seeFigure 20-34).

The Presenceof UnreplicatedDNA prevents E n t r yi n t o M i t o s i s

The spindle-assembly checkpoint prevents enrry into anaphaseuntil every single kinetochore of every chromatid is properly associatedwith spindle microtubules. If even a single kinetochore is unattached to a spindle microtubule, anaphaseis inhibited. Clues about how this checkpointoperates initially came from isolation of yeast mutants in the presenceof benomyl, a microtubule-depolymerizing drug. Low concentrations of benomyl increasethe time required for yeast cells to assemblethe mitotic spindle and attach kinetochores to microtubules. \X/ild-type cells exposed to benomyl do not begin anaphase until these processesare completed and then proceed on through mitosis, producing normal daughter cells. In contrast, mutants defectivein the spindle-assemblycheckpoint proceed through anaphasebefore assemblyof the spindle and attachment of kinetochores is complete; consequently,they mis-segregatetheir chromosomes,producing abnormal daughter cells that die. Analysis of these mutants identified a protein called Mad2 (mitotic arrest defective2) and other proteins that regulate Cdc20, the specificity factor required to target the APC/C to securin(seeFigure 20-35, Z). Recallthat APC/CCdc20-mediatedpolyubiquitination of securin and its subsequent degradation is required for activation of separaseand entry into anaphase (see Figure 20-23). Mad2 has been shown to associatewith kinetochoresthat are unattachedto microtubules. Kinetochore-bound Mad2 rapidly exchanges

Cells that fail to replicate all their chromosomesdo not normally enter mitosis. Operation of the intra-S-phase cbeckpoint control involves the recognition of unreplicated DNA and stalledDNA replicationforks, which causesinhibition of MPF activation (seeFigure 20-35, [). Genetic studies in the yeastsand biochemical studies with Xenopus egg extracts demonstrated that the ATR and Chkl protein kinasesinhibit entry into mitosis by cells that have not completedDNA synthesis.The associationof ATR with replication forks is thought to activare its protein kinase activity, leading to phosphorylationand activation of the Chkl kinase.Active Chkl then phosphorylatesand inactivatesthe Cdc25 phosphatase(Cdc25C in vertebrates).which otherwise removesthe inhibitory phosphatefrom mitotic CDKs. As a result, the mitotic cyclin-CDK complexesremain inhibited and cannot phosphorylate targets required to initiate mitosis. ATR continues to initiate this protein kinase cascadeuntil all replication forks complete DNA replication and disassemble. This mechanismmakes the initiation of mitosis dependent on the completion of chromosome replication. This dependencyor requirement that a cellcyclephasemust be completedbeforethe next phasecan be initiated is a critical aspect of checkpoint function required 888

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with a soluble form of Mad2 that inhibits all the Cdc20 in the cell. When microtubules attach to kinetochores,the kinetochores releasethe bound Mad2 and ceasethe processby which the inhibitorS soluble form of Mad2 is produced. However, when even a single kinetochore is unattached to microtubules from the opposite spindle pole of its sister,sufficient soluble inhibitory Mad2 is produced at the unattached kinetochore to inhibit all the Cdc20 in the cell. The current model for how this regulatory mechanismfunctions (Figure 20-36) was suggestedby X-ray crystallography and NMR data revealingthe structuresof interacting proteins involved in the process.The model is supported by directed mutagenesisstudiesguided by thesestructures,biochemical

studiesof the protein-proteininteractions,and microscopic proteins.This elestudiesin living cellsusing GFP-labeled checkpointcanaccount gantmodelfor the spindle-assembly for the ability of a singleunattachedkinetochoreto inhibit all the cellularCdc20until the kinetochorebecomespropwith spindlemicrotubules. erly associated

(a)

(b)

Checkpoint activation

Proper Segregation of Daughter Chromosomes ls Monitored by the Mitotic Exit Network properly'telophase have segregated Once chromosomes The variouseventsof telophaseand subsecommences. quent cytokinesis,collectivelyreferredto as the exit from

Checkpoint inactivation

Cytoplasmic pool of Mad2

Madl

E

Releaseof Madl-Mad2 tetramer from kinetochore Attachment completed

/\AllJ--,^\ l p 3 tl c ) l l ( c l p 3 t

ilrv

|t l U UM a d l Mad2 in open conformation (C J Mad2 in closedconformation

Thespindle20-36 Modelfor Cdc20Regulation. FIGURE has isactiveuntileverysinglekinetochore assembly checkpoint (a)TheMad2protein properly to spindle microtubules. attached in two conformations, one "open"(redsquares) andthe other exists "closed"(orange circles). According to thecurrentmodel,Mad2in to canbindeitherMadl or Cdc20.Binding the openconformation whichis converts Mad2to theclosed conformation, Madl or Cdc2O stablyboundto theseproteinsCdc20boundbythe closed in thesame TwoMad2oroteins conformation of Mad2is inactive. but closedMad2andopenMad2can do not interact, conformation from througha siteon Mad2distinct bindto eachothertransiently with eitherMadl or Cdc20Madl andthe the onethatassociates of Mad2forma tetramer thatbindsto closed conformation viathe Madl subunit(E). Mad2in the unattached kinetochores canbindtransiently to the Mad2in the closed openconformation (Z) This conformatron boundto Madl at the kinetochore with the closedMad2stimulates openMad2to binda interaction with a Cdc20OpenMad2canbindCdc20onlywhileit isinteracting the openMad2proteinto theclosed closedMad2 Thisconverts it to dissociate fromthe Mad2in theclosed causing conformation, (B) Thestableinteraction of closed at the kinetochore conformation Cdc20frombindingto theAPC/C Mad2with Cdc20prevents transiently Further, theclosedMad2boundto Cdc20caninteract (4), causing it to bind with anotherMad2in theopenconformation

Microtubules

thisMad2to theclosed Thisconverts anotherCdc20molecule. boundto Cdc20ThisnewlyformedclosedMad2conformation pair, fromthefirstMad2-Cdc20 dissociates Cdc20complex (5) ThusfreeMad2in the complexes generating two Mad2-Cdc20 to closedMad2boundto isquicklyconverted openconformation (6). of closedMad2that The source repeats cycle Cdc20asthis istheclosedMad2boundto Madl inrtiates thischainreaction how a singleunattached explaining with a kinetochore, associated of allthe Cdc20in the cell cancauseinactivation kinetochore (b) complexes. of closedMad2-Cdc20 throughtheformation (green) the causes to kinetochores of microtubules Attachment Mad2in the displaced of the Madl/Mad2tetramer. disolacement bindsand with openMad2,but rather, cannotinteract tetramer p31'o'"t,whichthenbindsMad2in anotherprotein, activates a releasing activeCdc20(Z) However, complexes, Mad2-Cdc2O can boundto kinetochores smallnumberof Madl-Mad2tetramers shown bythe mechanism complexes generate enoughMad2-Cdc20 have the activityof p31. Onceall kinetochores in (a)to overcome of all Madl-Mad2 the release causing to microtubules attached activeCdc20,which predominates, releasing p31activity tetramers, andproteasomal in polyubiquitination resulting bindsto theAPC/C, fromA andtheonsetof anaphase. [Modified of securin degradation Cell 2O05' seealsoK Nasmyth, Biol15:214; DeAntoniet al, 2005,Curr. 12O:739 | N C E L L - C Y C LREE G U L A T I O N C H E C K P O I N TI S

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mitosis, require inactivation of MPF. As discussedearlier, dephosphorylation of the APC/C specificity factor Cdhl by the Cdcl4 phosphataseleads to degradationof mitotic cyclins and loss of MPF activity late in anaphase(seeFigure 20-10). During interphaseand early mitosis, Cdc14 is sequesteredin the nucleolus and inactivated.The spindleposition checkpoint, which monitors the location of the segregating daughter chromosomes at the end of anaphase, determines whether active Cdc14 is released from the nucleolusto promote exit from mitosis (seeFigu r e 2 0 - 3 5 ,B ) . Operation of this checkpoint in S. cereuisiaedepends,in part, on a set of proteins referred to as the mitotic exit network. Regulation of Cdc14 activation operatessimilarly in most eukaryotes.In the fission yeastS. pombe, formation of the septum that divides daughter cells is regulated by proteins homologous to those that constitute the mitotic exit network in S. cereuisiae.Genes encoding similar proteins have been found in higher organisms where the homologs function in an analogous checkpoint that leads to arrest in late mitosis when daughter chromosomes do not segregate properly. A key component of the mitotic exit network is a small (monomeric) GTPase, called Teml (Figure 20-37). This member of the GTPase superfamily of switch proteins controls the activity of a protein kinase cascadesimilarly to the way Ras controls MAP kinase pathways (Chapter 16). During anaphase,Teml becomesassociatedwith the spindle pole body (SPB)closesrto the daughter cell bud. (The SPB, from which spindle microtubules originate, is equivalent to the centrosome in higher eukaryotes.) At the SPB, Teml is maintained in the inactive GDP-bound state by a specific GAP (GTPase-activatingprotein). The GEF (guanosine nucleotide-exchangefactor) that activates Teml is localized to the cortex of the bud and is absent from the mother cell. Another protein, Kin4 protein kinase, is localized to the mother cell cortex and is absent from the bud. \fhen spindle microtubule elongation at the end of anaphase has correctly positioned segregatingdaughter chromosomes into the bud, Teml comes into contact with its GEF and the Teml-GAP becomes phosphorylated and inhibited. As a consequence,Teml is converted into its active GTP-bound state. The terminal kinase in the cascade triggered by Teml.GTP then phosphorylates the nucleolar anchor that binds and inhibits Cdc1.4,releasingthe Cdc14 phosphatase into the cytoplasm and nucleoplasm in both the bud and mother cell (Figure 20-37, [). Once active Cdc1.4 is available, a cell can proceed through telophase and cytokinesis. The mitotic exit network is a good example of the dependency of one cycle phase on completion of the previous phase. Telophase and cytokinesis cannot initiate until the chromosome segregationmechanism carries daughter chromosomesinto the bud. This is becauseinitiation of telophase depends on the activation of Cdc14, and the activation of Cdc14 requires that Teml be pushed all the way to the bud cortex.

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FIGURE 20-37The spindle-position checkpoint.Cdc14 phosphatase activityis required for the exitfrom mitosis.Iop: In 5. cerevisiae, duringinterphase andearlymitosis, Cdc14issequestered (purple) andinactivated in the nucleolus Inactive Teml.GDP polebody(SPB) associates withthe spindle nearest to the budearly in anaphase with theaidof a linkerprotein(blue)andis maintained in the inactive stateby a specific GAP(GTPase-accelerating protein, yellow)lf chromosome ([), extension segregation occursproperly of thespindle microtubules inserts the daughter SPBintothe bud, causrng Temlto comein contactwith a specific GEF(guanine nucleotide-exchange factor)localized to the cortexof the bud (orange), andthe inactivation of theTeml-GAPThisconverts inactive Teml'GDP to activeTemlGTBwhichtriggers a proteinkinase cascade leading to release of activeCdc14andexitfrommitosislf thespindle apparatus failsto placethedaughter SPBin the bud(Z), the SPBencounters Kin4(cyan) localized to the mothercellcortex Thisactivates theTeml-GAfmaintaining Temlin the inactive GDpboundstateandCdc14remains associated with nucleoli. Arrestin latemitosis results. G Pereiraand E Schiebel, 2001, [Adaptedfrom Cun OpinCellBiol.13:7621

If daughter chromosomesfail to segregateinto the bud, Teml does not encounter the Teml-GEF. Instead. the Kin4 kinase associatedwith the mother cell cortex maintains the Teml-GAP in an activated state. Tem1, consequently,remains in its inactive, GDP-bound state, Cdc14 is not releasedfrom the nucleolus, and mitotic exit is blocked (Figare 20-37, Z). Kin4 is not required in cells that segregare their chromosomes properl5 but only in the small fraction of cells that fail to do so, giving rhem more time to push the daughter chromosomes into the bud. In S. cereuisiae.the

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error rate for mis-segregationof chromosomesis (1 in 105 cell divisions.

Cell-Cycle Arrest of Cellswith DamagedDNA Dependson Tumor Suppressors The proteins of the DNA-damage checkpoint sense DNA damage and block progression through the cell cycle until the damage is repaired. Damage to DNA can result from chemical agentsand from irradiation with ultraviolet (W) light or T-rays. Arrest in G1 and S prevents copying of damaged bases,which would fix mutations in the genome. Replication of damaged DNA also promotes chromosomal rearrangementsthat can contribute to the onset of cancer.Arrest in G2 allows DNA double-strandedbreaks to be repaired before mitosis. If a double-strandedbreak is not repaired, the broken distal portion of the damagedchromosome is not properly segregatedbecauseit is not physically linked to a centromere,which is pulled toward a spindle pole during anaphase. As we discuss in detail in Chapter 25, inactivation of tumor-suppressor genescontributes to the development of cancer.The proteins encoded by several tumor-suppressor genes,including ATM and Chk2, normally function in the DNA-damage checkpoint. Patients with mutations in both copies of ATM or Chk2 develop cancers far more frequently than normal. Both of these genes encode protein kinases. DNA damage due to W light is sensedby proteins that signal the presenceof UV-damaged DNA to the ATM kinase,activating it. Activated ATM then phosphorylatesand activates Chk2, which then phosphorylatesthe Cdc25A phosphatase,marking it for polyubiquitination by an ubiquitin-protein ligase and subsequent proteasomal degradation. Recall that removal of the inhibitory phosphate from mammalian CDK2 by Cdc25A is required for onset of and passagethrough the S phase, mediated by cyclin E-CDK2 and cyclin A-CDK2. Degradation of Cdc25A resulting from activation of the ATM-Chk2 pathway in G1 or S-phasecells thus leadsto G1 or S arrest (seeFigure 20-35, Eil and EE). A similar pathway consisting of the protein kinases ATR and Chkl leads to phosphorylation and polyubiquitination of Cdc25A in responseto ^y-irradiation.As discussedearlier for the intra-S-phasecheckpoint, Chkl also inactivates Cdc25C, preventing the activation of CDK1 and entry into mltosls. Another tumor suppressor,p53 protein, contributes to arrest of cells with damaged DNA. Cells with functional p53 arrest in G1 and G2 when exposed to 1-irradiation, whereascells lacking functional p53 do not arrest in G1. Although the p53 protein is a transcription factor, under normal conditions it is extremely unstable and generally does not accumulateto high enough levelsto stimulate transcription. The instability of p53 results from its polyubiquitination by a ubiquitin-protein ligase called Mdm2 and subse-

quent proteasomal degradation. The rapid degradation of p53 is inhibited by ATM and ATR, which phosphorylate p53 at a site that interferes with Mdm2 binding. This and other modifications of p53 in response to DNA damage greatly increase its ability to activate transcription of specific genesthat help the cell cope with DNA damage.One of these genes encodes p2lttn, a generalized CKI that binds and inhibits all mammalian cyclin-CDK complexes.As a result, cells are arrested in G1 and G2 until the DNA damage is repairedand p53, and subsequentlyp21cIP,levelsfall (see

Figure20-35,!d-!d). Under some circumstances,such as when DNA damage is extensive,p53 also activatesexpressionof genesthat lead to apoptosis,the processof programmed cell death that normally occurs in specificcells during the developmentof multicellular animals. In vertebrates,the p53 responseevolved to induce apoptosis in the face of extensiveDNA damage, presumably to prevent the accumulation of multiple mutations that might convert a normal cell into a cancercell. The dual role of p53 in both cell-cyclearrest and the induction of apoptosis may account for the observation that nearly all cancercellshave mutations in both allelesof the p53 geneor in the pathways that stabilizep53 in responseto DNA damage (Chapter 25). The consequencesof mutations in p53, ATM, and Chk2 provide dramatic examples of the significanceof cell-cyclecheckpointsto the health of a multicellular organism. I

Checkpointsin Cell-CycleRegulation r Checkpoint controls function to ensure that chromosomesare intact and that critical stagesof the cell cycle are completed before the following stageis initiated. r The intra-S-phasecheckpoint operatesduring S and G2 to prevent the activation of MPF before DNA synthesisis complete by inhibiting the activation of CDK1 by Cdc25C

(seeFigure20-35,Itrl. r The spindle-assemblycheckpoint, which prevents premature initiation of anaphase, utilizes Mad2 and other proteins to regulate the APC/C specificity factor Cdc20 that targets securin for polyubiquitination (seeFigure 2035, Z, and Figure 20-361. r The spindle-position checkpoint prevents telophaseand cytokinesis until daughter chromosomes have been properly segregated,so that the daughter cell has a full set of chromosomes(seeFigure20-35, E). r In the spindle-position checkpoint, the small GTPase Teml controls the availability of Cdc14 phosphatase, which in turn activatesthe APC/C specificity factor Cdhl that targets B-type cyclins for degradation, causing inactivation of MPF (seeFigure20-1,0). r The DNA-damage checkpoint arreststhe cell cycle in responseto DNA damageuntil the damageis repaired.Three

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types of tumor-suppressor proteins (ATIWATR, Chk'J,/z, and p53) are critical to this checkpoint. r Activation of the ATM or ATR protein kinases in responseto DNA damage due to UV light or ^y-irradiation leads to arrest in G1 and the S phase via a pathway that leads to loss of Cdc25A phosphatase activity. A second pathway from activated ATM/R stabilizes p53, which stimulates expression of p21cIP. Subsequentinhibition of multiple CDK-cyclin complexes by p21cIP causes prolonged arrest in G1 and G2 (seeFigure 20-35, ![-!!|). r In responseto extensive DNA damage, p53 also activatesgenesthat induceapoptosis.

Meiosis:A SpecialTypeof Cell Division In nearly all diploid eukaryotes, meiosis generateshaploid germ cells (eggsand sperm),which can then fuse with a germ cell from another individual to generatea diploid zygote that developsinto a new individual. Meiosis is a fundamental aspect of the biology and evolution of all eukaryotesbecauseit results in the reassortmentof the chromosome setsreceived from an individual's two parents. Both chromosome reassortment and recombination between parental DNA molecules during meiosis guaranteesthat each haploid germ cell generatedwill receive a unique combination of gene alleles that is distinct from each parent as well as from every other haploid germ cell formed. The mechanismsof meiosisare analogousto those of mitosis. However, severalkey differencesin meiosis allow this processto generatehaploid cells with incredible genetic diversity (seeFigure 5-3). In this section, we will discussthe parallels between molecular mechanisms of mitosis and meiosis, as well as the mechanistic differencesresponsible for the significant distinctions betweenthesetwo fundamental processes of cell division.

Key FeaturesDistinguishMeiosisfrom Mitosis (Figure Duringmeiosis 20-38),a singleroundof DNA replication is followed by two cycles of cell division, termed meiosis I and meiosis 11,each distinct from the mitotic divisions of somatic cells. Figure 20-39 summarizesthe distinctions between mitosis and meiosis. In G2 and prophase of meiosis I, the two replicated chromatids of each chromosome (Figure 20-38, step Il) are associatedwith each other by cohesin complexes along the full length of the chromosome arms, just as they are following DNA replication in a mitotic cell cycle (seeFigure 20-21, G2 phase).A major difference between meiosis and mitosis is that in prophase of meiosis I, homologous chromosomes(i.e., the maternal and paternal chromosome 1, the maternal and paternal chromosome 2, etc.) pair with eachother, a processknown as synapsls (Figure 20-39, row 4). This forms a biualent chromo-

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cellshavetwo copiesof each > FIGURE 20-38 Meiosis.Premeiotic (2n),onederived parentandone fromthe paternal chromosome parent.Forsimplicity, fromthe maternal the paternal andmaternal homologs arediagrammed StepE: All of onlyonechromosome duringthe S phasebeforethe firstmeiotic chromosomes arereplicated givinga 4n chromosomal complement. Cohesin complexes division, (notshown)linkthe sisterchromatids composing eachreplicated chromosome alongtheirfulllengthsStep[: Aschromosomes replicated homologs condense duringthefirstmeioticprophase, eventbetween becomepairedasthe resultof at leastonecrossover Thispairing of replicated a paternal anda maternal chromatid iscalledsynapsis. At metaphase, homologous chromosomes shown of onechromosome associate with microtubules here,bothchromatids pole,buteachmember emanating fromonespindle of a homologous pairassociates with microtubules emanating from chromosome poles. l, thehomologous opposite Step$: Duringanaphase of meiosis eachconsisting of two chromatids, arepulledto chromosomes, yieldsthetwo daughter oppositespindlepolesStep4: Cytokinesis ll withoutundergoing DNA cells(now2n),whichentermeiosis replication. At metaphase of meiosis ll, shownhere,thechromatids chromosome associate with spindle composing eachreplicated microtubules fromopposite spindlepoles,astheydo in mitosis. Steps of chromatids to opposite spindlepolesduring E and 6: Segregation generates the secondmeioticanaphase followedby cytokinesis haploid germcells(1n)containing onecopyof eachchromosome. Micrographs I andmetaphase ll in developing on the leftshowmeioticmetaphase gametes fromLilium(Lily)ovulesChromosomes arealignedat the plate.lPhotos metaphase courtesy of EdReschke/Peter Arnold, Inc] some, or tetrad, composedof four homologous chromatids, two maternal and two paternal. Significantly,at least one recombination event occurs betweena maternal and a paternal chromatid in every tetrad (Figure 20-39, row 5). The crossing over of chromatids produced by recombination can be observed microscopically in the first meiotic prophase and metaphase as structures called chiasmata (singular, cbiasma).In contrast, no pairing betweenhomologous chromosomes occurs during mitosis, and recombination between nonsisterchromatids is rare. Another key difference between mitosis and meiosis is that in the metaphaseof meiosis I, the kinetochores at the centromeresof sisterchromatids attach to spindle fibers emanating from the same spindle pole, rather than from opposite spindle poles as in mitosis. However, the kinetochoresof the maternal and paternal chromosomes of each tetrad attach to spindle microtubules from opposite spindle poles (Figure 20-38, step Z; Figure 20-39, row 6). Also, cohesion betweenthe full length of the sisterchromatid arms is maintained throughout metaphaseof meiosisI. This is in contrast to mitosis, where cohesionbetween sisterchromatid arms is Iost during prophase in mitotic cells from most organisms, so that cohesion is maintained only in the region of the centromere during mitotic metaphase (Figure 20-21,; Figure 20-39, row 7). Becausenonsister chromatids have recombined at least once by metaphaseof meiosis I, and because cohesion is maintained between the arms of sister chromatids, as the maternal and paternal chromosomes are pulled toward opposite spindle poles in metaphase, the

REGULATING T H E E U K A R Y O T I C E L LC Y C L E

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homologous chromosomes are held together by the chiasmata between them and cohesion of the sister chromatids distal to the crossover (Figure 20-40). During anaphaseof meiosis I, securin degradation releasesseparase,which then cleavesthe cohesin rings holding the chromosome arms together as during mitosis (Figure 20-23). However, during meiosis I, cohesin rings at the centromere are not cleaved (Figure 20-39, row 8). This allows the recombined maternal and paternal chromosomesto separate,but eachpair of chromatids remains associatedat the centromere (Figure 20-38, step E; Figure20-39, row 8). In some organisms, meiosis II proceedswithout decondensationof the chromosomesand assemblyof a nuclear envelope. In other organisms, these typical interphase events 894

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occur, but the interphase is short and nuclear envelope retraction into the ER and chromosome condensationof meiotic prophase II follow rapidly. During metaphaseII (Figure 20-38, step Zl), as in mitosis, the kinetochoresof each sister chromatid attach to spindle microtubules from opposite spindle poles. Also, as during mitosis, cohesion between the chromatid arms is lost and maintained only in the region of the centromere. lfhen the final kinetochore is properly attached to a spindle microtubule, anaphaseII occurs (Figure 20-38, step 5), followed by telophaseII and cytokinesis to generatefour haploid germ cells (Figure 20-38, step 6). For each chromosome, at least two of the haploid germ cells have recombinant chromosomesgeneratedfrom recombination between maternal and paternal chromosomes during

REGULATING THE EUKARYOTIC C E L LC Y C L E

Centriole ( s p i n d l ep o l e ) Spindle microtubules Centromeres

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FIGURE 20-40 Cohesionbetween homologous chromosomesin meiosisI metaphase.Connections between chromosomes duringmeiosis I aremosteasily visualized in organisms with acrocentric centromeres, suchasthegrasshopper. The kinetochores at the centromeres of sisterchromatids attachto spindle microtubules pole,with the emanating fromthe samespindle (red)andpaternal (blue)chromosomes kinetochores of the maternal attaching to spindle microtubules poles. fromopposite spindle The maternal andpaternal chromosomes areattached at thechiasmata formedby recombination between themandthe cohesion between sisterchromatid armsthatpersists throughout meiosis I metaphase. Notethatelimination of cohesion between sisterchromatid armsis allthatis required for the homologous chromosomes to separate at anaphaselAdapted fromL V Paliulis andR B Nicklas, 2OOO, ] CellBiol 15O:1223 1

prophase of meiosis I (Figure 20-38, step Z). Thus the recombination between nonsister chromatids that occurs in prophase of meiosis I has at least two functional consequencesrFirst, it holds homologous chromosomestogether during meiosis I metaphase(Figure 20-40). Second,it contributes to genetic diversity among individuals of a species by ensuring new combinations of geneallelesin different individuals. Genetic diversity also arisesfrom the independent reassortmentof maternal and paternal homologs during the meiotic divisions.

Repressionof G1Cyclinsand a Meiosis-specific ProteinKinasePromotePremeioticS Phase ln S. cereuisiaeand S. pombe, depletion of nitrogen and carbon sourcesinduces diploid cells to undergo meiosis,yielding haploid spores(seeFigure 1-6). This processis analogous to the formation of germ cells in higher eukaryotes.Multiple yeast mutants that cannot form spores have been isolated, and the wild-type proteins encodedby thesegeneshave been analyzed.These studieshave identified specializedcell-cycle proteins required for meiosis.

Under starvation conditions, expressionof G1 cyclins in S. cereuisiaeis repressed,blocking the normal progression of G1 in cells as they complete mitosis. Instead, a set of early meiotic proteins is induced. Among these is Ime2, a protein kinase closely homologous to CDKs that performs the essential G1 cyclin-CDK functions required to enter S phase: (1) phosphorylation of the APC/C specificity factor Cdh1, inactivating it so that B-type cyclins can accumulate, (2) phosphorylation of transcription factors to induce genesrequired for S phaseincluding DNA polymerasesand S-phasecyclins and CDKs, and (3) phosphorylation of the S-phaseinhibitor Sic1, leading to releaseof active S-phase cyclin-CDK complexes and the onset of DNA replication in meiosisI. The cell uses Ime2 during meiosis rather than the standard G1 cyclin-CDKs so that its protein kinase activity can '\)7hile the transcription and translabe regulated differently. tion of G1 cyclins required for activity of G1 cyclin-CDKs is repressed in nutrient-starved cells, the transcription and translation of Ime2 is activated.Also, the Ime2 kinase activity is regulateddifferently than for the G1 cyclins. It doesnot require a cyclin partner for kinase activity and is not regulated by the sameprotein kinasesand phosphatasesthat regulate the activities of G1 cyclin-CDKs. The mechanismby which DNA replication is suppressed between meiosisI and II is currently an active area of investigation. Following meiosis I anaphase,MPF activity does not fall as low as it doesfollowing mitotic anaphase.This intermediatelevel of MPF activity is required for normal meiosis II. It appearsthat MPF activity falls low enough to allow partial or completecytokinesis,but not low enough to allow dephosphorylation of DNA replication initiation factors. Presumably,DNA replication does not occur between meiosis I and II in part becauseDNA replication initiation factors are maintained in a hyperphosphorylated form that cannot assembleprereplication complexes on DNA (see Figure 20-30). A second rise in MPF activity occurs that is required for formation of the meiosis II spindle. After all sister kinetochores have attached to microtubules from opposite spindle poles, the activity of Cdc20 is derepressed, separaseis activated and cells proceed into meiosis II anaphase(Figure 20-38, step El), telophase,and cytokinesis, to generatehaploid germ cells.

Cohesin Recombinationand a Meiosis-Specific SubunitAre Necessaryfor the Specialized S e g r e g a t i o ni n M e i o s i sI Chromosome As discussedearlier, in metaphaseof meiosis I, both sister chromatids in one (replicated) chromosome associatewith microtubules emanating from the same spindle pole, rather than from opposite poles as they do in mitosis. Two physical links between homologous chromosomesare thought to resist the pulling force of the spindle until anaphase: (a) crossing over between chromatids, one from each pair of homologous chromosomes, and (b) cohesin cross-links between sister chromatids distal to the crossover point (see Figure 20-40). T Y P EO F C E L LD l v l 5 l O N M E l O S l SA : SPECIAL

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Evidencefor the function of recombination during meiosis in S. cereuisiaecomesfrom the observation that when recombination is blocked by mutations in proteins essential for the process, chromosomes segregaterandomly during meiosis I; that is, homologous chromosomes do not necessarily segregateto opposite spindle poles. Such segregation to opposite spindle poles normally occurs because both chromatids of the maternal and paternal homologous chromosome pairs associatewith spindle fibers emanating from opposite spindle poles (seeFigure 20-38, step B, and Figure 20-40). As discussedabove, this in turn requiresthat homologous chromosomespair during prophaseof meiosisI. Consequently,the finding that mutations that block recombination also block proper segregationin meiosis I implies that recombination is required for synapsisof homologous chromosomesin S. cereuisiae. Unlike the mitotic anaphase(Figure 20-41a), at the onset of meiotic anaphaseI, the cohesincross-linksbetween chromosome arms are cleavedby separase,allowing the homologous chromosomesto separate,but cohesincomplexesat the centromereremain linked (Figure 20-41,b,top).The maintenance of centromeric cohesion during meiosis I is necessary for the proper segregationof chromatids during meiosis II. Studieswith a S. pombe mutant have shown that a specialized cohesin kleisin subunit (seeFigure 20-23), Rec8, maintains centromeric cohesionbetweensisterchromatids during meiosis I. Expressedonly during meiosis, Rec8 is homologous to the cohesinsubunit that closesthe cohesinring in the cohesincomplex of mitotic cells.Immunolocalization experiments in S. pombe revealed that during early anaphaseof meiosisI, Rec8 is lost from chromosomearms but is retained at centromeres.However, during early anaphaseof meiosis II, centromeric Rec8 is degraded by separase,so the chromatids can segregate,as they do in mitosis (Figure 20-41b, bottom). S. cereuisiaeRec8 has been shown to localize and function similarly to S. pombe Rec8, and homologs of Rec8 also have beenidentified in higher organisms.ConsequentlS understanding the regulation of Rec8-cohesin complex cleavageis central to understanding chromosome segregation in meiosisI. Micromanipulation experiments during grasshopper spermatogenesisindicated that chromosome-associated factors protect centromeric Rec8 from cleavage during meiosis I but not during meiosis II. These experiments also demonstrated that the attachment of sister kinetochores to microtubules emanating from the same spindle pole during meiosis I, as opposed to attachment to spindle fibers from opposite spindle poles in meiosis II and mitosis, also results from factors associated with the chromosomes (Figure 20-42). Thus crossing over, Rec8, and special kinetochoreassociatedproteins appear to function in meiosis in all eukaryotes.

SpecialPropertiesof Rec8Regulatelts Cleavage i n M e i o s i sI a n d l l The mechanismthat protects S. cereuisiaeRecS from degradation at centromeresduring meiosisI is similar to the mech895

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A FIGURE 20-41 Cohesinfunctionduring mitosisand meiosis. (a)Duringmitosis, generated sisterchromatids by DNAreplication in the S phaseareinitially associated by cohesin complexes alongthe full lengthof thechromatids Duringchromosome condensation, (yellow)becomerestricted cohesincomplexes to the regionof the centromere at metaphase, asdepicted here.Onceseparase creaves the kleisin cohesin subunit(seeFigure 20-23),sisterchromatids can (b)In prophase separate, marking the onsetof anaphase of meiosis l, recombination betweenmaternal andpaternal chromatids produces parental synapsis of homologous chromosomes. By metaphase the chromatids of eachreplicated chromosome arecrosslinkedby cohesin complexes alongtheirfull lengthRec8, a meiosis-specific homologof the mitotickleisin, iscleaved in chromosome armsbut not in the centromere, allowinghomologous pairsto segregate chromosome to daughter cellsCentromeric Rec8 iscleaved duringmeiosis ll,allowingindividual chromatids to segregate to daughter cells.[Modified fromF.Uhlmann ,2001 , CurrOpin CellBiol 13:754)

anism that protects kleisin cohesin subunits at centromeres during mitosis. Recall that during mitotic prophase, protein kinasesactivated by the mitotic cyclin-CDKs phosphorylate cohesinsin the chromatid arms, causing them to dissociate, eliminating cohesion in chromatid arms by metaphase in most organisms. However, cohesion at the centromeres is maintained because a specific isoform of protein phosphatase 2A (PP2A) is localized to centromeric chromatin and keeps cohesin in a hypophosphorylated state that does not dissociate from chromatin (see Figure 20-22). Then, when the last kinetochore is properly associatedwith spindle microtubules, Cdc20 is derepressedand associateswith the APC/C, causing polyubiquitination of securin. This releases separase activity, which cleaves the kleisin whether it is

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activate the spindle checkpoint. Cells in nocodazole become arrestedin early mitosis becausethey cannot form a spindle, and thus all kinetochoresremain unattached.To determineif XnfT is required for a functional spindle checkpoint, Xenopus egg extracts, arrested in metaphase,were subjectedto various protocols (see the following figure): untreated (-nocodazole), or treated with nocodazoleand either mockdepleted (preimmune) or immuno-depleted of XnfT (ctXnfT). The extracts were then treated with Ca2* to overcome arrest, and aliquots of the extracts were assessedat various times for cyclin B, as shown on the Nfesternblot beIow. \fhat can you conclude about XnfT from thesedata?

Time(min) 0

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Roux, K. J., and B. Burke. 2006. From pore to kinetochoreand back: regulatingenvelopeassembly.Deu. Cell ll:276-278. a universaltrigger for sister \7irth, K. G., et al. 2005. Separase: chromatid disiunction but not chromosomecycle progression./. Cell Biol. 172:847-860. Yanagida,M. 2005. Basicmechanismof eukaryotic chromosome segregation. Phil. Trans.R. Soc.Lond. B Biol, Sci. 360:609-621. Cyclin-CDK and Ubiquitin-Protein Ligase Control of S-phase Bell, S. P.,and A. Dutta. 2002. DNA replication in eukaryotic cells.Ann. Reu.Biochem. 7l:333-37 4. Deshaies,R. J. 1999. SCF and Cullin/Ring H2-basedubiquitin ligases.Ann. Reu.Cell Deuel.Biol. 15:435467. Diffley, J. F.2004. Regulationof earlyeventsin chromosome replication.Curr. Biol 14:R77 8-R7 86. Nakayama, K. L, and K. Nakayama. 2005. Regulationof the cell cycle by SCF-type ubiquitin ligases.Semin. Cell Deu. Biol. 16:323-333. Reed,S. l. 2006. The ubiquitin-proteasomepathway in cell cycle control. ResubsProbl. Cell Dffer. 42:147-L81. Cell-CycleControl in Mammalian Cells

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References Overview of the Cell Cycle and lts Control Morgan, D. O. 2005. The Cell Cycle:Principlesof Control. New SciencePress. Nasmyth, K. 2001. A prize for proliferation. Cell 107:689-701,. Control of Mitosis by Cyclins and MPF Activity Doree, M., and T. Hunt. 2002.From Cdc2 to Cdkl: when did the cell cycle kinaseioin its cyclin partner?J. Cell Sci. Il5:246L-2464. Masui, Y. 2001. From oocytematuration to the in vitro cell cycle: the history of discoveriesof Maturation-PromotingFactor (MPF) and CytostaticFactor (CSF).Differentiation 69:1-t7. Cyclin-Dependent Kinase Regulation During Mitosis Nurse, P.2002. Cyclin dependentkinasesand cell cyclecontrol (Nobel lecture\.Chembiochem. 3 :596-603. Molecular Mechanisms for Regulating Mitotic Events Hirano, T. 2005. Condensins:organizingand segregatingthe genome.Cut Biol. 15:R255-R275. Kline-Smith,S. L., S. Sandall,and A. Desai.2005. Kinetochorespindlemicrotubule interactionsduring mitosis. Curr. Opin. Cell Biol.77:3546. Meyer, H. H. 2005. Golgi reassemblyafter mitosis:the AAA family meetsthe ubiquitin iamrly.Biochim. Biophys. Acta 1744:t08-11.9. Nasmyth, K., and C. H. Haering. 2005. The structureand function of SMC and kleisin complexes.Ann. Reu.Biochem. 74:595-648. Nigg, E. A. 2001. Mitotic kinasesas regulatorsof cell division and its checkpoints.Nature Reu.Mol. Cell Biol.2:21-32. Peters,J. M. 2006. The anaphasepromoting complex/cyclosome:a machinedesignedto destroy.Nature Reu.Mol. Cell Biol. 7:644-656.

Barr, F. A. 2004. Golgi inheritance:shakenbut not stirred./. Cell Biol. t64:95 5-9 58. DePamphilis,M. L., et aI.2006. Regulatingthe licensingof DNA replication origins in metazoa.Curr. Opin. Cell Biol. 18:231-239. Ekholm, S. V., and S. L Reed.2000. Regulationof G(1) cyclindependentkinasesin the mammalian cell cycle.Curr. Opin. Cell Biol. t2:675-684. Machida, Y.J., J. L. Hamlin, and A. Dutta. 2005. Right place, right time, and only once: replication initiation in metazoans.Cell 123:1.3-24. Porter,L. A., and D. J. Donoghue.2003. Cyclin 81 and CDKI: nuclearlocalizationand upstreamregulators.Prog. Cell Cycle Res. 5:335-347. Sears,R. C., and J. R. Nevins. 2002. Signalingnetworks that link cell proliferation and cell fate.J. Biol. Chem. 277:11.617-1.1.620. Sherr,C. J.2001,.The INK4a/ARF network in tumour suppression.Nature Reu.Mol. Cell Biol. 2:731.-737. Checkpoints in Cell-CycleRegulation Bartek,J., C. Lukas, and J. Lukas. 2004' Checkingon DNA damagein S phase.Nature Reu.Mol. Cell Biol. 5:792-804. Cheeseman,I. M., and A. Desai.2004' Cell division: feeling tenseenough?Nature 428:32-33. Gottifredi, V., and C. Prives.2005. The S phasecheckpoint: when the crowd meets at the fork. Semin. Cell Deu. Biol16:355-368. Hartwell, L. H. 2002. Yeast and cancer (Nobel lecture). BiosciRep.22:373-394. Kastan,M. B., and J. Bartek. 2004' Cell-cyclecheckpointsand cancer.Natur e 432:316-323. Kitagawa, R., and M. B. Kastan.2005' The AlM-dependent DNA damage signaling pathway. Cold Spring Harbor Symp' Quant. Biol. TO:99-I09. Lambert, S., and A. M. Carr. 2005. Checkpointresponsesto replication fork barriers.Biochimie 87:59I-602. Lew, D. J., and D. J. Burke. 2003. The spindleassemblyand spindleposition checkpoints.Ann. Reu' Genet.37:25l-282. McGowan, C. H., and P. Russell'2004.The DNA damage response:sensingand signaling.Curr. Opin. Cell Biol. 16:629-633. Nasmyth, K. 2005. How do so few control so many? Cell 120:739-746. REFERENCES

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Pereira,G., and E. Schiebel.2001. The role of the yeastspindle pole body and the mammalian centrosome in regulating late mitotic events.Curr. Opin. Cell Biol. 13:762-769. Seshan,A., and A. Amon. 2004. Linked for life: temporal and spatial coordination of late mitotic events.Curr. Opin. Celt Biol. 16:4148. Stark, G. R., and !7. R. Taylor. 2006. Control of the G2lM transition. Mol. Biotechnol. 32:227-248. Stegmeier, F., and A. Amon. 2004. Closing mitosis: the functions of the Cdc14 phosphataseand its regulation.Ann. Reu.Genet. 38:203-232. Takeda,D. Y., and A. Dutta. 2005. DNA replication and progression through S phase. Oncogene 24:2827-2843.

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Uhlmann, F. 2003. Separaseregulation during mitosis. Biochem, Soc. Symp. 7 02243-251.. Meiosis: A Special Type of Cell Division Marston, A. L., and A. Amon. 2004. Meiosis:cell-cyclecontrols shuffle and deal. Nature Reu. Mol. Cell Biol. 5:983-997. Petronczki,M., M. F. Siomos,and K. Nasmyth. 2003. Un m6nage a quatre: the molecular biology of chromosome segregationin meiosis.Cell 112:423440. 'Watanabe, Y.2004. Modifying sisterchromatid cohesionfor meiosis.I. Cell Sci. 117:401,74023.

cLASStC

EXPERIMENT

2O

FROMTHESEA:THEDISCOVERY CELLBIOLOGYEMERGING OF CYCLINS T. Evanset al., 1983,Ce//33:391.

From the first cell divisions after fertilization to aberrant divisions that occur in cancers, biologists have long been interested in how cells control when they divide. The processesof cell division have been separated into stages known collectively as the cell cycle. While studying early development in marine invertebrates in the early 1980s, Joan Ruderman and Tim Hunt discovered the cyclins, key regulators of the cell cycle.

tein synthesis from the maternal mRNA is required at the earlieststages of development.Ruderman and Hunt, while teaching a physiology course at the Marine Biological Lab in'Woods Hole, Massachusetts,began a set of experiments designed to uncover the genesthat were expressedat this point as well as the mechanismby which this burst of protein synthesis was controlled.

The Experiment Background The question of how an organism develops from a fertilized egg continues to drive a large body of scientific research. \(hereas such research was classically the concern of embryologists, the developing understanding of gene expressionin the 1980s brought new approaches to answer this question. One such approach was to examine the pattern of gene expression in both the oocyte and the newly fertilized egg. Ruderman and Hunt were among the biologists who took this approach to the study of early development. Biologists had well characterized the early development of a number of marine invertebrate systems. During the early stages of development, the embryonic cells grow synchronously, which allows an entire population of cells to be studied at the same stageof the cell cycle. Researchershad established that a large portion of the mRNA in the unfertilized oocyte is not translated. Upon fertilization, these maternal mRNA are rapidly translated. Previous studieshad shown that when fertilized eggs are treated with drugs that inhibit protein synthesis, cell division could not take place. This suggestedthat the initial burst of pro-

In a collaborative project, Ruderman and Hunt looked at regulation of gene expression in the fertilized egg of the surf clam Spisulasolidissima.Ifhereas it was known that overall protein synthesis rapidly increasedupon fertilization, they wanted to find out whether the proteins expressedin the earliest stageof development,the two-cell embryo, were different from those expressedin the unfertilized egg. $7hen either oocytes or two-cell clam embryos are treated with radioactively labeled amino acids,the cell takes up the amino acids, which are subsequently incorporated into newly synthesized proteins. Using this technique, Ruderman and Hunt monitored the Pattern of protein synthesisby breaking open the cells,separatingthe proteinsusing SDS-polyacrylamidegel electrophoresis (SDS-PAGE),and then visualizing the radioactively labeled proteins by autoradiography. \fhen they compared the pattern of protein synthesis in the oocyte with that in the two-cell embryo, they saw that three different proteins that were either not expressed or expressedat an extremely low level in the oocyte were highly expressedin the embryo. In a subsequentstudy,Ruderman examined the pattern of protein expression in the oocytes of the

starfish Asterias forbesi as they mature. She again observedthe increased expressionof three proteins of similar size to those that she and Hunt had seenin surf clam embryos. Soon afterward, in a third studS Hunt examined the changesin protein expressionduring the maturation and fertilization of sea urchin oocytes. This time he performed the experiment in a slightly different manner. Rather than treating the oocytes and embryo with radioactively labeled amino acids for a set time period, he labeled the cells continuously for more than 2 hours, removing samples for analysis at 10minute intervals. Now, he could monitor the changes in protein expression throughout the early stagesof development. As had been shown in other organisms,the pattern of protein synthesis was altered when the sea urchin oocyte was fertilized. Three proteinsrepresentedby three prominent bands exon an autoradiograph-were not in the but embryos, pressedin the intensity of the Interestinglr oocytes. over time; changed bands one of these the band was intense at the early time points, then barely visible after 85 minutes. It increasedin intensity again between 95 and 105 minutes. The intensity of the band, representing the amount of the protein in the cell, appearedto be oscillating over time. This suggestedthat the protein had been quickly degradedand then synthesized agarn, Becausethe time frame of the experiment coincided with early embryonic cell divisions, Hunt next asked whether the synthesis and destruction of the protein was correlated with progressionof the cell cycle. He examined a portion of cells from each time point under a microscope, counting the number of cells dividing at each time

OF CYCLINS C E L LB I O L O G YE M E R G I N GF R O M T H E S E A :T H E D I S C O V E R Y

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< FIGURE 1 Thisfigure comparesthe changinglevelsof sea urchincyclin(drawn in blue)with a control protein (drawn in purple)as early embryoniccellsprogressthrough the cell cycle.Theoverall levelof cyclinincreases overtime,andthenit is rapidly destroyed asthe cellsapproach Thispatternappears division. to repeatthrougheachcelldivisionMeanwhile, the overall levelof thecontrolproteincontinues to increase throughout thetimeperiod of the experiment[Adapted fromI Evans et al, 1983,Ce//33:391 ]

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2 hrs point where samples had been taken for protein analysis. Hunt then correlated the amounr of the protein present in the cell with the proporrion of cells dividing at each time point. He noticed that the level of expression of one of the proteins was highest before the cell divided and lowest upon cell division (seeFigure 1), suggestinga correlation with the stage of the cell cycle. \flhen the sameexperiment was performed in the surf clam, Hunt saw that two of the proteins that he and Ruderman had describedpreviously displayedthe same pattern of synthesisand destruction. Hunt called theseproteins cyclins to reflect their changing expression through the cell cycle.

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Discussion The discovery of the cyclins heralded an explosion of investigation into the cell cycle. It is now known that these proteins regulate the cell cycle by associating with cyclin-dependentkinases, which in turn regulate the activities of a variety of transcription and replication factors, as well as other proteins involved in the complex alterations in cell architecture and chromosome structure that occur during mitosis. In brief, cyclin-CDK complexes direct and regulate through the cell cycle. As with so many key regulators of cellular functions, it was soon shown that the cyclins discovered in sea urchins and surf clams are conservedin eukaryotes

R E G U L A T T NTGH E E U K A R Y O T TC CE L LC Y C L E

from yeastto man. Sincethe identification of the first cyclins, scientistshave identified at least 15 other cyclins that regulate all phasesof the cell cycle. In addition to the basicresearchinterest in these proteins, the cyclins' central role in cell division has made them a focal point in cancer research. Cyclins are involved in the regulation of severalgenesthat are known to play prominent roles in tumor development. Scientists have shown that at least one cyclin, cyclin D1, is overexpressed in a number of tumors. The role of these proteins in both normal and aberrantcell division continuesto be an active and exciting area of researchtoday.

CHAPTER

CELLBIRTH, AND LINEAGE, DEATH

All nucleiare cerebellum. Cellsbeingborn in the developing labeledin red;the greencellsaredividingand migratinginto internallayersof the neuraltissue [Courtesyof TalRaveh, MatthewScott,and JaneJohnsonl

uring the evolution of multicellular organisms, new mechanismsaroseto diversify cell types,to coordinate their production, to regulate their size and number, to organize them into functioning tissues,and to eliminate extraneous or aged cells. Signaling befween cells becameeven more important than it was for single-celledorganisms.The mode of reproduction also changed,with some cells becoming specializedas germ cells (e.g., eggs, sperm), which give rise to new organisms, as distinct from all other body cells, called somatic cells. Under normal conditions somatic cells will never be part of a new individual. The formation of working tissues and organs during development of multicellular organisms dependsin part on specific patterns of mitotic cell division. A series of such cell divisions akin to a family tree is called a cell lineage. A cell lineage traces the birth order of cells, the progressive restriction of their developmental potential, and their differentiation into specialized cell types (Figure 21.-1.).CeIl lineagesare controlled by cell-intrinsic (internal) factorscells acting according to their history and internal regulatorsas well as by cell-extrinsic (external) factors such as cell-cell signals and environmental inputs. A cell lineage begins with stem cells, unspecializedcells that can potentially reproduce themselvesand generate more-specialized cells indefinitely. Their name comes from the image of a plant stem, which grows upward, continuing to form more stem, while sending off leaves and branches to the side. A cell lineage ultimately culminates in formation of terminally differentiated cells such as skin cells, neurons, or

muscle cells. Terminal differentiation generally is irreversible, and the resulting highly specializedcells often cannot divide; they survive, carry out their functions for

cells is more limited than that of the stem cells from which they arise. (Although some researchersdistinguish between precursor and progenitor cells' we will use these terms interchangeably.) Once a new precursor cell type is created, it often produces transcription factors characteristic of its fate. These transcription factors coordinately activate' or repress'

OUTLIN E The Birth of Cells:Stem Cells,Niches, and Lineage

906

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Cell-TypeSpecificationin Yeast

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Regulationof AsymmetricCell Division

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Cell Death and lts Regulation

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A FIGURE 21-1 Overviewof the binh, lineage,and deathof cells.Following growth,cellsare"born" asthe resultof symmetric or asymmetric celldivision(a)Thetwo daughter cellsresulting from symmetric division areessentially identical to eachotherandto the parental cell Suchdaughter cellssubsequently canhavedifferent fatesif theyareexposed to different signals. Thetwo daughter cells resulting fromasymmetric division differfrombirthandconsequently havedifferent fatesAsymmetric division commonly is preceded by the localization (greendots)in onepartof of regulatory molecules the parentcell (b)A series of symmetric and/orasymmetric cell divisions, calleda celllineage, givesbirthto eachof the specialized celltypesfoundin a multicellular organism. Thepatternof cell lineage canbe undertightgenetic controlprogrammed celldeath occurs duringnormaldevelopment (eg , in thewebbingthatinitially develops whenfingersgrow)andalsoin response to infection or poison. A series programmed of specific events, calledapoptosis, is activated in thesesituations batteriesof genesthat direct the differentiation process.For instance,a few key regulatory transcription factors createthe different mating types of budding yeastand similarly, a small number of such factors produced in sequencetrigger the steps in forming differentiated muscle cells from precursors. 'We discussboth theseexamplesin this chapter. Typically we think of cell fates in terms of the differentiated cell types that are formed. A quite different cell fate, programmed cell death, also is absolutely crucial in the formation and maintenanceof many tissues.A precisegenetic regulatory system, with checks and balances,controls cell death just as other geneticprograms control cell differentiation. In this chapter,then, we considerthe life cycle of cellstheir birth, their patterns of division (lineage),and their death. These aspectsof cell biology convergewith developmental biology and are among the most imporrant processes regulated by the signaling pathways discussedin earlier chapters.

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C E L LB I R T H ,L I N E A G EA , ND DEATH

Many descriptions of cell division imply that the parental cell gives rise to two daughter cells that look and behaveexactly like the parental cell: that is, cell division is symmetric, and the progeny retain the same properties as the parental cell. But if this were always the case,none of the hundreds of differentiatedcell types presentin complex organismswould ever be formed. Differencesamong cells can arise when two initially identical daughter cells diverge upon receiving distinct developmentalor environmental signals.Alternatively, the two daughter cellsmay differ from "birth," with each inheriting different parts of the parental cell (seeFigure 21-1). Daughter cells produced by such asymmetric cell division may differ in size,shape, andlor composition, or their genes may be in different statesof activity or potential activity. The differencesin these internal signalsconfer different fates on the two cells. Here we discusssome generalfeaturesof how different cell types are generated,culminating with the best-understood complex cell lineage, that of the nematode Caenorhabditis elegans.In later sections,we focus on examples of the molecular mechanisms that determine particular cell types in yeast,Drosophila, and mammals.

Stem CellsGive Riseto Both Stem Cellsand D i f f e r e n t i a t i n gC e l l s Stem cells, which give rise to the specializedcells composing the tissues of the body, exhibit several patterns of cell division (Figure 21.-2).A stem cell may divide symmetrically (a) Maintainstem-cell population @

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FIGURE 21-2 Patternsof stem-celldivision.Divisions of stem cells(yellow) mustmaintain population, thestem-cell sometimes increase the numberof stemcells,andat the righttimeproduce differentiating cells(green)(a)Stemcellsthatundergoasymmetric produce divisions onestemcellandonedifferentiating cell Thisdoes not increase the population of stemcells(b)Somestemcellsin a population maydividesymmetrically to increase theirpopulation, whichmaybe usefulin normaldevelopment or duringrecovery from injury, whileat the sametimeothersaredividing asymmetrically asin (a) (c)In a thirdpattern, somestemcellsmaydivideasin (b),while at the sametimeothersproduce two differentiating progeny. fromS Morrison rt41:1068 andJ Kimble, [Adapted 2006,Nature I

to yield two daughter stem cells identical to itself. Alternatively, a stem cell may divide asymmetrically to generate a copy of itself and a derivative stem cell that has morerestrictedcapabilities,such as dividing for a limited period of time or giving rise to fewer types of progeny compared with the parental stem cell. A pluripotenl (or multipotent) stem cell has the capability of generating a number of different cell types, but not all. For instance, a pluripotent blood stem cell will form more of itself plus multiple types of blood cells,but never a skin cell. In contrast,a unipotent stem cell dividesto form a copy of itself plus a cell that can form only one cell type. For example stem cells in the intestinecontinuously reproducethemselves,while the other daughtercell differentiatesinto an intestinalepithelialcell, as we discussin greaterdetail below In many cases,asymmetric division of a stem cell generatesa progenitor cell, which embarks on a path of differentiation, or even a terminally differentiating cell. The two critical properties of stem cells that together distinguish them from all other cells are the ability to reproduce themselvesindefinitely, often called self-renewal, and the ability to divide asymmetrically to form one daughter stem cell identical to itself and one daughter cell of more restrictedpotential. Many stem-celldivisions are symmetric, producing two stem cells, but at some point some progeny need to differentiate. In this way, mitotic division of stem cells can either enlargea population of undifferentiated cells or maintain a stem-cell population while steadily producing a stream of differentiating cells. Although some types of precursorcellscan divide symmetrically to form more of themselves,they do so only for limited periods of time. Moreover, in contrast to stem cells,if a precursor cell divides asymmetrically,it generatestwo distinct daughter cells, neither of which is identical to the parental precursorcell. The fertilized egg, or zygote,is the ultimate totipotent cell becauseit has the capability to generateall the cell types of the body. Although not technicallya stem cell because it is not self-renewing,the zygote does give rise to cells with stem-cell properties. For example, the early mouse embryo passesthrough an eight-cellstagein which each cell can form every tissue;that is, they are totipotent. Thus the subdivision of body parts and tissuefates among the early embryonic cells has not irreversibly occurred at the eight-cellstage.At the 15-cell stage,this is no longer true; some of the cells are committed to particular differentiation paths.

Restricted Cell FatesAre Progressively D u r i n gD e v e l o p m e n t The eight cells resulting from the first three divisions of a mammalian zygote (fertilized egg) all look the same' As demonstratedexperimentally in sheep,each of the cells has the potential to give rise to a completeanimal. Additional divisions produce a mass, composed of =64 cells, that sepa-

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Skull Head,skeletalmuscle Skeleton Dermisof skin Connectivetissue Urogenitalsystem Heart B l o o d ,l y m p h c e l l s Spleen

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21-3 Fatesof the germ layersin animals.Someof A FIGURE arelisted of thethreegermlayers thetissuederivatives

rates into two cell types: trophectoderm' which will form extra-embryonic tissueslike the placenta, and the inner cell mass, which gives rise to the embryo proper' The inner cell mass eventually forms three germ layers, each with distinct fates. One layer,the ectoderm,will make neural and epidermal cells: another. the mesoderm,will make muscle and connective tissue; the third layer' the endoderm, will make gut epithelia (Figure21-3). Once the three germ layers are established,they subsequently divide into cell populations with different fates' For instance,the ectoderm becomesdivided into those cells that are precursorsto the skin epithelium and those that are precursors to the nervous system. There appears to be a progressive restriction in the range of cell types that can be formed from stem cells and precursor cells as development proceeds.An early embryonic stem cell' as we've seen' can lo.- .u.ty type of cell, an ectodermal cell has a choice between neural and epidermal fates, while a keratinocyte precursor can form skin but not neurons. Another restriction that occurs early in animal development is the setting aside of cells that will form the germ line-the stem cells and precursor cells that eventually will

is widespread(though not universal) among animals' In contrast, plants do nothing of the sort; meristems,growing tips of ,ooi, and shoots,can often give rise to germ-line cells and there is no germ-line lineageset aside early' One consequenceof the early segregationof germ-line cells is that the loss or rearrangement of genesin somatic cells will not affect the inherited genome of a future zygote' Nonetheless,although segmentsof the genomeare rearranged and lost during developmentof lymphocytesfrom hematopoietic precursors, most somatic cells seem to have an intact genome, equivalent to that in the germ line (Chapter 24)' Evidence that at least some somatic cells have a complete and functional genomecomesfrom the successfulproduction

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of cloned animals by nuclear-transfercloning. In this procedure, the nucleus of an adult (somatic) cell is introduced into an egg that lacks its nucleus; the manipulated egg, which contains the diploid number of chromosomes and is equivalentro a zygote,thenis implanted into a foster mother. The only sourceof geneticinformation to guide development of the embryo is the nuclear genome of the donor somatic cell. The frequent failure of such cloning experiments, however, raises questions about how many adult somatic cells do in fact have a complete functional genome.Even the successes,like the famous cloned sheep ,,Doll%', often have medical problems. The extent to which diffeientiated cells harbor fully functional genomesis still not fully understood. A cell could, for example, have an intact genome,but be unable to properly reactivate certain genes due to inherited chromatin states. These observationsraise two important questions:How are cell fates progressively restricted during development? Are these restrictions irreversible?In addressingthese questions, it is important to remember that a cell's capabilitiesin its normal in vivo location may differ from what it is capable of doing if manipulated experimentally. Thus the observed limits to what a cell can do may result from natural regulatory mechanismsor may reflect a failure to find conditions that reveal the cell's full potential. Although our focus in this chapter is on how cells become different, their ability to r.-"in the samealso is critical to the functioning of tissuesand the whole organism. Non-dividing differentiated cells with particular characteristics often retain these features for many decades.Stem cells that divide regularly, such as a skin stem cell, must produce one daughter cell with the properties of the parental cell, retaining its characterisric composition, shape,behavior,and responsesto specificexternal signals. Meanwhile, the other daughter cell, with its own distinct inheritance as the result of asymmetric cell division, embarks on a particular differentiation parhway, which may be determined both by the signalsthe cell receivesand by

intrinsic bias in the cell's potential, such as the previous activation of certain genes.

The Complete Cell Lineageof C. e/egansls Known In the development of some organisms,cell lineagesare under tight genetic control and thus are identical in all individuals of a species.In other organisms the exact number and arrangement of cells vary substantially among different individuals. The best-documentedexample of a reproducible pattern of cell divisions comes from the nematode C. elegans.Scientistshave traced the lineage of all the somatic cells in C. elegansfrom the fertilized egg to the mature worm by following the development of live worms using Nomarski differential interference contrast (DIC) microscopy (Figure 21 -4). About L0 rounds of cell division, or fewer, create the adult worm, which is about 1 mm long and 70 pm in diameter.The adult worm has 959 somatic cell nuclei (hermaphrodite form) or 1031 (male).The number of somatic cells is somewhat fewer than the number of nuclei because some cells contain multiple nuclei (i.e., they are syncytia). Remarkably, the pattern of cell divisions starting from a C. elegans fertllized egg is nearly always the same. As we discuss later in the chapter, many cells that are generated during development undergo programmed cell death and are missing in the adult worm. The consistency of the C. elegans cell lineage does not result entirely from each newly born cell inheriting specific information about its destiny.That is, their birth cells are not necessarily..hard wired" by their own internal inherited instructions to follow a particular path of differentiation. In some cases, various signals direct initially identical cells to different fates, and the outcomes of these signals are consistent from one animal to the next. The first few cell divisions in C. elegansproduce six different founder cells, each with a separate fate as shown in

Video:C. elegansCrawli

A FfGURE 21-4 Newly hatchedlarva of C. elegans.Someof the959somatic-cell nucleiin the hermaphrodite formare visualized in thismicrograph obtained by differential interference

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contrast(DlC)microscopy, sometimes calledNomarski microscopy. Themosteasily seenarethe intestinal nuclei, whichappearasround discs. J E Sulston andH.R Hovilz,1977, [From Devel. Biol.56:110 I

Figure 2t-5a, b. The initial division is asymmetric, giving rise to P1 and the AB founder cell. Further divisions in the P lineageform the other five founder cells. Someof the signals controlling division and fate asymmetry are known. For example, Wnt signalsfrom the P2 precursor control the asymmetric division of the EMS cell into E and MS founder cells. 'Wnt signaling (seeFigure 1.6-32)is also used in other asymmetric divisions in worms. Someof the embryonic cells function as stem cells, dividing repeatedlyto form more of themselvesor another type of precursor cell, while also generating differentiated cells that give rise to a particular tissue. The completelineageof C. elegansis shown in Figure 21-5c. This

organism has beena powerful model systemfor geneticstudies to identify the regulators that control cell lineagesin time and space.

HeterochronicMutants ProvideCluesAbout Controlof Cell Lineage Intriguing evidencefor the geneticcontrol of cell lineagehas come from isolation and analysisof heterochronic mutants. In thesemutants, a developmentalevent typical of one stage of development occurs too early (precocious development) or too late (retarded development). An example of the

of C. elegansEmbryogenesis

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AB Neurons EMS Hypodermis P h a r y n g e am l uscle E MS Bodymuscle Gut Bodymuscle P h a r y n g e am l uscle Neurons Glands P4D S o m a t i cg o n a d Germcells Other

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21-5 C. eleganslineage.(a)Patternof the firstfew A FfGURE of the to formation with P0(thezygote) andleading starting divisions (yellow isasymmetric, highlight). Thefirstdivision sixfoundercells in the P divisions producing P1andAB,a foundercell.Further generate the otherfivefoundercellsNotethatmorethan lineage or type(e 9., muscle canleadto thesametissue onelineage to it isthe precursor TheEMScellisso namedbecause neurons). with beginning The lineage and mesoderm. the endoderm mostof whicharesetaside cells, the P4cellgivesriseto allof the germ-line

Somaticgonad giveriseto All theotherlineages asin mostanimals. veryearly, of the of thefirstfew divisions cells.(b)Lightmicrographs somatic asin part thefoundercellswith cellslabeled embryothatgenerate (c)Full of organelles (a).Thetextureof the cellsshowsthe presence tissues of the some showing worm, the of body entire of the lineage few relatively cellundergoes formedNotethatanyparticular (b)fromEinhard Schierenberg fewerthan15.lPart typically divisions, K6lnl Universitet Institut, Zoologisches

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former is premature occurrenceof a cell division that yields a cell that differentiatesand a cell that dies; as a result, the Iineagethat should have followed from the dead cell never happens.An example of the latter is the delayed occurrence of a lineage,causing juvenile structures to be produced, incorrectly, in more mature animals. In both cases,the character of a parental cell is, in essence,changed to the character of a cell at a different stageof development. The study of heterochronicgeneshas been important to understandingthe mechanismsof developmentand generegulation. One example of precocious development in C. elegans comes from loss-of-function mutations in the lin-14 gene, which cause premature formation of a certain neural precursor,the PDNB neuroblasr (Figure 2t-6a). The lin-14 geneand severalothers found to be defectivein heterochronic worm mutants encode RNA-binding or DNA-binding proteins, which presumably coordinate expression of other genes. Two other genes (lin-4 and let-7\ discovered in heterochronic C. elegansmutants were initially puzzlingbecause they appearedto encodesmall RNAs that do not encodeany

protein. To discover the products of these genes,scientists first determinedwhich piecesof genomic DNA could restore gene function, and therefore proper cell lineage,to mutants defective in each gene. They then did the same thing with genomic DNA from the corresponding genomic regions of different speciesof worm. Comparison of the "rescuing" fragments from the different species revealed that they shared common short sequenceswith little protein-coding potential. The short RNA molecules encoded by lin-4 and let-7 were subsequently shown to inhibit translation of the mRNAs encoded by lin-14 and other heterochronic genes (Figure 21-6b). These small RNAs, called micro RNAs (miRNAs), are produced by RNA polymerase II and are complementary to sequencesin the 3' untranslated parts of target mRNAs. The miRNAs direct post-transcriptional silencing of mRNAs by hybridizing to them and blocking translation or stimulating degradation (see Figure 8-25). Temporal changesin the producion of lin-4, let-7, and other miRNAs during the life cycle of C. elegansserve as a regulatory clock for cell lineage.

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FIGURE 21-5 Timingof celldivisionsduringdevelopmentof RNAto the 3' untranslated (UTRs) regions of lin-14andlin-28 C. elegans.(a)Thepatternof celldivision for the V5 cellof C. elegans mRNAs prevents translation of thesemRNAs intoproteinThis isshownfor normal(wild-type) wormsandfor a heterochronic occurs following thefirstlarval(11)stage,permitting development to mutantcalledlin-14.InIhe lin-14mutant,the patternof celldivision proceed to the laterlarvalstagesStarting in thefourthlarvalstage (redarrows) thatnormally occurs onlyin thesecond larval (14),production stage(12) of let-7RNAbegins.tt hybridizes to lin-14,tin-28, occurs in thefirstlarvalstage(11),causing the pDNBneuroblast to be andlin-41mRNAs, preventing theirtranslation proteinisan LIN-41 generated prematurely. Inthemutant,theV5 cellbehaves duringL1 inhibitorof translation of thelin-29mRNA,so the appearance of /eflikecell"X" (purple) normally doesin 12.Theinference isthatthe LIN- 7 RNAallowsproduction protein, of LIN-29 whichisneeded for 14 proteinprevents L2-type celldivrsions, although precisely how it generation of adultcelllineages LIN-4mayalsobindto rn-4l RNA doesso is unknown.(b)Twosmallregulatory RNAs,/rn-4andlet-7, at laterstagesOnlythe 3' UTRs of the mRNAs aredepicted. lAdapted seryeascoordinating timersof geneexpression Bindingof thelin_4 from B J Reinhartet al , 2000, Nature403:901 I 910

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miRNAs have been identified in many other animals including vertebratesand insects.More than 300 are encoded in the human genome,perhapsas many as a thousand. Since production of miRNAs is temporally and spatially regulated, they are likely to control a broad range of events,perhapsincluding qimed events as in C. elegans.How production of theseregulatory miRNAs is temporally controlled is not yet known, but they have turned out to play many roles in regulating geneexpression(Chapter 8).

CulturedEmbryonicStem CellsCan Differentiateinto VariousCellTypes Embryonic stem (ES) cells can be isolated from early mammalian embryos and grown in culture (Figure 2'J.-7a).CuItured ES cellscan differentiateinto a wide range of cell types, either in vitro or after reinsertion into a host embryo. When grown in suspensionculture, human ES cells first differentiate into multicellular aggregates,called embryoid bodies, which resembleearly embryos in the variety of tissuesthey form. When these are subsequentlytransferred to a solid medium, they grow into confluent cell sheetscontaining a variety of differentiatedcell types including neural cellsand pigmented and non-pigmented epithelial cells (Figure 21-7b).

Under other conditions, ES cells have been induced to differ'What entiate into precursors for various types of blood cells. propertiesgive ES cells their remarkable plasticity?A variety of actors play a role: signaling proteins, DNA methylation, micro RNAs, transcription factors, and chromatin regulators can all affect which genesbecomeactive (Chapters7 and 8). During the earlieststagesof embryogenesis,as the fertilized egg begins to divide, both the paternal and maternal DNA becomes demethylated (see the discussion of DNA methylation in Chapter 7). This happens becausea key maintenancemethyltransferase(Dnmtl), which normally is present in the nucleus, is transiently excluded from the nucleus.During the first few cell divisions the pattern of methylation is reset,erasingearlier epigeneticmarking of the DNA and creating a condition where cells have greater potential for diverse pathways of development. Mice engineeredto lack Dnmtl die as early embryos with drastically undermethylated DNA. ES cells prepared from such embryos are able to divide in culture, but in contrast to normal ES cells cannot undergo in vitro differentiation. The properties of mouse ES cell are critically dependent on the action of three transcription factors produced shortly aker ferttlization: Nanog, Sox2, and Oct4. The genesthat are bound by thesefactors have been identified using chromatin

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IG < E X P E R I M E N TFA L U R2E1 - 7 Embryonicstem (ES)cellscan be maintainedin cultureand canform differentiatedcell types.(a)Human aregrownfrom cleavage-stage blastocysts produced by in vitrofertilization. embryos fromthe Theinnercellmassisseparated and tissues extra-embryonic surrounding platedontoa layerof fibroblast cellsthathelp cells cells.Individual the embryonic to nourish of EScells, andformcolonies arereolated for manygenerations whichcanbe maintained andcanbe storedfrozen.(b)In suspension into humanEScellsdifferentiate culture. calledembryoid aggregates multicellular bodies(fop) Afterembryoidbodiesare solidmedium, to a gelatinized transferred cell furtherintoconfluent theydifferentiate a varietyof differentiated sheetscontaining celltypesincludingneuralcells(middle),and cells epithelial pigmented andnonpigmented (a)and(b)adapted (bottom).[Parts fromJ S 19:193.1 Stem Ce//s etal. 2001, Odorico

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rmmunoprecipitation experiments (see Figure 7-37). Each protein is found at more than a thousand chromosomelocations. At about 350 locations, all three proteins are found. DNA microarrays have revealedwhich genesare active in ES cells.About half of the 350 loci where all three transcription factors accumulateare at or near genesthat are transcribedin ES cells.The target genesregulatedby thesetranscription factors encode a wide variety of proteins, including the Oct4, Nanog, and Sox2 proteins themselves,signalingcomponenrs of the BMP and JAICSTAT pathways, and chromatin factors. Chromatin regulators that control gene transcription (Chapter 7) are also important in ES cells. ln Drosophila, polycomb group proteins form complexes that maintain gene repressionstatesthat have been previously established by DNA-binding transcription factors. Two mammalian protein complexes related to the fly Polycomb proteins, PRC1 and PRC2, are produced in ES cells. Early mouse embryos lacking components of PRC2 have abnormal development of the inner cell mass (the embryo proper), and ES cells cannot be made from embryos lacking PRC2 functions. The PRC2 complex of proteins acts by adding methyl groups to Iysine27 of histone H3, thus altering chromatin structure to repressgenes.Remember that this type of regulation is distinct from methylation of DNA. The possibility of using stem cells therapeutically to restore or replace damaged tissueis fueling much research on how to recognize and culture these remarkable cells from embryosand from various tissuesin postnatal(adult) animals. For example, if neurons that produce the neurotransmitter dopamine could be generatedfrom stem cellsgrown in culture, it might be possibleto treat people with Parkinson'sdisease who have lost such neurons. For such an approach to succeed, a way must be found to direct a population of embryonic or other stem cells to form the right type of dopamine-producing neurons, and rejection by the immune system must be prevented. One way to prevent immune rejection is to use adult stem cells from a patient to produce therapeutic cells for that same patient. This is exactly what is done at present in some bone marrow transplants, as we shall seebelow. However it is not yet possibleto isolate adult stem cells with similar capabilities for most other tissues.In animal experiments, embryonic stem cells have proven considerablymore adept than adult stem cells at forming a variety of tissues.One approach for exploiting the advantagesof ES cells while reducing immunological rejection may be to insert a nucleusfrom a patient into the environment of an embryonic cell, replacing the endogenous nucleus with one that will confer patient-specific properties

Recent work has been directed at exploring whether embryonic or adult stem cells can be induced to differentiate into cell types that would be useful therapeutically. For example, mouse ES cells have been treated with inhibitors of phosphatidylinositol-3 kinase, a regulator in one of the 912

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phosphoinositide signaling pathways (Chapter 16). The treated EScells differentiate into cells that resemblepancreatic B cells in their production of insulin, their sensitivity to glucose levels,and their aggregationinto structuresreminiscent of pancreas structures. Implantation of these differentiated cells into diabetic mice restored their growth, weight, glucose levels, and survival rates to normal. Many important questions must be answered before the feasibility of using human stem cells for such purposes can be assessedadequately.I Apart from their possiblebenefit in treating disease,ES cells have already proven invaluable for producing mouse mutants useful in studying a wide range of diseases,developmental mechanisms, behavior, and physiology. By techniques describedin Chapter 5, it is possible to eliminate or modify the function of a specificgene in ES cells (seeFigure 5-40). Then the mutated ES cells can be employed ro produce mice with a gene knockout (seeFigure 5-41). Analysis of the effectscausedby deleting or modifying a gene in this way often provides clues about the normal function of the geneand its encodedprotein. \7e will now examine the properties and regulation of some postnatal (adult) stem cells, descendantsof ES cells, that build the various organs and tissuesin animals.

Adult Stem Cellsfor Different Animal Tissues O c c u p yS u s t a i n i n gN i c h e s Many differentiatedcell types are sloughedfrom the body or have life spans that are shorter than that of the organism. Diseaseand trauma also can lead to loss of differentiated cells. Since differentiated cells generally do not divide, they must be replenished from nearby stem-cell populations. Postnatal (adult) animals contain stem cells for many tissues including the blood, intestine, skin, ovaries and testes,muscle, and liver. Even some parts of the adult brain, where little cell division normally occurs, have a population of stem cells. In muscle and liver, stem cells are most important in healing, as relatively little cell division occurs in the adult tissuesat other times. Stem cells need the right microenvironment to maintain themselves.In addition to intrinsic regulatory signals-like the presenceof certain regulatory proteins-stem cells rely on extrinsic regulatory signals from surrounding cells to maintain their status as stem cells. The location where a stem-cellfate can be maintained is called a stem-cellniche by analogy to an ecologicalniche, which is a location that supports the existenceand competitive advantageof a particular organism. The right combination of intrinsic and extrinsic regulation, imparted by a niche, will create and sustain a population of stem cells. In order to investigate or use stem cells, they must be found and characterized. It is often difficult to identify stem cells precisely becausethey may lack distinctive shapesor gene expression.Much of the time, many stem cells do not divide particularly rapidly, being held in reserve, dividing slowly if at all, until stimulated by signals that convey the need for new cells. For example, an inadequate oxygen

supply can stimulate blood stem cellsto divide, and injury to the skin can stimulate regenerativecell division starting with the activation of stem cells.Somestem cells,however, including those that form the continuously shed epithelium of the intestine,are continuously dividing, usually at a slow rate. One approach for identifying stem cells in a mixed cell population depends on their relatively slow rate of division. In this approach, a type of pulse-chaseexperiment, cells are provided with a brief pulse of labeled DNA precursors,such as bromodeoxyuridine(BrdU), then later examined to seewhich cellsare labeled.After the BrdU pulse, cells that are not dividing will not be labeled at all, and rapidly dividing cellswill dilute the BrdU label with normal unlabeled nucleotides(the chase).Stem cells, in contrast, will incorporate BrdU during their slow division process. Since they divide relatively rarely, stem cells will retain the BrdU label longer than most other cells, marking them as label-retaining cells. This sort of label retention is often a useful way to identify stem cells.

In the fly ovary, the niche where oocyte precursors form and begin to differentiateis located next to the tip of the germarium (Figure 21-8a). There are two or three germline stem cellsin this location next to a few cap cells,which create the niche by secretingtwo transforming growth factor p (TGFp) proteins (Dpp and Gbb) and Hedgehog(Hh) protein (Figure21-8b). Thesesecretedprotein signalswere introduced in Chapter 16. Vhen the stem cells divide, they produce two daughters,one of which remains adjacentto the cap cells and is therefore a stem cell like the mother cell. The other daughter divides to produce two cystoblast cells that will differentiate into germ-line cells. The cystoblast cellsembark on a path of differentiationbecausethey are too far from the cap cells to receivethe cap cell-derived signals,including Hh, Dpp, and Gbb, and direct cell-cell ( a ) S t e mc e l l sa n dn i c h e si n f l y g e r m a r i u m Inner sheath

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Germ-line Stem Cells The germ line is the line of cells that produces oocytes and sperm. It is distinct from the somatic cells that make all the other tissuesbut are not passed on to progeny.The germ line, like somatic-celllineages,has stem cells. Stem-cellnicheshave been especiallywell defined in studiesof germ-linestemcellsinDrosophila and C. elegans. Germ-line stem cells are presentin adult flies and worms and the location of the stem cells is well known. The stem cells were identified by BrdU label retention.

> FfGURE21-8 Drosophilagermariumand the signalsthat of the germarium createits stem-cellniches.(a)Cross-section stem stemcells(yellow) andsomesomatic showingfemalegerm-line fromthem andthe progeny cellsderived cells(gold)in theirniches (green), which stemcellsproduce cytoblasts Thegerm-line folliclecells intooocytes; thesomatic stemcellsproduce differentiate (brown)thatwill maketheeggshellThecapcells(darkgreen)create a n dm a i n t a itnh en i c h ef o r g e r m - l i nset e mc e l l sw, h i l et h ei n n e r stemcells(b) the nichefor somatic sheathcells(blue)produce pathways of germ-line stem thatcontrolthe properties Signaling molecules-the TGFBproteins DppandGppas cellsThesignaling of theseligands to by capcellsBinding wellasHh- areproduced of the on the surfaceof a stemcellresultsin repression receptors MadandMed Repression of factors, bamgenebytwo transcription of bam activation stemcellsto renew,whereas barnallowsgerm-line promotes proteins, ArmandZpg,which differentiation Twosurface physically in linkcapcellsandstemcells,arealsoimportant nicheCellsout of reachof Arm andZpg maintaining thestem-cell ratherthanrenewMicroRNAsareincreasingly differentiate including ascritical regulators of celldifferentiation, recognized germ-line an essential cellsSomeof thembindto the Piwiprotein, pathways germ-line regulator in bothcapandstemcells(c)Signaling of somatic stemcells.TheWnt signal thatcontrolthe properties (Wg)isproduced by the innersheathcellsandis received Wingless (Fz)on a somatic receptor stemcell Hh issimilarly bythe Frizzled signal produced, andisreceived by the PtcreceptorBothreceptors stemcells resulting in self-renewal of somatic to controltranscription CellDevelBiol21:605 fromL LiandT.Xie,2005,Ann Rev. I lAdapted

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Germ-line stem cell lnner Cap cell . S e c r e t e sH h s i g n a l , . R e c e i v e sH h t h r o u g h s h e a t h c e l l . Secretestwo TGF0 Ptc receptor,promoting . Secretestwo s i g n a l s ,W g s i g n a l sD , p p& G b b . self-renewal. . R e c e i v e sD p p a n d . P r o d u c e sA r m andHh. . Produces G b b t h r o u g hT G F B and Zpg surface r e c e p t o rs u b u n i t sI a n d Arm protein proteins. . H a sP l W l p r o t e i n ll, promoting on its self-renewal. surface. in the nucleus. . TGFFprotein c a u s e signals activationof Mad and Med transcription factors to repress bam gene and allow self-renewal. . P r o d u c e sA r m a n d Z p g surface proteins,which interactwith themselves on the cap cell. . H a s P l W lp r o t e i ni n t h e nucleusto promote stem cell fate.

Somatic stem cell . ReceivesWg signals through the Fz receptors, promoting self-renewal. . R e c e i v e sH h s i g n a lt h r o u g h the Ptc receptor, Promotlng self-renewal. . P r o d u c sA r m , which lnteracts with Arm on i n n e rs h e a t h cell.

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interactions mediated by the cell-surfaceproteins Arm and Zpg, which together direct a cell to remain a stem cell. Both cap cells and germ-line stem cells produce Piwi proteins, which bind micro RNAs. Piwi proteins and their bound miRNAs regulate gene expression and control germ-line cell development in a wide variety of animals as well as stem-celldevelopmentin plants. Thus they consti, tute an ancient mechanism of developmentalregulation. Separatesomatic stem cellsin the germarium produce follicle cellsthat will make the eggshell.The somatic stem cells have a niche too, created by the inner sheath cells, which produce Wingless(Wg) protein-a fly Wnt signal-and Hh protein (Figure 2l-8c). Thus two different populations of stem cells can work in close coordination to produce different parts of an egg. Micro RNAs control the division properries of Drosophila female germ-line stem cells. The Dicer prorein, a doublestranded RNase, produces micro RNAs (seeFigure 8-25). Germ-line stem cells with dicer mutations fail to pass successfullythrough the G1 to S transition of the cell cycle; as a result, the population of stem cells and therefore oocytes is depleted.The absenceof miRNA function causes,directly or indirectly, increasedfunction of the p21127cyclin-dependent kinaseinhibitor. As discussedin Chapter 20, p21127normally restricts G1 to S transitions by regulating cyclin E-CDK complexes.Thus the net effect of the absent miRNA function, which permits increasedp21,127activity, is to restrict cell division. In worms the long tubelike arms of the gonads have tips where a cell called a distal tip cell crearesa stem-cellniche (Figure21-9).The transmembraneprotein Delta, produced by the distal tip cell, binds to the Notch receptor on the

Distal tip cell

Germ-line stem cell

Mitotic stem cell

Meiotic stem cell

FfGURE 21-9 C. elegansgerm-linestem-cellniche.A crosssection of thetip of a gonadarmshowsstemcellsin theirnicheand progeny derived fromthem Thesingledistaltip cell(green) in each gonadarmcreates andmaintains the nrcheSelf-renewing mitotic stemcellsproduced bythe germ-line stemcellsconvert to meiosis whentheymovebeyondthe rangeof the Deltasignalfromthedistal tip cell.Duringthesestages, thecellsareonlypartially separated by ("Y" shapes) membranes andaretherefore a syncytium lAdapted from L Li and T. Xie, 2005, Ann Rev.Cell Devel.Biot.21:605l

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germ-line stem cells. The DeltaA{otch signaling pathway (see Figure 1,6-36) promotes mitotic division of worm germ-line stem cells, thus creating more stem cells. Meiosis (i.e., germ-line differentiation) is blocked by the Delta signal until the stem cells move beyond the range of the signal from the distal tip cell. Mutations that activare Notch in the germ-linestemcells,evenin the absenceof Delta signal, cause a gonadal tumor with massive numbers of extra germ-line stem cells, due to excessivemitosis and little merosls. The identification and characterization of Drosophila and C. elegansgerm-line stem cells were important because they showed convincingly the existenceof stem-cell niches and permitted experiments to identify the niche-made signals that causecells to become and remain stem cells. Thus a stem-cell niche is a set of cells and the signals they produce, not just a location. Identification of specificmolecules that maintain the stem-cell state in Drosophila and C. elegans brought an unexpected bonus: Some of these molecules are also used to form a stem-cell niche and control stem-cell fates in mammals. For example, germ-line stem cells in the mouse testis are dependenton a TGFB signaling protein (GDNF) derived from somatic cells. Each seminiferous tubule contains exactly one germ-line stem cell, which divides asymmetrically to re-create itself and to produce a spermatogonial cell. This cell proliferates and its progeny becomespermatocytes,which go through the extraordinary differentiation processthat builds a sperm. The niche is created by a specializedregion of the Sertoli cell along with a myoid cell and a basementmembrane produced by the myoid cell, though many details of the molecular signals remain to be explored. Skin/Hair Stem Cells in Mammals Epithelial stem cells that give rise to skin and hair in mammals are located in hair follicles and in the basal layer of the epithelium between follicles. In the hair follicle, the stem cells occupy a niche called the bulge (Figure 21-10a). The stem cells divide asymmetrically to produce more stem cells and to make precursor cells of at least two kinds. One type of precursor will rise toward the surface of the skin and form keratinocytes, the major cell type of skin, which is a multilayered epithelium (the epidermis). Other cells emergefrom the stem cells to become hair-matrix progenitors that move down deeper in the hair follicle and form a complex set of structures including the hair itself. The molecular regulatorsin the skin stem-cellniche are incompletely known. However, as in flies, TGFB signals, arising from mesenchymalcells that surround the bulge cells, and a Wnt signal, arising from the dermal papilla, are important in controlling stem-cell renewal and differentiation into either skin or hair (Figure 21-10b). Evidence '$fnt for the importance of signaling came from manipulations of the expression of B-catenin, a protein that helps link certain cell-cell junctions to the cytoskeleton (see Figure 1,9-12)and also functions as a signal transducerin the

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FIGURE 21-10Skin/hairstem-cell nichein mammalsand the signalsthat controlit. (a)A hairfollicle showing stemcells(yellow) in thebulgeandinterfollicular stemcells(lightgreen) amongthebasal epithelial cells(green) outside thehairfollicle. Theprogeny of bulge stemcellsmigrate downto contribute to hairformation nearthe dermalpapillaThedarkgreencellshaveyetto beginterminal differentiation; theyaretransient amplifying cellsthatarestilldividing but cannotgenerate stemcells.Notethatonlythe basallayerof epithelial cellsadjacent to thebasement membrane areshown. Overlaying thesebasalcells,outsideof thefollicle,areseveral layers (b)Signaling of differentiating keratinocytes. eventsin the stem-cell nicheThesource of eachsignal isindicated in parentheses. A Wnt signalpromotes formation of newhaircells, but in thebulge-home (Dkk.Wif. andsFRP) of the stemcells-at leastthreeWnt inhibitors blockdifferentiation andpreserve the stem-cell stateAwayfromthose inhibitors, the lackof Wnt signaling allowsdifferentiation of stemcells intoskincells(keratinocytes). BMBwhichbelongs to theTGFB family proteins, isproduced of signaling by mesenchymal cellsadjacent to the bulge.Duringdevelopment, whennewhairisto begrown,thedermal papilla makesNoggin, whichblocks the BMPsignal, andmoreWnt, whichovercomes the inhibitors, allowingstemcellsto differentiate into haircells. BMPs havecomplex andincompletely understood rolesin skindevelopment, andtheirfunctions change withthestageof development fromF.M Wattetal,2006,CuffOpinGenetDevel. [Adapted 16:51 8,andL LiandI Xie,2005, Ann,ReuCellDevel. Biol.21:605 l

'!fnt

pathway (seeFigure L6-32). Activation of B-catenin changesthe fate of cells from epidermis (skin) to hair. In contrast, removal of B-cateninfrom the skin of engineered mice eliminates formation of hair cells. Epithelial stem cells then form only epidermis, not hair cells. Thus B-catenin acts as a switch that controls which type of precursor arises from epithelial stem cells. \fnt signals also have stimulatory effects on cell division, which can be

restrained by'Sfnt pathway inhibitors, such as DKK and sFRP,that are present in the bulge-a Battle of the Bulge of sorts. Newly formed keratinocytes move toward the outer surface, becoming increasingly flattened and filled with keratin intermediate filaments (Chapter 18). It normally takes about 15-30 days for a newly "born" keratinocyte in the lowest Iayer of the skin to differentiate and move to the topmost layer. The "cells" forming the topmost layer are actually dead and are continually shed from the surface. In addition to keratinocytes, skin contains dendritic epidermal T cells, an immune-system cell that produces a certain form of the T-cell receptor (Chapter 24). Ifhen dendritic epidermal T cells are geneticallymodified so they do not produce T-cell receptors,wound healing is slow and lesscomplete than in normal skin. Normal healing is restored by addition of keratinocyte growth factor. The current hypothesisis that when dendritic epidermal T cells recognizeantigenson cells in damaged tissue,they respond by producing stimulating proteins, such as keratinocyte growth factor, that promote the production of more keratinocytes and wound healing. Many other signals-including Hedgehog, calcium, and transforming growth factor a (TGFct)control the production of skin cells from stem cells. Discovering how all these signalswork together to control growth and stimulate healing will advanceour understandingof diseasessuch as psoriasisand skin cancer and perhapspave the way for effectivetreatments.I Intestinal Stem Cells In contrast to epidermis,the epithelium lining the small intestine is a single cell thick (see Figure 19-8). This thin layer is enormously important for keeping toxins and pathogens from entering our bodies; it also transports nutrients essentialfor survival from the intestinal lumen into the body (seeFigure 11-29). The cells of the intestinal epithelium continuously regenerate from a stem-cell population located deep in the intestinal wall in By identifying the Iabelpits called crypts (Figure 21,-1,1'a). retaining cells in the intestinal epithelium, researchersdetermined that the stem cells are located precisely four or five cells above the bottom of a crypt. The niche is created at the level of the by mesenchymalcells that abut the crypts 'Wnt signal, a BMP stem cells. These cells produce a (TGFB) signal, and possibly a ligand for the Notch receptor on stem cells (Figure 21-1'1b). Overproduction of Bcatenin in intestinal cells leads to excess proliferation of 'Sfnt those cells, as though they were receiving too much signal (which stabilizesB-catenin).Blocking the function of B-catenin by interfering with the TCF transcription factor that it activates abolishes the stem cells in the intestine. Thus $fnt signaling, acting through p-catenin, plays a critical role in maintaining the intestinal stem-cell population. BMP has the opposite effect, promoting differentiation and restraining the effect of Wnt.

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Podcast:Stem Cellsin the lntestinal Epithelium (b) Directionof cell migration

Proliferationzone (c) Intestinalstem cell signalingpathways

I Mesenchymalcells

Intestinal stem cells produce precursor cells that proliferate and differentiate as they ascendthe sides of crypts to form the surface layer of the finger-like gut projections calleduilli, acrosswhich intestinal absorption occurs. Pulsechaselabeling experiments with BrdU have shown that the time from cell birth in the crypts to the loss of cells at the tip of the villi is only about 2 to 3 days. Thus enormous numbers of cells must be produced continually to keep the epithelium intact. The production of new cells is preciselycontrolled: too little division would eliminate villi and lead to breakdown of the intestinal surface; roo much division would create an excessivelylarge epithelium and also might be a step toward cancer.Indeed, mutations that inappropriately activate Wnt signal transduction are a major contributor to the progression of colon cancer, as we shall see in Chapter 25. Neural Stem Cells The great interestin the formation of the nervous system and in finding better ways to prevent or treat neurodegenerativediseaseshas made the characterization of neural stem cells an important goal. The earliest stagesof vertebrate neural development involve the rolling up of ectoderm to form the neural tube, which extends the length of the embryo from head to tail (Figure 2l-12a).

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< FIGURE 21-11 lntestinalstem-cellnicheand projections the signalsthat control it. (a)Finger-like of the innersurface of the intestine calledvilliare dividedby deeppitscalledcryptsThesingle-cell thick protects epithelium usfrominfection andallows selective of usefulnutrients intothe blood transoort (b)An intestinal stream. cryptshowingthe intestinal stemcells(yellow), theirproliferating mitoticprogeny (lightgreen), (darkgreen)in thefinal andprecursors stagesof differentiation Mesenchymal cells,shownin blue,createthisstem-cell niche.Paneth cells(orange), locatedat the baseof the crypt,secrete antimicrobial defenseproteinscalleddefensins. Theseproteinsform poresin bacterial membranes, leading to deathof the bacteria. Signaling eventsin thestem-cell niche.Wnt promotestem-cell signals fates,offsetting BMPsignals thatpushtowarddifferentiation. Nogginrestrains the proliferation, BMPsignals andthuspromotes stem-cell whereas Dkkrestrains Wnt signaling whengrowthis not needed.TheNotchreceptorisalsoinvolved, althoughitsligandon mesenchymal cellsisunknown. fromL LiandT.Xie,2005,Ann.ReuCellDevel lAdapted Biol.21:6051

Initially the neural tube is composed of a single layer of cells, the neural stem cells (NSCs); these will give rise to the entire central nervous system (brain and spinal cord). Labeling and tracing experimentshave shown where neural cells are born and where they go after birth. The most active region of cell division is the subuentricular zone, which has the properties of a stem-cellniche and is named for its proximity to the central fluid-filled uentricle. The embryonic neural stem cells that line the ventricle divide can symmetrically, produce two daughter stem cells side-by-side (Figure 21.-1.2b),or asymmetrically, produce a cell that remains a stem cell and another that migrates radially outward. The migrating cells are often transient amplifying (TA) cells, which in turn divide to form neural precursors called neuroblasts. Once formed, TA cells and neuroblasts migrate radially outward and form successive layers of the neural tissue in an inside-out order, whereas the stem cells remain in contact with the ventricle (seeFigure 21-12b). Newly formed cells therefore traverse the layers of preexisting cells before taking up residenceon the outside. Tracing experiments with viruses have shown that a neuroblast can produce two daughters,one a neuron and one a glial cell. In these experiments, altbrary of defective

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< FfGURE21-12 Formationof the neuraltube and division a portionof the of neuralstem cells.(a)Earlyin development, fromthe restof the embryonic ectodermrollsup and separates c e l l sT h i sf o r m st h e e p i d e r m (i sg r a ya) n dt h e n e u r atlu b e( b l u e ) betweenthe two, neuralcrestcellsform and Nearthe interface t o c o n t r i b u tteo s k i np i g m e n t a t i onne, r v ef o r m a t i o n , t h e nm i g r a t e c r a n i o f a c si akle l e t o nh,e a r vt a l v e sp,e r i p h e r na el u r o n sa,n do t h e r for whichwe are a rodof mesoderm Thenotochord, structures provides that affectcellfatesin the signals named(chordates), n e u r atlu b e( C h a p t e2r2 ) f h e i n t e r i oor f t h e n e u r atlu b ew i l l v e n t r i c l eN s eural become a s e r i eos f f l u i d - f i l l ecdh a m b e rcsa l l e d d d j a c e nt ot t h ev e n t r i c l ei ns t h es u b v e n t r i c u l a r s t e mc e l l sl o c a t e a r a d i a l loyu t w a r dtso z o n ew i l ld i v i d et o f o r mn e u r o ntsh a t m i g r a t e (b) Neural stemcellsin the system. layers of the nervous form the (top),alongtheir zonecandividesymmetrically subventricular a p i c a l - b a saaxli st,o g i v er i s et o s i d e - b y - s iddaeu g h t esrt e mc e l l s , stemcellscan Alternatively, both in contactwith the ventricle. e n ed a u g h t etrh a ti sa d i v i d ea, l o n gt h e i ro t h e ra x i st,o p r o d u c o s t e mc e l la b l et o s e l f - r e n eawn da d a u g h t ecre l l c, a l l e da t r a n s i e n t a n dd i f f e r e n t i a t e a m p l i f y i n(gT A c) e l l t, h a tb e g i n tso m i g r a t e is (bottom)A keydifference betweenthe two patternsof division fromL R wolpert of the mitoticspindle[Adapted the orientation Press 2ded, Oxford University of Development, l et al 2OO2Principles

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retroviruses,each able to infect only once and containing a unique DNA sequence,was prepared (Figure 21-13a). Any cell infected by a singlevirion would give rise to a clone of cells that all carry that particular virus' DNA sequence.In this way, all the cellsthat derivedfrom a singleneural stem cell or TA cell can be identified as a clone (Figure 21-13b). T h e r e s u l r so f t h e s et r a c i n g e x p e r i m e n t sw e r e s u r p r i s i n g . First, some neuronswere found to have migrated considerable distanceslaterallS abandoning their radial migration to the outer cortical layer. Second,in some cases,a single neuron and singleglial cell, sharedthe sameviral DNA sequence.A neural precursorhad evidentlybeeninfectedand had then divided once to give rise to two quite different cell types. Most mammalian brain cells stop dividing by adulthood but some cells in the subventricularzone and at least one other part of the brain continue to act as stem cells and generatenew neurons (Figure 21,-1,4).In the subventricular zone of adults, the neural stem cells are astrocytes, a somewhat confusing nomenclature since more

traditionally "astrocyte" meant atype of glia cell. Evidently neural stem cells are a subsetof astrocytesnot previously r e c o g n i z e df o r t h e i r s p e c i a l s t e m - c e l l q u a l i t i e s . N e u r a l stem cells have some properties of astrocytes,such as producing glial fibrillary acidic protein (GFAP), but they also can divide asymmetrically to renew themselves and to produce TA cells. The subventricular stem-cell niche is created by mostly unknown signals from the ependymal cells that form a layer just inside the neural tube (lining the ventricle) and by endothelial cells that form blood vesselsin the vicinity (Figure 21-14b). The endothelial cells, and the basal lamina they form, are in direct contact with neural stem cells and are believed to be essentialin forming the niche. Each neural stem cell extends a single cilium through the ependymal cell layer to directly contact the ventricle. Though the function of the cilium is unknown, it may function as an antenna for receiving signals that would otherwise be inaccessibleto the neural stem cell. The signalsthat create the niche are not completely characterized,but there is evidencefor a blend of factors including FGFs, BMPs, IGR VEGR TGFo, and BDNF. The BMPs appear to favor astrocyte differentiatron over neural differentiation, and over-expressionof IGF (insulinlike growth factor) causesmice to develop with abnormally large brains. Later stagesof neural developmentare d i s c u s s e di n C h a p t e r 2 3 . Hematopoietic Stem Cells Like the intestinalepithelium the blood is a continuously replenishedtissue.The stem cells

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(a) Crosssectionof whole developingbrain

Distinctive oligonucleotide sequence

(b)

A EXPERIMENTAL FIGURE 21-13 Retrovirusinfectioncan be usedto tracecelllineage.(a)Engineered viralgenome. Thelong (LTRs) terminalrepeats arestandard retroviral repeats. Viralproteins required for infection areencoded in thegagandin A-envgenes PLAP isan introduced genefor an alkaline phosphatase Detection of thisenzyme by histochemical staining isusedto determine which cellscarrya virusTheoligonucleotide sequence, synthesized by providing randomnucleotides, isdifferent in eachvirusandcanbe amplified by PCR,usingprimers for sequences thatarein allviruses (purplearrows), andthensequenced A library of morethan107 distinct viruses wasmade Because theseviruses lackthegenes required for production of newvirionsin infected cells,each defective viruscaninfectonlyonce.(b)Tissue section showingcells infected with defective virusesTheDNAfromeachstained cloneof cellscanbe extracted andamplified by PCRto determine the sequence of the infecting virus.Cellsdescended fromthesame initially infected cellwill havethe sameoligonucleotide sequence, whereas separate infection events willgivedifferent sequences. [From J A G o l d e ne t a l , 1 9 9 5 ,P r o c N a t ' l .A c a d S c i U S A 9 2 : 5 7 0 4 1

that give rise to the different types of blood cells are located in the bone marrow in adult animals. All types of blood cells derive from a single type of pluripotent hematopoietic stem cell, which givesrise to the more-restrictedmyeloid and lympboid stem cells (Figure 21-15). Although myeloid and lymphoid stem cells are capable of self-renewal, each type is committed to one of the two major hematopoietic lineages. Thus these cells function as both stem cells and precursor c el l s . After hematopoietic stem cells form, numerous extracellular growth factors called cytokines regulateproliferation and differentiation of the precursor cells for various blood-cell lineages.Each branch of the blood-cell lineage tree has different cytokine regulators, allowing exquisite control of the production of specificcellstypes. For example, after a bleeding injury, when all blood cells are needed, multiple cytokines are produced. But when a 918

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Ependymal cells Neuroblast Transit-amplifying c el l s Lateral ventricle

NSC

Expansion Neuroblast

A FIGURE 21-14 Neuralstem-cellniche.(a)Cross-section of the developing nervous system showing ventricle, the lateral a fluid{illed spaceinsidethe neuraltube Theareajustsurrounding theventricle, calledthe subventricular zone,isthesiteof stemcellsfromwhich neuralprecursors arise(b)Theneuralstemcells(yellow), a subset of (blue)andadjacent astrocytes, arein contactwith bloodvessels ependymal cells(pink),bothof whichprovide signals or direct populationNeuralstemcells contacts thatmaintain the stem-cell (NSC) divideto renewthemselves population andto forma dividing (TA)cells(lightgreen;seeFigure21-12b).IA of transitamplifying (darkgreen), cellsin turndivideto formneuroblasts whichgiverise to neuronslAdapted fromL LiandT.Xie,2005,Ann Rev. CellDevelBiol 2 1 : 6 0l 5

person is traveling at high altitude and needs additional erythrocytes,erythropoietin-a cytokine that acts only on erythrocyte precursors-is produced. Erythropoietin activates several different intracellular signal-transduction pathways, leading to changes in gene expression that p r o m o t e f o r m a t i o n o f e r y t h r o c y t e s( s e eF i g u r e 1 6 - 6 ) . I n contrast GM-CSR a different cytokine, stimulates production of granulocytes, macrophages,eosinophils, and megakaryocytes.

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A FIGURE 21-15 Formationof blood cellsfrom hematopoieticstem cellsin the bone marrow.Pluripotent stem (bluecurved cells(yellow) maydividesymmetrically to self-renew arrow)or divideasymmetrically to forma myeloidor lymphoid stem t r e e na) n da d a u g h t ecre l lt h a ti sp l u r i p o t e nl i tk et h e c e l l( l i g h g parental cell Althoughmyeloidand lymphoid stemcellsare capable of self-renewal, eachtypeiscommitted to oneof the two majorhematopoietic lineagesDepending on the typesand present, amountsof cytokines the myeloidand lymphoid stemcells generate differentprecursor whichareincapable cells(darkgreen), of self-renewal. Precursor cellsarecalledcolony-forming cells (CFCs) because of theirabilityto form colonies containing the differentiated celltypesshownat right Thecolonies aredetected

that havehadtheirown hematopoietic on the spleenof animals r l l si n t r o d u c eF du r t h e r ce c e l l se l i m i n a t eadn dt h e p r e c u r s o proliferation, anddifferentiation commitment, cytokine-induced typesof bloodcells cellsgiveriseto the various of the precursor (red process areindicated this that support the cytokines Someof E: Eo : eosinophil; labels). GM : granulocyte-macrophage; B : B-cell; CFU= T : T-cell; mega: megakaryocyte; erythrocyte; : : factor;lL unit;CSF colony-stimulating colony-forming Tpo : SCF: stemcellfactor;Epo: erythropoietin; interleukin; factor;TGF: transforming TNF: tumornecrosis thrombopoietin; ligand: factor;FLT-3 growthfactor;SDF: stromalcell-derived fromM 3. [Adapted kinasereceptor tyrosine ligandfor fms-like ProcNat'lAcadSciUSA95:6573]1 1998, Socolovskyetal,

The hematopoietic lineageoriginally was worked out by injecting the various types of precursor cells into mice whose precursor cells had beenwiped out by irradiation. By observing which blood cells were restored in these transplant experiments,researcherscould infer which precursors (e.g., GM-CFC, BFU-E) or terminally differentiated cells (e.g., erythrocytes, monocytes) arise from a particular type of precursor. RemarkablS a single hematopoietic stem cell is sufficient to restore the entire blood system of an irradiated mouse. The first step in theseexperimentswas to separatethe different types of hematopoietic precursors.

This separationis possiblebecauseeach type of precursor produces unique combinations of cell-surface proteins that can serve as type-specific markers. If bone marrow extracts are treated with fluorochrome-labeledantibodies for these markers, cells with different surface markers can be separated in a fluorescence-activatedcell sorter (see F i g u r e9 - 2 8 ) . The frequency of hematopoietic stem cells is about 1 cell per 104 bone marrow cells.Activation of the Hoxb4 gene in embryonic stem cells drives the formation of hematopoietic stem cells. (As describedin Chapter 22, Hoxb4 also plays a C E L L SN, l C H E SA, N D L I N E A G E T H E B I R T HO F C E L L S : s T E M

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role in pattern formation along the head-to-tail body axis.) The Bmi geneis also required for self-renewalof hematopoietic stem cells, and of neural stem cells as well. This geneencodes a polycomb-type chromatin regulator protein that repressescertain genesincluding some Hox genes,a group of developmentally important genes discussedin Chapter 22. Bmil is a component of the PRC1 protein complex that we discussedabove in relation to embryonic stem cells. Thus membersof the polycomb group of proteins are important in both embryonic and adult stem cells. Like other stem cells, hematopoietic stem cells are residents of a niche. The niche is formed by spindle-shapedcells on the surface of the bone in the bone marrow. N-cadherin fastensthe stem cells to theseniche cells.A Delta-like ligand produced by niche cells signals to Notch receptors on the stem cells, and several other growth factor-receptor pairs stimulate their self-renewaland differentiation into mveloid or lymohoid stemcells. Stem cells can become cancerous. For example, Ieukemia is a cancer of the white blood cells. This type of canceris marked by two types of cells:leukemictumor cells, which arise from differentiated white blood cells and have limited growth abilities,and the more dangerous leukemic tumor stem cells with unlimited growth abilities. The leukemia tumor stem cells,which are capableof initiating a new tumor on their own, are present in a human tumor about once for every million dividing leukemic cells. Thus the bulk of the leukemic cells are not able to grow a new tumor. Therefore to provide an enduring cure, treatments must ensure the death or limited mitosis of the tumor stem cells. This is particularly challenging because many cancer stem cells divide slowly or not at all for substantial periods, making them selectively resistant to chemotherapy drugs and irradiation-both treatments that target rapidly dividing cells. To date, bone marrow transplants, a treatment for leukemia and other blood disorders, represent the most successfuland widespreaduse of stem cells in medicine.In 1959 a patient with end-stage(fatal) leukemia was irradiated to destroy her cancer cells. She was transfusedwith bone marrow cells from her identical twin sister, thus avoiding an immune response,and was in remission for three months. This promising beginningled to present-day treatments that can often lead to a complete cure for Ieukemia. The stem cells in transplanted bone marrow can generate neq functional blood cells in patients with certain hereditary blood diseasesand in cancerparientswho have received irradiation andlor chemotherapy. Both chemotherapy and irradiation destroy the bone marrow cells as well as cancer cells. Bone marrow transplants go beyond eliminating cancer cells as done with irradiation. Instead.an immune attack on the leukemic cells can be mounted by the injected donor cells. More than two dozen diseasesare now commonly treated with bone marrow transplantation. These include

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leukemias and anemiasof various types, lymphomas, severe combined immunodeficiency, and certain autoimmune disorders. The effectivenessof bone marrow transplants varies among diseasesand patients from minor improvement to full cures.Much researchis under way to employ other types of stem cells in the treatment of diseasesinvolving nonblood tissues.I You have probably noticed that all the molecular regulators of stem cells are familiar signal proteins rather than dedicated regulators that specialize in stem-cell control. Each type of signal is usedrepeatedlyto control cell fates and proliferation. Theseare ancient signaling systems,at least a half billion years old, for which new useshave emergedas cells, tissues,organs, and animals have evolved new variations. \7e will seemany more examplesof such evolutionary conservation in the next chapter on development.

MeristemsAre Nichesfor Stem Cellsin PostnatalPlants Stem cells in plants are located in meristems,populations of undifferentiatedcells found at the tips of growing shoots. Shoot apical meristems (SAMs) produce leaves and shoots as well as more stem cells that constitute the nearly immortal meristems.Meristems can persist for thousands of years in long-lived speciessuch as redwood trees and bristlecone pines. As a plant grows, the cells "left behind" the meristemsare encasedin rigid cell walls and can no longer grow. SAMs can split to form branches, each branch with its own SAM, or be converted into floral meristems (Figure 21-16). Floral meristemsgive rise ro the four floral organs-sepals, stamens, carpels, and petals-that form flowers. Unlike SAMs, floral meristems are gradually depletedas they give rise to the floral organs. A meristem is a stem-cellniche, but much remains to be learned about how the niche is created and maintained. Numerous genes have been found to regulate the formation, maintenance, and properties of meristems. Many of thesegenesencodetranscription factors that direct progeny of stem cells down different paths of differentiation. For instance,a hierarchy of regulators, particularly transcription factors, controls the separation of differentiating cells from SAMs as leaves form; similarlS three types of regulators control formation of the floral organs from floral meristems (seeFigure 22-36). In both cases,a cascadeof gene interactions occurs, with earlier transcription factors causing production of later ones. At the same time, cells are dividing and the differentiating ones are spreading away from their original birth sites. One signal rhat creates the plant niche is ZwillelPinhead, which encodesa protein related to the Piwi protein that supports stem-cell niches in animals (seeFigure 21-8). These are "argonaute" family proteins, which repress genes in response to certain small RNA molecules.I

(a) Regionsof shoot apical and floral meristems

(b) Fatesof cells in L2 layer

but may have different fates if they are exposed to different external signals (seeFigure 21-1). r Pluripotent stem cellscan produce more than one type of descendantcell, including in some casesa stem cell with a more-restricted potential to produce differentiated cell types. r Embryonic developmentof C. elegansbeginswith asymmetric division of the fertilized egg (zygote).The lineageof all the cells in adult worms is known and is highly reproducible (seeFigure21-5). r Short regulatory RNAs (micro RNAs) control the timing of developmentalcell divisions by preventing translation of mRNAs whose encoded proteins control cell lineages(see Figure2L-5). r Cultured embryonic stem cells (ES cells) are capable of giving rise to many kinds of differentiated cell types. They are useful in production of geneticallyaltered mice and offer potential for therapeuticuses.Specifictranscription factors and chromatin regulators are important in giving ES cellstheir unusualproperties. r Stem cells are formed in niches that provide signals to maintain a population of nondifferentiating stem cells.The niche must maintain stem cells without allowing their excessproliferation and must block differentiation. r Germline cells give rise to eggsor sperm. By definition' all other cells are somatic cells.

(a)In A FIGURE 21-16 Cellfates in meristemsoI Arabidopsrs. sections, with theselongitudinal cellnucleiarerevealed by staining propidium iodide,whichbindsto DNA.Iop:Theshootapical (SAM)produces meristem shoots,leaves, and moremeristem. Flower production occurswhenthe meristem from leaf/shoot swrtches production to flowerproduction, with an increase in the concomitant (FMs), numberof meristem cellsto formfloralmeristems asshown here Middle:Cellsin a SAMexhibitdifferentfatesand behaviors. zone(PZ,green)to produce Cellsdividerapidlyin the peripheral leaves andin the ribzone(Rib,blue)to produce centralshoot structures. Cellsin thecentralzone(CZ,red)dividemoreslowly, producing an ongoingsource of meristem andcontributing cellsto Thelayers coloredblue, the PZandRib.Eottorn: of the meristem, (cloned) pinkandyellowhere,areeachderived fromthe same precursor cell.Scale bars,50 pm (b)Colorcodingshowsthefatesof positions cellsin different in the L2 layer. Thecolorcodedoesnot to that in part(a).[Part(a)fromE Meyerowitz, 1997, Cell correspond micrographs courtesy of ElliotMeyerowitz Part(b)afterC Wolpert 88:299; of Development,2d University Press et al, 2002,Principles ed, Oxford l

r Populations of stem cells associatedwith the gonads, skin, intestinal epithelium and most other tissuesregenerate differentiated tissuecells that are damagedor sloughed or becomeaged (seeFigures21'-8-21-11',21,-14)' r In the blood cell lineage, different precursor types form and proliferate under the control of distinct cytokines (see Figure 21-1.5).This allows the body to specifically induce the replenishmentof some, or all, of the necessarytypes. r Stem cells are prevented from differentiating by specific controls that operatein the niche. A high level of B-catenin, a component of the tJfnt signaling pathway' has been implicated in preservingstem cells in the skin and intestine by directing cells toward division rather than differentiation states. r Plant stem cells persist for the life of the plant in the meristem. Meristem cells can give rise to a broad spectrum of cell types and structures(seeFigure 21-1'61'

Specificationin Yeast Cell-Type The Birth of Cells:Stem Cells,Niches,and Lineage r In asymmetriccell division, two different types of daughter cells are formed from one mother cell. In contrast, both daughter cells formed in symmetric divisions are identical

In the previous section, we saw that stem cells and precursor/progenitor cells produce progeny that embark upon specific differentiation paths. The elegant regulatory mechanisms of differentiation are referred to as cell-type specification Specification usually involves a combination

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of external signalswith internal signal-transductionmechanisms like those describedin Chapters 15 and 15. The transition from an undifferentiated cell to a differentiating one often involves the production of one or a small number of transcription factors. The newly produced transcription factors are powerful switches that trigger the activation (and sometimesrepression)of large batteriesof subservientgenes. Thus an initially modest change can causemassivechanges in geneexpressionthat confer a new character on the cell. Our first example of cell-type specification comes from the budding yeast, S. cereuisiae.We introduced this useful unicellular eukaryote way back in Chapter 1 and have encountered it in several other chapters. S. cereuisiaeforms three cell types: haploid a and ct cells, and diploid a/ct cells. Each type has its own distinctive set of active genes;many other genesare active in all three cell types.In a parrern common to many organisms and tissues,cell-type specification in yeast is controlled by a small number of transcription factors that coordinate the activities of many other genes.Similar regulatory featuresare found in the responsesof higher eukaryotic cells to environmental signals and in the specification and patterning of cells and tissues during development (Chapter 22). DNA microarray studies have provided a genome-wide picture of the fluctuations of geneexpressionin the different cell types and different stagesof the S. cereuisiaelife cycle (seeFigure 5-29 for an explanation of the DNA microarray technique). Among other things, these studies identified 32 genesthat are transcribedat more than twofold higher levels in o. cells than in a cells.Another 50 genesare transcribed at more than twofold higher levelsin a cells than in crcells.The products of these 82 genes,which initially are activated by cell-type specificationtranscription regulators,convey many of the critical differencesbetweenthe two cell types. The results clearly demonstratethat changesin expressionof only a small fraction of the genome,in this case(2 percent of the =5000 yeast genes,can significantly alter the behavior and properties of cells.Transcription of a much larger number of genes,about 25 percent of the total assayed,differed substantially in diploid cells compared with haploid cells. These differencesin expressionpatterns make sensesince a and ct cells are very similar (hence, expression of relatively few genes differ between them), whereas haploid and diploid cellsare quite different.

Mating-TypeTranscriptionFactorsSpecify Cell Types Each of the three S. cereuisiaecell types expressesa unique set of regulatory genesthat is responsiblefor all the differencesamong the three cell types. All haploid cells express certain haploid-specificgenes;in addition, a cells expressaspecific genes, and o. cells express cr-specificgenes.In a"/a diploid cells, diploid-specific genes are expressed,whereas haploid-specific,a-specific,and a-specific genesare not. As illustrated in Figure 21,-17,three cell type-specific transcription factors (o.L,u2, and a1) encoded atthe MAT locus act in combination with a general transcription factor called

922

CHAPTER 21

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C E L LB t R T H L , t N E A G EA, N D D E A T H

(a) a cell

----:: \

J- MArE-T

(b) ocell

ll

MATU2

MATaI

+CC+>@] (c) a/crcell

MATU2

MATU|

FIGURE 21-17Transcriptional controlof celltype-specific genesin S. cerevisiae. Thecodingsequences carriedat theMAT locusdifferin haploida anda cellsandin diploidcells.Threetypespecific transcription factors(ct1,o2, anda1)encoded attheMAT locusactwith MCM1,a constitutive transcription factorproduced by allthreecelltypes,to produce patternof gene a distinctive expression in eachof the threecelltypes.asg : a-specific geneVmRNAs; genes/mRNAs; cr59: a-specific hsg : haploidspecific Aenes/mRNAs.

MCMI, which is expressedin all three cell types, to mediate cell type-specific gene expressionin S. cereuisiae.Thus the actions of just three transcription factors can set the yeast cell on a specific differentiation pathway culminating in a particular cell type. From the DNA microarray experiments we know one effect of these key players: the activation or repressionof many dozensof genesthat control cell charactenstrcs. MCM1 was the first member of the MADS family of transcription factors to be discovered.(MADS is an acronym for the initial four factors identified in this family.) The DNA-binding proteins composing this family dimerize and contain a similar N-terminal MADS domain. In Section21.3 we will encounter other MADS transcription factors that participate in development of skeletal muscle. MADS transcription factors also specify cell types in floral organs (see Figure 22-35). Acting alone, MCM1 activatestranscription of a-specificgenesin a cells and of haploid-specificgenesin both ct and a cells (seeFigure 21,-17a,b).In haploid o cells,

(a) a cells

cr-specificURS

MCMl dimer

No tanscriptionof a-specificgenes

(b) o cells

cr-specific URS

a-specificURS

Transcription of a-specific genes

s2 dimer

Transcription of a-specific genes

a-specificURS

however,the activity of MCM1 also is determined by its association with the o.1.or a2 transcription factor. As a result of this combinatorial action, MCM1 promotes transcription of a-specific genesand repressestranscription of a-specific genesin cr cells. Now let's take a closer look at how MCM1 and the MAT-encoded proteins exert their effects.

M C M l a n d a 1 - M C M 1C o m p l e x e A s ctivate GeneTranscription In a cells,homodimeric MCM1 binds to the so-calledP box sequencein the upstream regulatory sequences(URSs)of aspecificgenes,stimulating their transcription (Figure 21-18a). Transcription of o.-specificgenesis controlled by two adjacent sequences-the P box and the Q box-located in the URSs associatedwith these genes.Although MCM1 alone binds to the P box in a-specificURSs,it does not bind to the P box in a-specific URSs. Thus a cells do not rranscribe aspecificgenes. In ct cells, which produce the ct1 transcription factor encoded by MATa, the simultaneous binding of MCM1 and cr,1to PQ sites occurs with high affinity (Figure 21-18b1. This binding turns on transcription of cr-specific genes. Therefore, a-specific transcription is a simple matter of a single transcription factor binding to its target genes,while ct-specifictranscription requires a combination of two factors-neither of which can activate target genesalone.

a2-MCM1 and c2-a1 ComplexesRepress Transcription Highly specificbinding occurs as a consequenceof the interaction of o2 with other transcription factors at different sites in DNA. Flanking the P box in each a-specificURS are two cr2-binding sites. Both MCM1 and a2 can bind independently to an a-specificURS with relatively low affinity. In cr cells, however, highly cooperative, simultaneous binding of both ct2 and MCM1 proteins to thesesitesoccurs with high affinity. This high-affinity binding repressestranscription of

No transcription ol a-specific genes

< FIGURE 21-18 Activityof MCMl in a and a yeastcells.MCMl bindsasa dimerto anda-specif ic upstream the Psitein ct-specific (URSs), regulatory sequences whichcontrol transcription of o-specific aenesanda-specific (a)In a cells,MCMl genes,respectively. genes. stimulates transcription of a-specific MCMl doesnot bindefficiently to the Psitein o-specrfic URSs in the absence of o1 protein (b)In o cells,theactivity of MCMl ismodified withcr1or o2 Theo1-MCM1 by itsassociation complex stimulates transcription of a-specific genes, whereas theo2-MCM1complex blocks genes. transcription of a-specific Theo2MCMl complex alsoisproduced in diploid cells,whereit hasthesameblocking effect (see on transcription of a-specific Aenes i l o u r ez t - t / c ) .

a-specific genes,ensuring that they are not expressedin e cellsand diploid cells (seeFigure 21.-1.8b,right).MCM1promotes binding of a2 to an a-specificURS by orienting the two DNA-binding domains of the ct2 dimer to the a2-binding sequencesin this URS. Sincea dimeric cr2molecule binds to both sitesin an cr-specificURS, each DNA site is referred to as a half-site. The relative positions of both half-sitesand their orientation are highly conserved among different aspecificURSs. Combinations of transcription factors create additional specificity in gene regulation. The presenceof numerous c2binding sitesin the genome and the "relaxed" specificity of ct2 protein may expand the range of genesthat it can regulate. For instance,in a/ct diploid cells, cr2 forms a heterodimer with a1 that repressesboth haploid-specificgenesand the geneenThe example of a2 suggests coding a1 (seeFigure 21,-1,7c). that relaxed specificitymay be a generalstrategyfor increasing the regulatory range of a single transcription factor.

PheromonesInduceMating of a and a Cellsto Generatea Third CellType An important feature of the yeast life cycle is the ability of haploid a and c cells to mate, that is, attach and fuse giving rise to a diploid ala cell (seeFigure 1-5). Each haploid cell type secretesa different mating factor, a small polypeptide pheromone, and expressesa cell-surfaceG protein-coupled receptor that recognizesthe pheromone secretedby cells of the other type. Thus a and a cells both secreteand respond Binding of the mating factors to pheromones(Figure 21,-1,9). to their receptorsinducesexpressionof a set of genesencoding proteins that direct arrest of the cell cycle in G1 and promote attachment/fusion of haploid cells to form diploid cells. In the presenceof sufficient nutrients, the diploid cells will continue to grow. Starvation, however, induces diploid cells to progressthrough meiosis,each yielding four haploid spores.If the environmental conditions becomeconduciveto vegetative growth, the spores will germinate and undergo mitotic division. C E L L . T Y PS E P E C I F I C A T I OI N Y E A S T

923

TT

a factor\ at

a

a

a-factorreceptor

Cell-cyclearresl Morphogenesis

mate. The STE12 gene encodesa transcription factor that binds to a DNA sequencereferred to as the pheromoneresponsiveelement,which is presentin many different a- and a-specificURSs.Binding of mating factors to cell-surfacereceptors induces a cascadeof signaling events, resulting in phosphorylation of various proteins including the Ste12protein (seeFigure l6-28a). This rapid phosphorylation is correlated with an increasein the ability of Ste12 to stimulate transcription. It is not yet known, however, whether Ste12 must be phosphorylatedto stimulate transcription in responseto pheromone. Interaction of Ste12 protein with DNA has been studied most extensively at the URS controlling transcription of STE2, an a-specific gene encoding the receptor for the cr pheromone. Pheromone-inducedproduction of the o receptor encoded by STE2 increasesthe efficiency of the mating process.Adjacent to the a-specificURS in the STE2 geneis a pheromone-responsive element that binds Ste12. When a cells are treated with ct pheromone, transcription of the STE2 geneincreasesin a processthat requiresSte12protein. Ste12 protein has been found to bind most efficiently to the pheromone-responsiveelement in the STE2 URS when MCM1 is simultaneouslybound to the adjacent P site. I(e saw previously that MCM1 can act as an activator or a repressor at different URSs depending on whether it complexeswith a1 or o.2.In this case,the function of MCM1 as an activator is stimulated by the binding of yet another transcription factor, Ste12, whose activity is modified by extracellular signals.

ruucrear tusion J

Cell-Type Specification in Yeast D i p l o i dc e l l

+Nutient7/ Mitotic growth

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oo oo Sporulation

A FIGURE 21-19 Pheromone-induced matingof haploidyeast cells.Thea cellsproduce o matingfactoranda-factorreceptor; the a cellsproduce a factorando.-factor receptorBinding of the mating factorsto theircognatereceptors on cellsof theopposite typeleads to geneactivation, resulting in matingandproduction of diploid cellsInthe presence of sufficient nutrients, thesecellswillgrowas diploidsWithoutsufficient nutrients, cellswill underqomeiosis and formfour haploid spores. Studies with yeast mutants have provided insights into how the a and ct pheromones induce mating. For instance, haploid yeast cells carrying mutations in the sterile 12 (STE12) locus cannot respond to pheromones and do not 924

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r Specificationof each of the three yeast cell types-the a and cr haploid cells and the diploid a"/acells-is determined by a unique set of transcription factors acting in different combinations at specific regulatory sites in the yeast genome (seeFigure 21,-1,7). r Some transcription factors can act as repressorsor activators depending on the specificregulatory sitesthey bind and the presenceor absenceof other transcription factors bound to neighboringsites. r Binding of mating-type pheromones by haploid yeast cells activates expressionof genesencoding proteins that mediate mating, thereby generatingthe third yeastcell type (seeFigure 21-19).

Specification and Differentiation of Muscle An impressivearray of molecular strategies,some analogous to those found in yeast cell-type specification,have evolved to carry out the complex developmentalpathways that characterizemulticellular organisms.Muscle cells have been the focus of many such studiesbecausetheir developmentcan be studied in cultured cells as well as in intact animals. Early

advancesin understandingthe formation of muscle cells (myogenesis)came from discovering regulatory genes that could convert cultured cells into muscle cells. Then mouse mutations affecting those geneswere created and studied to learn the functions of the proteins encoded by these genes, following which scientistshave investigatedhow the muscle regulatory genescontrol other genes. Recent microarray studies have looked for genes whose transcription differs in various subtypesof muscle in mice. These studies have identified 49 genes out of the 3000 genesexamined that are transcribed at substantially different levelsin red (endurance)muscle and white (fast response) muscle. Clues to the molecular basis of the functional differences between red and white muscle are likely to come from studying those 49 genesand their products. Here we examine the role of certain transcription factors in creating skeletal muscle in vertebrates.These muscle regulators illustrate how coordinated transcription of sets of target genescan produce differentiated cell types and how a cascadeof transcriptional eventsand signals is necessaryto coordinate cell behaviors and functions.

EmbryonicSomitesGive Riseto Myoblasts Vertebrate skeletal myogenesis proceeds through three stages:determination of the precursor muscle cells, called myoblasts,which commits them to a muscle cell fate; proliferation and in some casesmigration of myoblasts; and their terminal differentiation into mature muscle (Figure 21,-20). In the first stage,myoblasts arise from blocks of mesoderm cells, called somites,that are located next to the neural tube in the embryo. Specificsignalsfrom surrounding tissueplay an important role in determining where myoblasts will form in the developing somite. At the molecular level, the "decision" of a mesoderm cell to adopt a muscle cell fate reflectsthe activation of genesencodingparticular transcription factors. As myoblasts proliferate and migrate, say, to a developing limb bud, they becomealigned, stop dividing, and fuse to form a syncytium (a cell containing many nuclei but sharing 'We a common cytoplasm). refer to this multinucleate cell as a myotube. Concomitant with cell fusion is a dramatic rise in the expressionof genesnecessaryfor further muscle development and function. The specific extracellular signals that induce determination of each group of myoblasts are expressedonly transiently. These signalstrigger production of intracellular factors that maintain the myogenic program after the inducing signalsare gone. We discussthe identification and functions of these myogenic proteins, and their interactions, in the next severalsections.

M y o g e n i cG e n e sW e r e F i r s tl d e n t i f i e di n S t u d i e s with CulturedFibroblasts Myogenic genesare a fine example of how transcription factors control the progressivedifferentiation that occurs in a

Dermomyotome (givesrise to dermis of skin and muscle precursors)

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21-20 Threestagesin developmentof vertebrate A FIGURE spheres of embryonic areepithelial muscle. Somites skeletal becomedetermined cells,someof which(themyotome) mesoderm (tr). Afterthe fromothertissues signals afterreceiving asmyoblasts proliferate andmigrateto the limbbudsandelsewhere myoblasts (Z), theyundergo intomultinucleate terminaldifferentiation (B). Keytranscription factors cells,calledmyotubes muscle skeletal programarehighlighted in yellowSee that helpdrivethe myogenic a l s oF i g u r2e1 - 2 3 .

cell lineage. In vitro studies with the fibroblast cell line designated C3H 1.0TV2have played a central role in dissecting the transcription control mechanisms regulating skeletal myogenesis.When incubated in the presenceof 5azacytidine (a cytidine derivative that cannot be methylated), C3IH l\Tyz cells differentiate into myotubes. Upon entry into cells, S-azacytidineis converted to 5-azadeoxycytidine triphosphate and then is incorporated into DNA in place of deoxycytidine. Becausemethylated deoxycytidine residuescommonly are present in transcriptionally inactive DNA regions, replacementof cytidine residueswith a derivative that cannot be methylated may permit activation of genespreviously repressedby methylation. The high frequency at which azacytidine-treated C3H 10Ty2 cells are converted into myotubes suggestedto early workers that reactivation of one or a small number of closely linked genesis sufficient to drive a myogenic program. To test this hypothesis, researchersisolated DNA from C3H 10T\/z cells grown in the presence of S-azacytidine and transfectedit into untreated cells. The observation that 1 in 104 cells transfected with this DNA was converted into a myotube is consistentwith the hypothesisthat one or a small set of closely linked genesis responsiblefor converting fibroblasts into myotubes. Subsequentstudies led to the isolation and characterization of four different but related genes that can convert S P E C I F I C A T I OANN D D I F F E R E N T I A T I OONF M U S C L E

925

> EXPERIMENTAL FIGURE 21-21Myogenicaenesisolatedfrom azacytidine-treated cellscan drive myogenesiswhen transfectedinto other cells.(a)WhenC3H'10T%cells(afibroblast cellline)aretreated with azacytidine, theydevelop intomyotubes at highfrequency. Toisolatethe genesresponsible for converting azacytidine-treated cellsinto myotubes, allthe mRNAs fromtreated cellsfirstwereisolated fromcellextracts on an oligo-dT column. Because of theirpoly(A) tails,mRNAs areselectively retained on this column. TheredmRNAs represent molecules in azacytidineenriched treatedcells; the pinkonesareallothermRNAsStepsI andE:The isolated mRNAs wereconverted to radiolabeled cDNAs.StepB: Whenthe cDNAsweremixedwith mRNAsfromuntreatedC3H10f1/z (lightred)produced cells,onlycDNAs derived frommRNAs by both azacytidine-treated cellsanduntreated Theresulting cellshybridized. double-stranded DNAwasseparated fromthe unhybridized cDNAs (darkblue)produced onlyby azacytidine-treated cells.StepZl: The cDNAsspecific for azacytidine-treated cellsthenwereusedasprobes to screen a cDNAlibrary fromazacytidine-treated cells(seeFigure5-16). At leastsomeof the clonesidentified with theseprobescorrespond to genesrequired (b)Eachof thecDNAclones for myogenesis identified in part(a)wasincorporated intoa plasmid carrying a strongpromoter. StepsI and E: C3H10T1/z cellswerecotransfected with each plasmid recombinant plusa second plasmid carrying a geneconferring resistance to an antibiotic calledG418;onlycellsthat haveincorporated the plasmids willgrowon a mediumcontaining G418.Oneof the selected clones, designated myoD,wasshownto driveconversion of C3H10f1/z cellsintomuscle cells, identified bytheirbinding of labeled antibodies against protein(stepE) [See myosin, a muscle-specific RL Davis etal,1987,Cell51:987 l

C3I{ 10T1/z cells into muscle. Figure 21-21 oudines rhe experimental protocol for identifying and assaying one of these genes,called the myogenic determination (myoD) gene. C3H 10Ty2 cells transfectedwith myoD cDNA and those treatedwith S-azacytidineboth formed myotubes.The myoD cDNA alsowas able to converra number of other cultured cell linesinto muscle.Basedon thesefindings, the myoD genewas proposedto play a key role in muscledevelopment.A similar approach identified three other genes-myogenin, myfS, and mrf4-that also function in muscledevelopment.

Two Classesof RegulatoryFactorsAct in Concertto Guide Productionof MuscleCells The four myogenic proteins-MyoD, Myf5, myogenin, and MRF4-are all members of the basic helix-loop-helix (bHLH) family of DNA-binding transcription factors (see Figure 7-26). Near the center of these proteins is a basic DNA-binding (B) region adjacentto the HLH domain, which mediatesdimer formation. Flanking this central DNA- binding/dimerizationregion are tvvo activation domains. We refer to the four myogenic bHLH proteins collectively as muscle regulatory factors, or MRFs (Figure21-22a). bHLH proteinsform homo- and heterodimersthat bind to a 6-bp DNA site with rhe consensussequenceCANNTG (N : any nucleotide). Referred to as the E box, this sequenceis presentin many different locations within the genome (on a purely random basis the E box will be found every 256 nucleotides).Thus some mechanism(s)must ensurethat MRFs 926

C H A P T E R2 1

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C E L LB I R T H ,L I N E A G EA , ND DEATH

(a) Screenfor myogenicgenes .\A'iNNNAAANA .vttuwtAiAniA

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lsolatecDNAscharacteristic of azacytidine-treatedcells

(b) Assay for myogenicactivityof myoD cDNA

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Transfectwith a olasmid carryingmyoD cDNA and a plasmid conferringresistance to G418 Selecton G418-containing m e d i u mf o r c e l l st h a t tookup bothplasmids

(a) Structure of muscle-regulatoryfactors {MRFs) DNAbinding/dimerization H z N Transactivationl B

I

HLH lTransactivation

cooH

MEF-interactinq domain (b) Structure of myocyte-enhancingfactors (MEFs) DNAbinding/dimerization HzN

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tion with myogenin (Figure21-22b). The synergisticaction of the MEF homodimer and MRF-E2A heterodimer is thought to drive high-levelexpressionof muscle-specificgenes. Knockout mice and Drosophila mutants have been used to explore the roles of MRF and MEF proteins in conferring myogenic specificityin intact animals, extending the work in cell culture. These experimentsdemonstratedthe importance of three of the MRF proteins (MyoD, Myf5, and myogenin) and of MEF proteins for distinct steps in muscle development (see Figure 21,-20).The function of the fourth myogenic protein, Mrf4, is not entirely clear.

cooH

M R F - i n t e r a c tdi nogm a i n FIGURE 21-22 Generalstructuresof two classes of (muscle transcriptionfactorsthat participatein myogenesis. MRFs regulatory factors), suchasmyogenin andMyoD,arebHLH(basic helixproteins produced loop-helix) onlyin developing muscleMEFs (myocyte-enhancing factors), whichareproduced in several tissues in addition to developing muscle, belong to theMADSfamily. The myogenic activity of MRFs isenhanced bytheirinteraction with MEFs Thedomain structures of theproteins areshown,including (B),helix-loop-helix (HLH), transactivation, basic MADSandMEF domains,

specificallyregulatemuscle-specific genesand not other genes containing E boxesin their transcription-controlregions.One clue to how this myogenicspecificityis achievedwas the finding that the DNA-binding affinity of MyoD is tenfold greater when it binds as a heterodimercomplexedwith E2A, another bHLH protein, than when it binds as a homodimer.Moreover, in azacytidine-treatedC3H 10Ty2 cells,MyoD is found as a heterodimercomplexed with E2A, and both proteins are required for myogenesisin these cells. The DNA-binding domains of E2A and MyoD have similar but not identicalamino acid sequences, and both proteins recognizeE box sequences. The other MRFs also form heterodimerswith E2A that have properties similar to MyoD-E2A complexes. This heterodimerization restricts activity of the myogenic transcription factors to genesthat contain at leasttwo E boxeslocated closeto each other. Since E2A is expressedin many tissues,the requirement for E2A is not sufficient to confer myogenic specificity.Subsequent studies suggestedthat specific amino acids in the bHLH domain of all the MRFs confer myogenic specificity by allowing MRF-E2A complexesto bind specificallyto another family of DNA-binding proteins called myocyte enhancing factors, or MEFs. MEFs were consideredexcellent candidatesfor interaction with MRFs for two reasons.First, many muscle-specificgenescontain recognition sitesfor both MEFs and MRFs. Second, although MEFs cannot induce myogenic conversionof azacytidine-treated C3H 10T% cells by themselves,they enhancethe ability of MRFs to do so. This enhancementrequires physical interaction between a MEF and MRF-E2A heterodimer. MEFs belong to the MADS family of transcription factors and contain a MEF domain, adjacent to the MADS domain, that mediatesinterac-

Differentiationof Myoblastsls Under Positive and NegativeControl Powerful developmentalregulators like the MRFs cannot be allowed to run rampant. In fact, their actions are ctrcumscribedat severallevels.First, production of the muscleregulators is activatedonly in mesodermcellsin responseto locally acting signals,suchas Hedgehog,'Wnt,and BMP, that are produced at the right time and place in the embryo. Other proteinsmediateadditional mechanismsfor assuringtight control over myogenesis:chromatin-remodelingproteins are needed to make target genesaccessibleto MRFs; inhibitory proteins can restrict when MRFs act; and antagonistic relations between cell-cycle regulators and differentiation factors like MRFs ensurethat differentiatingcellswill not divide, and vice versa.All thesefactorscontrol when and where musclesform. Activating Chromatin-Remodeling Proteins MRF proteins control batteriesof muscle-specificgenes,but can do so only if chromatin factors allow access.Remodeling of chromatin, which usually is necessaryfor gene activation, is carried out by large protein complexes(e.g.,S'$fVSNFcomplex) that have ATPaseand perhaps helicaseactivity. These complexes recruit histone acetylasesthat modify chromatin to make genesaccessibleto transcription factors (Chapter 7). The hypothesis that remodeling complexes help myogenic factors was tested using dominant-negativeversions of the ATPaseproteins that form the cores of thesecomplexes.(Recall from Chapter 5 that a dominant-negativemutation producesa mutant phenotype even when a normal allele of the '$fhen genescarrying these dominantgene also is present.) negative mutations were transfectedinto C3H 10T72 cells, the subsequentintroduction of myogenic genes no longer converted the cells into myotubes. In addition, a musclespecific gene that is normally activated did not exhibit its usual pattern of chromatin changesin the doubly transfected C3H t0T1/z cells. These results indicate that transcription activation by myogenic proteins dependson a suitable chromatin structure in the regions of muscle-specificgenes. MEF2 recruits histone acetylasessuch as p300/CBR through another protein that serves as a mediator, thus activating transcription of target genes.Chromatin immunoprecipitation experimentswith antibodies against acetylated histone H4 show that the acetylatedhistone level associated with MEF2-regulatedgenesis higher in differentiatedmyotubes than in myoblasts (seeFigure 7-37). A N D D I F F E R E N T I A T IO OF NM U S C L E SPECIFICATIO

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Inhibitory Proteins Screeningfor genesrelated to myoD led to identification of a related protein that retains the HLH dimerization region but lacks the basic DNA-binding region and henceis unable to bind to E box sequencesin DNA. By binding to MyoD or E2A, this protein inhibits formation of MyoD-E2A heterodimersand hencetheir high-affinity binding to DNA. Accordingly, this protein is referred to as Id, for inhibitor of DNA binding. Id prevents cells that produce MyoD andE2A from activating transcription of the musclespecific gene encoding creatine kinase. As a result, the cells remain in a proliferative growth state. When these cells are induced to differentiate into muscle (for instance, by the removal of serum, which contains the growth factors required for proliferative growth), the Id concentration falls. MyoD-E2A dimers now can form and bind to the regulatory regions of target genes, driving differentiation of C3H 10Ty2 cellsinto myoblast-likecells. The role of histone deacetylases, which inhibit transcription, in muscle developmentwas revealedin experimentsin which scientists first introduced extra myoD genesinto cultured C3H 10T1/zcells to raise the level of MyoD. This resulted in increasedactivation of target genesand more rapid differentiation of the cells into myotubes. However, when genesencodinghistonedeacetylases also were introduced into the C3H 10Ty2 cells, the muscle-inducingeffect of MyoD was blocked and the cellsdid not differentiateinto myotubes. The explanation for how histone deacetylases inhibit MyoDinduced muscledifferentiation came from the surprisingfinding that the musclegeneactivator MEF2 can bind, through its MADS domain, to a histone deacetylase.This interaction, which can preventMEF2 function and muscledifferentiation, is normally blocked during differentiation becausethe histone deacetylaseis phosphorylatedby a Ca2*lcalmodulin-dependent protein kinase;the phosphorylateddeacetylasethen is moved from the nucleus to the cytoplasm. Taken together,theseresults indicate that activation of muscle genesby MyoD and MEF2 is in competition with inactivation of musclegenesby repressivechromatin structures. Cell-CycleProteins The onsetof terminal differentiation in many cell types is associatedwith arrest of the cell cycle, most commonly in G1, suggestingthat the transition from the determined to differentiated state may be influenced by cell-cycle proteins including cyclins and cyclin-dependent kinases(Chapter20). For instance,certain inhibitors of cyclin-dependentkinasescan induce muscledifferentiationin cell culture, and the amounts of these inhibitors are markedly higher in differentiating muscle cells than in nondifferentiating ones in vivo. Converseln differentiation of cultured myoblasts can be inhibited by transfecting the cells with DNA encodingcyclin D1 under the control of a constitutively active promoter. Expression of cyclin D1, which normally occursonly during G1, is induced by mitogenic factors in many cell types and drives the cell cycle (seeFigure 20-32). The ability of cyclin DL to prevent myoblast differentiation in vitro may mimic aspectsof the in vivo signals that antagonize the differentiation pathway. The antagonism between negative and positive regulators 928

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of G1 progression is likely to play an important role in controlling myogenesisin vivo.

C e l l - C e lS l i g n a l sA r e C r u c i a fl o r D e t e r m i n a t i o n a n d M i g r a t i o no f M y o b l a s t s As noted akeady,after myoblasts arisefrom somites,they must move to their proper locations and form the correct attachments as they differentiate into musclecells (Figure 21,-23).Expression of myogenic genes often occurs after elaborate events that tell certain somite cells to delaminate from the somite epithelium and guide their subsequentmovementsto muscle assemblysites. A transcription factor, Pax3, is produced in the subsetof somite cells that will form muscle.Pax3 appearsto be at the top of the regulatory hierarchy controlling muscleformation in the body wall and limbs. Myoblasts that will migrate, but not cells that remain behind, also produce a transcription factor called Lbx1. If Pax3 is not functional, Lbxl transcripts are not produced and myoblastsdo not migrate. Both Pax3 and Lbxl can affect expressionof myoD. The departure of myoblasts from somitesalso dependsupon reception of a secreted protein signal appropriately called scatter factor, Qr hepatocyte grocuth factor (9F/HGF). This signal, which is produced by embryonic connectivetissuecells (mesenchyme) in the limb buds, attracts migrating myoblasts, thus directing them to their proper destination. Production of SF/HGF is previously induced by still other secretedsignals.If the SF/HGF signal or its receptor on myoblasts is not functional, somite cells will produce Lbxl but not go on to migrate; thus no muscleswill form in the limbs. Expressionof the myogenin and mrf4 genesdoes not begin until migrating myoblasts approach their limb-bud destinations(seeFigure 21.-201.

Dermamyotome (gives rise to dermis of skin and to muscle)

N e u r a lt u b e Myoblasts (migratefrom the myotome to form skeletaland limb muscles)

Epidermis

o

Notochord

Sclerotome (givesriseto skeletal structures suchas vertebrae) FIGURE 21-23 Embryonicdeterminationand migrationof myoblastsin mammals.Afterformation of the neuraltube,each somiteformssclerotome, whichdevelops intoskeletal structures, and dermomyotome, whichgivesriseto the dermisof theskinandto the muscles. Lateral myoblasts migrate fromthe dermomyotome to the limbbud;medialmyoblasts develop intothetrunkmuscles. The givesriseto the connective remainder of a dermomyotome tissueof the skin [Adapted fromM Buckingiam, 1992,Trends Genet8:1441

Vertebrate myogenesis myogenin mRNA

E2,A(general)

^. /V\^+

6;;A I mrfal?l

\ -

Myf5, MyoD*

f

-1 MYogenin

I {others) | Muscleprecursor genes

Precursor determination

Differentiation

< FfGURE 21-24 Comparisonof genes that regulatevertebratemyogenesisand factors bHLHtranscription fly neurogenesis. functions in determination of haveanalogous precursor cellsandtheir neuralandmuscle intomature subseouent differentiation the andmuscle cellsIn bothcases, neurons genes proteinsencodedby the earliest-acting (/eft)areunderboth positiveand negative controlby otherrelatedproteins(bluetype) fromY N Janand Seetextfor details.[Adapted L Y Jan.1993,Cell75:827 |

Fly neurogenesis

genes

Emc

precursor genes

'We

have touched on just a few of the many external signalsand transcription factors that participate in development of a properly patterned muscle. The function of all these regulatory molecules must be coordinated both in spaceand in time during myogenesis.

bHLHRegulatoryProteinsFunctionin Creation of Other Tissues Four bHLH transcription factors that are remarkably similar to the myogenic bHLH proteins control neurogenesisin Drosophila. Similar proteins appearto function in neurogenesis in vertebrates and perhaps in the determination and differentiation of hematopoieticcells. The neurogenicDrosophila bHLH proteins are encoded by an -100-kb stretch of genomic DNA, termed the achaete-scutecomplex (AS-C), containing four genesdesignated achaete (ac), scute (sc), letbal of scute (l'sc), and asense1'a/.Analysis of the effects of loss-of-function mutations indicate that the Achaete (Ac) and Scute (Sc) proteins participate in determination of neuronal stem cells, called neuroblasts in flies, while the Asense (As) protein is required for differentiation of the progeny of these cells into neurons. (Note that the term neuroblastsrefers to stem cells in flies but to precursor cells in mammals.) These functions are analogousto the roles of MyoD and Myf5 in muscle determination and of myogenin in differentiation. Two other Drosophila proteins, designated Da and Emc, are analogous in structure and function to vertebrate E2A and Id, respectively. For example, heterodimeric complexes of Da with Ac or Sc bind to DNA better than the homodimeric forms of Ac and Sc. Emc, like Id, lacks any DNA-binding domain; it binds to Ac and Sc proteins, thus inhibiting their associationwith Da and binding to DNA. The similar functions of these myogenic and neurogenic proteins are depicted in Figure 21-24.

A family of bHLH proteins related to the Drosophila Achaete and Scute proteins has been identified in vertebrates. One of these,called neurogenin, controls the formation of neuroblasts. In situ hybridization experiments showed that neurogenin is produced at an early stagein the developing nervous system and induces production of NeuroD, another bHLH protein that acts later (Figure 21'-25a). Injection of large amounts of neurogeniz mRNA into Xenopas embryos further demonstratedthe ability of neurogenin to induce neurogenesis(Figure 21,-25b).The function of neurogenin is analogous to that of the Achaete and Scute in Drosophila; likewise, NeuroD and Asensemay have analogous functions in vertebratesand Drosophila.

Specification and Differentiation of Muscle r Development of skeletal muscle begins with the signalinduced determination of certain mesodermcellsin somites as myoblasts. Following their proliferation and migration, myoblasts stop dividing and differentiate into multinucleate muscle cells (myotubes) that express muscle-specific proteins (seeFigure 21'-20). r Four myogenic bHLH transcription factors-MyoD, myogenin, Myf5, and MRF4, collectively called muscleregulatory factors (MRFs)-associate with E2A and MEFs to form large transcriptional complexesthat drive myogenesisand expressionof muscle-specificgenes. r Dimerization of bHLH transcription factors with different partners modulates the specificity or affinity of their binding to specificDNA regulatory sites,and also may prevent their binding entirely. r The myogenic program driven by MRFs dependson the SITVSNF chromatin-remodeling complex, which makes targetgenesaccessible. S P E C I F I C A T I OANN D D I F F E R E N T I A T I OONF M U S C L E

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neurogenin mRNA

neuroDmRNA

B-tubulin mRNA

neurogeninmRNA

A EXPERIMENTAL FIGURE 21-25tn situ hybridizationand injectionexperimentsdemonstratethat neurogeninacts (a)Sections before NeuroDin vertebrateneurogenesis. of rat neuraltubeweretreatedwith a probespecific for neurogenrn mRNA (/eft)or neuroDmRNA(nght)Theopenspacein the centeristhe ventricle, andthe cellsliningthiscavityconstitute the subventricular layerAll the neuralcellsarebornin thesubventricular layerandthen migrate outward(seeFigure 21-12), As illustrated in these micrographs, neurogenin mRNAisproduced in proliferating neuroblasts in the subventricular layer(arrow), whereas neuroD

mRNAis present in migrating neuroblasts thathaveleftthe (b)Oneof thetwo cellsin earlyXenopus ventricular zone(arrow). embryos wasinjected with neurogenrn mRNA(inj)andthenstained with a probespecific for neuron-specific mRNAs encoding B-tubulin (/eft)or NeuroD(right).Theregionof the embryoderivedfromthe uninjected cellservedasa control(con)Theneurogenrn mRNA induced a massive increase in the numberof neuroblasts expressing neuroDmRNAandneurons expressing mRNAin the region B-tubulin of the neuraltubederived fromthe injected cell lrromQ Maetal, 1996,Cell87:43; courtesyof D J Anderson l

r The myogenic program is inhibited by bindingof Id protein to MyoD, which blocks binding of MyoD to DNA, and by histone deacetylases,which repress activation of target genesby MRFs.

may yield unequal daughter cells, for example, one that remains attachedto a stalk and one that developsflagella used for swimming. Essential to asymmetric cell division is polarization of the parental cell and then differential incorporation of parts of the parental cell into the two daughters(Figure 21-26). A variety of molecular mechanismsare employed to createand propagate the initial asymmetry that polarizes the parental cell. In addition to being different, the daughter cells must often be placed in a specific orientation with respectto sur'When rounding structures. stem cells divide asymmetrically, the cell that remains in contact with niche signalswill persist as a stem cell. Therefore, the mitotic spindlesand cell polarity must be aligned with the overall tissueso that differentiating cellsmove off in the right direction, and so that at least one daughter remains in the stem-cellniche to perpetuatethe stem-cellpopulation. This phenomenonis exemplified in the division of neural stem cells during embryonic development ( s e eF i g u r e2 1 - 1 2 b ) . We begin with an especiallywell-understood example of asymmetric cell division, the budding of yeast cells, and move on to recently discoveredprotein complexesimportant for asymmetric cell divisions in multicellular organisms.Nfe seein the yeast example an elegant systemthat links asymmetric division to the processof controlling cell type.

r Migration of myoblasts to the limb buds is induced by scatter factor/hepatocytegrowth factor (SF/HGF), a protein signal secretedby mesenchymalcells (seeFigure 21, 23). Myoblastsmust expressboth the Pax3 and Lbxl transcription factors to migrate. r Terminal differentiation of myoblasts and induction of muscle-specificproteins do not occur until myoblasts stop dividing and migrating. r Neurogenesisin Drosopbila dependsupon a set of four neurogenicbHLH proteins that are conceptuallyand structurally similar to the vertebrate myogenic proteins (see Figure21-24 ). r A related vertebrate protein, neurogenin, is required for formation of neural precursors and also determines their fate as neurons or glial cells.

Regulationof Asymmetric Cell Division During embryogenesis,the earliest stagein animal development, asymmetriccell division often createsthe initial diversity that ultimately culminares in formation of specific differentiatedcell types. Both stem cells and precursor cells can divide asymmetricallyvia similar mechanisms,although the details vary with the tissue. Even in bacteria, cell division

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YeastMating-TypeSwitchingDependsupon AsymmetricCell Division S. cereuisiaecells use a remarkable mechanismto control the differentiation of the cells as the cell lineage progresses. 'Sfhether a haploid yeast cell exhibits the ct or a maring rype

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A FIGURE 21-26 Generalfeaturesof asymmetriccelldivision. Various mechanisms canleadto asymmetric distribution of (red proteins cytoplasmic components, suchasparticular or mRNAs parental dots),thereby forminga polarized cell Division of a polarized cellwill be asymmetric if the mitoticspindle isoriented so cytoplasmic aredistributed unequally thatthe localized components if thespindle is to thetwo daughter cells,asshownhere.However, positioned relative differently to the localized cytoplasmic components, cells. division of a polarized cellmayproduce equivalent daughter

is determined by which genesare present at the MAT locus (seeFigure 2t-1,71.As describedin Chapter 7, the MAT locus in the S. cereuisiaegenome is flanked by two "silent," transcriptionally inactive loci containing the alternative o or a sequences(see Figure 7-33\. A specific DNA rearrangement brings the genesthat encodethe cr-specificor a-specific transcription factors from thesesilent loci to the active MAT locus where they can be transcribed. Interestingly,some haploid yeast cells can switch repeatedly between the cr and a types. Mating-type switching occurs when the a allele occupying the MAT locus is replacedby the a allele,or vice versa.The first step in this processis catalyzed by HO endonuclease,which is expressedin mother cells but not in daughtercells.Thus mating-typeswitching occursonly in mother cells (Figure 21-27). Transcription of the HO gene is dependenton the S\fVSNF chromatin-remodelingcomplex (seeFigure 7-431,the samecomplex that we encounteredearlier in our discussionof myogenesis.Daughter yeastcellsarising by budding from mother cells contain a protein called Ashlp (for Asymmetric synthesisof HO) that prevents recruitment of the SWUSNFcomplex to the HO gene,thereby preventingits transcription.The absenceof Ashl from mother cellsallows them to transcribethe HO gene. Recentexperimentshave revealedhow the asymmetry in the distribution of Ashl betweenmother and daughter cells is established.ASHL mRNA accumulatesin the growing bud that will form a daughter cell due to the action of a myosin motor protein (Chapter 17). This motor protein, called

21-27 Switchingof matingtype in haploidyeast A FIGURE by buddingformsa largermothercell(M)andsmaller cells.Division (D), bothof whichhavethe samematingtypeasthe cell daughter Themothercellcanswitchmating cell(crin thisexample) original typeduringG, of the nextcellcycleandthendivideagain,producing on transcription depends a type.Switching two cellsof theopposite of Ashl protein. onlyin the absence of the HOgene,whichoccurs cannot Ashl protein, cells,whichproduce daughter Thesmaller theydivideto form switch;aftergrowingin sizethroughinterphase, cellsandarrowsindicate cell.Orange a mothercellanddaughter switchevents

Myo4p, moves the ASHL mRNA, as a ribonucleoprotein complex, along actin filaments in one direction only, toward the bud (Figure 21,-281.Two connector proteins, termed She2p and She3p (for S$7l5p-dependentHO expression) tether the ASH1 mRNA to the Myo4p motor protein. By the time the bud separatesfrom the mother cell, the mother cell is largely depletedof ASHI mRNA and thus can switch mating type in the following G1 before additional ASH1 mRNA is produced and before DNA replication in the S phase. Budding yeastsuse a relatively simple mechanismto create molecular differencesbetween the two cells formed by division. In higher organisms,as in yeast,the mitotic spindle must be oriented in such a way that each daughter cell receivesits own set of cytoplasmic components. Genetic studies in C. elegansand Drosophila have revealedthe key participants, a first step in understandingat the molecular level how asymmetriccell division is regulatedin multicellular organisms.To illustrate thesecomplexities,we focus on asymmetric division of neuroblastsin Drosophila'

ProteinsThat RegulateAsymmetryAre Localizedat OppositeEndsof Dividing Neuroblastsin DrosoPhila Fly neuroblasts,which are stem cells, arise from a sheet of ectoderm cells that is one cell thick. As in vertebrates' the Drosophila ectoderm forms both epidermis and the nervous system, and many ectoderm cells have the potential to O F A S Y M M E T R I CC E L LD I V I S I O N REGULATION

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Video: ASHl mRNA Localization > FIGURE 21-28 Model for restrictionof mating-type switchingto mother cellsin S. cerevisiae. Ashl protein prevents a cellfromtranscribing theHOgenewhoseencoded proteininitiates the DNArearrangement that results in matingtypeswitching froma to cror a to a Switching occurs onlyin the mothercell,afterit separates froma newlybuddeddaughter cell, because of the presence of Ashl proteinonlyin the daughter cell Themolecular basisfor thisdifferential localization of Ashl isthe one-way transport of ASHImRNAintothe bud.A linkingprotein, She2p, bindsto specific 3' untranslated sequences in theASHI mRNAandalsobindsto She3pproteinThisproteinin turnbinds to a myosinmotor,Myo4p,whichmovesalongactinfilaments into the bud [See S KoonandB J Schnapp, 2001,Curr. Biology 1 1 : R 1I6 6

assumeeither a neural or epidermal fate. Under the control of genes that become active only in certain cells, some of the cells increasein size and begin to loosen from the ectodermal layer. At this point, the delaminating cells use the DeltaArlotch signaling pathway to mediate lateral inhibition of their neighbors, causing them to retain the epidermal fate (seeFigures 16-35 and 22-42). The delaminating cells move inside and becomesphericalneuroblasts,while the prospective epidermal cells remain behind and close up to form a tight sheet.This processgenerates50 neuroblasts

ASHI mRNA

\,.--

Bud

VV

in each body segment, which will give rise to about 700 neurons per segment. Once formed, the neuroblastsundergo asymmetric divisions,at each division recreatingthemselvesand producing a ganglion mother cell (GMC) on the basal side of the neurobIast (Figure 21-29). A single neuroblast will produce several GMCs; eachGMC in turn forms two neurons.Dependingon where they form in the embryo and consequent regulatory events,neuroblastsmay form more or fewer GMCs. NeurobIasts and GMCs in different locations exhibit different

EctodermafCell Divisionsin the DrosophilaEmbryo > F|GURE 21-29 Asymmetriccelldivisionduring Drosophila neurogenesis. Theectodermal sheet(tr) of theearlyembryo givesriseto bothepidermal cellsandneuralcells.Neuroblasts, the stemcellsfor the fly neryous system, areformedwhenectoderm cellsenlarge, separate fromthe ectodermal epithelium, andmove intothe interiorof the embryo(Z-4). Eachneuroblast thatarises divides asymmetrically to recreate itselfandproduce a ganglion mothercell,or GMC(E) Subsequent divisions of a neuroblast produce moreGMCs,creating a stackof theseprecursor cells(6) EachGMCdivides (Z). Neuroblasts onceto giveriseto two neurons andtheirneuronal descendants canhavedifferent fatesdepending on theirlocationThemicrograph showsan asymmetrically dividing Drosophila neuroblast. Theapicalend(red)willforma newneuroblast andthe basalend(blueandred)willforma GMC.Themicrotubules arelabeled in green[photograph courtesy of Dr.C Q Doe,University of Oregon l

E

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Surface epidermis It tnt"rio,.nervous system -,|

patterns of gene expression, an indicator of their fates. Analysis of fly mutants led to the discovery of key proteins that (1) establishapical-basalpolarity in neuroblasts,(2) align the mitotic spindle of dividing neuroblastswith their polarity, and (3) direct formation of daughtercellswhose fate and size differ from that of neuroblasts.Genetic studies of asymmetric cell divisions in the C. elegansearly embryo independently led to the discovery of important cell division asymmetry proteins. The machinery controlling asymmetric cell division is highly conservedand readily recognizedfrom worms to flies to mammals, indicating conservationof protein functions for more than half a billion years. Basal and apical protein complexes congregate during each neuroblast division, disperse,and then localize again for the next round of division. Four protein complexes, which we denote as MPSB, BPR DSL, and IPLG, govern the entire process(Figure 21,-30\: t BPP, an apical complex also known as the PAR complex, is responsiblefor defining the end of the cell that will remain a neuroblast.It comprisesBazooka and Par6, both of which containPDZ domains. and aPKC. an atypical isoform of protein kinase C. t IPLG, a secondapical complex, is composedof lnscuteable (Insc),Partner of inscuteable(Pins),Locomotion defects (Loco), and G;, a heterotrimericG protein (Chapter 15). This complex is critical for orienting the spindleduring asymmetric divisions. r DSL, a complex that is fairly evenly distributed around the cell, is composedof Discs-large(Dlg), Scribble(Scrib),

Proteincomplexesr I

see

IPLG

MPSB

*

K/ INTERPHASE NEUROBLAST

+

Neuroblast

GMC

ANAPHASE NEUROBLAST

21-30 Localizedproteincomplexesthat control A FIGURE neuroblast, the BPP asymmetriccelldivision.(a)Inthe Drosophila in ectoderm cellsandin delaminating islocalized apically complex (steps isalso complex 21-29)TheIPLG neuroblasts Il-B in Figure (notshown) fairly isdistributed localized. TheDSLcomplex apically by BPP, theMPSB to regulation In response aroundthecells. evenly into localizes to thebasalside,whereit willbe incorporated complex (GMC). genes in that encode ganglion Mutations mother cell the andaretherefore polarized proteins celldivision disruptasymmetric filaments transport alongcytoskeletal fatal.Motorprotein-mediated 2001, C Q DoeandB Bowerman, theMPSB basalcomplex. localizes [See 7005,Curr.OpinCellBiol.17:4751 andA Wodarz, Curr.OpinCellBiol 13:68,

and Lethal giant larvae (Lgl). Lgl reversibly associateswith the cytoskeleton.The DSL complex is mostly employed in localizing basal proteins. t MPSB,a basalcomplex, confersGMC cell fate. It includes the coiled-coil scaffold protein Miranda, the homeodomainclasstranscription factor Prospero, the RNA-binding protein Staufen,and a translational repressorprotein called Brain tumor (Brat). Vith the key players in neuroblast asymmetric division introduced. let's examine their functions more closely' Apical Complexes and Spindle Orientation For localized protein complexesto be differentially incorporated into two daughter cells, the plane of cell division must be appropriately oriented. In dividing fly neuroblasts, the mitotic spindle first aligns perpendicularto the apical-basalaxis and then turns 90 degreesto align with it at the same time that the basal complexesbecome localizedto the basal side (Figure 21.-31).The apical IPLG and BPP complexes,which are already in place before spindle rotation, control the final orientation of the spindle. This is supported by the finding that mutations in any of the components of these complexes eliminate the coordination of the spindle with apical-basal polarity, causingthe spindle orientation to becomerandom. The two apical protein complexeshave different roles in spindle orientation. First, the BPP complex responds to extrinsic cues from the overlying ectoderm to form an apical crescent at late interphase. In this way' the BPP complex aligns neuroblastpolarity with the surrounding tissueso that the apical side of the neuroblast is always next to the ectoderm. Second,the BPP complex recruits the IPLG complex to the apical cortex, and the BPPcomplex anchors one spindle pole to the apical cortex, thereby aligning the spindle along the apical-basalaxis. The direct link with the spindle is mediated by the NuMA protein, which joins the Pins protein of the IPLG complex to microtubules. The IPLG complex is sufficient for anchoring the spindle and promoting asymmetric division. Basal Complex and the Determination of GMC Fate During fly neuroblast divisions,the MPSB complex becomes localized to the basal cortex prior to each division and remains there while the basal part of the neuroblast becomesa new GMC (seeFigure 21,-30)'Miranda provides a scaffold for the other three proteins in the complex (Prospero' Staufen, and Brat) and is needed to muster them near the basal plasma membrane.After each division, MPSB proteins in the basallylocated daughtercell inhibit neuroblastproperties and confer GMC properties. Prospero negatively reguIatestranscription of cell-cyclegenes'which remain active in the dividing neuroblast. Brat post-transcriptionally inhibits the transcription factor Myc, a positive regulator of cell division and negativeregulator of cell size.In this way Brat helps keep the GMC small and restrainsits division- Genetic studies lrovide support for such involvement of Prospero and Brat in determinationof GMCs. For example,brat mutations O F A S Y M M E T R I CC E L LD I V I S I O N REGULATION

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Video: Mitotic Spindle Asymmetry in Drosophila Neuroblast Cell Division

l

A EXPERIMENTAL FIGURE 21-31Time-lapse fluorescence imaging revealsrotation of the mitotic spindlein asymmetricalfydividing neuroblasts.EarlyDrosophila embryos wereinjected with a hybridgenecomposed of thegeneencoding greenfluorescent protein(GFP) fusedto the geneencoding Tau,a proteinthatbindsto microtubules At thetop aretime-lapse images of a singledividingneuroblast in a ilveembryo. Thebasalsideisat the top,andtheapicalsideat the bottom At time0, equivalent to

cause GMCs to enlarge into neuroblastsand keep dividing, whereas loss of ProsperocausesGMCs to remain small but maintain neuroblast-stylegeneexpressionand proliferation. How doesthe MPSBcomplex becomelocatedbasallyprior to each neuroblast division? The answer is more complex than Ashlp localizationin yeast.Both apical BPPand uniform cortical DSL complexesare involved. The BPPcomplex controls MPSB localization. The atypical protein kinase C (apKC), a component of the BPPcomplex, phosphorylatesand thus inactivates the Lgl protein, a component of the DSL complex. Lgl is required to bring MPSB proteins ro the basal cortex. Since aPKC is located at the apical end of the cells, Lgl is active only in basalregions.The restriction of active Lgl to basalcortex explains how MPSB proteins are brought to the basal cortex where they causeone daughter cell to becomea GMC. How doesactive, basalLgl control localization of MpSB? Although the full story is nor yer known, genetic and biochemical studies show that actin, myosin II, and myosin VI are involved. For instance,drug-induced disruption of actin microfilaments blocks MPSB targeting to the neuroblastcortex. Myosin VI binds Miranda (the "M" of MpSB) directly and is also required for basaltargeting of the MpSB complex. Asymmetry of Daughter Cell Size A notable feature of neuroblast asymmetric division is the pronounced difference in the sizeof neuroblastsand GMCs. This cell-sizedifference is regulatedby the Pinsand Go1componentsof the IpLG complex. The IPLG complex is brought to the apical cortex by virtue of the association of its Insc component with the Baz componentof the BPPcomplex. \fhen a neuroblastdividesto produce a GMC and a neuroblast,the GMC is usually considerably smaller. The spindle is oriented, as we have described,in the apical-basaldirection, and at metaphaseof the neuroblast division, the two halves of the spindle are about 934

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prophase, thetwo centrosomes arevisible on opposite sidesof the cell.These functionasthespindle poles;asmitosis proceeds, the microtubules formingthe mitoticspindle areassembled fromthe poles (seeFigure18-34). (at 32,64,and80 seconds), In successive images the bipolarspindle canbeseemto formandrotate90 degrees to alignwith theapical-basal axis,asschematically depicted at the bottom.[From JA Kaltschmidt etal, 2000,Nature CellBiol.2:7; courtesy ofJ Kaltschmidt andA H, Brand, Wellcome/CRC lnstitute, Cambridge University.l

equal in size.However the two centrosomes,one at each pole of the spindle, behave differently. The basal centrosome, marking the pole where the GMC will form, has few astral microtubules,while the apical centrosomeenlargesand grows a bushy mass of astral microtubules that make that pole of the dividing neuroblast considerably larger (Figure 21-32). INTERPHASE NEUROBLAST

ANAPHASE NEUROBLAST

Centrosome Astral microtubules Neuroblast

-+ Cytoskeleton: I

nctin microfilaments

I

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GMC microtubules

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A FIGURE 21-32 Orientationof mitoticspindleand difference in daughtercell size in asymmetricdivisionof neuroblasts. Interactions between spindle microtubules andthe lpLGapical complex (red)liejustunder orientthespindle. Actinmicrofilaments thecellsurface (blue)radiate at alltimes.Microtubules fromthe centrosome duringinterphase andthenassemble intothe mjtotic spindle, attached to theduplicated centrosomes, duringcelldivision. Notethe biased location of thecentrosome duringinterphase, at the apicalendof thecell Theasymmetry in daughter cellsizebegins with differential assembly of astralmicrotubules, whichareconsiderably shorter andlessabundant in thebasalendof thedividing cell,which formsthedaughter ganglion mothercell(GMC). fromC e, [Adapted DoeandB Bowerman, 2001,Curr.OpinCellBiol 13:681

Genetic tests have shown redundant control of the cellsize asymmetry betweenthe two daughter cells. If either the BPP apical complex or the IPLG apical complex is functional, the cells formed will be the usual small GMC and large neuroblast. In contrast, a double mutant with defects in both the BPPand IPLG complexes(e.g.,a double pins and baz mutant) produces two daughter cells that are equal in size. Double mutations that inactivate the Gg and G" subunits, but not the Go1subunit, of the Gi protein component of the IPLG complex, causenumerous astral microtubules to form on both centrosomes.Over-production of the G9 protein has the opposite effect-no astral tubules on either centrosome. From thesegeneticanalyseswe may conclude that a normal function of the G9 protein is to selectivelyprevent assembly of astral microtubules at the basal centrosome. This regulation would involve the action of Gp/G" subunits that are not part of the apical IPLG complexes.Indeed, Gp is uniformly distributed all around the neuroblast cortex. Heterotrimeric G proteins like the one in the IPLG complex often are controlled by a G protein-coupled receptor that, when activated, dissociatesthe trimer by binding G. and releasingactive GB/G, subunits (Chapter 15). No sign of a G protein--coupledreceptor has been found in the search for proteins controlling neuroblast asymmetry.Instead Pins and Loco, componentsof the IPLG complex, substitutefor a receptor in triggering dissociation of the inactive heterotrimeric G protein. Pins and Loco are partially redundant; mutating both causesdefectsequivalentto mutation of either the Gs or G, subunit. Pins and Loco bind to GDP'G.1and act like guanine nucleotidedissociationinhibitors, thereby keeping G6 associatedwith GDP and allowing G6'GDP and GB/G.'to act upon their (as yet unknown) targets.As would be expectedfor a typical G protein cycle,a GTPase-activating protein (GAP) and a GDP exchangefactor (GEF) have also been found to regulate neuroblast division asymmetry.The GAP reaction inactivates the G protein by breaking down GTP to GDP, while the GEF reaction rechargesthe G protein for activity by bringing in a new GTP. The mechanism by which the G protein component of the IPLG complex regulatesthe activity of the centrosomeremains unknown. Summary of Asymmetry-Determining Protein Complexes The initial courseof eventsin polarizing and organizing asymmetric cell division can be summarized as having three phases:(1) establishmentof cell polarity, (2) alignment of the mitotic spindle with cell polarity, and (3) specification of distinct sibling fates. For phase 1, the BPP complex is already apically located '$fhen some of those cells sink beneath in all ectoderm cells. the surfaceto becomeneuroblasts,the apical localization of BPPpersists.The IPLG complex becomesapically located after the BPP complex. Acting through the evenly distributed DSL complex, the two apical complexesdirect the basal localization of MPSB. For phase2, the orientation of the spindle relative to the ectoderm is an indirect outcome of the apically located BPP complex, which links the already oriented ectoderm to the IPLG complex. Mitotic spindle microtubules are joined to

the apical IPLG complex by the fly NuMA protein, thus orienting the spindle. For phase 3, as asymmetric cell divisions commence, each neuroblastrenewsitself while budding off a smaller daughter GMC in the interior (basal) direction. Different sibling fates are specified by proteins incorporated into the daughter cells. Neuroblasts have stem-cell properties and are determined to retain that stem-cell fate by aPKC, a patt of the BPP complex that promotes neuroblast self-renewal.Brat and Prospero, componentsof the MPSB complex, are located in the smaller interior (basal)daughter cell and promote GMC differentiation. The G protein component of the IPLG complex activates astral microtubule assembly in a dividing neuroblast, determining the size of the two poles and hence of the daughter cells. Since IPLG is apically localized, the apical daughter cell (a neuroblast)is larger than the basal daughtercell (a GMC). From this summary,we can seehow a set of protein complexes coordinates multiple critical events during asymmetric cell division: localization and activation of asymmetry regulators, differential segregationof cell fate-determining regulatory proteins, orientation of the spindle, and generation of different sizeddaughter cells.

Regulation of Asymmetric Cell Division r Asymmetric cell division requires polarization of the dividing cell, which usually entails localization of some cytoplasmic components, and then the unequal distribution of thesecomponentsto the daughter cells (seeFigure 21'-26). r In the asymmetric division of budding yeasts' a myosindependenttransport systemcarries ASH1 mRNA into the bud (seeFigure21'-28). r Ashl protein is produced in the daughter cell soon after division and prevents expression of HO endonuclease, which is necessary for mating-type switching. Thus a daughter cell cannot switch mating type, whereas the mother cell from which it arisescan (seeFigure 21'-27). r Asymmetric cell division in Drosophila neutoblastsdepends on two apical protein complexes (BPP' IPLG), a basal complex (MPSB), and an evenly distributed complex (DSL). The basal proteins are incorporated into the ganglion mother cell (GMC) and contain proteins that determine cell fate (seeFigure 21,-30). r Asymmetry factors exert their influence at least in part by controlling the orientation of the mitotic spindle, so that asymmetricallylocalizedproteins and structuresare differentially incorporated into the two daughter cells (see Figure 21,-32). r The atypical protein kinase C (aPKC) in the BPP apical complex phosphorylatesthe LGL protein, a component of the DSL complex, but can do so only in the apical region, since that is where BPP is located. Nonphosphorylated LGL, which therefore exists only in basal regions, is active in bringing MPSB to the cortex where it requires the actin cytoskeletonto be anchored. O F A S Y M M E T R I CC E L LD I V I S I O N REGULATION

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r The general processof asymmetric cell division and the protein complexes controlling it are highly conserved through evolutionary tlme.

majority of cells generatedduring brain developmentsubsequently die. Cellular interactionsregulatecell death in two fundamentally different ways. First, most if not all cellsin multicellular organismsrequire signalsto stay alive. In the absenceof such survival signals,frequently referredto as trophic factors,cells activatea "suicide" program. Second,in some developmental contexts, including the immune sysrem,specific signals induce a "murder" program that kills cells.lfhether cellscommit suicide for lack of survival signals or are murdered by killing signalsfrom other cells, death is mediated by a common molecular pathway. In this section,we first distinguish

CellDeathand lts Regulation Programmedcell death is a counter-intuitivebut essential cell fate. Cell death keeps our hands from being webbed, our embryonic tails from persisting,our immune sysrem from respondingto our own proteins, and our brain from being filled with uselesselectricalconnections.In fact, the

Video:CellsUndergoingApoptosis'&) (b)

Mildconvolution C h r o m a t i nc o m p a c t i o n and margination Condensationof cytoplasm

B r e a k u po f n u c l e a re n v e l o p e N u c l e a rf r a g m e n t a t i o n Blebbing C e l lf r a g m e n t a t i o n

Phagocytosis

Apoptotic body Apoptoticcell Phagocyticcell

FIGURE21-33 Ultrastructural features of cell death by apoptosis.(a)Schematic drawingsillustrating the progression of morphologicchangesobservedin apoptoticcells Earlyin apoptosis, densechromosome condensation occursalongthe nuclearperiphery. Thecellbodyalsoshrinks,althoughmostorganelles remainintact Laterboth the nucleusand cytoplasmfragment,forming apoptotic

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bodies, whicharephagocytosed bysurrounding cells(b)photomicrographs comparing a normalcell(fop)andapoptotic cell(bottom) Clearly visiblein the latteraredensespheres of compacted chromatin asthenucleus (a)adapted begins to fragmentlPart fromJ Kuby, 1997, lmmunology,3ded,W H Freema& n C o , p 5 3 P a r t ( b ) f r o mM . J A r e n d s and A H Wyllie,1991,lnt'\. Rev.Exp Pathol.32:2231

programmed cell death from death due to tissueinjurg then considerthe role of trophic factors in neuronal development, and finally describe the evolutionarily conserved effector pathway that leadsto cell suicideor murder.

P r o g r a m m e dC e l lD e a t hO c c u r sT h r o u g h Apoptosis The demiseof cells by programmed cell death is marked by a well-definedsequenceof morphological changes,collectively referredto as apoptosis,a Greek word that means "dropping off" or "falling off," as leavesfrom a tree. Dying cells shrink and condenseand then fragment, releasingsmall membranebound apoptotic bodies, which generally are engulfed by other cells (Figure 21-33; see also Figure 1-19). The nuclei condenseand the DNA is fragmented.Importantly, the intracellular constituents are not releasedinto the extracellular milieu where they might have deleteriouseffectson neighboring cells.The stereotypicalchangesin cells during apoptosis, like condensation of the nucleus and engulfment by surrounding cells, suggestedto early workers that this type of cell death was under the control of a strict program. This program is critical during both embryonic and adult life to maintain normal cell number and composition. The genesinvolved in controlling cell death encodeproteins with three distinct functions: r "Killer" proteins are required for a cell to begin the apoptotrc process.

'$fhen neurons grow to make developing nervous system. sometimesover muscles, or to neurons connectionsto other will eventually grow than more cells considerabledistances, in the spinal located are bodies cell neurons' survive. The processes extend far their ganglia, while cord and adjacent prevail make connections Those that regions. these outside die. fail to connect that those and survive; In the early 1900s the number of neurons innervating the periphery was shown to dependupon the sizeof the tissueto which they would connect, the so-called "tatget field." For instance, removal of limb buds from the developing chick embryo leads to a reduction in the number of sensoryneurons and motoneurons innervating the bud (Figure 21,-34). ( a ) A m p u t a t i o no f d e v e l o p i n gl i m b b u d

O p t i cc u p and lens

Chickembryo

S p i n a lc o r d Limbbud

S p i n a lc o r d (crosssection)

Motor neurons

r "Destruction" proteins do things like digest DNA in a dying cell. r "Engulfment" proteins are required for phagocytosisof the dying cell by another cell. At first glance, engulfment seemsto be simply an after-death cleanupprocess,but some evidencesuggeststhat it is part of the final death decision.For example, mutations in killer genes always preventcells from initiating apoptosis,whereasmutations that block engulfmentsometimesallow cells to survive that would normally die. That is, cells with engulfment-gene mutations can initiate apoptosis but sometimesrecover.Engulfment involves the assemblyof a halo of actin around the dying cell, triggeredby apoptosisproteinsthat activateRac, a monomeric G protein that helps regulate actin polymerizatron (seeFigure 17-42).A signalon the surfaceofthe dying cell also stimulates a receptor on neighboring cells, which initiates membrane changesleading to engulfment. In contrast to apoptosis,cellsthat die in responseto tissue damageexhibit very different morphologicalchanges,referred to as necrosis.Typically,cells that undergo this processswell and burst, releasingtheir intracellular contents, which can damagesurroundingcellsand frequentlycauseinflammation'

NeurotrophinsPromoteSurvivalof Neurons The earlieststudiesdemonstratingthe importance of trophic factors in cellular development came from analysesof the

(b) Transplantation of extra limb bud

Motorneuron generation:

100o/o

100o/o

100o/o

100o/o

I Motoon"rronapoptosis Y

Motor neuron survival:

50Yo

lOYo

50Yo

75o/o

21-34 The survivalof motor FIGURE A EXPERIMENTAL neuronsdependson the sizeof the muscletarget field they of a limbbudfromonesideof a chick innervate.(a)Removal in the number in a markeddecrease embryoat about2 5 daysresults embryo, side.In an amputated on the affected of motorneurons on bothsides aregenerated of motorneurons normalnumbers remain manyfewermotorneurons (middldLaterin development, the than on limb missing the with on the sideof the spinalcord percent motor the of 50 (bottom). about only that Note side normal survive(b) normally aregenerated thatoriginally neurons of an extralimbbudintoan earlychickembryo Transplantation on thestdewith effect,moremotorneurons produces the opposite fromD side normal the than on [Adapted tissue target additional Connections, of Neural Theory A Trophic 1988,BodyandBrain: Purves, andT M Jessell, J H Schwartz, andE R Kandel, Press, University Harvard of NeuralScience,4thed , McGraw-Hill,,p 1054, Figure53-11 l 2OOO.Principles C E L LD E A T HA N D I T S R E G U L A T I O N

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Conversely, grafting additional limb tissue to a limb bud leads to an increasein the number of neurons in corresoonding regions of the spinal cord and sensoryganglia. InJeed, incremental increasesin the target-field size are accompanied by commensurateincremental increasesin the number of neurons innervating the target field. This relation was found to result from the selectivesurvival of neurons rather than changes in their differentiation or proliferation. The observation that many sensoryand motor neurons die after reaching their peripheral target field suggestedthat these neurons compete for survival factors produced by the target tISSUC.

Subsequentto these early observations,scientistsdiscovered that transplantation of a mouse sarcoma tumor into a chick led to a marked increasein the numbersof certain types of neurons.This finding implicated the tumor as a rich source of the presumed trophic factor. To isolate and purify this factor, known simply as nerve growth factor (NGF), scientistsusedan in vitro assayin which outgrowth of neurites from sensory ganglia (nerves)was measured. Neurites are extensionsof the cell cytoplasmthat can grow to become the long wires of the nervous system, the axons and dendrites(seeFigure23-2).The later discoverythat the submaxillary gland in the mouse also produceslarge quantities of NGF enabledbiochemiststo purify and to sequenceit. A homodimer of two 118-residuepolypeptides,NGF belongs to a family of structurally and functionally related trophic factors collectively referred to as neurotrophins. Brainderived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) also are membersof this protein family. Neurotrophins bind to and activare a family of recepror tyrosine kinases called Trks (pronounced .,tracks',). (The general structure of receptor tyrosine kinasesand the intracellular signaling pathways they activate are covered in Chapter 16.) Each neurotrophin binds with high affinity to one Trk receptor: NGF binds to TrkA; BDNR to TrkB; and NT-3, to TrkC. NT-3 can also bind with lower affinity to

both TrkA and TrkB. Binding of these factors to their receptors provides a survival signal for different classesof neurons. As neurons grow from the spinal cord to the periphery, neurotrophins produced by target tissues bind to Trk receptors on the growth cones of the extending axons, promoting survival of neurons that successfullyreach targets.In addition, lerrotrophins bind to a distinct type of receptor called p75Nr* lNtR : neurotrophin recepior) but with lower affinity. However, p75*t* forms heteromultimeric complexeswith the different Trk receptors;this association increasesthe affinity of Trks for their ligands. Depending on the cell type, binding of NGF and BDNF to p75*t' in the absence of TrkA may promore cell death rather than prevent it. (The phenomenon of multiple neurotrophins interacting with multiple similar receprors is comparable to EGF-like ligands and their HER receptors,illustrated in Figure 1,6-18). To critically addressthe role of the neurotrophins in development, scientistsproduced mice with knockout mutations in each of the neurotrophins and their receptors.These studiesrevealedthat different neurotrophins and their corresponding receptorsare required for the survival of different classes of sensory neurons (Figure 21,-35). For instance, pain-sensitive (nociceptive) neurons, which express TrkA, are selectivelylost from the dorsal root ganglion of knockout mice lacking NGF or TrkA, whereas TrkB- and TrkCexpressing neurons are unaffected in such knockouts. In contrast, TrkC-expressing proprioceptive neurons, which detect the position of the limbs, are missing from the dorsal root ganglion in TrkC and NT-3 mutanrs.

A Cascadeof CaspaseProteinsFunctionsin One Apoptotic Pathway Neurotrophins and other signals that keep cells alive act upon an evolutionarily conservedcell-deathcontrol system. Key insights into the molecular mechanismsregulating cell

> EXPERIMENTAL FTGURE 21-35 Different WildType classesof sensoryneuronsare lost in knockoutmicelackingdifferenttrophic Spinal M echano receotors factorsor their receptors. In animals lacking cord nervegrowthfactor(NGF)or its receptor Dorsalroot TrkA, ganglion (pain-sensing) smallnociceptive (blue) neurons thatinnervate theskinaremissingThese Propioceptive neurons express TrkAreceptor neurons andinnervate NGF-producing targets. In animals lacking Nociceptive (NT-3) eitherneurotrophin-3 or itsreceptor neurons TrkC,largepropioceptive (red) neurons innervating muscle spindles aremissingMuscle produces NT-3andthe propioceptive neurons express TrkCMechanoreceptors (orange), anotherclass of sensory neurons in the dorsal rootganglion, areunaffected in thesemutants. [Adaptedfrom W D Snldet I 994, Cett77:627 ]

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Video: ProgrammedCell Death in C. elegansEmbryonic Development 21-36 Mutationsin the ced-3 FIGURE < EXPERIMENTAL gene block programmedcell death in C. elegans.(a)Newly mutantlarvacarrya mutationin theced-7gene.Because hatched of deadcells,highly engulfment in thisgeneprevent mutations (arrows), their facilitating deadcellsaccumulate refractile in boththe (b)Newlyhatched larvawith mutations visualization deadcellsin of refractile ced-l andced-3genes.Theabsence that no celldeathsoccurredThus thesedoublemutantsindicates HM celldeath.lFrom for programmed CED-3proteinisrequired Ellisl Hilary courtesyof 1986, Ce//91:818; H R Horvitz, Ellisand

death came from genetic studies using C. elegans. Of the 947 nongonadal cells generated during development of the adult hermaphroditeform, 131 cellsundergo programmed cell death. Specific mutations have identified four genes whose encoded proteins play an essentialrole in controlling programmed cell death during C. elegans development: ced-3, ced-4, ced-9, and egl-1.In ced-3 or ced-4 mutants, for example,the 131 "doomed" cells survive (Figure 21,-36).The mammalian proteins that correspond most closely to the worm CED-3, CED-4, CED-9, and EGL-1 proteins are indicated in Figure 21-37. In discussingthe worm proteins we will include the mammalian names in parenthesesto make it easier to keep the relationships clear. The confluenceof geneticstudiesin worms and studies on human cancer cells first suggestedthat an evolutionarily conservedpathway mediatesapoptosis.The first mammalian apoptotic gene to be cloned, bcl-2, was isolated from human follicular lymphomas. A mutant form of this gene, created in lymphoma cells by a chromosomal rearrangement,was shown to act as an oncogenethat promoted cell survival rather than cell death (Chapter 25). The chromosomerearrangementioins the coding region of the bcl-2 gene to an immunoglobulin gene enhancer. The combination results in over-production of BcI-2 protein that keepscancer cells alive when, otherwise, they would becomeprogrammed to die. The human Bcl-2 protein and

worm CED-9 protein are homologous' and a bcl-2 transgene can block the extensive cell death found in ced-9 mutant worms even though the two proteins are only 23 petcent homologous. Thus both proteins act as regulators that suppressthe apoptotic pathway (seeFigure 21'-37)'ln addition, both proteins contain a single transmembrane domain and are localized to the outer mitochondrial, nuclear, and endoplasmic reticulum membranes, where they serveas sensorsthat control the apoptotic pathway in responseto external stimuli. As we discussbelow, other regulators promote apoptosis. In the worm apoptotic pathway' CED-3 (caspase9) is required to destroy cell components during apoptosis. CED-4 (Apaf-1) is a protease-activating factor that causes autocleavageof (and by) the CED-3 precursor protein, creating an active CED-3 proteasethat initiates cell death (seeFigure 21.-37).Cell death does not occur in ced-3 and ced-4 single mutants or in ced-9/ced-3 do'lble mutants' whereas all cells die during embryonic Iife in ced-9 mutants' so the adult form never derrilops.Thesegeneticstudiesindicate that the CED3 and CED-4 are "killer" proteins required for cell death' that CED-9 (Bcl-2) suppressesapoptosis,and that the apoptotic pathway can be activatedin all cells.Moreover, the absenceof cell death in ced-9/ced-3double mutants suggests that CED-9 acts "upstream" of CED-3 to suppressthe apoptotic pathway. The mechanismby which CED-9 (Bcl-Z)controls CED-3 (caspase9) is now known. CED-9 protein, which is normally tethered to the outside of mitochondria, forms a complex with CED-4 (Apaf-l), thereby preventing activation of CED3 by CED-4. As a result, the cell survives.This mechanism fits with the genetics,which shows that the absenceof CEDt has no effeit if CED-3 is also missing (ced-3/ced-9double mutants have no cell death). The crystal structure of the trimeric CED-4/CED-9 complex revealsa huge contact surface between each of the two CED-4 molecules and the single CED-9 molecule (Figure 21'-38it.The large contact

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(a) Nematodes

(b) Mammals A p o p t o t i cs t i m u l i

EGL-1 disrupts the CED-4/CED-9 complex comes from the crystal structure of EGL-1 (Bid/Bim) complexedwith CED-9 (Bcl-2).In this complex,the BH3 domain forms rhe key part of tEGLTI ( B H 3o n r y ) the contact surfacebetweenthe two proteins. CED,9 has a difA I F | | HTRA2/OMI ferent conformation when bound by EGL-1 than when bound by CED-4. This finding suggeststhat EGL-1 binding distorts EndoG CED-9, making its interactionwith CED-4 lessprobable and Iess stable. Once EGL-1 causesdissociationof the CED4ICED-9 complex, the releasedCED-4 dimer dimerizesagain to make a tetramer, which then activates CED-3. Cell death soon follows (Figure 21-39). Evidence for similar events has beenfound in human cultured cells. Evidencethat the steps describedhere are sufficient for caspaseactivation comes from experiments in which the eventswere reconstitutedin vitro (i.e., in solution) with purified components. CED-3, CED-4, a truncated CED-9 that lacked its transmembranemitochondrial membrane anchor, and EGL-1 were purified, as was a CED-4/CED-9 complex. Effectors [tAP;-] Purified CED-4 (Apaf-1) was able to acceleratethe autocatalysisof purified CED-3 (caspase9), but addition of the truncated CED-9 (Bcl-2) to the reaction mixrure inhibited C e l l u l a tra r g e t s the autocleavage.When the CED-4/CED-9 complex was mixed with CED-3, autocleavagedtd not occur, but addition Apoptosis of EGL-1 to the reaction restored CED-3 autocleavage. The effector proteins in the apoptotic pathwaS the cas,,c.\vJ-.r pases,are named becausethey contain a key cysteineresiduein the catalytic site and selectivelycleaveproteins at sitesjust Cterminal to aspartateresidues.Caspases work as homodimers. A FIGURE 21-37 Evolutionaryconservation of apoptosis pathways.Simrlar proteins, shownin identical play colors, corresponding rolesin bothnematodes andmammals(a)ln nematodes, the proteincalledEGL-1 bindsto CED-9on the surface of mitochondria; thisinteraction releases CED-4 fromthe CED9/CED-4 complex. FreeCED-4 thenactivates autoproteolysrs of the caspase CED-3, whichdestroys cellproteins to driveapoptosis These relationships areshownasa geneticpathway, with EGL-linhibiting CED-9, whichin turn inhibits CED-4ActiveCED-4 a/B-fold activates CED_3 (b)In mammals, homologs of the nematode proteins andother CED-4 proteins regulate apoptosts. TheBcl-2proteinlssimilar to CED_9 in To promoting cellsurvival by preventing activation Apaf-1,whichis mitochondrial s i m i l at o r C E D - 4T w oB H 3 - o npl yr o t e i n B memDrane s ,i da n dB i m ,i n h i b iBt c l _t2o Helical allowapoptosis. Apoptotic stimulidamagemitochondria, leading to oclp-fold domain release of several proteins thatstimulate celldeath.In parrrcurar, cytochrome c released frommitochondria activates Apaf_1, whichin turnactivates caspase-9 Thisinitiator caspase thenactivates effector Helical caspases-3 and-7, eventually leading to celldeath Seetextfor domain discussion of othermammalian (SMAC/DIABLO proteins andlAps) thathaveno nematode homologs[Adapted fromS J Riedl andy Shi,

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surface makes the associationhighly specific, but in such a

BH3 domain. SinceEGL-1 lacks most of the other domains of CED-9, EGL-1 is calleda BH3-only protein. The closestmammalian BH3-only proteins are Bim and Bid. Insieht into how 940

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Winged helix

FIGURE 21-38Structureof the CED-4/CED-g proteincomplex. Thecrystal structure hastwo CED-4 molecules associated withone CED-9 molecule (darkblue)serves TheC-terminus of CED-9 to tether thecomplex to themitochondrial membrane CED-4 iscomposed of (CARD, fourdomains o/g folds,wingedhelixdomain, andanother helical domain)EachCED-4 molecule hasa boundATpanda Mgr* ionwhicharevisible asa cluster of orangeandblueatomswithineach subunit[Based onN Yanetal, 2005, Nature 437:831 I

EGL-1

cED-4 dimer

I D-g/EGL-1 Mitochondrion

A

FEm-t-y-tr"s*l

FfGURE 21-39 Activationof CED-3proteasein C. elegans. thattrigger protein, to signals in response whichisproduced EGL-1 with CED-9 on dimerfromitsassociation CED-4 celldeath,displaces (tr) ThefreeCED-4 with dimercombines of mitochondria thesurface (E), whichcatalyzes of the theconversion to forma tetramer another (anenzymatically precursor of a protease) inactive zymogen CED-3 (B). Thiseffector to protease thenbegins caspase intoactiveCED-3 leading to cell apoptosis, andthusinitiate destroy cellcomponents 437|831 fromN Yanetal, 2005,Nature death(4) [Adapted I

with one domain of each stabilizing the active site of the other. The principal effector caspasein C. elegansis CED-3, while humans have 15 different caspases.AII caspasesare initially made as procaspasesthat must be cleavedto become active. Such proteolytic processingof proproteins is used repeatedlyin blood clotting, generationof digestiveenzymes' and generationof hormones.In vertebrates,initiator caspases (e.g.,caspase-9)are activated by autoproteolysisinduced by other types of proteins (e.g., Apaf-l), which help the initiators to aggregate.Activated initiator caspasescleaveeffector caspases(e.g.,caspase-3)and thus quickly amplify the total caspaseactivity level in the dying cell. The various effector caspasesrecognizeand cleaveshort amino acid sequencesin many different target proteins. They differ in their preferred target sequences.Their specific intracellular targets include proteins of the nuclear lamina and cytoskeletonwhose cleavage leadsto the demiseof a cell. In mammals and flies but not worms, apoptosisis regulated by severalother proteins(seeFigure21-37, right).For instance,a family of inhibitor of apoptosisproteins (IAPs)' provides another way to restrain both initiator and effector IAPs haveone or more zinc-bindingdomainsthat can caspases. bind directly to caspasesand inhibit their proteaseactivity. (Baculovirus, a type of insect virus, produces a protein that thus preventingan insimilarly binds to and inhibits caspases, stop a viral infection besuicide to fected cell from committing inhibition of caspasesby The viruses be made.) can fore new needs to undergo a cell problem when creates a IAPs, however, since again, picture once the enter Mitochondria apoptosis. they are the source of a family of proteins, called SMAC/DIABLOs, that inhibit IAPs. After cell injury, SMAC/DIABLOs

releasedfrom mitochondria bind to IAPs in the cytosol' thereby blocking the IAPs from binding to caspases.By relieving IAPmediated inhibition, SMAC/DIABLOs promote caspaseactivity and cell death. Three other mitochondria-associated proteins-Ht ra2l Omi serine protease' apoptosis-inducingfactor (AIF), and endonucleaseG-also help to kill cells upon their releasefrom mitochondria following cell injury. Htra2l Omi cleavesIAPs, thus relieving their restraint of apoptosis' Since this regulation is catalytic, Htta2lOmi is a more powerful antagonist of IAPs than is SMAC/DIABLO. AIR a flavoprotein that normally acts as a NADH oxidase, is cleaved by Droteasesand moves to the nucleus where it causeschromoio-e .o.rd.tsation and DNA fragmentation. These effectsare so not all apoptosisinvolvescaspases' caspase-independent'

Pro-ApoptoticRegulatorsPermitCaspase Activation in the Absenceof TrophicFactors Having introduced the major participants in the apoptotic pathway, we now take a closer look at the workings of the miiochondrial membrane proteins that regulate apoptosis' Although the normal function of CED-9 andBcl-2 is to suppress the cell-death pathwaS other intracellular regulatory proteins promote apoptosis.The first pro-apoptotic regulator to be identified, named Bax, is related in sequenceto CED-9 and Bcl2. Overproduction of Bax induces cell death rather than protectingcellsfrom apoptosis,as CED-9 and Bcl-2 do. Thus this

the intracellular signaling pathways regulating them. Some Bcl-2 family members preserveor disrupt the integrity of the outer mitochondrial membrane, thereby controlling releaseof mitochondrial proteins such as cytochrome c' In normal healthy cells' cytochrome c is localized betweenthe inner and outer mitochondrial membrane,but in cellsundergo-

ducesapoptosis.A variety of death-inducing stimuli causeBax to move from the cytosol to the outer mitochon-ono-.ti drial membranewhere they oligomerize' Bax homodimers, but not Bcl-2 homodimers ot Bcl-2lBax heterodimers, permit influx of ions through the mitochondrial membrane' It remains unclear how this ion influx triggers the releaseof cytochrome c' The effect of Bcl-2 family members on the permeability of the mitochondrial outer membrane has been mimicked in

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of Bcl-2 family membersappearsto reflect their generalability to alter mitochondrial membranes.In addition to increasedpermeability, mitochondria normally undergo dramatic changesin their nerwork morphology by fusion and fission during the cell-death process. Bcl-xl, a vertebrate member of the Bcl-2 family, and CED-9 introduced into mammalian cells, can induce mitochondrial fusion. Thus theseproteins appear to have profound abilities to engineer the properties of outer mitochondrial membranes. Once cytochrome c is releasedinto the cytosol, it binds to Apaf-1 (the mammalian homolog of CED-4) and promotes activation of a caspasecascadeleading to cell death (seeFigure 21-37, right).ln the absenceof cytochrome c, monomeric Apaf-1 is bound to dATp. After binding cytochrome c, Apaf-1.cleavesits bound dATp into dADp and undergoes a dramatic assembly process into a disc-shaped heptamer,a 1,.4megadaltonwheel of death called the apoptosome (Figure 21-40). The apoptosomeservesas an acrivation machine for initiator and effector casDases.

SomeTrophicFactorsInduceInactivationof a Pro-ApoptoticRegulator We saw earlier that neurotrophins such as nerve growth factor (NGF) prorect neurons from cell death. In the absenceof trophic factors, however, the nonphosphorylated form of a pro-apoptotic protein called Bad is associatedwith Bcl-2l

Bcl-xl at the mitochondrial membrane (Figure 21-41a). Binding of Bad inhibits the anti-apoptotic function of Bcl-2/ Bcl-xl, thereby promoting cell death. Phosphorylated Bad, however,cannot bind to Bcl-2lBcl-xl and is found in the cytosol complexed to the phosphoserine-bindingprotein 14-33. Hence, signaling pathways leading to Bad phosphorylation would be particularly attractive candidates for transmitting survival signals. A number of trophic factors including NGF have been shown to trigger the PI-3 kinase signaling pathway, leading to activation of protein kinase B (seeFigure 16-30). Activated protein kinase B phosphorylatesBad at sitesknown to inhibit its pro-apoptotic activity. Moreover, a constitutively active form of protein kinase B can rescue cultured neurotrophin-deprived neurons, which otherwise would undergo apoptosis and die. These findings support the mechanism for the survival action of trophic factors depicted in Figure 21-41b. In other cell types, different trophic factors may promote cell survival through post-translational modification of other componentsof the cell-deathmachinery. Another mechanism by which neurotrophins can affect apoptosis, this time positively, involves p75tt*, the lowaffinity neurotrophin receptormentionedabove.This protein can either promote or inhibit apoptosisdependingon the cellular context. In certain neurons, neurotrophin signals such as BDNF stimulate apoptosis by acting through p75NrR. In these neurons, cleavageof p75MR by a membrane-bound

E

Cytochromec releasefrom mitochondriabinds Apaf-1 dATP hydrolysis

E

Procaspase9 recruitment to Apaf-1

___+

Caspase3 and IAP recruitmentto Apaf-1 Apoptosome

A FIGURE 21-40Assemblyof the mammalianapoptosome.In theabsence of apoptosis triggers, Apaf-1exists in the cytosot asan inactive monomer boundto dATpApaf-1contains multiple WD4O repeats, a dATP-binding CED-4 domain,anda CARDdomain.Step istriggered, [: Whenapoptosis damage to mitochondria allows release of cytochrome c, whichbindsto Apaf-1Thisinteraction leads to hydrolysis of the bounddATpto dADpanda changein the conformation of Apaf-1.Stepf,l: In itsextended conformation, 942

.

cHAprER 21

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Apaf-1assembles intoa seven-subunit complex, theapoprosome Step$: Interaction of theapoptosome withthe initiator procaspase_ 9 stimulates theautocleavage anddimerization of the procaspase, whichis necessary for itsactivity. Activecaspase-9 thenactsupon effectorcaspases suchascaspase-3 Inhibitorof apoptosis proteins (lAPs) arealsoboundbythe apoptosome, although theirexact actionstherearenot understoodlAdapted lromZf. Schafer andS Kornbluth, 2006,Devel. Cell10:5491

(b) Presenceof trophic factor: Inhibition of caspaseactivation

(a) Absence of trophic factor: Caspaseactivation '-

Trophicfactor receptor

E

Plasmamembrane

Death.@^ r-l /

Cleavageof substrates

/

6;;b /

pathwaysleadingto 21-41 Proposedintracellular A FIGURE cell to trophic factor-mediated by apoptosis or cell death of a trophicfactor, survivalin mammaliancells.(a)Inthe absence pro-apoptotic proteinBadbindsto the anti-apoptotic the soluble intothe mitochondrial proteins whichareinserted Bcl-2andBcl-xl, proteins (tr). Badbindingprevents theanti-apoptotic membrane protein. pro-apoptotic with Bax,a membrane-bound frominteracting in the channels Baxformshomo-oligomeric As a consequence, an as-yet-unknown ionflux[Z] Through membrane that mediate c intothe of cytochrome thisfluxleadsto the release mechanism,

whereit bindsto the adapterproteinApaf-1(p), promoting cytosol, thatleadsto celldeath(Z|) (b)In somecells, cascade a caspase Pl-3kinase bindingof a trophicfactor,suchasNGF(Il) stimulates protein B kinase of activation the downstream to leading activity, Badthenformsa (PKB), Bad Phosphorylated whichphosphorylates in the withthe 14-3-3protein(U ). WithBadsequestered complex proteins caninhibitthe Bcl-2lBcl-xl the anti-apoptotic cytosol, c of cytochrome the release of Bax(B), therebypreventing activity and B Pettman from cascade. the caspase of [Adapted andactivation 20:633 Neuron l C E Henderson,1998,

releasesthe receptor'sintracellular proteasecalled^y-secretase, domain, which is associatedwith a DNA-binding protein called NRIF. The cleavageof p75MR leadsto the ubiquitination of NRIF and its movement to the nucleus where it stimulates is apoptosis,perhaps by regulating transcription.1-Secretase cleavage the intramembrane protease that catalyzes the same of the receptor Notch, thus activating it, and also of amyloid precursor protein (APP) in the genesisof Alzheimer'sdisease (seeFigures1.6-36and L6-37).

tor (TNFa), which is releasedby macrophages,triggers the cell death and tissue destruction seen in certain chronic inflammatory diseases (Chapter 24). Another important death-inducingsignal,the Fasligand' is a cell-surfaceprotein produced by activated natural killer cells and cytotoxic T iymphocytes. This signal can trigger death of virus-infected cells, some tumor cells, and foreign graft cells. Both TNF and Fas ligand act through cell-surface "death" receptorsthat have a singletransmembranedomain and are activatedwhen ligand binding brings three receptor molecules into close proximity. The trimeric receptor complex attracts a protein called FADD (Fas-associateddeath domain), which servesas an adapter to recruit and in some way activatecaspase-8,an initiator caspase'in cellsreceiving a death signal. The death domain found in FADD is a sequence that is present in a number of proteins involved in apoptosis.Once activated,caspase-8activatesother caspases

Tumor NecrosisFactorand RelatedDeath SignalsPromoteCell Murder by Activating Caspases Although cell death can arise as a default in the absenceof survival factors, apoptosis can also be stimulated by positively acting death signals.For instance,tumor necrosisfac-

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and the amplification cascadebegins.To test the ability of the Fas receptor to induce cell death, researchersincubated cells with antibodiesagainstthe receptor.Theseantibodies,which bind and cross-link Fas receptors,were found to stimulate cell death, indicating that activation of the Fasreceptoris sufficient to trigger apoptosis.

Cell Death and tts Regulation r All cells require trophic factors to prevent apoptosisand thus survive. In the absenceof these factors, cells commit suicide. r Genetic studies in C. elegansdefined an evolutionarily conservedapoptotic pathway with three major components: membrane-boundregulatory proteins, cytosolic regulatory proteins, and effector proteasescalled caspasesin vertebrates(seeFigure 21-37). r Once activated,apoptotic proteasescleavespecificintracellular substratesleading to the demise of a cell. proteins (e.g., CED-4, Apaf-1), which bind regulatory proteins and caspases,are required for caspaseactivation (seeFigures 21-39 and21-40\. r Pro-apoptotic regulator proteins (e.g., Bax, Bad) promote caspaseactivation, and anti-apoptotic regulators (e.g., Bcl-2) suppressactivation. Direct interactions between pro-apoptotic and anti-apoptotic proteins lead to cell death in the absenceof trophic facors. Binding of extracellular trophic factors can trigger changesin these interactions,resulting in cell survival (seeFigure 21-41). r The Bcl-2 family contains both pro-apoptotic and antiapoptotic proteins; all are single-passtransmembrane proteins and engagein protein-protein interactions. Bcl-2 molecules can restrain the release of cytochrome c from mitochondria, inhibiting cell death, while pro-apoptotic factors stimulate membrane breakdown that allows cytochrome C to escape,bind to Apaf-1, and thus activatecaspases. r Binding of extracellular death signals, such as rumor necrosisfactor and Fas ligand, to their receptors activates an associatedprotein (FADD) that in turn triggers the caspasecascadeleading to cell murder.

Cell birth, lineage, and death, which lie at the heart of the

a y,ardlong, pulsating multinucleate muscle cells,exquisitely light-sensitiveretina cells,ravenousmacrophagesthai recognize and engulf germs, and all the hundreds of other cell types. Regulators of cell lineageproduce this rich variety by controlling two critical decisions: (1) when and where to 944

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activate the cell cycle (Chapter 20) and (2) whether the rwo daughter cells will be the same or different. A cell may be just like its parent, or it may embark on a new path. Cell birth is normally carefully resrricted to specific localesand times, such as the basallayer of the skin or the root meristem. Liver regenerateswhen there is injury, but liver cancer is prevented by restricting unnecessarygrowth at other times. Cell lineageis patterned by the asymmetric distribution of key regulatorsto the daughter cellsof a division. Some of theseregulators are intrinsic to the parent cell, becoming asymmetrically distributed during polarization of the cell; other regulators are external signals that differentially reach the daughter cells. Asymmetry of cells becomes asymmetry of tissues and whole organisms. Our left and right hands differ only as a result of cell asymmetry. Somecells persist for the life of the organism, but others such as blood and intestinal cells turn over rapidly. Many cells live for awhile and are then programmed to die and be replaced by others arising from a stem-cellpopulation. programmed cell death is also the basisfor the meticulous elimination of potentially harmful cells, such as autoreacriveimmune cells, which attack the body's own cells, or neurons that have failed to properly connect. Cell-death programs have also evolved as a defenseagainst infection, and virusinfected cells are selectivelymurdered in responseto death signals.Viruses,in turn, devote much of theiieffort to evading host defenses.For example, p53, a transcription factor that sensescell stressesand damage and activatestranscription of pro-apoptotic membersof the b cl-2 genefamilS is inhibited by the adenovirusE1B protein. It has been estimated that about a third of the adenovirus genome is directed at evading host defenses.Cell death is relevant to toxic chemicals as well as viral infections; malformations due to poisons often originatefrom excessapoptosis. Failures of programmed cell death can lead to uncontrolled cancerous growth (Chapter 25). The proteins that prevent the death of cancer cells therefore become possible targets for drugs. A tumor may contain a mixture of cells, some capable of seedingnew tumors or continued uncontrolled growth, and some capable only of growing in place or for a limited time. In this sensethe tumor has its own stem cells, and they must be found and studied, so they become vulnerable to medical intervention. One option is to manipulate the cell-death pathway by sending signals that will make cancer cells destroy themselves. Much attention is now being given to the regulation of stem cells in an effort to understand how dividing populations of cells are created and maintained. This has clear implications for repair of tissue: for example, to restore damagedeyes,torn cartilage,degeneratingbrain tissue,or failing organs. One interesting possibility is that some populations of stem cells with the potential to generateor regeneratetissue are normally eliminated by cell death during later development. If so, finding ways to selectivelyblock the death of these cells could make regenerationmore likely. Could the elimination of such cells during mammalian developmentbe the difference between an amphibian capable of limb regeneration and a mammal that is not?

KeyTerms apoptosis937 apoptosome 942 asymmetriccell division 905 Bcl-2 famtly 941. BPP complex 933 940 caspases 905 lineage cell death signals943 determination 925 differentiation 905 ectoderm 907 embryonicstem (ES)cells911 endoderm 907 ganglion mother cell

(GMC)e32 germline907 heterochronicmutants 909 MAT locus 922 mating factor 923

920 meristems 907 mesoderm microRNAs (miRNAs)910 MPSBcompIex933 muscleregulatoryfactors (MRFs)926 neurotrophins938 nuclear-transfer cloning908 pluripotent907 precursor(progenitor) cells905 somaticcells905 stemcells905 stem-cellniche912 transientamplifying(TA) cells905 totipotent 907 trophic factors935

mating-type switching 931

Review the Concepts 1. What two properties define a stem cell? Distinguish between a totipotent stem cell, a pluripotent stem cell, and a precursor (progenitor) cell. 2. Where are stem cells located in plants? $7hereare stem cells located in adult animals?How doesthe concept of stem cell differ between animal and plant systems? 3. ln 1997, Dolly the sheepwas cloned by a techniquecalled somatic cell nuclear transfer (or nuclear-transfercloning). A nucleus from an adult mammary cell was transferred into an egg from which the nucleus had been removed. The egg was allowed to divide several times in culture, then the embryo was transferred to a surrogate mother who gave birth to Dolly. Dolly died in 2003 after mating and giving birth herself to viable offspring. \Uhat does the creation of Dolly tell us about the potential of nuclear material derived from a fully differentiatedadult cell?Doesthe creationof Dolly tell us anything about the potential of an intact, fully differentiated adult cell? Name three types of information that function to preserve cell type. \Which of these types of information was shown to be reversibleby the Dolly experiment? 4. The roundworm C. eleganshas proven to be a valuable model organism for studiesof cell birth, cell lineage,and cell death.'What properties of C. elegansrender it so well suited for these studies?Vhy is so much information from C. elegans experimentsof use to investigatorsinterestedin mammalian development? 5. How are retroviruses used in tracing experiments that map cell lineages?

6. In the budding yeast S. cereuisiae,what is the role of the MCM1 protein in the following? a. transcription of a-specificgenesin a cells b. blocking transcription of ct-specificgenesin a cells c. transcription of a-specificgenesin ct cells d. blocking transcription of a-specificgenesin ct cells 7. ln S. cereuisiae, what ensures that a and a cells mate with one another rather than with cells of the same mating type (i.e.,a with a or ct with a)? 8. Exposure of C3H L0Tlz ceIls to 5-azacytidine, a nucleotide analog, is a model systemfor muscle differentiation. How was S-azacytidinetreatment used to isolate the genes involved in muscle differentiation? 9. Through the experiments on C3H 1'0Ty2 cells treated with 5-azacytidine,MyoD was identified as a key transcription factor in regulating the differentiation of muscle. To what generalclassof DNA-binding proteins doesMyoD belong? How do the interactions of MyoD with the following proteins affect its function? (alE2A, (b) MEFs' (c) Id. L0. The mechanismsthat regulate muscle differentiation in mammals and neural differentiation in Drosophila (and probably mammals as well) bear remarkable similarities. 'What proteins function analogousto MyoD, myogenin, Id, and E2A in neural cell differentiation in Drosophila? Based on these analogies, predict the effect of microinjection of myoD mRNA on the developmentof Xenopus embryos. 11. Predict the effect of the following mutations on the ability of mother and daughter cells of S. cereuisiaeto undetgo mating-type switching following cell division: a. loss-of-functionmutation in the HO endonuclease b. gain-of-function mutation that renders HO endonuclease gene constitutively expressed independent of SIUTI/SNF c. gain-of-function mutation in SVUSNF that renders it insensitiveto Ashl 12. Asymmetric cell division often relies on cytoskeletalelements to generareor maintain the asymmetricdistribution of cellular factors. In S. cereuisiae, what factor is localized to the bud by myosin motors? In Drosophila neuroblasts,what factors are localized apically by microtubules ? 13. How do studiesof brain developmentin knockout mice support the statementthat apoptosis is a default pathway in neuronal cells? 'S7hat morphologic features distinguish programmed 14. necrotic cell death? TNF and Fas ligand bind and cell death to trigger cell death. Although the receptors cell-surface death signal is generatedexternal to the cell, why do we consider the death induced by these moleculesto be apoptotic rather than necrotic? 15. Predict the effects of the following mutations on the ability of a cell to undergo apoptosls: a. mutation in Bad such that it cannot be phosphorylated by protein kinase B (PKB) b. c.

overexpressionof Bcl-2 mutation in Bax such that it cannot form homodimers R E V I E WT H E C O N C E P T S

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One common characteristicof cancer cells is a loss of function in the apoptotic pathway. Vhich of the mutations listed above might you expect to find in some cancer cells? 16. How do IAPs (inhibitor of apoptosis proteins) interact with caspasesto prevent apoptosis?How do mitochondrial proteins interactwith IAPs to preventinhibition of apoptosis?

Analyze the Data The immortal-strand hypothesispostulatesthat when a stem cell divides asymetrically to produce one "new" stem cell and one progenitor cell, the new stem cell receivesthe sister chromatids containing the oldest strand of DNA (the immortal strand). The other daughter cell, a progenitor cell that eventually gives rise to differentiated cells. receivesthe sister chromatids containing more recent DNA strands (see diagram below). If the immortal-strand mechanismactually operates,it would prevent the accumulation of mutations in adult stem cells that otherwise would occur during each round of DNA replication.

hypothesis, researchersrecently conducted the following studies: a. Satellite cells were isolated from mouse muscle fibers and cultured in vitro in the presenceof BrdU, a nucleotide analog that is incorporated into DNA during replication. After 4 days in BrdU, all satellitecells in the culture were extensively labeled with BrdU (pulse), as expected if these cells underwent symmetric divisions. The cells were then incubated for 18 hours in the absenceof BrdU (chase), a period of time that correspondsto approximately two cell divisions in these cells. The images below show two examples of muscle satellite cells undergoing division after this 18-hr incubation in the absenceof BrdU. The blue labeling (Hoechst) shows total DNA, the red labeling shows where BrdU-containing DNA is located.

Phase

Oldest strand Stem cell

ord strand New strand

I

s-ntaru Phase

J Mitosis

Asymmetric c e l ld i v i s i o n

Stem cell

Progenitorcell

Muscle satellitecells are progenitorsfor myoblastsand are the source of cells that result in muscle growth after birth and muscle repair after in;'ury. The satellite cells can replenish themselves,suggestingthat they also have properties of stem cells. To test the immortal-strand 946

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The majority of the cells appear like the dividing cell in the top panel, but about 1.5 percent of dividing cells appear like that in the lower panel. Can you explain theseobservations? Given that mice have 40 chromosomes,could the segregation of the BrdU-labeled sister chromatids, observedin the lower panel, have occurred by chance? b. Satellitecells were subjectedto a BrdU pulse-chase experiment similar to that describedin part (a) above and then were assessedfor the production of Numb, a protein whose presence or absence allows two daughter cells to adopt different developmentalfates.The micrographs below show a dividing cell stainedfor Numb (green)and for BrdUcontaining DNA (red). \fhat do you expect to be the outcome of the daughter cell that acquiresNumb? How might you determine if Numb is involved in generatingco-segregation of the older DNA strands?

c. Suppose you conducted a pulse-chaseexperiment using an establishedcultured cell line, but asymmetric divisions like that observedin the lower panel in part (a) above were not observed.Explain this result.

References The Birth of Cells: Stem Cells, Niches, and Lineage Aurelio, O., T. Boulin, and O. Hobert. 2003. Identificationof spatial and temporal cuesthat regulatepostembryonicexpressionof axon maintenancefactors in the C. elegansventral nervecord. Deuelopment13U599-6'1.0. Buszczak,M., and A. C. Spradling.2006. Searchingchromatin for stem cell identity. Cell 125:233-236. Chopra, V.S.,and R. K. Mishra. 2005. To SIR with Polycomb: linking silencingmechanisms.Bioessays27:1,19-121,. Copelan,E.A. 2006. Hematopoieticstem-celltransplantation. N. Engl. J. Med.354:1.81.3-1.826. Edenfeld,G., J. Pielage,and C. Klambt.2002. Cell lineagespecification in the nervous system.Curr. Opin. Genet.Deuel. 122473477. Feinberg,A. P.,R. Ohlsson,and S. Henikoff. 2005. The epigenetic progenitor origin of human cancer.Nature Reu.Genet. T:21,-33. Golden,J. A., S. C. Fields-Berr5and C. L. Cepko. 1995.Construction and characterizationof a highly complex retroviral library for lineageanalysis.Proc. Nat'|. Acad. Sci.U SA 9225704-5708. Hatfield, S.D., et al. 2005. Stemcell division is regulatedby the micro RNA pathway.Nature 435:974-978. Hochedlinger,K., and R. Jaenisch.2006. Nuclear reprogramming and pluripotency.Nature 441:1.061.-1067. Hori, Y., et aL.2002.Growth inhibitors promote differentiation of insulin-producingtissuefrom embryonic stem cells.Proc. Nat'|. Acad. Sci. USA 99:161.05-1.6710. Huelsken,J., et al. 2001. B-Catenincontrolshair follicle morphogenesisand stemcell differentiationin the skin. Cell 105:533-545. Li, L., and T. Xie. 2005. Stemcell niche: structureand function. Ann. Reu.Cell Deuel. Biol.2L605-631. Marshman, E., C. Booth, and C. S. Potten.2002. The intestinal epithelialstemcell. Bioessays24;9I-98. Morrison, S.J., and J. Kimble. 2005. Asymmetric and symmetric stem-celldivisions in developmentand cancer.Nature 441:1,068-1.074. Orkin, S. H. 2000. Diversificationof haematopoieticstemcells to specificlineages.Nature Reu.Genet. l:57-64. Reinhart,B. J., et al. 2000. The 21-nucleotide/er-7RNA regulates developmentaltiming in Caenorhabditis elegans.Nature 4032901,-906. SanchezAlvardo, A.2006. Planarianregeneration:its end is its beginning.Cell 124:241-245. Shafritz,D.A., et al. 2006. Liver stemcellsand prospectsfor liver reconstitutionby transplantedcells.Hepatology 43(2 Suppl 1):S89-9 8. Watt, F. M., C. Lo Selso,and V. Silva-Vargas.2006.Epidermal stem cells:an update. Curr. Opin. Genet.Deuel. 16:518-524. '!7u, H., and Y. E. Sun. 2006. Epigeneticregulation of stemcell differentiation.Pediatr.Res.59(4 Pt 2):21R-25R. Cell-Type Specification in Yeast Bagnat,M., and K. Simons.2002. Cell surfacepolarization during yeastmating. Proc. Nat'|. Acad. Sci.USA 99214183-14188. Coic, E., G-F.Richard, and J. E. Haber.2006. Cell cycle-dependentregulationof Saccharomyces cereuisiaedonor preference during mating-typeswitchingby SBF (Swi4/Swi6)and Fkh1. Mol. Cell Biol. 26:.5470-5480. Cosma,M. P.2004. Daughter-specificrepressionof Saccharomyces cereuisiaeHO: Ashl is the commander.EMBO Rep. 5:953-957.

Dittmar, G. A., et aL.2002.Role of a ubiquitin-like modification s. Science29522442-2446. in polarizedmorphogenesi Dohlman, H. G., and J. W. Thorner. 2001. Regulationof G protein-initiatedsignaltransductionin yeast:paradigmsand principles. Ann. Reu.Biocbem. 70:703-7 54. Hall. I. M.. et a\.2002. Establishmentand maintenanceof a heterochromatindomain. Science297:2232-2237. Kirchmaier,A. L., and J. Rine. 2005. Cell cyclerequirementsin Mol. Cell cereuisiae. assemblingsilent chromatin in Saccharomyces Biol. 26:852-862. Lau, A., H. Blitzblau, and S. P. 8e11.2002.Cell-cyclecontrol of the establishmentof mating-type silencing in S. cereuisiae.Genes Deuel. 16:2935-2945. Miller, M. G., and A. D. Johnson.2002' White-opaqueswitching in Candida albicans is controlled by mating-type locus homeodomain proteinsand allows efficientmating. Cell 110:293-302. Takizawa,P. A., and R. D. Vale. 2000' The myosin motor, Myo4p, binds Ashl mRNA via the adapterprotein, She3p.Proc. Nat'l. Acad. Sci.USA 97:5273-5278. Specification and Differentiation of Muscle Bailen P.,T. Holowacz, and A. B. Lassar.2001. The origin of skeletal muscle stem cells in the embryo and the aduk. Curr. Opin. Cell Biol.13:679-689. Berkes,C.A, and S.J. Tapscott.2005. MyoD and the transcriptional control of myogenesis.Semin.Cell. Deuel.Biol- 162585-595. Buckingham,M., S. Meilhac, and S. Zaffuan.2005. Building the mammalian heart from two sourcesof myocardial cells.Nature Reu. Genet.6:826-835. Dhawan, J., and T. A. Rando. 2005. Stemcells in postnatal actimyogenesis:molecularmechanismsof satellitecell quiescence, vation and replenishment.TrendsCell Biol. t5:666-673. Gustafsson,M. K., et aI.2002. Myf5 is a direct target of longrange Shh signalingand Gli regulationfor musclespecification. G enesDeuel.16z11'4-126. McKinsey,T. A., C. L. Zhang, and E. N. Olson. 2002. Signaling chromatin to make muscle.Curr. Opin. Cell Biol.74:763-772. Pipes,G. C., E. E. Creemers,and E. N. Olson. 2006. The myocardin family of transcriptional coactivators:versatileregulators of GenesDeuel.2O:1545-1'555. cell growth, migration,and myogenesis. Yan, 2., et al. 2003. Highly coordinatedgeneregulationin mouseskeletalmuscleregeneration./. Biol. Chem.278:8826-8836. Regulation of Asymmetric Cell Division Bellaiche,Y., and M. Gotta. 2005' HeterotrimericG proteins and regulationof sizeasymmetryduring cell division. Curr' Opin. Cell Biol. 17:658-663. Betschinger, J., and J. A. Knoblich. 2004.Dare to be different: asymmetric ..ll dirritiott in Drosopbila, C. elegansand vertebrates. Curr. Biol. 74:R67 4-68 5. Betschinger, J., K. Mechtler,and J. A. Knoblich. 2003. The Par complex directs asymmetric cell division by phosphorylating the cytoskeletalprotein Lgl. Nature 422:326-330. Betschinger, J., K. Mechtler,and J. A. Knoblich' 2006. Asymmetric segregationof the tumor suppressorbrat regulatesself-renewal in Drosopbila neural stem cells. Cell 124:1241-1253. and -independBhalerao,S., et al. 2005.Localization-dependent in Drosopbila. specification ent roles of numb contribute to cell-fate Curr.Biol. 15:l 583-l 590. Bowman, S. K., et al.2006.The Drosophila NuMA Homolog Mud regulatesspindleorientation in asymmetriccell division. Deuel. Cell 102731-742. Cleary,M. D., and C. Q. Doe. 2005. Regulationof neuroblast competence:multiple temporal identity factors specifydistinctneu.o.rr^l f"t", within a single early competencewindow GenesDeuel. 20:429434. REFEREN CE

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Cowan, C.R., and A. A. Hyman. 2004. Asvmmetriccell division in C. elegans:cortical polarity and spindlepositioning.Ann. Reu. Cell D euel.Biol. 20:4274 53. Fichelson,P.,et al. 2005. Cell cycle and cell-fatedetermination rn Drosophila neural cell lineages.Trends Genet. 212413420. Heidstra,R., D.'Welch,and B. Scheres.2004. Mosaic analyses using marked activation and deletionclonesdissectA rabidopsisSCARECROW action in asymmerriccell division. GenesDeuel. iS|De+-D69. Helariutta, Y., et al. 2000. The SHORT-ROOT geneconrrols radial patterning of the Arabidops,sroot through radial signaling.Cel/ 101:555-557. Hutterer, A., et al. 2004. Sequentialroles of Cdc42. Par-6. aPKC,and Lgl in the establishmintof epithelialpolarityduring Drosophila embryogenesis . Deuel. Cell 6:845-854. Kipreos,E. T. 2005. C. eleganscell cycles:invarianceand stem cell divisions.Nature Reu.Mol. Cell Biol.6:766-776. Lee, C-Y., K. J. Robinson,and C. Q. Doe. 2006.Lg|, Pins and aPKC regulateneuroblastself-renewalversusdifferentiation.Nature 439:594-598. Lee, C-Y., et al. 2006. Brat is a Miranda cargo protein that promotes neuronal differentiationand inhibits neuroblastself-renewal. Deuel. Cell 1O2441,449. Lu, H., and D. Bilder.2005. Endocyticcontrol of epithelialpolariry and proliferation in Drosophila. Nature Cell Biol. 7:1,232-1239. Nance,J. 2005. PAR proteins and the establishmentof cell polarity during C. elegansdevelopment.Bioessays27:126-135. O'Donnell, K.A., et al. 2005. c-Myc-regulatedmicro RNAs modulate E2F1 expression.Nature 4352839-843. Petritsch,C., et al. 2003. The Drosophila myosin VI Jaguaris required for basalprotein targetingand correct spindleorientation in mitotic neuroblasrs.Deuel. Cell 42273-28I. . !!"ll P.J., et al. 2003. A polarity complex of mPar-5and atypical PKC binds, phosphorylatesand regulatesmammalian Lgl. Na-ture Cell Biol. 5:30'l-308. Shapiro,L., H. H. McAdams, and R. Losick. 2002. Generatins and exploiting polarity in bacteria.Science298:1942-1946. Siegrest,S. E., and C. Q. Doe. 2005. Microtubule-inducedpins/ Go; cortical polarity in Drosopbila neuroblasts.Cell 123:1323-1,335. Siegrest,S. 8., and C. Q. Doe. 2005. Extrinsic cuesorient the cell division axis in Drosophila embryonic neuroblasts.Deuelopment 1332529-536. Siller,K. H., C. Cabernard,and C. Q. Doe. 2006.The NuMArelatedMud protein binds Pins and regulatesspindleorientation in Drosophila neuroblasrs.Nature Cell Biol. 8:594-500. 'Wang, H., and 17. Chia. 2005. Drosophila neuralprogenitor po_ larity and asymmetricdivision. Biol. Cett 97:63-74. Wodarz, A. 2005. Molecular control of cell polarity and asvmmetric cell division tn Drosophila neuroblasts.i"rr. Opln. Celt Biol. L7:475481. . Zarnescu,D. C., et al. 2005. FragileX protein funcrionswith lgl and the par complex in flies and mice.Deuil. Cell 8:43-52. Zgurski, J. M., et al. Asymmetricauxin responseprecedes asymmetricgrowrh and differentiationof asymmetricieafI and asymmetricleaf2 Arabidopsrsleaves.Plant Cell 17:77-91. Cell Death and lts Regulation Aderem, A. 2002. How to eat somethingbiggerthan your head. Cell ll0:5-8. Alvarez-Garcia,I., and E. A. Miska. 2005. Micro RNA functions in animal developmentand human disease.Deuelo\ment 132:46534662.

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Ambrose,V. 2003. Micro RNA pathways in flies and worms: growth, death, fat, stress,and timing. Cell 113:673-676. Baehrecke,E. H. 2002. How death shapeslife during development. Nature Reu. Molec. Cell Biol. 32779-787. Bao, Q., S. T. Riedl, and Y. Shi. 2005. Structureof Apaf-1 in the auto-inhibited form: a critical role for ADP. Cell Cycle 8: 1001-1003. Brennecke,J., et al. 2003. bantam encodesa developmentally regulated micro RNA that controls cell proliferation and regulates the proapoptotic genehid,in Drosophila. Cell 113z25-36. Cory, S., and J. M. Adams. 2002. The Bcl2 family: regularors of the cellular life-or-death switch. Nature Reu. Cancer 2: 647-656. Estaquier,J., and D. Arnoult. 2006. CED-9 and EGL-I: a duo also regulating mitochondrial network morphology. Molec. Cell 2l:730-732. Hay, B. A., and M. Guo. 2005. Caspase-dependent cell death in Drosophila. Ann. Reu.Cell Deuel. Biol.22:623-650. Green,D. R., and G. Kroemer.2004.The pathophysiologyof mitochondrial cell death. Science305:626-629. 'Weinberg. 2002. Taking the study of cancer Jacks, T., and R. A. cell survival to a new dimension.Cell 11l:923-925. Kinchen,J. M., et al. 2005. Two pathwaysconvergeat CED-10 to mediate actin rearrangementand corpse removal in C. elegans. Nature 434:93-99. Kinchen,J. M., and M. O. Hengartner.2005. Talesof cannibalism, suicide,and murder: programmedcell death in C. elegans. Curr. Top. Deuel. Biol. 65 1,45. Lakhani, S. A., et al. 2006. Caspases3 and 7:key mediatorsof mitochondrial eventsof apoptosis.Science10:847-851. Marsden, V. S., and A. Strasser. 2003. Control of apoptosisin the immune system:Bcl-2, BH3-only proteins and more. Ann. Reu. Immunol. 2l:71-'1,05. Penninger,J. M., and Kroemer,G. 2003. Mitochondria, AIF, and caspases-rivaling for cell death execution. Nature Cell Biol. 5:97-99. Riedl, S.J., and Y. Shi. 2004. Molecular mechanismsof caspase regulation during apoptosis. Nature Reu. Molec. Cell Biol. 5:897-907. Schafer,2.T., and S. Kornbluth. 2006.The apoptosome:physiological, developmental,and pathologicalmodesof regulation. Deuel. Cell 10:549-561,. Vaccari, T., and D. Bilder. 2005. The Drosophila tumor suppressor vps25 preventsnonautonomousoverproliferationby regulating notch trafficking. Deuel. Cell 9:687-698. Xu, P, M. Guo, and B. A. Hay. 2004. Micro RNAs and the regulation of cell death. TrendsGenet.20:6L7-624. Yan, N., et al.2004. Structural,biochemical,and functional analysesof CED-9 recognitionby the proapoptotic proteins EGL-1 and CED-4. Molec. Cell 24999-1006. Yan, N., et al. 2005. Strucrureof the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans.Nature 437 :831-837 . Yan, N., and Y. Shi. 2005. Mechanisms of apoptosis through structural biology. Ann. Reu. CelI Deuel. Biol.2L: 35-56. Zuzarte-Luis, V., and J. M. Hurle. 2002. Programmedcell death in the developing limb. Int'\. J. Deuel. Biol. 46; 871-876.

CHAPTER

Froma singlecellto a humanembryo.Twentyhoursafterthe fertilization of a humanegg,the maleand femalepronucleiare aboutto fuseand combinethe geneticinformationfrom fatherand mother.Forty-six dayslaterthe embryo,2 cm long,is beginningto developorgansand tissues, nourishedby bloodenteringthrough the umbilicalcord ICourtesy of TheLennartNilsson AwardBoard]

ust as notes and chords blend into a symphong genes, proteins, and cells act as an integrated system during embryogenesis,the development of an embryo. Signals flow within and between cells, and massivewaves of gene expressionallow thousands of types and shapesof cells to form (Chapters 7, 15, 16). For normal embryogenesis,the cell cycle must be regulated, as describedin Chapter 20, so that cell growth and division occur at the right times and places;cell lineageslike those describedin Chapter 21 must be organized in time and space; mechanisms described in Chapters 17-19 must organizecells into tissues,organs, and whole bodies; and cell death (Chapter 2L) must be programmed so that the webbing-not the fingers-is removed. In this chapter we explore the regulation of early-stage animal embryos to observe developmental mechanisms in context.'We concentrateon insectsand mammals,with some examplesfrom other animal species,as well as plants. After a brief summary of early development,we describehow eggs and sperm are made, how fertilization occurs, and the special genetic properties of early mammalian cells. Then we look at the earliestcell divisions in mammalian development and the creation of different layers of tissues.The formation of repeating segmentsin animal embryos and the genesthat eventually causethose segmentsto differ are discussednext. !7e also examine severalparticularly informative aspectsof later animal development, including formation of the leftright asymmetry of the bodS control of cell fates in the early nervous system, and the patterning of limbs. As we cover various topics, we will see how different experimental approaches-lineage tracing, genetic screens,mosaic animals, manipulations of signaling proteins, and transplantation-

THEMOLECULAR CELLBIOLOGYOF DEVELOPMENT

have been used to discover and analyze key molecular and cellular eventsthat build animals. Let's start by thinking about a simple situation in which a sheet of cells has formed through cell division, but all the cells are identical. To form a working tissue,each cell has to do its iob. Somemay divide, some may bend, some may send out a signal. Each cell must somehow learn its location and fate, and start to differentiate appropriately' Differentiation may entail activation of certain genes,production of particular proteins, an increase or decrease in cell division, changed shape,changed surfaceproperties and adhesion to other cells, the releaseof secretedsignals,the acquisition of electrical activity, polarization along one or more axes,

OUTLINE 22J

Highlightsof DeveloPment

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and Fertilization Gametogenesis

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22.3 Cell Diversityand Patterningin Early Vertebrate Embryos

22.4 Control of Body Segmentation:Themesand Variationsin Insectsand Vertebrates

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22.5 Cell-TypeSpecificationin EarlyNeural Development

22.6 Growth and Patterningof Limbs

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migration, or a combination of any of these. A mistake in any aspectof cell differentiation early in developmentcan be fatal to the organism. The fascination of developmentalcell biology lies in discovering how the integrated system of development works and why it is so successfuldespitevariations in environment, inherited genes, cell numbers, and nutrition. At the same time, this field offers a new way to explore evolution and human origins: how animal forms and speciesarose,are maintained, and change. In addition, many diseasesare most readily understood in the context of normal developmental processesgone awry. And it all beginswith a singlecell!

Highlightsof Development Single-celledorganismscan go through complex developmental lfe cycles during which their shapesand behaviors may changedramatically. An example is the life cycle of the malariaplasmodiumdiscussedin Chapter 1 (seeFigure 1-4). In this chapter,however,we concentrateon developmentof multicellularanimals.The earlieststagesof animal development accomplishseveralcrucial goals:combination of maternal and paternalgenomesin a new organism;an increase in the number of cells; formation of three main layers of cells, the first step in creation of different cell and tissue types; and the laying out of the main organization of the embryo-front to back, head to tail, and left to rieht.

DevelopmentProgresses from Egg and Sperm t o a n E a r l yE m b r y o The development of a new organism begins with the fusion of male and female gametes:an egg (oocyte), carrying a set of chromosomesfrom the mother, and a sperm, carrying a set of chromosomes from the father. The gametes,or sex cells, are haploid becausethey have gone through meiosis and thus contain only one set of chromosomes(seeFigure 5-3). They combine in a processcalledfertilizarion,creating the initial single cell, the zygote, that has two sets of chromosomes (one maternal and one paternal) and is therefore diploid. The zygote beginsto divide in a processcalled cleavage, which produces a mass of cells that often look rather similar to eachother (Figure22-1).The progenyof theseinitial cells will gradually become different, forming all the organsand tissues. Early in embryogenesisthe cells divide into two distinct sets: germ-line cells, which will give rise to gametes, and somatic cells,which will form most of the body but are not passedon to future progeny. Germ-line cells are carriers of genetic alterations and, in some cases,inherited disease states, but anything that happens to the DNA of somatic cells cannot be passedon to future generations. In order for a functioning organism to develop, the embryo must becomepolarized. That is, cells on one side (leftright or head-tail or front-back) behavedifferently than cells on another side. The information that sets the oolarities of Firstcleavage 2-cell stage 30 hours Oviduct

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CHAPTER 22

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eventsoccurThetimingisindicated in termsof hoursor daysafter release of an eggfromthe ovary.[Adapted fromT.WSadler, Langman's MedicalEmbryology,9Ihed , LippencottWilliamsand Wilkins,Figure2-1 1 l

THEMOLECULAC R E L LB | O L O G YO F D E V E L O P M E N T

the embryo may be provided in the form of localized proteins or RNA moleculesin the egg or the location where the sperm entersthe egg,which can causelocal changesthat begin the process of polarization. Alternatively, a random asymmetry of the early embryo may become fixed as the embryo's final polarity. To createthe initial tissuelayers, sheetsof cells fold and some cells move inward, a processcalled gastrulation. The embryo becomesmore three-dimensionaland consistsof three germ layers: the ectoderm ("outer skin"), the mesoderm ("middle skin"), and the endoderm("inner skin"). Although the concept of three germ layersis really an oversimplification of the more complex truth, eachlayer givesrise to particular tissuesand structures,even in rather diverse animals, and thus the terms have had enough value to persist (seeFigure 21-3). The cell movementsthat generatethe germ layers require that the surface properties of cells change to release or cement connections between cells. Cells in the early embryo can form either loose-knit massesclassifiedas mesenchyme or sheets called epithelia. In the course of development,cells can go back and forth betweenthesetwo states.Epithelia are usually polarized, one side of the sheet being describedas basal and the other apical. Early in development,cells begin to signal using the various types of secretedproteins discussedin Chapters 15 and 16. For signalingto be useful in creating new cell types,some cells must make the signal, while others make the receptor. Cells that are able to senseand respond to a signal are described as competenr.The polarization of the early embryo that begins to make cells different allows some cells to become signal producers and other cells to become signal receiverslsome cells both produce and receivesignals.Induction is the processwhereby a signal sent from one cell or set of cells influencesthe fates of other cells.For example, a signal may induce precursor cells to form neural tissue. Embryonic cells are constantly sampling their local environment to see how it conforms to their genetically programmed expectations.The absenceof an expectedsignal or the presenceof an inappropriate one can causea cell to die, divide, move, change identity, or change shape. One signal can inform a cell about its location along the dorsal-ventral (back-front) body axis, while another tells the cell its anteriorposterior (head-tail)position. Yet a third signal could tell the cell to commence mitosis. A cell can integrate multiple signals and thus respond simultaneouslyto different signals.In some cases,a cell's final responsewill not be a simple summation of the multiple signals.Instead,one signal may modify the cell's responseto another signal. In addition to local signals, long-distancesignals such as hormones, which can reach much or all of a developingor mature animal, coordinate the timing of events, stimulating growth or triggering other changes. Some developmental signals act in a concentrationdependentmanner.That is, the receivingcellsrespond in one way to high signal levelsand in a different way to low signal levels.Generally,cells close to the source of a signal will be exposedto high levels,and cellsfarther away to lower levels. Thus the location of the source, the location of cells that

have the relevant receptor, and the ability of the signal to move through tissuewill govern where particular responses to a concentration-dependentsignal occur.

As the EmbryoDevelops,Cell LayersBecome T i s s u e sa n d O r g a n s Molecular cell biology underlies the ways individual cells differentiate into different cell types, say those forming a kidney or the skin. Critical to cell differentiation is activation of specificgenes,such as the genesthat encodevarious keratins, the fibrous proteins of the skin. In addition to the differential gene expressionthat createsand charactetizesa type of cell, the formation of tissuesand organs dependson the proper arrangements of cells. For example, the lightsensitivecells of the eye must be arrayed on the retina surface, and muscle cells must be arranged so that they can move the lens and focus the image on the retina. Thus formation of organs and tissues depends on communication between cells as well as movements and rearrangementsof cells. The branching patterns of blood vessels,and the proper connectionsbetweennervesand muscles,are possible becausedevelopingcells communicate. The process of forming all the working parts of the body-the heart, liver, kidney, gonads, lungs' etc'-is known as organogenesis. Organogenesis often involves bending and folding layers of cells. An epithelial sheet of cellscan bend if cells along a stripe down the middle all constrict their apical end, while the other end is relaxed. Such coordinated changesin cytoskeletalshape and organization are crucial in organogenesis,as are signalsbetween different tissue layers. Circulatory systems and hearts typically undergo organogenesisearlg since abundant oxygen must be deliveredto every cell. Distinct cell types from distinct lineagescollaborate to form an organ. The skin has epidermis from the ectoderm and dermis from the mesoderm; the lens of the eye derives from one cell lineage and retina from another; limbs are made of muscle and bone and nerves and skin. Signals between tissuesof different origins allow formation of proper structuresat the right times and places. The organization of whole tissues, known as pattern formation, underlies much of the beauty of the natural world: flowers, butterfly wings, faces,coral reef fish; warning colors, camouflage,and mimicry. Symmetricpatterns are common in animals: Our left hand mirrors our right hand, for instance.Both hands are made of the samestuff; only the pattern differs. Patterns often include repeating units like body segmentsor vertebraeor fingers. Variations on the repeats confer different functions, like opposable thumbs. Symmetry breaking, or asymmetric patterning, is crucial to developmentof many animals, including humans. For example, our growing heart twists in a particular helical way to begin forming heart chambers. Although this chapter concentrateson development of the embryo, developmentcontinues after birth of an animal. Many tissuescontinue to grow and develop until the adult staqe is reached.And, as discussedin Chapter 21, some H I G H L I G H TOSF D E V E L O P M E N T

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tissuessuch as skin, blood, hippocampus (part of the brain), intestinal lining, and the eyesof fish continuously regenerate even in adults, or regenerateafter injury (e.g.,liver). In many animals, ecologicalcomplexitiessuch as changingconditions or migrations trigger metamorphosls.Insectembryos,for instance, metamorphose to become larvae (juveniles),which subsequently metamorphose to become adults; salmon metamorphoseto adapt from fresh to salt water. Aging also can be viewed as a developmentalprocess.As an animal ages, many cell types undergo physiological changesthat underlie changesin organ and tissuefunctions. That aging is a genetically programmed aspect of development is apparent from observationsof seeminglysimilar tissuesaging at different rates in different animal species.Aging can also be strongly influenced by environmental factors, so as in many developmentalprocessesthere is an interplay betweengenes,nutrition, and experience.

GenesThat RegulateDevelopment Are at the Heart of Evolution Evolution is a processof changein the forms and abilities of living organisms over generations.Just as no two humans are identical (even "identical" twins are not). differencesare found within any group of organisms.Occasionally the differencesconfer an advantage in reproduction. In fact the process of evolution has been employed by farmers and others for thousands of years, selectingplants and animals with useful properties and selectivelybreeding those. The enormous variation among strains of dogs, for example, has resulted from selection by humans over perhaps 10,000-20,000 years. The fossil record shows that natural selection, which has operated for hundreds of millions of years, has led to changes far greater than the differences among dogs. Changesin climate, geology, location and the presenceof other creatures created new ecological niches and animals evolved in ways that allowed life in many of them. Since the form and functions of any organism are controlled by an increasingly well-understood battery of development-regulatinggenes,changesin such genesmust account for the heritable changesthat underlie the evolution of different animals and plants. However, the mutations underlying inherited evolutionary changes,whether the selection was by humans or by natural causes,generally have been unknown in the pasr. This is beginning to changewith the current surge of knowledge about which genesmatter to the development of tissues and organs, along with the increasingavailability of DNA sequencesfrom many organisms. Theseadvancesare revealingthe molecular geneticdifferencesthat distinguish varietiesof plants and animal. Many changes in genes are harmful, and changes in genesthat control growth and/or development might be expected to have major deleteriouseffects.Indeed, many inherited human genetic syndromes (a syndrome ls a group of diseasefeatures that tend to occur together) involving birth defects or cancer have been linked to develoomental qenes. 952

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The conservation of most types of developmentallyimportant proteins across diverse animal speciesindicate that theseproteins existedin animal ancestorshalf a billion years ago. In many cases,particular proteins have remained engagedin generatinga particular organ or tissuetype over all that time, despite the dramatic morphological distinctions between,say,insect and mammalian hearts. The similarities in gene functions among animal specieshave allowed inferencesto be drawn about human gene functions and disease processesbasedon information from many different species. Evolution of animal form could in principle dependupon the emergenceof new genesand new proteins, but relatively few such casesare known. Instead changes in body form over generationsappear to be due primarily to alterations in hundreds of thousands of short regulatory DNA sequences that influence the transcription of genesand the processing of RNA. Most of the working protein "hardware" is similar in animals of vastly different morphology, but the "software" can changereadily and rapidly.

Highlights of Development r The paternal and maternal genomescome together in the fertilized egg. r Germ-line cells make eggs and sperm and some are inherited by progeny; somatic cells make all the rest of the body but are not passedon to progeny. r During embryogenesis,a largely symmetric fertilized human egg becomesan early embryo that is asymmetricalong the anterior-posterior(head-tail),left-right, and dorsalventral (back-front) axes (seeFigure22-1). r An early embryo forms three initial layers of cellsendoderm, mesoderm,and ectoderm-that give rise to different tissuesand organs. r Embryonic cells communicate with each other via secreted protein signals that bind to receptors on receiving cells, triggering changesin the receiving cells that lead to their differentiation into specificcell types. This process,in which one type of cell, or group of cells, sendsa signal that affectsthe fates of other cells, is called induction. r Organogenesisis the processof forming all the working tissuesand organs of the body. r Pattern formation is the process of organizing the shapes,sizes,and colors of tissuesand organs during development. r Many geneshave been identified that conrrol development, and damage to such genescan lead to birth defects, cancer,or tissuedegeneration, r Many rypes of development-regulatinggenesare evolutionarily conserved.Not only can the genesbe identified in a wide spectrum of animal types, but in many casesa gene plays a similar role in seeminglyvery different animals.This reflectsthe evolution of animals from common ancesrors.

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Gametogenesis and FertiIization lfhether in the comfort of a womb or the shelter of a shell. embryos confront similar challenges:starting with the right chromosomes,obtainingnutrition, organizinggrowth, making diversecell types, and creating pattern.

G e r m - l i n eC e l l sA r e A l l T h a t W e l n h e r i t The setting aside of germ-line cells early in developmenthas been hypothesizedto protect chromosomesfrom damage by reducing the number of rounds of replication they undergo or by allowing specialprotection of the cells that are critical to heredity. Whatever the reason, early segregationof the germ line is widespread (though not universal) among animals. In contrast, plants do nothing of the sort: Most meristems, the groups of dividing cells at the tips of growing shoots and roots, can give rise to germ-line cells. One consequenceof the early segregationof germ-line cells is that the loss or rearrangementof genesin somatic cells cannot affect the inherited genome of a future zygote. Fruit flies (Drosophila) were critical in working out many fundamentalaspectsof chromosomebehavior beginning almost a century ago. Now they are used to investigate development,genomics, and molecular cell biology, and a variety of mutant flies serveas models of human diseases.Thesemodel organismsalso are usefulfor examining gametogenesis, the creation of eggsloogenesisland sperm (spermatogenesis) . Drosophila oogenesisbeginswith a stem cell that divides asymmetricallyto generatea single germ-line cell (or simply germ cell), which divides four times to generate16 cells.One of thesecells will complete meiosis (seeFigure 5-3), becoming an oocyte; the other 15 cells become nwrsecells, which synthesize proteins and mRNAs that are transported through cytoplasmic bridgesto the oocyte (Figure22-2).The

proteins and mRNAs provided by the nurse cells are necessary for maturation of the oocyte and for the early stagesof embryogenesis;they also play a key role in organizing the main body of a fly. At least one-third of the Drosophila genome is represented in the mRNA contributed by the mother to the oocyte, a substantial dowry. Each group of 16 cells is surrounded by a singlelayer of somatic cellscalled the follicle, which depositsthe eggshell.The mature oocyte' or egg, is releasedinto the oviduct, where it is fertilized; the fertilized egg (the zygotel is then laid' In mammals,about 2,500 germ-linecell precursors,the primordial germ cells (PGCs) form during gastrulation and migrate to a part of the abdominal cavity's mesoderm where the gonad (ovary or testis) will eventually form' These primordial germ cells produce Kit protein, a cellsurface receptor for Steel, a mitogenic protein signal secreted from cells along the path of PGC migration. Thus, as the primordial germ cells migrate, they divide under the influence of Steel.After arriving at their destination in the gonads,primordial germ cells prepare for either oogenesis or spermatogenesls. As shown in Figure 22-3a, primordial germ cells in the developinghuman ovary continue dividing during months 2-7 of gestation to produce about six million primary oocytes. Of these, about 400,000 survive to puberty and about 500 will be ovulated during a lifetime. The primary oocytes begin meiosis but are arrestedat the first meiotic prophase. Because of this prophase arrest, primary oocytes are tetraploid, which provides extra copies of the genome to help provision the unusually large egg cell. Some oocytes remain in the first meiotic prophase for nearly 50 years.Puberty triggers rapid growth of primary oocytes,which will eventually be about 200 pm in diameter. Meiosis continues at ovulation, but is completed only after fertilization. Each meiosis yields a single mature oocyte, or egg cell.

D o r s a la p p e n d a g e

E g gm e m b r a n e Egg shell

Nascent eggshell

P o l a rg r a n u l e s Dorsal Anterior 4-

Posterior I

ventral

E E a r l yo o g e n e s i s

z

Mid-oogenesis

FfGURE 22-2 Drosophilaoogenesis.A singlegerm-cell givesriseto fifteennursecells(green) precursor anda singleoocyte ([) Theearlyoocyteisaboutthe same (yellow) earlyin oogenesis nursecells;thefollicle, a layerof somatic sizeasthe neighboring the oocyteandnursecellsThenursecellsbeginto cells,surrounds necessary for oocytematuration, mRNAs andproteins synthesize andthefolliclecellsbeginto formthe eggshellMidwaythrough (Z), the oocytehasincreased The in sizeconsiderably oogenesis (gray)The eggshell by thecompleted matureegg(B) issurrounded

Nucleus

E M a t u r ee g g

V i t e l l i n em e m b r a n e P e r i v i t e l l i n es p a c e

and synthesized but mRNAs nursecellshavebeendiscarded, early in the function cells nurse by the oocyte to the translocated regionof theegg in the posterior located embryoPolargranules The cellswillarise. markthe regionin whichgerm-line cytoplasm of the matureoocyte(e.g, the off-centerpositionof the asymmetry in the determination setsthestagefor the initialcell-fate nucleus) egg of the fertilization oviduct, into the release its After embryo An etal, 1993, fromA J F Griffiths embryogenesis triggers lAdapted p 643l andCompany, Analyso5thed, W H Freeman to Genetic lntroduction G A M E T O G E N E SA I SN D F E R T I L I Z A T I O N

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(a) Oogenesis

(b) Spermatogenesis

Embryo Embryo PGcs ,_\ /r-_{ t( l( )l multiplydurins \v7 mlgrarton \/ EntercouaryI Enters testisI v Y

After birth

| ruotu.ft'u,

I multiplication in embryo I Primary oocyte

< FIGURE 22-3 Mammaliangametogenesis. In bothmalesand females, the process of gameteformation beginsin theembryoand . iscompleted in theadult.(a)Oogenesis begins with proliferation of PGcs primordial germ cells(PGCs) in the earlyembryoandtheir ))multiplyduring migration accumulation at thesitewheretheovarywillform.Primary oocytes become arrested in meiosis I untilpuberty. Uponovulation, an oocyte completes meiosis I andarrests in meiosis Il.Meiosis ll iscompleted if theooryteisfertilizedEachmeiosis yields a singlehaploid oocyte; the otherproducts polarbodies, of meiosis become whicharenonfunctional. (b)Spermatogenesis alsobegins with PCGs multiplying in theembryo andaccumulating in the developing testis.Unlikeoocyteprecursors, (spermatogonia) spermprecursors arrestin G1of thecellcycleand do not commence meiosis untilafterbirth,Subsequent mitoticand meiotic yielding celldivisions areincomplete, haploid spermatids with (syncytia) bridgedcytoplasm Spermatids differentiate intomature (seeFigure spermin a process calledspermiogenesis 22-4)lAdapted fromL Wolpert et al, 2001,Principles of Development,2nd ed, Oxford University Press, Figure 12-18 l

3"x"#"" arrested in m e i o s i sI

Adult

I

I *

Spermatocyte

I Maturation primaryoocyte I of v Eggcoat Cortical granules of I Comptetion Ovulation I meiosisl, arrest in meiosis tl I Secondary oocyte

t I uetosis

v

Meiosis tl f Spermatids(syncytial

First polar body of Fertitizat;on I ComPletion merosrs tl +

Primary germ cells that migrate to the developing male testis have a completely different fate (Figure 22-3b). In the testis,they are arrestedin G1 of the cell cycle and no meiosis occurs.After birth thesearrestedcells, calledspermatogonia, resumemitosis. Their mitosis is a bit Deculiar.in that incomplete cytokinesiscausescells with briiged cytoplasm (syncytia, "same cells") to form. From the spermatogonia come primary spern'ratocytes, the cells that enter meiosis.still with 954

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cytoplasmic bridges. Each meiosis produces four haploid gametes,or spermatids,connectedto each other by cytoplasmic bridges. These bridges allow synchronization of germcell maturation and sharing of substancesbetweencells;thus the haploid cellsresulting from meiosiscan shareproducts of the unique X or Y chromosomeeachhaploid cell carries. Spermatids undergo a dramatic differentiation process (spermiogenesls)that generatesmature sperm cells (Figure 22-4).The Golgi apparatusmoves to one end of the cell to form the auosomal cap over the nucleus,while the flagellum beginsto form at the other end of the cell. Mitochondria coalescenear the base of the flagellum, ready to provide ATP for swimming. Much of the cytoplasm is extruded, the cytoplasmic bridgesare lost, and the nucleuscondensesto a mere shadow of its former self, driven by the arginine-rich protein protamine, which displaces the normal histones from the chromatin. The sperm is ready to go. In humans, formation of a sperm cell from a spermatocyte takes about two months. Human males produce about 108 sperm/day and more than 1012 in a lifetime. Human sperm are about 50 pm long, but Drosophila spermare considerably larger. Amazingly, the sperm in one fly speciesare about 5.8 cm long, which is twenty times longer than the male fly that produces them! A mature sperm consistsof a head, containing the acrosome and condensednucleus, and a flagellum tail. The tail contains a complex structure, the axoneme,composedof microtubules and dynein motor proteins whose movementspropel the sperm toward the egg (see Figures 18-29-18-31.).Mitochondria at the base of the tail provide energy in the form of ATP to power axoneme movement. Mutations that damage motor proteins in sperm flagella can cause sterility. In the inherited disease Kartagener's syndrome, for instance, defects in the dynein arms of the axoneme(sometimescausedby mutation of the dynein gene itself) cause immotile cilia and flagella. The consequencesinclude male infertility and situs inversus.

THEMOLECULAC R E L LB t O L O G yO F D E V E L O P M E N T

I Spermatid Nucleus itochondria

Flagellum Acrosomal vesicle

pairs, so an averagesize human chromosome of about 130 x 10o bp will have 130,000 differencesfrom its homolog. Add to this diversity the processof recombination (about one crossoverper chromosome) that gives rise to new combinations of sequences,and the diversity is striking. Male gametogenesisalso governs the sex of the next generation: Sperm carrying an X chromosome produce female offspring, while those carrying a Y chromosome produce male offspring.

F e r t i l i z a t i o nU n i f i e st h e G e n o m e o@,

Fertilization can be a spectacularevent, as when all the coral animals of the Great Barrier Reef of Australia release eggs and sperm on the same night once each year at a full moon. The process of fertilization, which results in union of an egg and sperm, comprisesseveralchallengingevents: penetrationof an egg by a singlesperm cell, assemblyof a diploid (no more and no less)genome,completion of the egg's meiotic division, and initiation of a proper program of gene activation. Sperm were first described in the late I Maturesperm 1600s, and eggs,being much largeq were recognized much Acrosomal earlier. Fertilization itself, however, was not directly observed and documented until the late 1800s. Pioneering studies on fertilization were done with sea urchin eggsand Nucleus Mitochondria Flagellum sperm becauseboth eggs and sperm are easy to isolate, grow in culture, and watch. Observation of the fusion of Head Tail the haploid pronuclei of the egg and sperm helped to dispel A FIGURE 22-4 Spermiogenesis. Thedifferentiation of a the earlier idea that sperm were contaminating animals (Il) intoa maturespermcell(@)involves spermatid a series of living in semen. dramatic morphological changesAt oneend,the acrosomal cap It is remarkable that a sperm is ever able to reach and formsoverthe nucleus, whichbecomes highlycondensed. At the penetrate an egg. In humans, for instance, each sperm is otherend,theflagellum elongates, andmuchof thecytoplasm competing with more than 100 million other sperm for a arounditsbaseis lost,leaving a sheathof mitochondria Although single egg, and it must swim a long distanceto approach not depicted, eachspermatid isconnected by cytoplasmic bridges to the egg. Moreover, the egg has multiple surrounding layers (seeFigure adjacent spermatids 22-3b)These aresevered during spermiogenesis, so eachmaturespermcellcanmoveindependently. that restrict sperm entry. Although sperm are streamlined for speedand swimming abilitS only a few dozen reach the of Biological Development, McGraw[Adaptedfrom K Kalthoff, 1996,Analysis egg in the oviduct (seeFigure 22-1'1.The flagellum of huHill,Figure 3 9, andL Wolpert et al, 1998,Principles of Development, 1sted, man sperm contains about 9000 dynein motors that flex OxfordUniversity Press, Figure 1221 l microtubules in the axoneme, causing successivebending that propels the sperm forward (seeFigure 18-31). Estimates of the force produced by a singledynein motor pro'1.-6 picoNewtons (pN). Since 1 pN is Situs inversus is a birth defect in which the heart and other tein range ftom organs are located on the wrong side of the body. This abenough to lift a red blood cell, a sperm clearly packs a lot normality results from the immotility of cilia that would of motor power. normally polarize the left-right axis of the body during gasThe acrosomal cap (or simplS acrosome),found at the t r u l a t i o n , a s w e s h a l l s e ei n S e c t i o n 2 2 . 3 . l tip of the sperm head, is a membrane-bound compartment specializedfor interaction with the oocyte. The acrosome's Severalcritically important events happen during gamembrane is just under the plasma membrane at the sperm metogenesis.In both sexesmeiosis reducesthe number of head; on the other side of the acrosome,its membraneis juxchromosomes to a haploid set. The sorting of chromotaposed to the nuclear membrane (seeFigure 22-41. lnside somesthat goes on is itself an extraordinary generator of the acrosome are soluble enzymesincluding hydrolasesand variation. Sincethere are 23 pairs of chromosomesin huproteases.Once a sperm approaches an egg, it must first mans and there is an equal chance of retaining either one penetrate a layer of cumulus cells that are derived from the of a pair in a particular gamete, 223 different possible ovarian follicle where the oocyte matured. The sperm then combinations of chromosomescould emerge from meioencounters the zona pellucida, a gelatinous extracellular sis. Among human populations, there is single nucleotide matrix, =6 p,m thick, that surrounds the egg (Figure 22-5a). sequenceheterogeneity about once in a thousand base The zona pellucidais composedlargely of three glycoproteins G A M E T O G E N E SA I SN D F E R T I L I Z A T I O N

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(a)

Zona pellucida

Polar body

Oocyte

< FIGURE 22-5 Gametefusionduringfertilization. (a)Mammalian eggs,suchasthe mouseoocyteshownhere,aresurrounded material, whichprovides bya ringof translucent thezonapellucida, a binding matrix for sperm. Thediameter of a mouse eggis=70pm,andthe productof zonapellucida is=6 pm thick.Thepolarbodyisa nonfunctional meiosisScalebar:30 p.m.(b)Inthe initialstageof fertilization, thesperm penetrates a layerof cumulus cellssunounding theegg([) to reachthe zonapellucida. Interactions betweenGalT, a proteinon thespermsurface, a glycoprotein in thezonapellucida, triggertheacrosomal reaction andZP3, (Z), whichreleases enzymes fromtheacrosomal vesicleDegradation of the zonapellucida by hydrolases andproteases released duringtheacrosomal reaction allowsthespermto beginentering theegg(B). Specific proteins recognition on thesur{aces of eggandspermfacilitate fusionof theirplasma membranes. Fusion andsubsequent entryof thefirstsperm (4 andS) triggerthe release nucleus intotheeggcytoplasm of Ca2* (orange) granules withintheoocyteCortical respond to the Ca2+surgeby fusingwith theoocytemembrane andreleasing enzymes thatacton the (a)courtesy zonapellucida to prevent bindingof additional sperm[Part of DougKlinePart(b)adapted f romL Wolpert etal, 2001,Principles of Development, Press, 2nded, Oxford Frgure 12-22l

a

Acrosomal reaction

E

E

Binding of sperm to zona pellucida Sperm recognizesZP3, a zona pellucida protein

Penetrationthrough zona pellucida

+-

Sperm nucleus enters egg cytoplasm

Releaseof cortical granules; Fusion of plasma membranes

E 956

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g

calIed 2P1., 2, and 3, the most interesting of which is ZP3 becauseit binds sperm. The sugar residues on ZP3 are neededfor the sperm to recognizethe oocyte; their removal preventsfertilization. The sugarsonZP3 are bound by beta(GalT) on the surface of the 1,4-galactosyltransferase-I sperm. \lhen ZP3 on the egg induces aggregationof multiple copiesof GalT on the head of one sperm, a G protein cascade in the sperm cell is triggered, resulting in exocytosisof the acrosomeand releaseof its contents onto the surfaceof the egg (Figure22-5b, stepsI and Z). This processcommonly is called the acrosomal reaction. Sperm lacking GalT are unable to bind ZP3 or undergo the acrosomal reaction, although other proteins of the sperm surface allow GalTdeficient sperm to still bind to the zona pellucida. The recognition of ZP3 is speciesspecific, thus preventing the formation of an inviable embryo with a sperm from one speciesand oocyte from another. As the releasedacrosomalenzymesdigestthe zona pellucida, the sperm can penetrate this barrier to its entry. The plasma membranes of the sperm and egg then touch and fuse, allowing the sperm nucleusto enter the egg (Figtre 225, steps B-E). Again, specific recognition proteins have been discovered that mediate membrane fusion, including CD9, an integrin in the egg plasma membrane, and Izumo, an immunoglobulin-domain protein in the sperm plasma membrane (Figure 22-6). Proteins such as Izumo and CD9 could becometargets for controlling fertility. Following mating of animals, sperm are in a race to approach and fuse with an egg. Under normal circumstances, many sperm cells are likely to reach each availableegg. The first sperm to successfullypenetratethe zona pellucida and fuse with an egg triggers a dramatic responseby the egg that prevents polyspermy, the entry of other sperm that would bring in excesschromosomes.After the first spermsucceedsin fusing with the surfaceof the oocyte, a flux of Ca2* flows through the oocyte at about 5-10 p,m/sec,starting from the site of sperm entry. One effect of this Caz* flux is to cause vesicleslocated just under the plasma membrane of the egg, called cortical granules,to releasetheir contents through the plasma membrane and form a shielding fertilization membrane that blocks other sperm from entering.In this cortical reaction,the movement of the granulesis controlled by a rich assemblyof actin microfilaments that forms from globular actin in responseto sperm entry. The Ca2* flux induced by spermtgg fusion also acts as an effective signal for beginning developmentof the zygote.Among the earliesteventsto occur is completion of meiosisby the egg (recallthat it is blocked in meiosisI, seeFigure22-3a). The haploid eggand spermnuclei can then fuse,yielding the diploid nucleusof the zygote. An oocyte contributes a considerabledowry to the newly formed zygote.In mammals and many other animal species, all the mitochondrial DNA in the zygotecomesfrom the egg; no sperm mitochondrial DNA survivesfertilization. Femalespecific mitochondrial DNA inheritance has been used to trace maternal heritage in human history, for example following early humans from their origins in Africa. Little or no transcription occurs during oocyte meiosis and the first embryo cleavages,so during this time the oocyte's RNA is

(a)

Phase

Hoechst33342

f{ F

\

+ \

(b)

Hoechst33342

(n

o

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FIGURE 22-5 A spermmembraneprotein EXPERIMENTAL mediatessperm-eggmembranefusion.lzumo,a proteinin the plasma of bothmouseandhumansperm,facilitates membrane (a)Hamster eggs spermandoocytemembranes fusionbetween with heterozygous wereinseminated of theirzonapellucida stripped lzumo-l- mousesperm.Phase-contrast lzumo*/ and homozygous spermSix by multiple showa singleeggsurrounded micrographs spermheads Hoechst 33342,whichstains hoursafterinsemination, blue,wasaddedto the medium.lnthelzumo*/-sperm,thewhite with the egg spermheadsreacting swelling indicate arrowheads with 33342.Nofusionwasobserved with Hoechst afterstaining /(b) hamster eggs zona-free In a second experiment, lzumo sperm. of humanspermin the presence withwild-type wereinseminated or controllgGantibodyAfter6 lzumo(anti-hlzumo) anti-human was 33342.Fusion with Hoechst werestained hours,the samples (whitearrowheads) but not in the in thecontrolsample observed spermdo not antibodyTheinactivated presence of anti-hlzumo 434:2341 N Inoue et al, 2005,Nature block.lFrom triggera polyspermy

crucial. Among the most abundant are histone mRNAs that have short poly-A tails and as a result are translated inefficiently. Translation of these histone mRNAs is regulated by a stem-loop binding protein (SLBP) that binds to nucleotidesin the 3' untranslated sequence.SLBP,regulated G A M E T O G E N E SA I SN D F E R T I L I Z A T I O N

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by phosphorylation, cycleswith the cell cycle so that histone mRNA is stabilized and translated preferentially during S phase.Other oocyte mRNAs are differentially activatedduring early embryonic development.

Genomiclmprinting ControlsGeneActivation Accordingto Maternal or Paternal Chromosome Origin One might expect that the two pronuclei contributed by the sperm and oocyte would have equal capacity to activare genes,except for the difference between the X and Y chromosomes. Yet even after fertrlization, when the paternally and maternally derived chromosomes are in the same nucleus,their male or female origin continues to have a lingering influence. This phenomenon has been revealed by experiments with mouse embryos. Diploid embryos can be constructed from two male pronuclei or two female pronuclei, which in theory should be adequate.However, zygotes with two male-derived haploid genomes form good extraembryonic tissuesbut highly defectiveembryos. Conversely, zygotes with two female-derivedhaploid genomes form quite good embryos but poor yolk sacsand placentas,which are extra-embryonic tissues. The explanation for these results is a process called genomic imprinting, which occurs during spermatogenesisand oogenesis.Imprinting is accomplishedby modifications of the chromatin, but not the DNA sequence,in the developing gametes so that only certain genesare accessiblefor subsequent activation and transcription. In humans, for example, the gene for insulinlike growth factor 2 (Igf2) is presenton both copies of chromosome 11.in the embryo, but is inactivated on the chromosome derived from the mother. Conversely,in some humans the lgf-2r geneon the male-derivedchromosome 5 is inactive, whereas the female-derived allele is active. Igf-2r encodes a receptor for lgf-2, which transports Igf-2 to the lysosome for degradation. Imprinting does not alter the nucleotidesequenceof DNA. For that reasoneither the maternal or paternal copy of every chromosome,if it endsup in a fertilized gamete,can function in development of progeny. Abnormalities in gene imprinting that cause defects in growth, a variety of inherited diseases,and cancer,highlight the importance of this process.The mechanismof imprinting involves differential methylation of DNA during germJine differentiation. The most common and important rype of methylation alters CpG dinucleotides,which occur about 30 million times in the mammalian genome. Commonly 50-80 percent of the C residuesin CpG dinucleotidesare modified by severalDNA methyltransferases. Most CpG methylations are erasedin primordial germ-linecells during embryonic development, thus allowing reactivation of imprinted genes. Subsequentre-methylationas germJine cellsdevelopdifferentially affects some genesdepending on whether a chromosome is going through oogenesisor spermatogenesis (i.e., whether the animal is male or female). Thus eggsand sperm end up with different, distinct imprints. After fertrlization, a second

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wave of demethylation occurs in cleavageand blastocyst-stage mouseembryos,though this doesnot affect imprinted genes. The reason imprinting has evolved is unclear.Relatively few genes,about eighty in mammals, are known to be imprinted. However, the genesregulated in this manner play a role in controlling embryonic growth and development.This observation suggestsa possible relation between growth control and the maternal or paternal origin of the genes.

Too Much of a Good Thing: The X Chromosome ls Regulatedby DosageCompensation Males have one X chromosome and females have two. Becauseof this differencein the number of X chromosomes in the two sexes,a femaleembryo potentially will make twice as much of each of the products encodedby X-chromosome genesas a male does. That double-doseturns out to be toxic, and a variety of mechanisms have evolved in many species,including humans, to restore a viable balance of sex-chromosomegeneexpresslon. About four days after ferilization, mammalian embryos consist of an outer sheathof cells, which will becomeextraembryonic tissuesuch as the placenta,and an inner mass of cells, which will form the embryo proper. At this stage,one of the rwo X chromosomes(eitherX-from the maternal parent or Xo from the paternal parent) in each of the inner cells of a female embryo becomeshighly condensedand transcriptionally inactive.Half the cellsare left with an active Xu chromosome; the other half, with an activeX- chromosome. X inactivation is a mechanism of dosagecompensatioz that ensures that males and femalesproduce roughly similar levelsof Xchromosome gene products. Once an embryonic cell undergoes X inactivation, the same X chromosome (X- or Xp) remains active and the other remains inactive in all the descendants of that cell. Thus all women are, in this sense,genetic mosaics,since half their cells have an activeX* and half have an active Xu. Becausethe two X chromosomesdiffer on averageat 1in 1000 basepairs, the cellsin femaleswill no longer be identical with respectto active X{inked genes.Males, in contrast, have the same active X chromosome in every cell. Mammalian dosagecompensation requires a region of the X chromosome called the X-inactivation center (Figure 22-7a). Vithin this region are tvvo genes,Xlsf and Tsix, that both encode long RNAs. Becausethe genesoverlap and are transcribed in opposite directions, their RNA products are antisensewith respect to each other (hencethe reversednames). Xlsr RNA, which is made from only one of the two X chromosomesin a female cell, actually coats the chromosome from which it is made. SinceXrsl RNA does not move to the other X chromosome,that X is not coated and as a result remains active. The mechanism for triggering expression of the Xlst gene from only one chromosome is incompletely understood. Cisacting control elementsin the X-inactivation center appear to detect the presenceof the two X chromosomesin female cells (Figure22-7b). A clue about the sensingprocesscomesfrom the observation that the two X-inactivation centersin a female

T H E M O L E C U L AC R E L LB | O L O G YO F D E V E L O P M E N T

(a) X-inactivationcenter (XlC)

coated X chromosome.This and other early changestrigger a seriesof chromatin modifications that lead to compaction and transcriptionalinactivity.

MouseX.aJ.];O

tel

Gametogenesisand Fertilization Tsix

r Early in mammalian embryogenesis,the primordial germ cells migrate to the gonadsand begin, but do not complete, the production of gametes(oocytesor sperm) in utero.

5kb

(b)Mechanism of dosagecompensation in females(2X)

I

XtCson two X chromosomes sense eachotherand pair.

Centromere

II

+ I

I In mammalian spermatogenesis,germ-line precursors form in the embryo and are stored in the testis; these diploid cells undergo meiosis after birth, forming haploid spermatids (see Figure 22-3b). Differentiation of spermatids (spermiogenesis)yields mature sperm, elongated cells with a long flagella and compressednucleus.

Xrstis transcribed on one X chromosome; Isrx on the other.

p Xrstnruncoatsthe chromosome fromwhichit is transcribed.

X

I n a c t i v eX

I

r In mammalian oogenesis,primary oocytes arrested in meiosis form in the embryo and are stored in the ovary; they complete meiosis, forming haploid mature oocytes (.ggs), following fertilization (seeFigure 22-3a1. Oocytes contain a pool of mitochondria, mRNAs, and other materials necessaryfor early development.

Active X

Changesin chromatinin Xlsf-coated X chromosomeinactivate most transcriptionfrom the chromosome.

FIGURE 22-7 Dosagecompensation in mammalianfemales. Earlyin embryogenesis, oneof thefemaleX chromosomes becomes highlycompacted in a special chromatin structure; thiscompacted, transcriptionally inactive chromosome iscalleda BarrbodyThe mechanism thatdetects the presence of two X chromosomes in the femaleembryoinvolves a regionon theX chromosome, theXinactivation pairingof center(XlC),diagrammed in (a).Thetransient the XlCson the maternal andpaternal X chromosomes in females initiates the process of dosage compensation, outlinedin (b).Since pairingandsubsequent eventsoccurrandomly in eachcell,halfthe cellswill havethe maternally derived X inactivated andthe other halfwillhavethe paternally derived X inactivated maleembryos Since haveonlyoneX chromosome no XICpairing canoccurandnoXrst RNAisproduced, hencethereis no X-chromosome inactivation cell transiently co-localize prior to one of the X chromosomes becoming inactive.This colocalization may result in (1) low expression of Tsix from the chromosome that will be inactivated, which leads to (2) a temporary chromatin-regulated silencing of Xist transcription, followed by (3) a surge of high Xlst expressionleading to inactivation of the chromosome. After production of Xlsz RNA, Polycomb-groupproteins modify Iysine 27 residues on histone H3 tails of the Xist-

r During fertilization, sperm initially recognize and bind to the gelatinous layer (zona pellucida) on the outside of the oocyte. This interaction, which involves glycoproteins (e.g.,ZP3l in the zona pellucidaand proteinson the sperm surface, triggers the acrosomal reaction in the sperm. Enzymesreleasedfrom the sperm acrosomedegradethe zona pellucida, leading to fusion of the sperm and egg plasma membranes(seeFigure22-5). r Fertilization must be restricted to a single sperm to prevent chromosome imbalances. Sperm-egg fusion induces changesin the oocyte that exclude entry of other sperm. r Although the sperm and oocyte each contribute a haploid genome,genomic imprinting restrictswhich allelesare active in each cell. In the caseof some imprinted genes,the paternal copy is active in the embryo and the corresponding maternalcopy is inactive,or vice versa. r Males survive with one X chromosome, but the two X chromosomesin femalescould lead to a double dose of Xgene products that is harmful. This situation is avoided in human females by inactivation of one of the two X chromosomesin every cell of an early embryo, a processcalled dosagecompensation (seeF igure 22-7 ).

CellDiversityand Patterning in EarlyVertebrateEmbryos Fertilization produces a single cell, the zygote, which divides rapidly. \Within a few days, the newly formed cells start sending and receiving various signals that largely determine their future fates. The first distinctions to appear are tlvo different

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A FIGURE 22-8 Cleavagedivisionsin the mouseembryo.There is littlecellgrowthduringthesedivisions, sothatthe cellsbecome

progressively smaller. Seetextfor discussion. Oxford University [Copyright Press, T.Fleming l

cell rypes and then the initial polarization of the embryo along its various axes. In time. further distinctions lead to formation of multiple cell layers from which tissuesand organs begin to form. Much of this early fate determination requires signaling systemsthat we discussedin Chapters15 and 16. The general theme is that a few cells will begin to produce a signal, and other cells will produce the receptors that make them responsive to that signal; signal reception then induces the production of certain transcription factors that regulate batteries of genesto control the fate of the receiving cell.

Whether a cell assumesa TE or ICM fate is determined by its location within very early embryos. This can be demonstratedexperimentally by placing a labeledcell on the outside or the inside of a very early embryo (Figure 22-9) Labeled cells placed on the outside form extra-embryonic tissues(TE fate) almost exclusively,and those insertedinside preferentially form embryo tissues (ICM fate). DNA microartay analysis of gene expression at each stage of early development reveal dramatic changes in which genes are expressedas the embryo progressesfrom the 2-cell to blastocyst stage.Even thesevery early embryos use'Wnt, Notch, and TGFB signaling pathways (Chapter 16). Both ICM and TE cells have the properties of stem cells: That is, each type can divide to renew itself and to start its own distinct lineagethat producesdiversepopulations of differentiated cells. The inner cell mass is the source of embryonic stem (ES) cells, which can contribute to any part of the embryo (seeFigure 21.-7).The isolation of thesecells as cell lines has enableddramatic advancesin mouse geneticmanipulations. The earliest known sign of the ICM fate is expression of the Oct4 gene, which is a critical regulator for maintaining cells in a plastic pluripotent state. As shown in Figure 22-9a,ICM cellsare locatedon one side of the blastocoel, while TE cells form a hollow ball around the inner cell mass and blastocoel. At this blastocyst stage,the TE cells are in an organized epithelial sheet, with cell-cell junctions between adjacent cells. In contrast, the ICM cells are a loose mass that can be described as mesenchyme,a term commonly applied to loosely organized and loosely attached cells. During development, cells often undergo an epithelial-mesenchymaltransition, or the reverse. Epithelial cells form sheets that act as a barrier, move in harmony, and have a clear polar character from one side of the sheet to the other. Mesenchymal cells are bold individuals, less responsive to peer pressure.With their ability to cut loose from each other, they can migrate as individual cells, seednew organs, form circulating blood cells, and adhere in a three-dimensional mass, such as the inner cell mass.

CleavageLeadsto the First Differentiation Events The zygote is the ultimate totipotent cell becauseit has the capability to generateall the cell types of the body. Fertilization is quickly followed by cleavage,cell divisions that take about one day each in the mouse. In mammals, cleavagedivisions occur before the embryo is implanted in the uterus wall (seeFigure 22-1).The condensed,transcriptionally inactive chromatin from the sperm returns to a more normal state by replacement of the sperm's special histones with normal histonesprovided by the oocyte. Initially the cells are fairly sphericaland loosely attached to each other (Figure 22-81.As demonsrratedexperimentally in sheep,each cell at the eight-cell stagehas the potential to give rise to a complete animal. Three days after fertilization the 8-cell embryo divides again to form the 15-cell morula (from the Greek for "raspberry"), after which the cell affinities increasesubstantially and the embryo undergoescompaction, a processthat dependsin part upon the surfacemolecule E-cadherin (Chapter 19). Compaction is driven by increased cell-cell adhesion and initially results in a more solid mass of cells, the compacted morula. Next, some cell-cell adhesionsdiminish, and fluid beginsto flow into an internal cavitS the blastocoel.Additional divisions produce a blastocyst-stageembryo, composed of =64 cells that have separated into two cell types: trophectoderm (TE), which will form extra-embryonic tissueslike the placenta, and the inner cell mass (ICM), which gives rise to the embryo proper.

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A EXPERIMENTAL FIGURE 22-9 Celllocationdetermines cell fate in the earlyembryo,(a)A 4-cellembryonormally into develops (TE) a blastocyst consisting of trophectoderm cellson theoutside and innercellmass(lCM)cellsinside(b)Inorderto discover whether positionaffectsthe fatesof cells,transplantation were experiments donewithmouseembryosFirst, recipient morula-stage embryos had cellsremoved to makeroomfor implanted cellsThendonormorulastageembryos weresoakedin a dyethatdoesnottransferbetween labeled cellsFinally, cellsfromthedonorembryos wereinjected into inneror outerregions of therecipient embryos, asshownin the

embryoisheldin placebya slightvacuum Therecipient micrograph fatesof the to theholdingpipette(c)Thesubsequent applied labeled cellsweremonitoredFor of thetransplanted descendants (16morula-stage although cellsaredepicted, 4-cellrecipient simplicity, The results, recipients donors and were used as both cell)embryos form showthatoutercellsovewhelmingly in thegraphs, summarized innercellmassbutalso andinnercellstendto become trophectoderm (a)and(c)adapted fromL trophectoderm formconsiderable lParts

T h e G e n o m e so f M o s t S o m a t i cC e l l s Are Complete

scriptionally active (Chapters 6 and 7). A cell could, for example, have an intact genome,but be unable to properly reactivate it due to inherited chromattn states. Further evidencethat the genome of a differentiated cell can revert to having the full developmental potential characteristic of an embryonic stem cell comes from experiments in which differentiated olfactory sensory neurons were genetically marked with green fluorescence protein (GFP) and then used as donors of nuclei. S7henthe nuclei from differentiated olfactory cells were implanted into enucleatedmouseoocytes,14 percentof the oocytesdeveloped into blastocyststhat produced GFP. These GFP-marked blastocystswere used to derive embryonic stem (ES) cell lines, which were then used to generatemouse embryos. After implantation into female mice, these embryos' derived entirely from olfactory neuron genomes' formed healthy mice. Thus the genome of a differentiated cell can be reprogrammed completely to form all tissues of a mouse.

What is happening to the genome during early embryonic stages?Although differentparts of the genomeare transcribed in different cells,the genomeitself is thought to be identicalin nearly all cells.One well-documentedexceptionoccursduring developmentof lymphocytesfrom hematopoieticprecursors. Segmentsof the genomeare rearrangedor lost during lymphocyte development,generating clones of lymphocytes whose genomesare unique (Chapter 24). Also, mature erlthrocytes (red blood cells) lack a nucleus and thus have no nuclear genome.Most somaticcells,however,appearto havean intact genome,equivalentto that in the germ line. Evidencethat at least some somatic cells have a complete and functional genome comes from the successfulproduction of cloned animals by nuclear-transfercloning. In this procedure,the nucleus of an adult (somatic)cell is introduced into an egg that lacks its nucleus;the manipulated egg, which contains the diploid number of chromosomesand is equivalent to a zygote, then is implanted into a foster mother. The only source of geneticinformation to guide development of the embryo is the nuclear genome from the donor somatic cell. The frequent failure of such cloning experiments raises questions about how many adult somatic cells do in fact have a complete functional genome.Even the successes, like the famous cloned sheep"Dolly," have some medical problems. Even if differentiated cells have a physically complete genome, clearly only portions of it are tran-

Wolpert et al , 2OO1, Principlesof Development,2nd ed , Oxford Press,Figure312 Part(b) from R L Gardnerand J Nichols,1991, Human Reprod6:25-35.1

GastrulationCreatesMultiple TissueLayers, Which BecomePolarized The embryonic cells composing the inner cell mass of the blastocysthave impressiveabilities, but they hardly look like an embryo. Soon after the blastocystimplants into the uterine wall, the loose-knit conglomerationof ICM cellsis turned into a multi-layered structurewith head-tail' front-back, and

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< FIGURE 22-10 Humandevelopmentfrom days7 to 11 after fertilization.lmplantation of the blastocyst intothewallof the uterusoccurs at about7 days(seeFigure22-1 for earlier stages). The ispolarized blastocyst in thatthe innercellmass(lCM)isat oneend (fluid-filled andthe blastocoel cavity) at theother,bothencased in trophectoderm cells, whicharenow knownasthe trophoblast By day7 (tr), the ICMcellsof the blastocyst havealready begunto (green) separate intotwo layers-thehypoblast andthe epiblast (blue). Theepiblast willformtheembryoproperandthe hypoblast (inaddition willformextra-embryonic structures to thoseformedby trophoblast) Next,thetrophoblast cellsproliferate andinvade the wallof the uterus(E), whichis necessary for theembryoto start receiving nourishment fromthe mother. Byday9 (B), theamniotic cavity(gray), whichformsbetween theepiblast andthetrophoblast, is enlargingBy10-11 days(4), theembryo(nowthesizeof a period on thispage)isfullyimplanted andisbeingsupplied bythe maternal (white)isderived circulatory systemTheextra-embryonic endoderm fromthe hypoblast andwillformtheyolksac,a remnant of our evolutionary history whenourancestors required significant amounts of yolkto nourish theembryoThetwo celllayers of the implanted embryoaretransformed intothe threegermlayers duringthe next stage,gastrulatron from5 F.Gilbert, 2006,Developmental [Adapted Biology, 8thed, Sinauer Press, Figure 11-33l

Epiblast

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left-right polarity. As depicted in Figure 22-10, the inner cell mass initially separatesinto two layers called the hypoblast and the epiblast. The epiblast will form the embryo proper. The hypoblast will form extra-embryonic structures that connect to the mother's circulation, Other extra-embryonic structureswill form from the trophectoderm, called the troph o blast after implantation. In the mouse, in which axis formation has been studied extensively,the first sign of the anterior-posterior axis becomes apparent at 5.5 days (2 weeks in humans). At this time a visible groove, the primitiue streak, forms on the surface of the epiblast in the region that will becomethe posterior of the embryo. Along the primitive streak, cells leavethe epiblast layer and move into the spacebetween the epiblast and hypoblast in the processof gastrulation (Figure 22-11). The first cells to invaginate form embryonic endoderm; 952

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FIGURE 22-11 Gastrulationin animals.Duringgastrulation, epiblast cellsmigrate to create thethreegermlayers: ectoderm, mesoderm, andendoderm. Cellsin the different layers havelargely (a)Shown distinct fatesandtherefore represent distinct celllineages hereisa sketchof a humanembryoabout16daysafterfertilization of an egg Thefirstcellsto movefromtheepiblast intothe rnterior (notshown)andappear formendoderm. Theydisplace the hypoblast asa layeralongthe bottomof the interior spaceThenextcellsto invaginate becomemesoderm Theremaining epiblast cellsbecome ectoderm(b)Scanning electron micrograph of a crosssection of a (a)adapted similar-stage embryo[Part fromN A Campbell andJ B Reece,2005, Biology,7lh ed , Pearson/Benjamin Cummings,Figure47 13 P a r t( b ) c o u r t e s yo f K a t h yS u l i k ,U n i v e r s i toyf N o r t hC a r o l i n a - C h a p eHli l ll

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later arrivals becomemesoderm.Cells that do not invaginate to become endoderm or mesoderm remain behind in the epiblast to becomeectoderm. The post-gastrulationembryo proper thus has three germ layers and is polarized along the dorsal-ventralaxis and the anterior-posterioraxis. The ectoderm will make neural and epidermal cells; the mesoderm will make muscle, connective tissue, and blood; the endoderm will make gut epithelia. Once the three germ layers are established,they subsequently divide into cell populations with different fates. There appearsto be a progressiverestriction in the range of cell types that can be formed from stem cells and precursorcells as development proceeds.An early embryonic stem cell, as we've seen,can form every type of cell; an ectodermal cell has a choicebetweenneural and epidermalfates;a keratinocyteprecursor can form skin but not neurons. These observations raise two important questions:How are cell fates progressivelyrestrictedduring development?Are theserestrictionsirreversible?Suchquestionsare often addressedwith transplant experiments,asking what a cell will do when moved to an ab'lDfill normal position in an embryo. it retain the fate of the old location, or adopt a fate appropriate to the new location? In addressingsuch questions,it is important to remember that what a cell in its normal in vivo location will do may differ from what a cell is capableof doing if it is manipulatedexperimentally. Thus the observedlimits to what a cell can do may result from natural regulatory mechanismsor may reflect a failure to find conditions that revealthe cell'sfull potential. The fates of different parts of the early embryo were determined in the early days of embryology by marking amphibian or chick embryo cells with ink and tracking them. More recently, chimeric animals composed of chicken and quail cells have been used to study cell-fate determination during embryonic development.Embryos composedof cells from both bird speciesdevelop fairly normally, yet the cells derived from each donor are distinguishableunder the microscope.Thus the contributions of the different donor cells to the final bird can be ascertained.In addition to testing which cells can form which types of tissue, the rigidity of cell-fate determination can be tested.'When cells from one germ layer are transplantedinto one of the other layers,they do not give rise to cells appropriate to their new location. Thus the endoderm, mesoderm, and ectoderm are not only morphologically distinct; they are firmly determined as different cell types with different fates. In vertebrates,secretedprotein signalsare involved in directing not only the initial formation of the germ layers but also their polarization along the body axis. In the remainder of Section22.3, we examine the role of secretedsignalsand their antagonistsin early development.

SignalGradientsMay InduceDifferent Cell Fates The most intensiveand revealingstudiesof gastrulation and formation of the initial tissueshave been done in the clawed toad Xenopus laeuis (more commonly describedas a frog). By dissecting and transplanting Xenopzs tissues,developmental biologists have identified powerful signalsthat direct cell fates in the early embryo. Some of these signals have

been found to function in similar ways in early mammalian embryos. The Xenopu.segg is huge, about 1 mm in diameter. After fertilization, this giant cell divides, initially without growth, to make successivelysmaller cells that vary in size.Once the embryo is a ball of cells,a blastocoelcavity openswithin the ball. The cells at one end of the embryo are large; this side of the embryo is called the uegetalpole. Cells at the oppositeend are smaller; this side is known as the dnimdl pole. Morphological and molecular markers clearly revealthe polarization of the embryo by the midblastula stage.For example,the proteins VegT and Vg-1 are molecular markers for the vegetal pole. On what will become the dorsal side ("back") of the embryo, the protein B-cateninaccumulatesto high levels.The position of B-cateninaccumulationis controlled by the site of sperm entry. As we have seenin Chapter 16, B-cateninis activated as a transcription factor by Vnt signals (seeFigure L6-32). Even in the absenceof a Wnt signal, accumulatedBcatenin can trigger the induction of specificgenes. The accumulationof B-cateninis the earliestknown indicator of the frog dorsal-ventral axis. More importantly, Bcatenin is essentialfor the formation of two signal-emitting centerson the dorsal side of the late blastula: the Nieuwkoop center and the BCNE (blastwla chordin and noggin expression) center (Figure 22-1,2). The Nieuwkoop center forms where Vegl Vg-1, and high levelsof B-cateninoverlap, that is, in the vegetalpart of the dorsal side.Someof the future endoderm is locatedin this sameregion. The Nieuwkoop center releasesNodal proteins, which are members of the transforming growth factor B (TGFB) family of secretedprotein signals (Chapter 16). The BCNE center,which forms where future neural-ectodermcells arise, is detectablethrough its expressionof genesencoding some transcription factors as well as Chordin and Noggin. Thesetwo secretedproteins are antagonistsof bone morphogenetic proteins (BMPs)' other membersof the TGFB family. Thus the BCNE centerseemsto be mainly involved in preventingBMP action' At the equator of the slightly later, gastrula-stageembryo, a central belt of cellsthat forms betweenthe ectoderm at the animal pole and the endoderm at the vegetal pole becomesthe mesoderm. During Xenopus gastrulation,the cellsforming the future endoderm and mesoderminvaginateby the samesort of process that occurs in mammalian embryos (seeFigure 22-111. 'We know now that the mesoderm is induced by sigTGFB signaling proteins. In the search particularly nals, for mesoderm-inducingfactors, researchersadded everything but the kitchen sink to early frog embryos. A remarkable range of moleculeswas found to induce mesoderm cells, including many that could not possibly be natural inducers.For a molecule to be considereda genuine' natural inducer,it has to meet three criteria: (1) the molecule has to be necessaryfor mesoderminduction to occur (based on interferenceor mutants); (2) it has to be sufficient to induce mesodermfrom cells not normally fated to be mesoderm (based on ectopic expressiontests, that is, expression in abnormal sites); and (3) it has to be produced at the right time and place in a normal embryo (basedon antibody staining or in situ hybridization). Few

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Endoderm Spemann organlzer

A FfGURE 22-12 Signalingcentersin the early Xenopus embryo.Cross-sections of the embryoat threestages areshown. Thethreegermlayers-endoderm (yellow), (red),and mesoderm (blue)-aredetermined ectoderm bythe gastrula stage(!) They (0 and Z), whicharepolarized derivefromearlier embryos byVegT (atranscription factor)andVg-1(aTGFBsignal) to formtheanimal

poleandby Nodalsignals fromthe Nieuwkoop centerto form ventral-to-dorsal cellsMesoderm is induced by signals comingfrom thevegetal regionto the centralembryoDorsal-ventral cellfates arealsocontrolled by signals fromthe Spemann organizer in the gastrulaSeetextfor furtherdiscussion. fromL Wolpert etal, [Adapted 2001 of Development, 2nded, Oxford Press, Figure 3-35l , Principles

molecules fit all these requirements, but some TGFp proteins do. In some cases,the induction of cell fates involves a bi, nary choice:In the presenceof a signal,the cell is directed down one developmentalpathway; in the absenceof the signal, the cell assumesa different developmental fate or fails to develop at all. Such signalscan work in a relay mode. That is, an initial signal inducesa cascadeof induction in which cellscloseto the signalsourceare inducedto assume specific fates; they, in turn, produce other signals to organize their neighbors(Figure22-13a). Alternativelg a signal may induce different cell fates, depending on its concentration. In this gradient mode, the fate of a receiving cell is determined by the amount of the signal that reachesit,

which is related to its distance from the signal source (Figve 22-13b). Any substancethat can induce different responsesdepending on its concentration is often referred to as a morphogen. The concentration at which a signal induces a specific cellular responseis called a threshold. A graded signal, or morphogen, exhibits severalthresholds,each one corresponding to a specificresponsein receiving cells. For instance,a low concentration of an inductive signal causesa cell to assume fate A, but a higher signal concentration causesthe cell to assumefate B. In the gradient mode of signaling, the signal is newly created,and so it has not built up to equal levels everywhere.AlternativelS the signal could be produced at one end of a field of cells and destroyedor inactivated at the other (the "sourceand sink" idea),so a gradeddistribution is maintained. Studieswith activin, a TGFB-type signaling protein that can alter cell fate in early Xenopus embryos,have provided insight into how cells determine the concentration of a graded inductive signal. Activin helps organize the mesoderm along the dorsal-ventral axis of an animal. Specific genesare used as indicators of the tissue-creatingeffects of signals such as activin. For instance,a low concentration of activin induces expression of the Xenopus brachyury (Xbra) genethroughout the early mesoderm.Xbra is a transcription factor necessaryfor mesoderm development. Higher concentrations of activin induce expression of the Xenopws goosecoid (Xgsc) gene. Xgsc protein is able to transform ventral into dorsal mesoderm;so the local induction of Xgsc by activin causesthe formation of dorsal, rather than ventral, mesodermal cells near the activin source. Using 35S-labeledactivin, scientists demonstrared that Xenopus blastulacellseachproduce some5000 type II TGFBlike receptors that bind activin. Findings from additional experiments showed that maximal Xbra expression was achieved when about 100 receptors were occupied. At a

( a ) R e l a ys i g n a l i n g

( b ) G r a d i e n st i g n a l i n g

A FfGURE22-'13Two modesof inductivesignaling.Inthe relay mode(a),a short-range signal(redarrow)stimulates the receiving cellto sendanothersignal(purple), andso on for oneor more roundsInthe gradient mode(b),a signalproduced in localized s o u r ccee l l s( p i n kc e l l sr)e a c h ense a r bcye l l si n l a r g ear m o u n ttsh a n theamounts reaching distantcellslf the receiving cellsrespond differently to different concentrations of thesignal(indicated by widthof thearrows), thena singlesignalmaycreatemultiple cell woe5 964

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concentration of activin at which 300 receptorswere occupied, cells began expressinghigher levels of Xgsc. Similar results were obtained with blastula cells experimentally manipulated to produce sevenfoldhigher levelsof the activin type II receptor. These findings indicate that blastula cells measure the absolute number of ligand-bound receptors rather than the ratio of bound to unbound receptors, and confirm the importance of signal concentration.

SignalAntagonistsInfluenceCell Fatesand TissueInduction The most dorsal of the mesodermcells in the early frog gastrula become a famous signaling center called the Spemann organizer (Figure 22-12, B). This organizer, reported by Spemannand Mangold in 1,924,can be transplanted into a host embryo, where it takes over part of the host embryo and causes the formation of a nearly complete new axis (Figure 22-1,4).The Spemannorganizer is formed under the influence of Nodal and perhaps other signals from the Nieuwkoop center. The Spemannorganizer is the source of a remarkable number of secreted signal antagonists. These include Chordin and Noggin (BMP antagonists);Frzb-1, Crescent, sFRP2,and Dkk-1 (!7nt antagonists);and the multi-signal antagonist Cerberus, which dampens the effects of lfnt and BMP and Nod,al signals. Ifhen Noggin is applied to embryos that lack a functional Spemann organizer, the embryos develop normally. This finding shows that Noggin all by itself can mimic the effect of the Spemann organizer.A ventral signaling center, at the opposite end of the mesoderm from the Spemann organizer,makes several signalsincluding BMP4 (a TFGB family signal). Thus the

FIGURE 22-14 AtransplantedSpemann EXPERIMENTAL organizerdirectsformation of a new body axis in host embryo. (Iop)Normalfrogembryoat swimming When tadpolestage.(Boftom) center,istransplanted organizer, a powerfulsignaling the Spemann (right,the intoan earlyhostembryosothat it now hastwo organizers implantisthewhitepatchat the bottom),muchof the mainneural (left).Seetextfor discussion axisisduplicated andmesodermal [From H. 2004, Ann. Rev. CellDevelBiol2O:2851. and Kuroda, E deRobertis

two signaling centers, one dorsal and one ventral, appear to battle each other for control of cell fates across the mesoderm. You have undoubtedly noticed that many of the moleculesinvolved in tissueformation in early embryos are signal antagonists. One effect of such an antagonist can be to sharpen or move a boundary between cell types. A signal coming from source cells is progressively less potent with distance; at some point, it falls below a threshold amount and is without effect. If a secretedantagonist comes from the opposite direction, it will block the action of the signal even in cells receivingabove-thresholdamounts. 'We seean example of this phenomenon in the formation of neural cells in the anterior of frog embryos. NormallS secreted TGFB proteins preuent the formation of neural cells near the animal pole of Xenopus embryo. When the animal pole is removed and placed into culture' it is called the "animal cap." Production of BMP4 (a TGFB family signal; see Chapter 151by an animal cap prevents formation of neural tissue in the culture. The effect of signals and other regulators on neural induction can be tested by exposing parts of the animal cap from Xenopus embryos to individual proteins and seeingwhether neural cells form. This type of in vitro experiment first revealedthe ability of Chordin to antagonize BMP4 and induce neural-cell identity. Only when BMP signaling is successfulcan non-neural cell types form. Together,thesedata led to a simple model in which Chordin prevents BMP4 from binding to its receptor. In principle, inhibition could occur by the direct binding of Chordin to BMP receptorsor to BMP moleculesthemselves.Biochemical studies demonstrated that Chordin binds BMP2 and BMP4 homodimers or BMP4/BMP7 heterodimers with high affinity (Ka : 3 x 10-10 M) and prevents them from binding to their receptors (Figure 22-75). Chordin-mediated inhibition of BMP signaling is relieved by Xolloid protein, a proteasethat specificallycleavesChordin in Chordin-BMP complexes,releasingactive BMP' Cerberus, another antagonist secretedfrom cells in the Spemann organizer,is named after the mythological guardian dog with three heads becauseit has binding sites for three different types of powerful signals-Wnt, Nodal, and BMP' The binding of thesesignalsby Cerberuspreventsactivation of their respectivereceptors. By inactivating \il(nt, Nodal, and BMP signals having roles in the development of the trunk and tail of the body, Cerberus promotes head development. ln Xenopus, neural induction seemsto be the default state that must be actively blocked in order for other cell types to develop. In chicks and mammals, however, fibroblast growth factor (FGF), BMPs, and Wnt appear to be necessarysignals for induction of neural tissue in the posterior embryo. In the anterior, the actions of BMP and'!7nt are prevented by antagonists(Figure 22-1'61.These antagonistsare produced in a region called the node, which is functionally equivalent to the Spemannorganizer in frogs. The node is a depressionat the anterior end of the primitive streak; during gaitrulation, the streak with the node leading it gradually expands forward toward the head.

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Exterior

Cytosol

( b ) R e l e a s eo f i n h i b i t i o nb v X o l l o i d

N os i g n a l i n g

Signaling

FfGURE22-15 Modulationof BMP4signaling in Xenopus by Chordinand Xolloid,(a)ChordinbindsBMp4,a TGFB-family proteinsignal, secreted andprevents it frombindingto itsreceptor SeeFigure16-4to reviewtheTGFBsignaling pathway(b)Xolloid

specifically cleaves Chordinin the Chordin-BMP4 complex, releasing BMP4in a formthatcanbindto itsreceptor andtriggersignaling

Sortingout all the signalsand antagonistsinvolvedin determining cell fates along the three body axes will require much additional research.Nonetheless,biologistsnow have identified most of the powerful regulators that causethe formation of different tissue types in the early embryo, including those that control differencesbetween the left and right sidesof the body.

the left-right axis. Antibody staining and in situ hybridization have been used to look at gene expressionin late gastrula embryos. One of the most striking and interestingfindings has been that some genesare active on only the left or only the right side of the embryo. These include genesencoding three secretedprotein signals:Sonic hedgehog,FGF, and Nodal (Figure22-17). Experimentsin which geneswith this type of expressionpattern were disrupted have shown that such genesare not merely responding to left-right information; they are part of the systemfor controlling the subsequent pattern of the embryo. An animal tissueor organ may exhibit left-right asymmetry in two ways: by forming primarily on one side of the body or by having an asymmetric

A Cascade o f S i g n a l sD i s t i n g u i s h eLs e f t from Right Having discusseddorsal-ventraland anterior-posterioraxis formation, we turn next to the geneticcontrols that organize

Wnts, BMPs,FGFs

BMBWnt antagonists

Posterior primitive streak

Anterior

CHAPTER 22

< FIGURE 22-16 Regulators of anterior-posterior cellfatesin the mouseembryo.Thelategastrula embryo, whichisactually curled,isdepicted hereasa flattened ovalwith threelayers, ectoderm, mesoderm, andendodermTheprimitive streak, where futuremesoderm cellsmovedinsideduringgastrulation, is indicated asa purpleareaon the surface of the oval Thenode(greencircle) isa signaling centerthatformsearlier andeventually becomes the (seeFigure notochord 22-39).Regulatory signals thatcontrol anterior-posterior patterning areindicated abovetheoval.Wnt,BMB andFGFsecreted proteins signaling aremadeby posterior tissues and directcellsto assume posterior-cell fatessuchasneuraltissueThe actions of BMPandWnt proteins areblocked in theanterior embryo proteins by antagonistic thataremadeby cellsin the nodeand accumulate there [Adaptedfrom S F.Gilbert,2006, DevelopmentalBiology 8 t h e d , S i n a u eP r r e s sF, i g u r e11 - 4 1l

Ectoderm Mesoderm Endoderm

966

[SeeS Piccoloel al , 1997, Cell 91:407]

|

THEMOLECULAC R E L LB t O L O G yO F D E V E L O P M E N T

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(al Drosophi Ia developmental stages

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E m b r y o n i cd e v e l o p m e n t -1 day T h r e e l a r v a ls t a g e s -4 days Third instar larva

Pupa

of Dorsal and its translocation into nuclei. As a result, Dorsal enters ventral nuclei at a high level' lateral nuclei at a modest level, and dorsal nuclei not at all (Figure 22-23)' Thus Dorsal acts in a graded fashion and has the property of a morphogen, even though this protein is not secreted'The differential entry of the Dorsal transcription factor into nuclei, which controls subsequentcell fates, is governed by a complex signalingprocessthat starts during oogenesisin the follicle cellsand is transmitted to the developingembryo' We will not pursue the details of dorsal-ventral patterning further and will instead concentrate on anterior-posterior patternlng. To decipher the molecular basis of cell-fate determination and patterning along the three body axes, investigators have cloned genesidentified in screensfor mutations that affect the body plan; determinedthe spatial and temporal patterns of mRNA production for each gene and the distributhe tion of the encodedproteins in the embryo; and assessed effectsof mutations on cell differentiation, tissuepatterning' and the expressionof other regulatory genes.The principles of cell-fatedetermination and tissuepatterning learned from Drosophila have proved to have broad applicability to animal development.

( b ) l m a g i n a l d i s c s ,p r e c u r s o r st o t h e a d u l t

TranscriptionalControl Specifiesthe Embryo's Anterior and Posterior 'We

turn now to determination of the anterior-posterior axis in the early fly embryo while it is still a syncytium' The processbeginsduring oogenesiswhen maternal mRNAs ptoiuced by nurse cells are transported into the oocyte and become localized in discrete spatial domains (see Figure

22-22 Major stagesin the developmentol Drosophila A FIGURE intoa e99develops and locationof imaginaldiscs.(a)Thefertilized in a few hoursThelarva,a cellularization andundergoes blastoderm throughthree in about1 dayandpasses form,appears segmented (instars) Pupation intoa prepupa. overa 4-dayperiod,developing stages of theadultflyfromthe takes=4-5days,endingwiththeemergence discsareset pupalcase.(b)Groups cellscalledimaginal of ectodermal thesegive Duringpupation, sitesin thelarvalbodycavity. asideat specific cellsgiveriseto bodypartsindicatedOtherprecursor riseto thevarious (a) structures. andotherinternal thenervous system, lPart adultmuscle, in E W etal,'1969, fromJ W Fristrom Part(b) adapted Kaye Suyama courtesy of UtahPress, University inBiology, onProblems Hanly. ed, ParkCitySymposium o Jdrl

to the oocyte as a dowry from the mother. The dorsal-ventral control systeminvolves differential transport of a transcription factor called Dorsal into the nuclei of the syncytial embryo. This transcription factor, related to the vertebrate NF-rB protein, is presentin its inactive form throughout the cytoplasm of the syncytial embryo. A signal proteln concentrated on the ventral side of the embryo triggers the NF-rB signalingpathway (seeFigure 1'6-35),leadingto activation

anterior cell fates. Bicoid protein is a homeodomain-type transcription factor that activates expression of certain anterior-specific genes discussed later' In the syncytial fly embryo, Bicoid away from f,rotein spreadsthrough the common cytoplasm end where it is produced from the localized ,h. "rrt.riot mRNA. As a result' a Bicoid protein gradient is established along the anterior-posterior axis of the syncytial embryo' Sincethe effectsof Bicoid are concentration-dependent'it is acting as a morphogen. Evidence that the Bicoid protein gradiint determines anterior structures was obtained ihro,tgh injection of synthetic bicoid mRNA at different locations in the embryo. This treatment led to the formation of anterior structures at the site of iniection, with progressively more posterior structures forming at increasing distancesfrom the iniection site' Another test was to make flies that produced extra anterior Bicoid protein; in these flies, the anterior structures expanded to occupy a greater proportion of the embrYo.

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(a) Wild type

Thresholdabove which fr4listgene is activated

-= o

I-iI I I Il"i"l"T"k Ventralmesoderm (b) Mutant-no Dorsal

Thresholdabove which fwrsf gene is activated

,:) c o a

o

(c) Mutant-nuclear Dorsal everywhere n U

Thresholdabove which fr4listgene is activated

o

All mesoderm

A gradientof the transcription ral cellfates in the early omologof vertebrate NF-xB(See Figure16-35)(a)Inwild-type embryos, moreDorsal proteinenters the nucleion theventral sideOnceinside a nucleus, Dorsal activates targetgenessuchasfwist,whichencodes a transcription factorthat directsmesoderm formationThedifferential entryof Dorsalthus

creates a dorsal-ventral polarityin twrstexpression and mesoderm induction. Inactive cytoplasmic NF-nB on thedorsal sidefailsto activatetwist.(b)A mutantlackingDorsalmakesno mesoderm cells. (c)Conversely, in a mutantthathasDorsal in thenucleiof allcells, all cellsdifferentiate asmesoderm[Adapted fromL Wolpert etal, principles of Development,2ndedition, Oxford Press,Figure5-14 l

orly in parallel (Figure 22-25a-c). Analysis of the hunchback gene revealed that it contains three low-affinity and three high-affinity binding sites for Bicoid protein. Experiments with syntheric genescontaining either all high-affinity or all low-affinity Bicoid-binding sitesdemonstratedrhat the affinity of the site determines the threshold concentration of Bicoid at which gene rranscription is activated (Figure 22-25d, e). In addition, the number of Bicoid-binding sites occupied at a given concentration has been shown to determine the amplitude, or level, of the transcription response. 972

CHAPTER 22

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THEMOLECULAC R E L LB | O L O G YO F D E V E L O P M E N T

llll;

Development overviewAnimation:GeneControlin Embryonic 1 5 0m i n

1 6 0m i n

1 8 0m i n

22-24 Maternallyderivedbicoid FIGURE < EXPERIMENTAL mRNAis localizedto the anterior region of early Drosophila to the with anterior shownarepositioned embryos.All embryos situ hybridization in experiment, In this the top leftanddorsalat for bicoidmRNA labeledRNAprobespecific with a radioactively 2 5-3.5 hoursafter sections on whole-embryo wasperformed f romthesyncytial thetransition Thistimeperiodcovers fertilization probe After excess gastrulation of to the beginning blastoderm (dark mRNA bicoid maternal to probe hybridized wasremoved, h iyc o i dp r o t e i ni sa s i l v egr r a i n sw) a sd e t e c t ebdy a u t o r a d i o g r a pB t r a n s c r i p t i foanc t o rt h a ta c t sa l o n ea n dw i t h o t h e rr e g u l a t o tros anterior of certaingenesin the embryo's controlthe expression photographs of courtesy 335:25; 1988, tVature PW Ingham, regionfFrom I n g h a m W P l

2 1 0m i n

Findings from studiesof Bicoid's ability to regulatetranscription of the hunchback geneshow that variations in the levels of transcription factors, as well as in the number or affinity of specificregulatory sequencescontrolling different targetgenes,or both, contribute to generatingdiversepatterns of gene expressionduring Drosophila development.Similar mechanismsare employedin other developingorganisms.

TranslationInhibitorsReinforceAnteriorPosteriorPatterning Cell fates at the posterior end of the fly embryo are specified by a different mechanism-one in which control is at the

Bicoid protein gradient in embryo

Copies of bicoidgene in mother

Promoter

Expressionpattern

translational level rather than the transcriptional level' As

uniformly distributed throughout the embryo, its translation is prevented in the posterior region by another maternally deiived protein called Nanos, which is localized to the posterior end of the embryo' The set of genesrequired for posterior locali zation of Nanos protein is also required for germ-line cells to form at the posterior end of the embryo' bn. of thesegenes(staufen)is necessaryfor developmentof primordial germ cells (PGCs) in zebrafish. Thus at Ieast so-e g.t--line regulators have existed since fish and flies had a common ancestor.

!

(a)

0

o

Anterior -> Posterior

hunchback (b)

(c)

(d)

1

2

2

(e) 2

S U U

Highaffinity Bicoidbinding site

Lowaffinity Bicoidbinding site

Synthetic High-affinityBicoidb i n d i n gs i t e s

Synthetic

--\rrz-

J-----------} IF

Low-affinityBicoidb i n d i n gs i t e s

22-25 MaternallyderivedBicoid FIGURE < EXPERIMENTAL hunchback(hb) gene embryonic of the controls expression (a-c) the numberof lncreasing axis' anterior-posterior the along gradient in theearly theBicoid bicoidgenesin motherflieschanged e m b r y ol ,e a d i n tgo a c o r r e s p o n d icnhga n g ei n t h e g r a d i e not f genein the proteinproduced from thehunchback Hunchback threehighpromoter contains hunchback genome. fhe embryo's flies sites.Transgenic Bicoid-binding affinityandthreelow-affinity promotercontaining genelinkedto a synthetic a reporter carrying sites(e)were sites(d)or four low-affinity eitherfour high-affinity gradrent in the protein Bicoid same the to response preparedIn by a promoter genecontrolled of the reporter embryo,expression moreposteriorly sitesextended Bicoid-binding high-affinity carrying sites.This low-affinity gene carrying of a reporter thandidtranscription that activates Bicoid of concentration thatthe threshold resultindicates the Bicoid-binding of the affinity on depends transcription hunchback fashion[Adapted othertargetgenesin a similar regulates site Bicoid Ce//68:201l from D St Johnstonand C NUssleln-Volhard,1992,

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Figure 22-26 lllustrates how translational regulation by Nanos helps to establish the anterior -+ posterior Hunchback gradient neededfor normal fly development.Translational repression of hunchback mRNA by Nanos depends (a)

Fenilized egg

Early embryo

Hunchback protein derived from maternalRNA

Nanos protein

(b) Nanos protein

h b m R N A( m a t e r n a l )

InsectSegmentationls Controlledby a Cascade of TranscriptionFactors

Anterior

Posterior

I +

Nano protei

I

-@en

;;-+AAAAAAA3' mRNA

Nanos promotes deadenylation

Translationof hb mRNA

No hb translation

I

v

I

I

v

v

Anterior development

Abdominal development

Hb protein

h b m R N A( m a t e r n a l )

posterior

Anterior

A FIGURE 22-26 Roleof Nanosproteinin excludingmaternally derivedHunchback(Hb) protein from the posteriorregionof Drosophilaembryos.(a)Bothnanos(blue)andhunchback (red) mRNAs derived fromthe motheraredistributed uniformly in the fertilized eggandearlyembryo. Nanosprotein, whichisproduced onlyin the posterior region, subsequently inhibits translation of maternal hb mRNAposteriorly. (b)Diffusion of Nanosproteinfromits siteof synthesis in theposterior _+ regionestablishes a posterior anterior Nanosgradient. A complex of Nanosandtwo otherproteins inhibits translation of maternal hb mRNA. Asa consequence, maternally derived Hbproteinisexpressed in a gradedfashion that parallels andreinforces the Hbproteingradient resulting fromBrcoid_ controlled transcription of theembryos hb gene(seeFigure 22_25) el al, 1997 [SeeC Wreden 124:30151 , Development 974

.

c H A p r E R2 2

|

on a specific sequencein the 3'-untranslated region of the mRNA, the Nanos-responseelements (NRE). Along with two other RNA-binding proteins, Nanos binds to the NRE in hwnchbac&mRNA. The results of genetic and molecular studies suggest that Nanos promotes deadenylation of hunchback mRNA and thereby decreasesits translation. In the absenceof Nanos, the accumulation of maternal Hb protein in the posterior region leads to failure of the posterior structures to form normally, and the embryo dies. Conversely,if Nanos is produced in the anterior, thereby inhibiting the production of Hb from both maternal and embryonic hunchback mRNA, anterior body parts fail to form, againa lethal consequence.Translational control due to the action of an inhibitor, mRNA localization, or both, may be a widely used strategy for regulating development. For instance, specific mRNAs are localized during the development of muscle cells, and during cell division in the budding yeastSaccharomycescereuisiae(seeFigure 2I-28).

In both insectsand vertebrates,the anterior-posterior(headtail) axis is divided into a set of repeats,or more preciselyrepeats with variations: vertebrae and associatedganglia in vertebrates,body segmentsin insects.Specificgenescontrol subdivision of the embryo into repeats, while other genes control the differences between repeats. As noted already, not all vertebraehave attached ribs, and only some of an insect'sbody segmentshave legs growing from them. Ve discussthe genescontrolling segmentationof insectsin this section and the rather different type of regulation underlying vertebrate segmentationin the next section. Then we delve into the genesthat control differencesbetween segmenrs. Once the gap geneshave been properly activated in the Drosophila embryo, the next stepson the road to body segmentation are controlled by a transcription-factor (TF) cascade in which one TF controls a gene encoding another TR which in turn conrrols expressionof a third TF. At each step, more than one genemay be regulated.Sucha TF cascadecan generatea population of cellsthar may all look alike but differ at the transcriptional level. TF cascadeshave both a temporal and a spatial dimension. At each step in a cascade.for instance.RNA polymeraseand ribosomescan take more than an hour to produce a transcription factor from its correspondingmRNA. Spatial factors come into play when cells at different positions within an embryo synthesizedifferent transcription factors. The rough outline of cell fates that is laid down in the syncytialfly embryo is refined into a systemfor preciselycontrolling the fates of individual cells.Discovery of the relevant regulators came from a genetic screenfor mutants with altered embryo body segments.In addition to hunchback, four other gap genes-Krilppel, knirps, giant, and tailless-are transcribed in specificspatial domains beginning about 2 hours after fertilization (Figure 22-27a). Expression of these genes, like that of hunchbacft,is regulatedfirst by marernal factors and then by cross-interactionsamong the gap genes.

T H EM o L E c u L A R c E L LB t o l o c y o F D E V E L o p M E N T

/z^s\ \\"2

of SegmentationGenesin a DrosophilaEmbryo Video:Expression

( a ) G a p - g e n ep r o t e i n s

( b ) H u n c h b a ca knd Krrippel

(c)

Hunchback

Kriippel

n rps

{|'

22-27 Gapgenesand pair-rule FIGURE A EXPERIMENTAL genesare expressedin characteristic spatialpatternsin early permeabilized werestained embryos Drosophilaembryos.Fixed, protein for a particular antibodies specific withfluorescence-labeled to the leftanddorsal with anterior shownarepositioned All embryos for individually embryos werestained at thetop (a)Thesesyncytial of byfourof thefivegapgenesTranscription encoded the proteins by Hunchback, IheKruppel,knirps,andgiant gapgenesis regulated to embryowasdoublystained andCaudal(b)Thissyncytial Bicoid, protein(green)The protein(red)andKruppel Hunchback visualize in part proteinvisible posterior hereisonlyweaklyvisible Hunchback ( a )d u et o t h ep l a n eo f f o c u sT h ey e l l o wb a n di d e n t i f i et h s er e g i o n i n w h i c hp r o d u c t i oonf t h e s et w o g a pp r o t e i nosv e r l a p (sc )I n t h i s eg meb r y oF, u s htia r a z u( b l u ea) n dE v e n - s k i p p e d blastoderm-sta genesffz andeve, (brown)proteins, by the pair-rule encoded to the in stripesEachstripecorresponds areexpressed respectively, primordial about14segments cellsof onebodysegmentAltogether

canbe of segmentation evidence areformedNomorphological of for the RNAor proteinproducts seenat thisstage,but staining plan body a segmented of genesreveals the beginnings pair-rule (lower primordia (d)Therelationship betweenthe earlysegment gene(darkgray),and of onepair-rule stripes expression embryo), thatareformed(upperlarva)is larvalsegments the eventual fromheadto segments, the different Thecolorsindicate depicted. larval The larva' to embryo from tail,andhowtheycorrespond develops Notethateachsegment externally. headis barelyvisible that isaboutfour cellswide(inthe head-tofroma prrmordium andabout60 cellsaroundHalfof the segments taildirection) geneandhalffrom the pair-rule fromcellsthat express develop a different express interstripes "interstripes" not; the do that the segments A : abdominal segments; gene.T : thoracic pair-rule et al, 1992,Cell59:23].Part(b)courtesy fromG Struhl IPart(a)adapted of Part(d)courtesy Lawrence of Peter of M LevinePart(c)courtesy

All the gap-geneproteins are transcription factors. Becausethese proteins are distributed in broad overlapping p e a k s ( F i g u r e2 2 - 2 7 b ) , e a c h c e l l a l o n g t h e a n t e r i o r - p o s t e rior axis contains a particular combination of gap-gene proteins that activates or repressesspecific geneswithin that cell. Indeed, something like a battle ensues,because some gap proteins repressthe transcription of genes encoding other gap proteins. Although they have no known extracellular ligands, some gap proteins resemblenuclear receptors, which are intracellular proteins that bind lipophilic ligands (e.g., steroid hormones) capable of crossing the plasma membrane. Most ligand-nuclear re-

ceptor complexes function as transcription factors (see Figure 7-50). The sequencesimilarity between gap proteins and nuclear receptors suggeststhat gap genesmay have evolved from genes whose transcription was controlled by signalsthat could cross membranes,such as the steroid hormones.The use of such signal-controlledgenes' rather than TF cascades,could explain how early cell-fate specificationoperatesin animals that do not have a syncytial stage. The fates of cells distributed along the anterior-posterior axis are specifiedearly in fly development.At the sametime' cells are responding to the dorsal-ventral control system'

N i p a mP a t e l

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Each cell is thus uniquely specifiedalong both axes. If each of the five gap geneswere expressedin its own section of the embryo, at just one concentration, only five cell types could be formed. The actual situation permits far greater diversity among the cells.The amount of eachgap protein variesfrom Iow to high to low along the anterior-posterioraxis, and the expressiondomains of different gap genes overlap. This complexity createscombinations of transcription factors that lead to the creation of many more than five cell types. RemarkablS the next step in Drosophila development generates a repeating pattern of cell types from the rather chaotic non-repearing pattern of gap-gene expressron domains.

stripes,separatedby "interstripes" where that pair-rule gene is not transcribed (Figure 22-27d). Murant embryos that lack the function of a pair-rule gene have their body segments fused together in pair-wise fashion-hence the name of_this class of genes.The expression stripes for each pairrule gene partly overlap with those of other pair-rule gines; so each gene must be responding in a unique way ro gapgene and other earlier regulators.

The transcription of pair-rule genesis controlled by transcription factors encoded by g"p and maternal genes. Becausegap and maternal genesare expressedin broad, nonrepeating bands, the question arises: How can such a non-repeating pattern of gene activities confer a repeating pattern such as the striped expressionof pair-rule genes?To answer this question, we consider the transcription of the euen-skipped(eue) genein stripe 2, which is controlled by the maternally derived Bicoid protein and the gap proteins Hunchback, Kri-ippel,and Giant. All four of thesetranscription factors bind to a clustered set of regulatory sires, or enhancer, located upstream of the eue promoter (Figure 22-28a). Hunchback and Bicoid activatethe transcription of eue in a broad spatial domain, whereas Kriippel and Giant represseue transcrrption, thus creating sharp posterior and anterior boundaries.The combined effectsof theseproteins, each of which has a unique concentration gradient along the anterior-posterior axis, initially demarcatesthe boundaries of stripe2 expression(Figure22-28b). Expression of the other eue stripes also depends on specificenhancers.Each stripe of eue expressionis formed in responseto a different combination of transcriptional regulators acting on a specificenhancer,so the non-repeating distributions of regulators createrepeating patternsof pair-rule gene repression and activation. If even one enhancer is bound by an activating combination of transcriptional

Video: EstablishingEve Expression in Drosophila Embryogenesis ffi (a) eye gene transcription regulation

Stripe2 e nn an c e r Start of transcription G i a n (t { )

B i c o i d( t ) A c t i v a t o r s( t )

!

(b) evestripe2 regulation H u n c h b a c(kt )

G i a n t( 0 )

evestripe2

K r i i p p e(l { )

I

< FfGURE 22-28 Controlol even-skipped(eve)stripe 2 in the Drosophilaembryo.Onlyoneof the eyegenestripesisrepresented. Withineacheuestripe,a segment boundary will laterform Thuseye functiongivesriseto halfthesegment boundaries in the embryo (a)Diagram of the 815-bpenhancer controlling transcrrption of the pair-rule geneeyein stripe2 Thisregulatory regioncontains binding sitesfor BicoidandHunchback proteins, whichactivate thetranscription of eve,andfor Giantand Kruppelproteins, whichrepress its transcription Theenhancer isshownwith allbindingsitesoccupied, but in an embryooccupation of siteswillvarywith position alongthe anterior-posterior axis(b)Concentration gradients of thefour transcription factorsthatregulate evestripe2 Thecoordinated effect (J) andtwo activators of the two repressors (t) determine the precise boundaries of thesecond anterior euestripeOnlyin the orange regionisthe combination of regulators correct for the eyegeneto be transcribed in response to thestripe2 controlelement. Further anterior, Giantturnseyeoff;furtherposterior, the levelof Bicoid activator istoo lowto overcome repression by Kruppel. Expression of otherstripes isregulated independently by othercombinations of transcription factorsthatbindto enhancers not depicted in part(a) [SeeS Smallet al , 1991, Genes& Devel.8:827 |

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regulators, the presenceof other enhancersin an inactive, "off" state (not bound to a regulator) will not prevent transcription. For instance,in Eve stripe 2, the right combination and amounts of Hunchback and Bicoid createan "on" state that activatestranscription even though other enhancersare present in the inactive state. In each stripe, at least one enhancer is bound by an activating combination of regulators. Note that this systemof genecontrol is flexible and could be used to produce non-repeating patterns of transcription if that was useful to an animal. Similar responsesto gap and maternal proteins govern the striped patterns of transcription of the two other pairrule genes,runt and hairy. Becausethe enhancersof rwnt and hairy respond to different combinations of regulators, the eue,runt, and hairy expressionstripespartly overlap one another, with each stripe for any one gene offset from a stripe for another gene. Subsequently,other pair-rule genes, including fusbi tarazu (ftz) and paired, become active in responseto the Eve, Runt, and Hairy proteins, which are transcription factors, as well as to maternal and gap proteins. The outcome of this transcription-factor cascadeis a pattern of overlapping stripes. The initial pattern of pair-rule stripes,which is not very sharp or precise, is sharpened by autoregulation. The Eve protein, for instance, binds to its own gene and increases transcription in the stripes, a positive autoregulatory loop. This enhancementdoes not occur at the edgesof stripes where the initial Eve protein concentration is low; so the boundary between stripe and interstripe is fine-tuned. The pair-rule genesdirect formation of the embryo's segment boundaries. Since each pair-rule gene is expressedin stripes and each stripe overlaps one segmentboundary, each pair-rule gene contributes to half the segment boundaries. Acting together,all the pair-rule genesform all the segment boundaries and also control other pattern elementswithin each segment.In early embryos each segmentprimordium is about four cells wide along the anterior-posterior axis, which corresponds to the approximate width of pair-rule expressionstripes.With pair-rule genesactive in alternating four-on four-off patterns, the repeat unit is about eight cells. Each cell expressesa combination of transcription factors that can distinguish it from any of the other sevencells in the repeat unit. Under the control of pair-rule proteins and the later-acting segment-polarity genes,the repeating morphology of segmentsbeginsto emerge;it is completedabout 10 hours after fertrlization. As cell-fate determination progressesin the fly embryo, a variety of signaling proteins begin to play a role. Theseinclude Hedgehogand'!7nt, which are encoded by segment-polaritygenesand are produced in stripes, one stripe within each segment, under the control of pair-rule gene products. The broader and earlier-formed stripes of pair-rule gene expressionoverlap in certain regions, and that's where particular combinations of pair-rule transcription factors give rise to the fine pattern of segment-polarity gene stripes. Note that the onset of signal-basedcontrols allows cells to respond to what their neighbors have done and make adjustments.Otherwise parts of the pattern might be missing or duplicated.

From the broad maternal gradient of Bicoid to the singlecell precisionof the segment-polaritygenes'the fly embryo is progressivelysubdivided into repeatingunits. One can readily imagine how changes in stripe-specific enhancers, amounts of particular transcription factors, and the range of signals during evolution could modify the pattern of segmentsin differentorganisms.

VertebrateSegmentationls Controlled by CyclicalExpressionof RegulatoryGenes Now we return to vertebratesto examine how segmentation in these animals compares with that in insects. After the three body axes have been establishedin a vertebrate embryo, dramatic changestake place along all of them. One of the most visible changesis the initiation of a repeating pattern that later gives rise to vertebrae and ribs. This is patterning along the anterior (head) to posterior (tail) axis. In mice and humans, the first sign of the vertebrae appears in the mesoderm that accumulatesunder the primitive streak. The mesoderm forms, you will recall, by an epithelialmesenchymal transition in which cells along the primitive streak cut loose and migrate inside (seeFigure 22-11').On each side of the midline axis, mesoderm composed of loose mesenchymalcellsbeginsto round up to form pairs of spherical epithelia caIIedsomites.Somitesinitially form at the anterior and successivelyappear pair by pair in the posterior direction (Figure 22-29a), giving them value as a way to stageembryos.This striking caseof a mesenchymal-epithelial transition has huge consequencesfor the embryo. From somitescome the vertebraeand ribs, the musclesof the body wall and limbs, and the dermis (inner skin) of the back. Without somites,we would be blobs. The mesodermthat has not yet formed somites is called

from the tail, a retinoic acid signal from the head, and Wnt and Notch signalswithin the presomitic mesoderm.The fgfS gene is expressedin the presomitic mesoderm where it is iorming near the posterior tip of the embryo. Because/gf8 mRNA is unstable, the highest level of FGF8 protein builds

(Figure22-29b). The most remarkable aspectof somite-formation regulation in vertebrates was first discovered in studies of the chicken gene hairyl, which is related to one of the Drosophila pair-rule segmentationgenes.In situ hybridization of developing somites in chick embryos showed that hairyl transcriptsare produced in cycles,with the duration of one cycle corresponding to the time it takes to form one somite (90 minutes in a chick, longer in mammals). A wave

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(a) Earlyembryo (5 pairs of somites)

(b)

Somites

Anterior

Late embryo (9 pairs of somites)

FGFSprotein Posterior

Presomitic mesoderm

Growth and maturation

Tailbud

A FIGURE 22-29 Progressive formationof somitesin human embryos.(a)Fivepairsof somites haveformedat theanterior of the earlier embryoon the left.Somiteformationproceeds towardthe posterior, andin the laterembryoon the right,ninepairshaveformed In bothmicrographs, the developing headison the leftandthetailbud on the right (b)Gradients of FGF8, proteinmadein thetail a secreted of hairyl expressionmoves from posterior to anterior in the presomitic (unsegmented)mesoderm. Subsequentinvestigations revealed a rather large number of genesthat undergo cyclesof expression,and all turned out to be related to the Notch or ril/nt signalingpathways. Mutations in either pathway cause drastic defects in somite formation. In humans. for instance, mutations affecting Notch pathway components causeAlagille syndrome and Jarcho-Levin syndrome, both of which are associatedwith malformed vertebrae. For both the Notch and Wnt pathways, feedback loops are established that cause temporal cycling of expression. For example, the hesT gene encodes a transcription factor involved in Notch signal transduction. rWhentranscription of hesTis stimulated by a FGF signal from the posterio; presomitic mesoderm, a burst of HesT protein production occurs (Figure 22-30a). HesT protein, in rurn, controls the expressionof target genesthat contribute to somite formation. BecauseHesT also acts as a repressorof its own gene, binding to its gene and turning it off, the HesT protein accumulates only until it reaches a high enough level to represses hes7 transcription. This negative autoregulation thus limits the duration of hes7 expression.Each part of the presomitic mesoderm does the same thing in turn: hes7

expressionjust as a somite forms, another burst in the cells that will form the subsequentsomite, and so on. If Notch or Wnt feedback loops are blocked, somites are highly abnormal, but the details of how both pathways control shaping of the somites are not known. 978

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bud,andretinoic acidfromthe head,controlsomiteformation from presomitic mesoderm, whichfirstarises in thetailbud Highlevels of FGF8prevent maturation of presomitic cellsintosomites in the posteriolwhereas highlevels of retinoic acidactsto stimulate formation (a)Kohei of somiteslPart Shiota/Congenital Anomaly Research Center, Kyoto University. Part(b)adapted fromA F.Schier; 2004,Nature 427i4031 'We

can now understand the two different strategiesfor controlling the formation of repeating body parts in insects and vertebrates. In Drosophila, the regulation differs for each body segment: different combinations of gap transcription factors, activated in specific regions along the anterior-posterior axis by maternal influences, regulate pairrule gene stripes, and pair-rule transcription factors in turn combine to regulatethe still finer stripesof segment-polarity gene transcription. Each stripe has a distinct regulatory history involving different gap or pair-rule proteins. Thus repeat formation is controlled by spatial differences.In contrast, repeating vertebrate somites are formed by the same regulatory process occurring again and again. A remarkable feedback system creates a cyclical clock that causes the genes responsible for building somites to be expressedin bursts. Thus repeat formation is controlled by temporal (time) differences.

DifferencesBetweenSegmentsAre Controlled by Hox Genes Despite the differencesin how repeating body parts are formed in insectsand vertebrates,the two groups of animals are reunified in employing the same family of genesto crearevariation among the repeats.Theseare the Hox genes,which control differences in cell identities and indeed the identities of whole parts of an animal along the anterior-posterior axis. Thesedifferencesare superimposed upon the underlying reperitive nature of some of the tissues.Hox genesencode highly related transcription factors containing the homeodomain motif (Chapter 7). Indeed,what unifies the whole group of Hox proteins is a similar DNA-binding homeodomain sequence;the proteins have little else in common. The homeodomain sequencesare also the basisfor classifying all the Hox genes.

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(a)Activationof hesTtranscription

(b) Sequentialinductionand autoregulationof hesT Signal Tail

Head

/1

22-30 Controlof cyclicalgene expressionin the A FIGURE is developingsomites.(a)An initialburstof hesZtranscription t r i g g e r ebdy a p o s i t i vsei g n a lp, r o b a b lFy G F SA s H e s Tp r o t e i n( a component) accumulates, it eventually bindsto its Notchpathway Thrsprocess is repeated, once own geneandturnsoff transcription performingsomite,in increasingly posterior regions of the presomitic

end, (b)TheFGF8 to be at the posterlor signalcontinues mesoderm to istriggered presomitic mesoderm moreposterior so progressively programs, to leading NotchandWnt expression cyclical activate of of transcription represent bursts somiteformationTheredcircles andthe components NotchandWnt signaling genesencoding loopsthatshutthemdown feedback negative

Mutations in Hox genesoften causehomeosis-that is, the formation of a body part having the characteristicsnormally found in another part at a different site. For example, some mutant flies develop legs on their headsinsteadof antennae. Lossof function of a particular Hox genein a locationwhere it is normally active leadsto homeosisif a different Hox genebethere;the resultis the formation of cellsand comesderepressed structures characteristic of the derepressedgene. A Hox gene that is abnormally expressedwhere it is normally inactivecan take over and impose its own favorite developmentalpathway on its new location (Figure22-3I).

mosomes is colinear with the order in which they are expressedalong the anterior-posterioraxis (Figure 22-32a). The fly Hox genesare located at two locations on the same chromosome but are effectively one cluster of eight Hox genes.At one end of the cluster are "head" genes,which are transcribed specificallyin the head and are necessaryfor formation of head structures.Next to them are genesactiYeand functional in the thorax. and at the other end of the cluster are abdomen genes.The arrangement reflects evolutionary gene duplications and is retained becausethe genes share regulatory sequencessuch as enhancers(Chapter 7). The expressiondomains of Hox genescan overlap' so the development of a particular body structure can depend upon more than one gene. ln Drosophila, the spatial pattern of Hox-gene transcription is regulatedby maternal, gap' and pair-rule transcription factors. The protein encodedby a particular Hox gene controls the organizationof cellswithin the region in which that Hox geneis expressed.For example,a Hox protein can direct

Organization of Hox Genes Classicalgeneticstudiesin Drosophila led to discoveryof the first Hox genes(e.g.,Aatennapedia and Ultrabithorax). Corresponding genes with similar functions (orthologs) have since been identified in most animal species.Each Hox gene is transcribed in a particular region along the anterior-posterior axis in a remarkable arrangement where the order of genesalong the chro-

Normal the LikeotherHoxgenes, 22-31 Hox-genephenotypes. FIGURE (lJbx)genecontrols of cellswithinthe lJltrabithorax the organization wing actsin preventing lt normally regionin whichit isexpressed. sothatnormalflieshavea singlepairof wings Mutations formation, in Hoxgenesoftenleadto the formationof a bodypartwhereit does

Ubx mutant not normallyexist,In the mutantshown here,the lossof Ubxfunction from the third thoracicsegmentallowswingsto form where normally 1978, IFromE B Lewis, thereare only balancerorganscalledhalteres. 1978, copyright Nafure, permission from by Reprinted Nature276i565 Limited Journals Macmillan l .

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( a )D r o s o p h i l a

3'

5' Antennapedia comptex

(b)Mouse

Bithoraxcomolex

Hoxa,chromosome6

3',

5'

Hoxb,chromosome11

Hoxc,chromosome15

Hoxd,chromosome2

FIGURE 22-32 Relationbetween Hox gene clustersin Drosophilaand mammals.(a)ThesingleDrosophrla Hoxcluster i ss p l i ti n t ot w o c h r o m o s o m|aolc a t i o nos n ; eg e n eg r o u pi st h e Antennapedia complex andtheotheristhe Bithorax complex. The genesthatcontrolheadformation areat the 3' endof thecluster (yellow/red shades), thosethatcontrolformation of theabdomen areat the 5' end(blue/green shades), andtheonesin-between controlthoracic (purple), structures asillustrated in thefly drawing, whrchshowswherethe differentgenesareexpressed (b)The arrangement of genesin the mouseandhumanHoxgeneclusters issimilar to that in Drosophila, buttherearefourclusters andeach of themis missing someof thesetof genesForexample, theclass 1 Hoxgenesof miceandhumans aresimilar to the /abgeneof Drosophila, basedon encodedproteinsequences Thereisa class1

or preventthe local production of a secretedsignalingprotein, cell-surfacereceptor,or transcription factor that is neededto build an appendageon a particular body segment.Drosophila Hox proteins control the transcription of target geneswhose encodedproteins determinethe diversemorphologiesof body segments.Much remains to be learned about how morphology is controlled by thesetarget genes,but some rargersencodepowerful Y/nt and TGFB signals.The associationof Hox proteins with their binding siteson DNA is assistedby cofactors that bind to both Hox proteins and DNA, adding specificity and affinity ro rheseinteracrions. How about vertebrates?In contrast to flies, which have a single Hox gene cluster, mammals have four copies of the Hox cluster, a-d, located on different chromosomes(Figure 22-32b). \Within each cluster, the genesare numbered 1, at the head end, to 13, at the tail end. The different copiesof a particular gene (e.g.,Hox4) in the four clustersare closer to each other in sequencethan to Hox genesof another numerical class. Although the mammalian Hox genes are clearly related to the fly Hox genes,there has been an expansion of fly abd-type genesin vertebrates(Hox classes8-13). 'Vfithin eachcluster some geneshave beenlost, evidently becausethe other two or three copies are sufficient. Experiments with 980

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genein Hoxa, b, andd clusters but not in thec clusterEvidently threeisenough.In contrast, the class4 geneis represented in all of the mammalian clusters. Anotherdifference frominsectto mammal isthat mammals have"extra"versions genes(class of the posterior 9 andup)that correspond to the abd-typegenesin flies The drawings of the fly andmouseembryoindicate whereHoxgenesare transcribed, fromwhichit canbe seenthat the orderhasbeen preserved duringthe roughlyhalfbillionyearssincetheyhada commonancestorThedrawings area simpilfication, sincein many cases a geneisexpressed with a sharpanteriorboundary aswellas a gradedpatterngoingtowardthe tail,andexpression patterns varybetweendifferenttissues[Adapted fromL Wolpert et al, 2001, Principles of Development, 2nded, Oxford University Press, Box4-4al

mouse "knockouts" missing one or more Hox genesfrom one numerical class show considerableredundancy among the 39 total genes. Evolution of Hox Gene Clusters Hox clusters are the most dramatic example of the conservation of gene groupings acrossa wide range of animals. Their organization is so striking that they serve as useful tools for studying evolution. The single Hox cluster in Drosophila is represented four times in mammals (seeFigure 22-32). Comparisons of the genome sequencesof a variety of vertebrates have revealed that the copies of the Hox clusters are far from perfect. During evolution, some specieshave lost one or more Hox genes;in other species,Hox geneshave become duplicated within a cluster. The transitions between different clusters and losses and gains of individual Hox genes among present-day organisms allow ancestral forms to be deduced (Figure 22-33). For example, in the time sincefrogs and humans had a common ancestor, about 370 million years ago, frogs have lost Hox genesb13 and d12. As more studies are done of how Hox genescontrol body morphology, it will be increasingly possible to relate evolurionary changesin Hox genesto pattern formation in the embryos

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Aa Ab Ba Bb

Aa++-rlHlll+ Ab Ba Bb Ca

Cb Da

cb Da

Db Spotted green pufferfish

Zebrafish

Lossof 11genes

Lossof 7 genes

-296 Mya

Aa Ab Ba Bb

-|..flIHl|I|I+

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6

c-.. n

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cb

cc

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Human

Westernclawedfrog

Hypotheticalancestor Lossof 31 genes -420 Mya

Genome duplication

-370 Mya

AfrHrHl|ill+ B+-Hl|llrrrl+

Loss of 2 genes

L...,'..'.....|-lr..Ji..Ji..@

D+-flilr-+}-+ Coelacanth A B

Lossof 1 gene -410 Mya

Lossof 1 gene

D Shark

-450 Mya

Lossof 5 genes

A tlffi

Loss of at least 1 gene

-528 Mya A B

c-----

D Hypothetical ancestor

Lossof 3 genes

c-.. Hypotheticalancestor

+ Exsistinggene sequenceknown + Hypotheticalgene sequencecurrentlyunknown + Describedpseudogene

22-33 Evolutionof vertebrateHox clusters.Genome FIGURE projects organisms, fromdifferent in whichallgenesaresequenced a sw e l la sf o c u s e sd t u d i eosf H o xg e n e sa, r ep r o v i d i nign s i g hitn t o TheHoxclusters shownherein of the Hoxclusters. the evolution of the darkgreen,blue,yellow,and redarebasedon sequencing a ryg a n i s mC g e n o m eosf t h e i n d i c a t epdr e s e n t - d o s .o m p a r i n g that the copiesof the from a varietyof vertebrates reveals sequences oneor moregenes In somespecies, Hoxclusters arefar fromperfect: withina havebeenlost,whiletn others,geneshavebeenduplicated conservation of DNAsequence Since thefossilrecordandstudies cluster. shown the relationships betweenthe organisms haveestablished

of Hoxgenesin to deducethe arrangement here,it is possible (genes an reconstruct and light colors) in ancestors hypothetical in the evolutionof the Hox outlineof the eventsthat happened in time(Mya: millionyearsago) distances Theapproximate clusters in red areindicated hada commonancestor sinceanypairof species not every here, so represented are type.Of course,not all ancestors As morestudiesaredoneof how Hoxgenes steocanbe deduced. possible to relate it will be increasingly controlbodymorphology, in Hoxgenesto patternformationin the changes evolutionary 2005,Trends andA Meyer, fromS Hoegg theycontrol.[Adapted embryos Genet21:.441 l

they control. This genomics approach to exploring evolution will yield more detailed biological histories as more genomesare sequenced.

trol target genes,and they use cofactors of the sametypes used by flies. Mutations affecting some of thesecofactors have been implicated in human cancer. As in flies, Hox-gene expressiondomains in early vertebrate embryos respond to seemingly invisible boundaries that correspond later to transitions between morphologically distinct repeating body units. Each Hox gene is expressedin some somites but not others (Figure 22-34)-They are expressedfirst in presomitic mesoderm,where they control the morphology of the vertebraethat will form later. Vertebrae are classified into groups according to their morphology and position along the anterior-posterior axis.

Functions of Vertebrate Hox Proteins VertebrateHox proteins control the different morphologies of vertebrae,of repeatedsegmentsof the hindbrain, and of the digits of the limbs. Mutations affecting some of the most posterior Hox genesin humans cause inherited syndromes that involve polydactyly (extra fingers or toes) and syndactyly (fused fingers or toes). Particular Hox genesare active in many other tissuestoo. As in flies, mammalian Hox proteins often act in combination to con-

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Wild type | tz

T13

FIGURE 22-34 Expression of the mouseHoxcl| gene in somites.Theanteriorboundary (arrow)is of Hoxcl0expression clearly visible in this9-day-old embryolFrom M Carapuco eral, 2005, Genes & Devel19:21161

The effectsof Hox mutations on vertebral developmenthave been studied with engineeredmouse mutants. In one of the most striking experiments, mice were produced that lack functional Hoxa10, HoxcL0, and Hoxd10 genes(thereis no Hoxb10). These mice exhibited a dramatic transformation of vertebral patterns. Lumbar and even sacral vertebrae, which normally have no ribs, developed with varying degreesof partial ribs (Figure 22-35). Probably evenmore Hox geneswould have to be changedfor a complete transformation. The inference is that the normal role of Hox10 transcription factors is to prevent rib development.

Hoxl0rnutant (lacksall Hox10a,c, and d genes)

T13

T12

Mutant L3

Hox-GeneExpressionls Maintainedby a Variety of Mechanisms 'Sfhen

Hox genes are turned on, their transcription must continue to maintain cell properties in specificlocations. As in the caseof the pair-rule gene euen-skipped,the regulatory regions of some Hox genescontain binding sitesfor their encoded proteins. Thus Hox proteins can help to maintain their own expressionthrough many cell generationsusing an autoregulatory loop. Another mechanism for maintaining normal patterns of Hox-gene expressionrequires proteins that modulate chromatin structure.Theseproteins are encodedby two classesof genesreferredto as the Trithorax group and Polycomb group. The pattern of Hox-gene expression is initially normal in Polycomb-groupmutants, but eventuallyHox-gene rranscnption is derepressedin placeswhere the genesshould be inactive. The result is multiple homeotic transformations,indicating that the normal function of Polycomb proteins is to keep Hox genesin a transcriptionally inactive state.The resultsof immunohistologicaland biochemicalstudieshave shown that Polycomb proteins bind to multiple chromosomal locations and form large complexescontaining different proteins of the Polycomb group. The current view is that the transientrepression of genesset up by patterning proteins earlier in development is "locked in" by Polycomb proteins. This stable polycomb-dependent repression may result from the ability of these proteins to assemble inactive chromatin structures (Chapter 7). Polycomb complexes contain many proteins,

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EXPERf MENTALFIGURE 22-35 Hoxl0 genesregulate vertebrashape.Separate knockoutmicewereconstructed, each l a c k i n g a p o r t ioof nt h e H o x l 0 a , c , odr g e n e E a c h otfh e s e genesis on a differentchromosomeIn eachcase,heterozygous micesurvived because theyhavea second,wild-type,copyof the gene.Themicewerecrossed to construct a strainheterozygous f o r m u t a t i o nast a l lt h r e eg e n e t i cl o c i C r o s s i ntgh e s em i c e t o g e t h egr e n e r a t esdo m eh o m o z y g o umsu t a n e t m b r y oIsa c k i n g b o t hc o p i e so f a l lt h r e eH o x 7 0g e n e sS. k e l e t o nf rso m 1 8 . 5d a y mutantembryos andwild-typeembryoswereisolatedand stained to revealthe detailsof the skeletons. Thesetop-viewand crosss e c t i o n ad li a g r a m sb,a s e do n t h e s t a i n e d s k e l e t o n isl l, u s t r a t eh e results. In wild-typemice,the thoracic(T)vertebrae haveribs, w i t h t h e m o s tp o s t e r i orri bo n T 1 3 .L u m b a(rL )v e r t e b r a(eb l u e (redbracket)do not haveribs. bracket) and sacral(S)vertebrate Mutantsthat arehomozygous for all threeHoxl0 geneshaveribs o n l u m b avr e r t e b r a(ee g , L 3 )t h a tw o u l dn o r m a l lhy a v eb e e n r i b l e s sa,n de v e no n s o m es a c r arli b s( e g , S 2 ) T . h ei n s e t s h o w crosssections of differentvertebrae and ribsto revealtheirshapes i n t h a td i m e n s i o n F.r o mt h e s er e s u l tw s e c a ni n f e rt h a t H o x 1 0 p r o t e i nas r en o r m a l lrye q u i r etdo s u p p r e srsi b f o r m a t i o o nn the d e v e l o p i nl g u m b a ar n ds a c r avl e r t e b r a e S.i n c et h e H o x l 0 g e n e s a r en o r m a l ley x p r e s s er ndt h i sr e g i o no f t h e e m b r y ot,h i s c o n c l u s i omna k e s e n s el f a n yo n eH o x l 0 g e n ei sf u n c t i o n i n g , t h e e x t r ar i b sa r en o t s e e no, r o n l yr a r e l ys e e n s, o t h eH o x l 0 g e n e sh a v ea t l e a s p t a r t i a l lrye d u n d a nf ut n c t i o n s[ A . d a p t ef rdo m D M W e l l i k a n d M R C a p e c c h i ,2 0 0 3 , S c i e n c e3 0 1 : 3 6 3 l

THEMOLECULAC R E L LB | O L O G YO F D E V E L O P M E N T

six stamensin whorl 3, and two carpels containing ovaries in including histone deacetylases, and appear to inactivatetranwhorl 4 (Figure 22-36a). These organs grow from a collection scription by modifying histonesto promote genesilencing. 'Whereas of undifferentiated, morphologically indistinguishablecells Polycomb proteins repressexpressionof certain called the floral meristem.As cellswithin the center of the floral Hox genes,proteins encoded by the Trithorax group of meristem divide, four concentric rings of primordia form segenes are necessaryfor maintaining expression of Hox quentially. The outer-ring primordium, which gives rise to the genes.Like Polycomb proteins, Trithorax proteins bind to sepals,forms first, followed by the primordium giving rise to the multiple chromosomal sites and form large multiprotein petals,then the stamenand carpel primordia. complexes,somewith a massof =2 x 106Da, about half the size of a ribosome. Some Trithorax-group proteins are hoFloral Organ-ldentity Genes Genetic studieshave shown mologous to the yeast S\fVSNF proteins, which are crucial that normal flower developmentrequires three classesof floral for transcriptional activation of many yeastgenes.Trithorax organ-identity genes, designatedA, B' and C genes.Mutations proteins stimulate gene expressionby selectivelyremodeling in these genesproduce phenotypesequivalent to those associthe chromatin structure of certain loci to a transcriptionally ated with homeotic mutations in flies and mammals;that is, one activeform (seeFigure7-43).The core of eachcomplexis an part of the body is replacedby another. In plants lacking all A, ATPase,often of the Brm classof proteins. There is evidence B, and C function, the floral organs develop as leaves(Figure that many or most genesrequire such complexes for tran22-36b,left). scription to take place. The loss-of-function mutations that led to the identification Many regulators of Hox-gene expressionhave been imof the A, B, and C gene classesare summarizedin Figure plicated in leukemia. Chromosomal translocationsthat fuse the genes encoding these regulators to novel sequences, 22-36b (rigbt\. On the basis of the various homeotic phenotypes observed,scientistsproposed a model to explain how the sometimes causing a gene encoding a chimeric protein to three classesof genescontrol floral-organ identity. According form, are frequently found in leukemia patients. Such futo this ABC model for specifying floral organs, class A genes sions, for instance, can create oncogenesthat cause white specify sepalidentity in whorl 1 and do not require either class blood cellsto grow uncontrollably(seeFigure 25-20l.Hox B or class C genesto do so. Similarly,class C genesspecify genes are active in blood cells, though Hox functions in those cells are incompletely understood. Homeotic genes-that is, geneslike the Hox genesthat control development of whole parts of the body-are also important in plant development,as we shall seenow. Again the homeotic genessuperimposea set of variations on an underlying repeat pattern. also postulatesthat A genesrepressC genesin whorls 1' andZ and, conversely C genesrepressA genesin whorls 3 and 4. t e q u i r e sS p a t i a l l y F l o w e rD e v e l o p m e nR To determineif the actual expressionpatterns of classA' RegulatedProductionof TranscriptionFactors B, and C genesare consistent with this model, researchers cloned these genesand assessedthe expression patterns of Gn It may seema long jump from animal segmentationto pattern their mRNAs in the four whorls in wild-type Arabidopsis plants, molecules that control in terms of the but @ plants and in loss-of-function mutants (Figure 22-37a, b). formation, many principles are similar. Like vertebraeor insect Consistent with the ABC model, A genesare expressedin segments,flowers have repeating parts. The basic mechanisms whorls 1. and 2, B genesin whorls 2 and 3, and C genesin controlling development in plants are much like those in whorls 3 and 4. Furthermore, in class A mutants' class C Drosophila: differential production of transcription factors, genesare also expressedin organ primordia of whorls 1 and controlled in spaceand time, specifiescell identities.Our un2; similarly, in class C mutants' class A genes are also exderstanding of cell-identity control in plants benefited greatly pressedin whorls 3 and 4. Thesefindings are consistentwith from the choice of Arabidopsisthaliana as a model organism. the homeotic transformations observedin thesemutants. This plant hasmany of the sameadvantagesas fliesand worms To test whether these patterns of expression are funcfor use as a model system:It is easyto groq mutants can be tionally important, scientists produced transgemc Araobtained, and transgenicorganismscan be made. We will focus bidopsis plants in which floral organ-identity genes were on certain transcription-control mechanismsregulating the expressedin inappropriate whorls. For instance, the introformation of cell identity in flowers. These mechanismsare duition of a transgenecarrying classB geneslinked to an strikingly similar to those controlling cell-type and anteriorA-classpromoter leads to the ubiquitous expressionof class posterior regionalspecificationin yeastand animals.I B genesin all whorls (Figure 22-37c). In such transgenics' whorl 1, now under the control of classA and B genes'develFloral Organs A flower comprises four different organs ops into petalsinsteadof sepals;likewise,whorl 4, under the called sepals,petals,stamens,and carpels,which are arranged control of both classB and classC genes,givesrise to stamens in concentric circles called whorls. Whorl 1 is the outermost; instead of carpels. These results support the functional imwhorl 4, the innermost. Arabidopsis has a completeset of floral portance of the ABC model for specifyingfloral identity' organs,including four sepalsin whorl 1, four petalsin whorl 2,

AND VERTEBRATES T :H E M E SA N D V A R I A T I O N Sl N I N S E C T S C O N T R O LO F B O D Y S E G M E N T A T I O N

983

Wild-typefloral organs S e p a l s( w h o r l 1 ) Petals (whorl 2) Stamens (whorl 3) Carpels ( w h o r l4 )

Loss-of-function homeoticmutations Whorl 1 Wild type

T

C l a s sA mutants

I

ClassB mutants

T

I

C l a s sC mutants

I

l

I

T

Sequencingof floral organ-identity genesrevealed that many encodeproteins belongingto the MADS family of transcription factors,which form homo- and hetero-dimers.Thus floral-organ identity may be specified by a combinatorial mechanismin which differencesin the activities of different (a) Wildtype wl

w2

4

z

I

homo- and heterodimericforms of various A, B, and C pro, teins regulate the expression of subordinate downstream genesnecessaryfor the formation of the different cell types in each organ. Other MADS transcription factors function in cell-typespecificationin yeast and muscle (Chapter 21).

(b) Lossof function

W3

wl

W4

< FfGURE22-36 Floralorgans and the effects of mutationsin organ-identitygenes.(a)Flowers of wild-typeArabidopsis thalianahavefour sepals in whorl1,four petalsin whorl2, sixstamens in whorl3, andtwo carpels in whorl4 Thefloral o r g a n sa r ef o u n di n c o n c e n t rw i ch o r l sa sd i a grammedat right.(b)lnArabidopsrs with mutations in allthreeclasses of floralorgan-identity genes, thefourfloralorgansaretransformed intoleaf(/eft)Phenotypic likestructures analysis of mutants identified threeclasses of genesthat control specification of floralorgansin Arabidopsis (right).Class A mutations affectorganidentityin w h o r l s1 a n d2 : s e p a l(sg r e e nb)e c o m ce a r p e l s ( p u r p l ea)n dp e t a l s( o r a n g eb)e c o m e stamens (red)Class B mutations causetransformation of whorls2 and3: petalsbecome sepals andstamens become carpels. In classC mutations, whorls3 and4 aretransformed: stamens becomepetals andcarpels become sepals[See D Wiegel andE M lvleyerowitz, 1994,Cell78:203l

w2

w3 W4

(c) B-genetransgenic

w1 w2

w3 W4

A_ se

pe

st

ca

pe

pe

st

st

B-

(se pe EXPERIMENTAL FtcURE22-37 Expression patternsof class A, B, and C genessupportthe ABCmodel of floral organ specification.Depictedherearethe observed patternsof expression thefloralorgan-identity genesin wild-type, mutant,andtransgenic Arabidopsis. Coloredbarsrepresent the A, B,and C mRNAsin each 984

CHAPTER 22

|

pe

se

whorl(W1,W2, W3, W4) Theobserved floralorganin eachwhorl is indicated asfollows:sepal: se;petals: pe;stamens: st; andcarpels: ca Seetextfor discussion D Wiegel andE M [See Meyerowitz, 1994,Cell78:203, andB A Krizek andE M Meyerowitz, 1996, Development 122:11 l

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had proper axis formation but failed to undergo neurulation. To demonstratethat miRNAs, and not someother moleculeaffected by Dicer, are responsiblefor the problem, scientistsattempted to rescuethe mutant fish embryos by injecting them with a prominent classof preformed miRNA duplexes.This succeeded to a remarkabledegree,restoringmuch of the defective neural development.k will be fascinating to learn in the future how miRNAs control neural tube morphogenesrs.

(a) Gradedinductionof differentcell types in the neuraltube b y S h h a n d B M Ps i g n a l s Dorsal Epidermis

Roof plate

Neural tube

Sensory n e ur o n s V1 neurons

S i g n a lG r a d i e n t sa n d T r a n s c r i p t i o F nactors SpecifyCellTypesin the NeuralTube and Somites

V2 neurons Somites

As we've seenalready,developmentalsignalscan act in relay fashion,a sort of bucket brigadein which eachcell receivesone signal and passeson another,or in graded fashion, in which differentconcentrationsof one signalinducedifferentcell fates (seeFigure22-13). Gradedsignals,or morphogens,are important in specifyingcell fatesin differentparts of rhe neural tube: motor neuronsin the ventral part, a variety of interneuronsin the lateral parts, and sensoryneuronsin the dorsal part. The differentcell typescan be distinguished,prior to morphological differentiation,by the proteinsthat they produce. Graded concentrationsof Sonic hedgehog(Shh),a vertebrate equivalent of Drosophila Hedgehog, determine the fatesof at leastfour cell types in the chick ventral neural tube. These cells are found at different positions along the dorsoventral axis in the following order from ventral to dorsal: floor-plate cells,motor neurons,V2 interneurons,and V1 interneurons.During development,Shh is initially produced at high levels in the notochord, which directly contacts the ventral-mostregion of the neural tube (Figure22-40a). The Shh from the notochord induces the most ventral neuraltube cells to form floor-plate cells, a type of non-neuronal glial cell (Chapter 23). Floor-platecells also produce Shh, forming a Shh-signalingcenter in the ventral-most region of the neural tube. Antibodies to Shh protein block the formation of the different ventral neural-tube cells in the chick, and thesecell types fail to form in mice homozygousfor mutations in the Sonic hedgehog(Shh) gene. To determine whether Shh-triggered induction of ventral neural-tubecellsis through a gradedor a relay mechanism,scientistsadded different concentrationsof Shh to chick neuraltube explants.In the absenceof Shh,no ventral cellsformed. In the presenceof very high concentrationsof Shh, floor-plate cellsformed; whereas,at a slightly lower concentrarion,motor 'When neuronsformed. the levelof Shh was decreasedanother twofold, only V2 neurons formed. And, finallg only V1 neurons developedwhen the Shh concentrationwas decreasedanother twofold. Thesedata stronglysuggestthat in the developing neural tube different cell types are formed in responseto a ventral -+ dorsal gradient of Shh, though exactly when in developmentthe signalhas its impact is unknown. The accumulating evidencefor gradientsdoesnot rule out additional relay signalsthat may yet be discovered. Cell fates in the dorsal region of the neural tube are determined by BMP proteins (e.g., BMP4 and BMPT), which belong to the TGFB family. Recall that TGFB-type signals

Motor neurons

F l o o rp l a t e

Notochord

( b ) R e s p o n s eos f n e u r a l - t u b cee l l st o g r a d e dS h h a n d B M P s i g n a l s a l o n gd o r s a l - v e n t r aalx i s PaxT

Dbxl

Dbx2

Nkx6.1 N kx6.1

lrx3

Pax6

Nkx2.2

FIGURE 22-40 Regulationof neural-cellfate in vertebrates. (Shh)secreted (a)Sonichedgehog induces by cellsin the notochord Shh, floor-plate Thefloorplate,in turn,produces development cell that induces additional whichformsa ventral-+ dorsalgradient (TGFPtype signals) secreted fatesIn the dorsalregion,BMPproteins fromroofplatecells. ectoderm cellsandsubsequently fromoverlying (b)Therelative by the of ShhandBMPareindicated concentrations by gradientsCellfatesin the neuraltubecanbe detected colored production factorsshown of allthetranscription the differential levels of Shhinduce between thegradientsHighto moderate of theNkx22 andNkx6/ genes(J) but blockproduction expression at thetop (T) Evenlow factorsindicated of thefivetranscription of Dbxl andPax7,bothgenes levelsof Shhcanblockexpression comingfromcellsin by BMPsignals ispromoted whoseexpression the dorsalneuraltube TheborderbetweenPax6andNkx22, and by mutually betweenDbx2andNkx61, isfurthersharpened the repressive interactions; eachproteinturnsoff the geneencoding cellfates of allthisisa setof different otherproteinTheoutcome (e g , motorneurons neurons) alongthe dorsal-ventral or sensory factors, a uniqueblendof transcription axisEachcelltypecontains of manyothergenesthat controlexpression whichpresumably T.M Jessell, 2000, properties on cellsthattype [See conferdistinctive Rev. Nature Genet'l:2Ol

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and their antagonists are critical in determining dorsal cell fatesin early frog embryos.A Drosophila TGFB signalcalled Dpp also functions in determiningdorsal cell fatesin early fly embryos.Indeed,TGFB signalingappearsto be an evolutionarily ancient regulator of dorsoventral patterning. In vertebrate embryos, BMP proteins secretedfrom ectoderm cells overlying the dorsal side of the neural tube promote the formation of dorsal cells such as sensory neurons (seeFigure 22-40a). Thus cells in the neural tube sensemultiple signals that originate at opposite positions on the dorsoventral axis. By integrating the signals from both origins, each cell embarks on a particular course of differentiation. The gradients of Shh and TGFB spreading out from the ventral and dorsal sides of the neural tube activate or repress the production of particular transcription factors in neural-tube cells at different positions along the dorsal-ventral axis (Figure 22-40b). When thesetranscription factors are first made, the boundariesbenveenthem are fuzzy,butsomeofthe boundaries sharpendue to mutually repressivecross-regulation. Similar mechanismsdetermine cell fates in the somites, which give rise to body wall muscle (myotome), the inner layer of skin called the dermis (dermatome), and the vertebrae (sclerotomel. These different cell fates are induced by signals from surrounding tissues.For instance, Shh coming from the notochord induces sclerorome.Thus the same signal. Shh. induces motor neurons in the neural tube and bone primordia in the somites. The receiving cells are preprogrammed to respond in distinct ways to the sameinducer dependingon their prior history. Somitesalso receivesignals from other directions, such as Wnt from the dorsal neural tube, that induce different subsetsof cells.

Most Neuronsin the BrainArise in the Innermost NeuralTubeand Migrate Outward The cortex of the brain is a thin sheet of cells organized into half a dozenlayers.This portion of our anatomy-required for the most-advancedthinking abilities-is the source of our greatest pride and feelings of superioriry for better or worse, over other living things. Development of the neural tube, which is initially a singlecell layer thick, into a brain requires both the generation of vast numbers of neq progenitor cells from stem cells and the organization of those new cells into layers. The new cellsform mostly in the subuentricularzone, the inner lining of the neural tube lying closestto the ventricles, which are the fluid-filled cavities inside the brain that arise from the interior of the neural tube. Cells take up their final positions in a simple inside-out order, forming the innermost Iayer first and then, progressively,the outer ones (seeFigure 21,-1,2).Thus neurons born late have to migrate past the older cells that have akeady taken up their stations.The migration of some cells involves inreractions with radial glia, support cells that elongate to span the entire distance from the subventricular zone to the outer layer of the cortex. In addition some cells undergo remarkable tangential migration at right anglesto the ventricle-to-surfaceplane. Scientistshave used timeJapse microscopy to observe the behaviors of migrating neurons. Their movemenr is not 988

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smooth or continuous. Instead, the cells move vigorously for a time, pauseand extend processesin what appearsto be a testing of the waters, and then resumemotion in either the original direction or another. In some casesneurons seemto move over each other; in other casesthey follow the processesof glia cells. Migrating cells are responding to a wide variety of guidance cues in the form of surface molecules and secretedsignals, while internally each cell undergoesdramatic shapechanges. Once neurons reach their final destinations,and often while they are in transit, they form long processesto communicate with other cells: the dendrites, which receive signals, and the axons, which transmit signals,sometimesover distancesgreater than a meter.Ve will discusshow the correct wiring pattern is built, to the extent that it is understood, in Chapter 23.

L a t e r a lI n h i b i t i o nM e d i a t e db y N o t c hS i g n a l i n g CausesEarlyNeuralCellsto BecomeDifferent The development of the nervous system provides examples of an important general mechanism for ensuring that all necessary structuresare built, but not in excessivenumbers. This mechanismcalled lateral inhibition, consistsin essenceof a cell communicating to surrounding cells "I am doing this, so you shouldmake somethingelse."Adjacentcellswith equivalentor near-equivalentpotential are in this way directed toward distinct fates. Genetic analysesof Drosophila neural development first revealed the role of the highly conservedNotch/Delta pathway in lateral inhibition. The Drosophila proteins Notch and Delta are the prototype receptor and ligand, respectively, in this signaling pathway. Both proteins are large transmembrane proteins whose extracellular domains contain multiple EGFlike repeats and binding sites for the other protein. Although Delta is cleavedto make an apparentlysolubleversion of its extracellular domain, findings from studies with genetically mosaic Drosophila have shown that the Delta signal reachesonly adjacentcells. Interaction between Delta and Notch triggers the proteolytic cleavage of Notch, releasing its cytosolic segment, which translocatesto the nucleus and regulates the transcription of specific target genes (seeFigure 16-36). ln particular, Notch signaling activates the transcription of Notch itself and repressesthe transcription of Deha, thereby intensifying the difference betweenthe interacting cells (Figure 22-41a). Notch-mediated signaling can give rise to a sharp boundary between two cell populations or can single out one cell from a cluster of cells (Figure 22-41b1. Notch signaling controls cell fates in most tissuesand has consequencesfor differentiation, proliferation, the creation of cell asymmetry, and apoptosis. Here we describetwo examples of Notch signaling in cell-fate determination. Loss-of-function mutations in the Notch or Deba genes produce a wide spectrum of phenorypes in Drosophila. One consequenceof such mutations in either gene is an increasein the number of neuroblasts in the central nervous system. In Drosophila embryogenesis,a sheet of ectoderm cells becomes divided into two populations of cells:those that move insidethe embryo develop into neuroblasts,which give rise to neurons; those that remain external form the epidermis and cuticle (see

T H E M O L E C U L A RC E L LB I O L O G YO F D E V E L O P M E N T

(a)

Intrinsically biased

Equivalent

< FfGURE 22-41 Amplilication of an initial bias to create lateralinhibition.(a)A differentcelltypesby Notch-mediated equivalent cellsmayariserandomly between two initially difference (/eft)Alternatively, bias(center) interacting cellsmayhavean intrinsic that have received different cells bias(right)Forinstance, or an extrinsic willbe intrinsically biased; those proteins celldivision in an asymmetric (orange) willbe extrinsically biased signals different that havereceived R e g a r d l eosfsh o wt h e s m a liln i t i abl i a sa r i s e sN, o t c hb e c o m e s promoting itsownexpression and predominant in oneof thetwo cells, production of itsligandDeltain thatcell.Intheothercell, repressing of the production Theoutcomeisreinforcement of Deltapredominates (b)Notch-mediated inhibition maycreate lateral smaliinitialdifference. fieldof cells, suchasalongtheedgeof in an initial a sharpboundary a centralcellfroma wing,or distinguish Drosophila the developing precursor establishment asin neural cluster of cells, surrounding

Extrinsically biased

I (b)

et al , 1999, Science234:710] [Adaptedfrom S Artavanis-Tsakonas

Field

Cluster

Ftgwe 21-29). As some of the cells enlargeand then loosen from the ectodermalsheetto becomeneuroblasts,they signalto surrounding cells to prevent their neighbors from becoming neuroblasts-a caseof lateralinhibition. Notch,/Deltasignaling is usedfor this inhibition; in embryoslacking the Notch receptor or its ligand,all the ectodermprecursorcellsbecomeneural. The role of Notch signalingin specifyingneural cell fates has beenstudiedextensivelyin the developingDrosophila peripheral nervoussystem.In flies, various sensoryorgans arise from proneural cell clusters,which produce bHLH transcription factors, such as Achaete and Scute,that promote neural cell fates.In normal development,one cell within a proneural cluster is somehow anointed to becomea sensoryorgctnpre-

cursor (SOP).In the other cellsof a cluster,Notch signaling leadsto the repressionof proneural genes,and so the neural fate is inhibited; thesenonselectedcellsgive rise to epidermis (Figure 22-42). Temperature-sensitivemutations that cause functional loss of either Notch or Delta lead to the development of additional SOPs from a proneural cluster. In contrast, in developingflies that produce a constitutively active form of Notch (i.e., active in the absenceof a ligand), all the cells in a proneural cluster developinto epidermal cells. To assessthe role of the Notch pathway during primary neurogenesisin vertebrates,scientistsinjected mRNA encoding different forms of Notch and Delta tnto Xenopus embryos. Injection of mRNA encoding the constitutively active cytosolic segment of Notch inhibited the formation of neurons. In contrast, injection of mRNA encoding an altered form of Delta that prevents Notch activation led to (b)

(a)

I n d u c t i o no f proneuralcluster

Determination

Differentiation

T] :11

Lower level of Emc

SOP

I

I

Cell fate: SOP

A FIGURE 22-42 Roleof Notch-mediatedlateral inhibition in (SOPs) formationof sensoryorganprecursors in Drosophila. (a)Extracellular signaling molecules andtranscription factors, encoded genes, pattern byearly-patterning controlthe precise spatiotemporal of proneural bHLHproteins suchasAchaete andScute(yellow)Most proteinthat cellswithinthefieldexpress Emc(orange), a related antagonizes Achaete andScute, A smallgroupof cells,a proneural produce proneural cluster, bHLHproteinsTheregionof a proneural fromwhrchan SOPwillformexpresses cluster lowerlevels of Emc, givingthesecellsa biastowardSOPformationInteractions among thesecells,mediated by Notchsignaling, leadsto accumulation of Notch-regulated proteins E(spl) repressor in neighboring cells(blue), restricting SOPformation to a singlecell(green)(b)Initially, achaete (ac)andotherproneural genesaretranscribed in allthecellswithina

andother proneural cluster, asareNotchandDeltaAchaete proneural promoteexpression of Delta.Whenonecell bHLHproteins (/eft),its moreAchaete slightly to produce at randombegins in Notchsignaling to stronger production leading of Deltaincreases, cells,the Notch cells(right)ln the receiving allitsneighboring Su(H), factordesignated pathway activates a transcription signaling genes. F(sp/) expression of E(spl) expression whichin turnstimulates (left)cell,allowinga neural,i,e.SOBfate stayslow in the high-Delta of proteins transcription repress specifically In the rightcell,the E(spl) in Achaete proneural genes resulting decrease The ac andother the initialrandom in Delta,thusamplifying leadsto a decrease of theseinteractions difference amongthe cellsAsa consequence asa SOP;allthe isselected cluster andothers,onecellof a proneural cells intoepidermal anddevelop otherslosetheirneuralpotential

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the formation of too many neurons. These findings indicate that in vertebrates,as in Drosophila, lateral inhibition mediated by Notch signaling controls neural precursor cell fates. Similarly in the nematode Caenorhabditis elegans, Notch signaling is used during vulva development to form distinct adjacentcell types by lateral inhibition. The Notch signalingpathway is usedduring the formation of many organs and tissues,but not always for lateral inhibition.

11 days

Cell-TypeSpecificationin Early Neural Development r The vertebrate nervous system developsfrom ectoderm cells, which are initially arranged in a sheetformed during gastrulation.Folding of this ectodermalsheet (the neural plate) first forms the neural tube in the processof neurulation (seeFigures 22-38 and 22-39).

13 days

r The notochord, a rod of mesodermlying underneath(ventral to) the neural tube, is a sourceof signalingproteins that induce cell fates in surrounding tissues.For example, Sonic hedgehog(Shh) from the notochord influencesthe fates of nearby cellsin the neural tube, somites,and endoderm.

1 4d a y s

r Shh is a morphogen that directs certain cell fates at high dosesand other cell fates at lower doses.It tends to promote ventral fates, like floorplate. Its influence is tempered by other signals such as BMP, a TGFB-type signal coming from overlying ectoderm that promotes dorsal neural fates (seeFigure 22-40). r The cellsof the neuraltube, which is initially one cell thick, proliferateand form neural precursorcellsthat migrate radially outward to form the layersof the brain and spinalcord. r During normal Drosophild neurogenesis, only some ectodermalcells form neurons;others form other ectodermderivedcellslike skin. The balancebetweencell types is regulated by lateral inhibition involving Notch signaling.In this process,cells that have taken on a neural fate rnstruct surrounding cellsnot to do so (seeFigures22-41 and 22-42).

Growthand Patterningof Limbs Vertebratelimb developmentprovides a beautiful example of how the actions of individual cellscombine to creareDarrern. Vertebratelimbs grow from small "buds" composedof an inner mass of mesodermcells surrounded by a iheath of ectoderm (Figure 22-43). Hindlimbs and forelimbs are obviously related, as are left and right limbs. If the limbs were broken down into their constituent moleculesor cells, the composition of all four would be nearly identical,yer their shapesdiffer in ways that are absolutely crucial to successfullife. Pattern formation-that is, organizing those moleculesand cells into a coherentwhole-is a central goal of studying the molecular cell biology of limb development.Where would birds be with four legs instead of a pair each of legs and wings? Birds have in fact beena prime experimentalanimal for studies on limb development,sincechick embryos can be readily accessedin the egg during the period of limb formatron. 990

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15 days

^ ,,o;; Limbdevetopmentin the mouse.Limbbuds, whichconsist of an innermass of mesoderm cells andouterlayerof ectoderm, format specific locations on theembryo s flank Outgrowth andpatterning alongthethreeaxesleads to formation of theallthe limbstructures L Wolpert etal, 2001,Principles of Development, 2nd [From ed, Oxford University Press, Figure 10-5l In testing various theories of limb development in the chick, researchershave subjectedembryosto transplantation, injectedthem with signalingproteinsor signal-producingcells, and introduced retrovirusesthat direct geneexpressionin abnormal sites.The continued growth of the animal in the egg then is observed to seethe effect of any particular treatment. Becauseit is relativelyeasyto reduceor eliminatea gene'sfunction in mice, geneticexperimentshave beenusefulin studying limb developmentin theseanimals.The resultsfrom the two experimentalsystemsare largelyin agreement.

H o x G e n e sD e t e r m i n et h e R i g h t P l a c e sf o r L i m b s to Grow The first event in vertebratelimb developmentis determination of where, along the head-tailaxis, limb growth will begin. 'We are not millipedes;decisionsmust be made.In both insects

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( a ) E f f e c to f r e d u c e do r n o F G F 1 0

22-44lhe effectof alteringFGF10 < EXPERf MENTALFIGURE functionon limb developmentin the mouse.(a)Geneknockout partof the fgf10 gene,wereconstructed by mice,lackinga f unctional a few days Thesephotographs of mouseembryos recombination for the fgf10 gene(+/-) are beforebirlhshowthat miceheterozygous impaired limb mice(-/-) haveseverely buthomozygous fairlynormal, mice(+/+) These with that in wild-type compared development growthfactor10 genetic of f ibroblast dataprovethe importance (b)Totest (FGF10), protein, for limbdevelopment signal a secreted limbdevelopment, experiments iscapable of triggering whetherFGF10 embryos areadvantageous weredoneThese withchickembryos operation to growaftera surgical theembryo cancontinue because protein implanted in theflankof weresurgically in FGF10 Beads soaked priorto limbdevelopment, in a region where a mid-stage chickembryo 0, butnotcontrol substances, developFGF'I no limbwouldnormally of a fifthlimb Theyoungchick theformation wasableto induce leg embryo,hasan extra(ectopic) shownhere,froma FGF1O-treated (a)fromMinetal, 1998, isshownIPart Onlyhalfof thechicksskeleton

(b) Effectof ectopicFGF10

Genes& Devel 12:3156 Part(b)from M J Cohn et al , 1995, CeilAO:739l

mesodermand initiates outgrowth of a limb from specificregions of the embryo's flank. Mouse mutants lacking FGF10 develop without limbs (Figure 22-44a). Conversely,implantation of a bead soakedin FGF10 at sitesin the flank of a chick embryo where a limb doesnot normally form causesan extra limb to grow (Figure 22-44b). These results demonstrate the remarkable inductive capabilitiesof FGF. Wnt signaling also plays a role in the initial outgrowth of limb buds.

Normal

and vertebrates,the Hox genescontrol where limbs are made. The mesodermthat givesrise to limb buds, called intermedidte mesoderm,is located adjacent to the somite mesoderm; mesoderm farther from somites is called laterdl plate mesoderm. Expressionof Hox genesin the intermediatemesoderm influencesthe position of limb-bud formation by controlling expressionof genes(e.g., Tbx and Pitxl) that encode other transcription factors in the lateral plate mesoderm.The Tbx and Pitxl proteins,in turn, control production of secretedsignals that are required for limb development. Among these signals are fibroblast growth factor 10 (FGF10), which is secretedfrom cells in the lateral plate

L i m b D e v e l o p m e nD t e p e n d so n I n t e g r a t i o n o f M u l t i p l e E x t r a c e l l u l aSr i g n a lG r a d i e n t s The three axes of a limb bud and developedlimb-anteriorposterior (thumb to little finger), dorsal-ventral(back of hand versuspalm), and proximal-distal (shoulder to fingers)-are shown in Figure 22-45. Early limb developmentis marked by fcrrmationof two important signalingcenters:the apical ectodermal ridge (AER), a region of surfaceectodermat the distal tip of the emerginglimb bud and the zone of polarizing actiutty (ZPA) in mesodermat the posterior end of the bud.

Anterior Mesoderm sells A E Rc e l l s Proximal€

+

Anterior

Distal

Posterior

Posterior Proximal

Limbbud A FfGURE22-45 fhe axes of the limb bud and the hand. A l i m b b u d ( l e f t )a n d f u l l y d e v e l o p e dl i m b ,a h a n d i n t h i s e x a m p l e ( r i g h t ) ,h a st h r e ea x e s :a n t e r i o r - p o s t e r i(ot h r u m bt o l i t t l ef i n g e r ) , p r o x i m a l - d i s t (asl h o u l d etro f i n g e r s )a, n d d o r s a l - v e n t r (abl a c ko f

Distal

h a n dv e r s u sp a l m ) I n t h e d e v e l o p i n lgi m bb u d ,t h e a p i c ael c t o d e r m a l r i d g e( A E R i)s a s o u r c eo f f i b r o b l a sgt r o w t hf a c t o r( F G Fs) i g n a l sa, n d g c t i v i t y( Z P A i)s a s o u r c eo f S o n i ch e d g e h o g t h e z o n eo f p o l a r i z i n a

(shh) GF L I M B S G R O W T HA N D P A T T E R N I NO

991

(b)

(a)

(c)

Fgfs induced by FGF10 _--> FGF10-----+

..+ rroilTerailon

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FGFS

maintained b y F G F S+ FGF4

Fgf4 induced by Shh

-'-\ Lateralplate mesoderm FgflO

Surfaceectoderm

;,

Shh

FgfB

f

rgtu+ rsts

FIGURE 22-46 lntegrationof three signalsin vertebrate limb developmentalong the proximal-distaland anteriorp o s t e r i o ra x e s .E a c hl i m bb u dg r o w so u t o f t h e f l a n ko f t h e embryo(a)A fibroblast growthfactor(FGF) signal,probably FGF10, comesfromthe mesoderm in specific regions of the embryo's flank, o n er e g i o nf o r e a c hl i m b F G F 1a0c t so n a l o c arl e g i o no f s u r f a c e ectoderm called t h ea p i c ael c t o d e r mrai ld g e( A E Rb)e c a u si et w i l l f o r ma p r o m i n e nr itd g e( b )T h ee c t o d e r m t h a tr e c e i v easF G F 1 0 e G F Ba,n o t h esr e c r e t esdi g n a A s i g n ai ls i n d u c etdo p r o d u c F l t the p o s t e r i oern do f t h e l i m bb u d ,F G F B i n d u c etsr a n s c r i p t i o fnt h e (Shh)gene(c)Shhsignaling Sonichedgehog induces transcription of thegeneencoding FGF4 in theAER,FGFB promote andFGF4 proliferation continued of themesoderm cells, causing outgrowth of thelimbbud Shhalsostimulates posterior thisoutgrowth andconfers characteristics partof the limb Development on theposterior along thedorsal-ventral axisdepends on a Wnt signal thatisnotshownhere In responseto FGF10,the AER beginsto secreteFGF8 and later FGF4. Both of thesesignalsdrive persistentdivision of mesodermcellsand thereforeconrinuedlimb outgrowth. Sonic hedgehog(Shh) is produced in the ZPA of the posterior limb bud (Figure22-46); indeed it is Shh secretionthat definesthe ZPA. The FGF signalsconfer a distal fate on cellsin the limb bud, and Shh confersa posteriorfate.If Shh is addedto the anterior part of the bud, the limb that eventuallyforms will have two posterior patternsof bonesin mirror-image,and no anterior. Along the dorsal-ventralaxis, a\Ynt7a signaldirectscells to form ventral cell types.The \7nt7a, FGF4, and FGF8 signals promote the transcription of Shh, and Shh signalingpromotes the transcription of the Fgf4 and FgfS genes.Thus the signals are mutually reinforcingin cellsthat are closeenough;cellstoo far from one of the reinforcing signalswill ceasemaking their own signal.In this wag the strengthand movementof signals is tied to the eventualsizeand shapeof the limb. The main developmentaltask in the formation of limbs and organs is to organize a few cell types (e.g.,mesenchyme, vascular,epithelial) into complex multicellular srructures.As we've just seen,cellsin the midst of the limb bud are exposed to gradients of multiple signals (FGFs, Shh, and Wnt) that provide the initial guidelinesfor this building process.By integratingthesesignals,eachcell is directedto expressspecific 992

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transcription factors dependingon its location relative to the three limb axes. The action of thesetranscription factors in turn control formation of the various parts of the limb. Since each of the limb-bud signals must be produced in exactly the right cells,there is much interest in learning how their genesare controlled. The Shh gene,for example,is transcribed only in the ZPA cells of the posterior limb bud in responseto FGF and other factors.Initial efforts to describethe transcriptional enhancer that controls Shh transcription in the limb bud failed. The mystery was solvedwhen four families were found with a high frequency of polydactyly, an inherited abnormality marked by the presenceof extra digits on the hands and feet. The mutation involved in each family mapped to a region of the genome in the vicinity of the human Shh gene,although the protein-codingparts of Shh were normal. Thesefindings strongly suggestedthat damageto the enhancercontrolling Shh transcription causedthe abnormal developmentresulting in polydactyly in thesefamilies. The Shh limb enhancer is quite remarkable in two respects.First, this regulatory sequenceis located inside the transcription unit of another gene. Second, it is located about one million base pairs away from the Shh promoter! This is the Guinness record for long-distanceregulation of gene transcription. Recent sequence comparisons have shown a high degree of sequencesimilarity throughout the =7-kb Sbh enhancerin mice and humans. A smaller region of sequencesimilarity, the core sequence,also occurs in ricefish (Figure 22-471. All four polydactylous families were found to have mutations that changed the sequenceof the Shh enhancer,as did two polydactylous mouse strains. These findings demonstrate the power of using the evolutionary conservation of DNA sequencesto understand gene regulation and accountfor human disease.

H o x G e n e sA l s o C o n t r o lF i n eP a t t e r n i n g of Limb Structures Once limb-bud growth and Shh production have gotten underway, Shh induces Hox-gene expression, starting at the posterior of the bud. Subsequently,expression of the Hox genes undergoes a complex series of changes and spreads more anteriorly along the bud. The transcription factors produced from Hoxd11-Hoxd13 in posterior cells in turn stimulate expressionof Shh in the posterior. As the bud grows during the period 9.0-10.5 days after fertilization, the domains of Hoxd-gene expressionspreadout (Figure 22-48). The Hox genesremain active in the distal limb bud, where digits will be formed. The combinations of Hox transcription factors in the cells of the developing limb bud, in their elaborateoverlapping patterns, are responsiblefor fine-scale control of limb pattern. Humans carrying murarions in Hoxd13 have an inherited limb malformation syndrome calledsynpolydactyly,marked by duplications of fingers and toes. This defect demonstratesthe crucial nature of Hoxd13 for normal limb development.In mice, deleting aIl Hoxa and Hoxd genescauseslimbs to develop as short stubs without any distal structures,confirming the importance of Hox genesin mouse limb development.

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r Outgrowth of the limb bud along the proximal-distal axis is driven by a FGF signal that emergesfrom the apical ectodermal ridge (AER) at the outermost part of the bud. Patterning of the bud along the anterior-posterioraxis depends upon Sonic hedgehog(Shh) signal produced in the zone of polarizing activity (ZPA) in the posterior bud (seeFigure 22-46). A lil/nt signal directs cells to form ventral cell types. 'Sfnt r Information from FGR Shh, and signals is integrated by cells,so that eachassumesits proper fate and role in the developing limb. r Shh regulatesexpressionof Hox genesin complex overlapping patterns during limb-bud development (seeFigure 22-48). The Hox genes are important for regulating detailed pattern in the limb, for example, of the different digits and the bone, muscle,and nerve they contain.

You have undoubtedly noticed that the sameclassesof signals and transcription factors are used again and again during development of a polarized embryo. There are three reasonsfor this. First, much to the amazementand relief of biologists,the number of different classesof signals and of transcription factors is not too large, perhaps 20 different types of signals for example. For each type of signal, the standard signaltransductionpathway is quite well known (Chapter 15). Variations on the standard are interesting, but it is often fairly safe to predict what will happen when a particular signal (e.g., BMP) reachesa cell. and how to detect and measurethe cell responseto the signal. Second,the protein classesand gene systems involved in development are, to a high degree, conservedin most animals.This conservationhas createda common "molecular genetic" languageamong biologistsworking on the development of many organisms, allowing them to share in discoveryand understanding.Third, the patterns of evolution of development are becoming clear. Understanding how developmentis controlled allows us to understandmuch better how distinct animal forms can arise from mutations that changethe actionsof developmentalregulators.What use is all this work, you might ask. Two answersseemobvious: understanding our origins and using our knowledge of animal developmentto prevent and treat human diseases. Darwin understood the importance of embryology to his theory and wrote extensively on the subject. But the genetic and molecular cell biology mechanismsunderlying evolution were completely unknown in his time. Now we have rich detail about how genescontrol animal form, and a flood of new information is pouring in. New fossils are found regularly of intermediate ancestral forms that link present-day animals. For example, fossils that appear to representcommon ancestersto whales and hippos have been found, as well as the creature Tiktaalik that looks like a fish with scales,but has four legs. Such ancient creaturesprovide clues about how changesin developmental processes-pattern forming processes-underlie evolution. As the networks of interactions that control development of eachparticular tissueare traced,we will be able to

understand how alterations of some components can change animal form and function. Among all the changesthat led to animal diversity-that made bats able to navigate by sonar and moths able to "sniff" single molecules and cheetahsthat can run at 60 mph-were changesthat made us what we are. Genetic damage to any of the developmental regulators are likely to lead to birth defects,cancer,degenerativedisease, and altered resistanceto infection. Indeed,all of theseconditions have been observed, and their relationship to faulty development established.Thus developmentalbiology is a rich sourceof new information about the causesof human disease. Since many proteins work in pathways, linking one developmental regulator to a diseasehas often led to identification of additional human genestied to the same disease.Apart from finding human diseasegenes,there is the very real prospect of applying our knowledge of development to spur tissue regeneration and promote faster, better healing. Regeneration of blood cells from bone marrow transplants that contain hematopoieticstem cellsis now a well-establishedprocedure. There is every likelihood that a flood of new therapies will emerge as manipulation of signals and other developmental proteins becomesmore precise and sophisticated. The ability to transfer knowledge from a wide variety of animal embryos to human biology, a triumph of evolutionary theory and practice, is a foundation for the next stagesof medical advances.

KeyTerms acrosomal reaction 957 apical ectodermalridge

lateral inhibition 988 maternal mRNAs 971

(AER)ee1 950 blastocyst

morphogen 964 morphogenesis969

cleavage950

neurulation 985

cortical reaction9ST

notochord 987 organogenesis951 pair-rule genes976

dosagecompensation system958 epithelial-mesenchymal transitions 950 floral organ-identity genes983 gametes950 gap genes972 gastrulation 951 germ layers 951 genomic imprinting 958 homeosis979 Hox genes978 induction 951

pattern formation 951 segment-polaritygenes 977 sensoryorgan precursor

(soP) e8e

somites977 Spemannorganizer965 syncytium970 transcriptioncascade 974 zoneof polarizingactivity (zPA) 991,

Reviewthe Concepts t. In differentiated cells, only selected genes are tranmost scribed.Yet, except for lymphocytes and erythrocytes' '$7hat evisomatic cells contain the same nuclear genome. denceshows this is true? R E V I E WT H E C O N C E P T S

995

2. Compare and contrast the action of morphogens involved in dorsal-ventral specificationin Xenopus laeuis and Dro soph ila melanogaster. 3. Using in situ hybridization with a probe specific for dorsal mRNA, where in the syncytial Drosophila embryo would you expect dorsal to be expressed?Using immunohistochemistry with an antibody specific for Dorsal protein, where would you expect to detect Dorsal protein? 4. A microarray analysisof wild-type fly embryos and dorsal mutant embryoswould be expectedto yield information on all genesregulated by Dorsal protein. \Why?Other than new genes regulatedby Dorsal, one would expectto seechangesrn regulation of previouslyidentifiedgenes.Expressionof which genes would be increasedor decreasedin dorsal mutants? 5. Deleting the 3' UTR of the bicoid genewould yield what phenotype in a mutant fly? \fhy? 6. How can the group of five gap genesspecify more than five types of cells in Drosophila embryos? 7. At which stage in embryological development do somitesform? $fhat factors affect their development? 8. Sfhat is homeosis?Give an example of a floral homeotic mutation and describethe phenotype of the mutant and the normal function of the wild-type geneproduct. 9. Hox-gene expressioncan be maintained through many cell generations. What molecular mechanismsare involved in this process?

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a. Northern blot analysis using a probe complementary to oskar mRNA or to rp49 mRNA was performed on mRNA isolated from egg chambers,which contain nurse cells and the developing oocyte, in wild-type and mutant flies. The results are shown in part (a) of the figure below. Micrographs of two egg chambersthat were subjectedto in situ hybridization with a probe directedagainstos&armRNA are shown in part (b). The egg chamber on the left is from a wild-type female;the egg chamber on the right is from an osAX female. Hybridization appearsas white staining in the micrographs.

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10. Describe an experiment with Xenopus embryos that demonstratesthe role of the Notch pathway in regulating the formation of neurons in vertebrates. 11. What is the evidencethat a gradient of Sonic hedgehog leads to developmentof different cell types within the chick neural tube? 12. Vhat evidence implicates fibroblast growrh factor 10 (FGF10)in limb development? 13. Synpolydactylyis an inherited human abnormality characterizedby duplicated fingers and toes. !(/hat type of muta'Sfhat tion could cause this abnormality? stage of development would be affected?

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Analyze the Data ln Drosophila. maternally produced mRNAs and proteins that determine the body axes are transported from nurse cells into the developingoocyte. During early oogenesis,one of thesematernal mRNAs, oskar,is dispersedthroughout the oocyte and is not translated. By mid-oogenesis,osAar mRNA is transported to the posterior pole of the oocyte where it is translated;the Oskar protein then initiates formation of most abdominal segments and the germJine cells.This developmentalprocessis defective in oskar nonsense (osANS) mutants. To further understand the role of oskar mRNA and irs protein in Drosopbila development,a new mutant (oskX) was generated (seeA. Jennyet a1.,2006,Deuelopment133:2827-2833).In this fly mutant, the oskar genecontains a transposableelement(TE) in its first exon (E1), as diagrammedhere.For simpliciry introns are representedas thin black lines separatingthe exons: 996

CHAPTER 22

I

\What do these data suggestabout the osftX mutation relative to the osftNS mutation? What is the purpose of probing for rp49 mRNA? b. Further study revealedthat femaleshemizygous (only one allele presentin the animal) for osANSlay eggsthat, when fertilized, develop into embryos lacking posterior structures.In contrast, females hemizygous for osAX produce ooyctes that begin to form but then degenerate.S7hatinformation do these observationsprovide about the function of Oskar protein? The following rescue experiments were undertaken. c. Severaltransgenescarrying different domains ofthe oskar gene were constructedand introduced individually into femaleshemizygous for the osAX mutation. The ability of femalesexpressing each transgeneto lay eggswas then monitored. In all transgeneconstructs,the oskar ptomoter was replacedwith a yeast inducible promoter (UAS). As diagrammed in part (a) of the

THEMOLECULAC R E L LB I O L O G YO F D E V E L O P M E N T

figure below, the first transgene construct includes the entire wild-type oskar genewith its three introns (UAS oskWT). For simpliciry introns are not depicted in the drawings. In the second construct, the wild-type oskar gene lacks its three introns (UAS oskAi(1,2,3)).In the third construct,3' untranslatedregion (UTR) of the wild-type oskar geneis replacedwith the 3' UTR from an irrelevant gene (UAS oskK1O). The final construct contains only the 3' UTR of the oskar gene (UAS osk3' UTR). The relative eggJayingability of the transgenicfemalesis shown in part (b). The white bar (*t"t) is a measureof the eggslaid on averageby nontransgenicwild-type females. (a) E1 E2 E3 E4 UAS

3'UTR

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Ashe, H. L., and J. Briscoe.2006.The interpretationof morphogengradients.Deuelopment133:385-394. Blitz,I. L., G. Andelfinger,and M. E. Horb. 2006. Germ layers to organs:usingXenopus to study "later" development.Semin.Cell Deuel.Biol. 17:1.33-145. Eggan,K., et al.2004. Mice cloned from olfactory sensoryneurons. Natwre 428:4449. Grimm, O. H., and J. B. Gurdon. 2002. Nuclear exclusionof Smad2is a mechanismleadingto loss of competence.Nature Cell Biol. 4:51.9-522. Gurdon, J.8.2006. From nucleartransfer to nuclearreprogramming:the reversalof cell differentiation.Ann' Reu' Cell Deuel' Biol.22:,1-22. Hoegg, S., and A. Meyer. 2005. Hox clustersas modelsfor vertebrategenomeevolution. TrendsGenet.2l:421424. Ingham, P. $7.,and M. Placzek.2006. Orchestratingontogenesis:variations on a theme by sonic hedgehog.Nature Reu.Genet. 7:841-850. Kamath, R. S., et al. 2003. Systematicfunctional analysisof the Caenorhabditis elegansgenome using RNAi. Nature 421:231:237. Kim, S. K., et al. 2001. A geneexpressionmap for Caenorhabditis elegans.Science293 :2087-2092. Kimmel, A. R., and R. A. Firtel. 2004. Breakingsymmetries: regulation of Dictyosteliwa developmentthrough chemoattractant Curr. Opin. Genet.Deuel' and morphogensignal-response. l4(5):540-549. Leptin, M. 2005. Gastrulationmovements:the logic and the nuts and boks. Deuel. Cell 8:305-320. O'Connor, M. B., et aL.2006.ShapingBMP morphogengradients in the Drosophila embryo and pupal wing. Deuelopment t33(2\:183-193. SchierA. F., and \7. S. Talbot. 2005. Molecular geneticsof axis formation in zebrafish.Ann. Reu.Genet.39:561'-61'3. Tomancak,P.,et al. 2002. Systematicdeterminationof patterns of gene expression dttrrng Drosophila embryogenesis.Genome Biol. 8.1-0088. 14. 3 (12):research0O8 Gametogenesis and Fertilization

tr E 1.00 5

Chow, J.C., et al. 2005. Silencingof the mammalian X chromosome.Ann. Reu.GenomicsHuman Genet.6:69-92. Hoodbhoy, T., and J. Dean.2004.Insights into the molecular basis € 0.80 of sperm-eggrecognition in mammals. Reproduction 127:417422. 6 Inoue, N., et al. 2005. The immunoglobulin superfamilyprotein is required for spermto fuse with eggs.Nature 434:234-238. 0.60 Izumo 3; Lee,J. T. 2005. Regulationof X-chromosomecountingby Tsix uJ Science309:768-77 L. and Xite sequences. 0.40 Navarro, P.,et al. 2005. Tsix transcription acrossthe Xist gene alterschromatin confirmation without affectingXlsf transcription: 0.20 imolicationsfor X-chromosomeinactivation. Genes6 Devel t9-.1474-1484. Sado,T., and A. C. Ferguson-Smith.2005. Imprinted X inacti0.00 vation and reprogrammingin the preimplantationmouseembryo. w1118 UAs UAS UAS UAS oskWT oskli(1,2,3) oskK10 osk3'UTR Human Molec. Genet. 1:R59-R64. Trasler,I. M.2006. Gameteimprinting: settingepigeneticpatOffspring from females expressingtransgenes1 or 2 were terns for the next generation.Reprod. Fertil. Deuel- 18:63-69. normal, whereas those arising from females expressing 'Wasserman, 'What P. M., et al. 2004. Egg-sperminteractionsat other infortransgene4 lacked abdominal segments. fertilization in mammals.Eur. J. Obstet. Gynecol.Reprod. Biol. mation can be deduced from these observations about the 1 15S:557-S60.

role of oskar in Drosophila development?

References Highlights of Development Anderson,K. V., and P.W. Ingham. 2003. The transformation of the model organism:a decadeof developmentalgenetics.Nature Genet. 33(Suppll:28 5-293 .

Cell Diversity and Patterning in Early Vertebrate Embryos Freeman,M., and J. B. Gurdon. 2002. Regulatoryprinciplesof developmentalsignaling.Ann. Reu.Cell Deuel.Biol. 18:515-539. Hirokawa, N., et al. 2005. Nodal flow and the generationof left-right asymmetry.Cell 125:3345 ' Olson, E. N. 2006. Generegulatory networks in the evolution and developmentof the heart. Science313:1'922-1927. REFEREN CES

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Shiratori,H., and H. Hamada. 2006.The left-right axis in the mouse:from origin to morphology.Deuelopment133:2095-2104. Stern,C. D. Evolution of the mechanismsthat establishthe embryonic axes.2006. Curr. Opin. Genet.Deuel. 16:41.3-41,8. Tam, P. P.,D. A. Loebel,and S. S. Tanaka.2006. Building the mousegastrula:signals,asymmetryand lineages.Curr. Opin. Genet. Deuel.16:419-425. Control of Body Segmentation: Themes and Variations in Insects and Vertebrates Akam, M. 1987. The molecular basisfor metamericpattern in the D r osoph ila embryo. D eueIopm ent l0l :1,-22. Andrioli, L. P.,et al. 2002. Anterior repressionof a Drosophila stripe enhancerrequiresthree position-specificmechanisms.Deyelopment 129:49314940. Aulehla,A., and B. G. Herrmann.2004. Segmenration in vertebrates:clock and gradientfinally ioined. Genesbeuel. 18:2060-2067. Carapuco,M., et al. 2005. Hox genesspecifyvertebraltypes in the presomiticmesoderm.Genes(t Deuel. 19:2116-2121. Chang,A. J., and D. Morisato. 2002. Regulationof Easteractivity is required for shapingthe Dorsal gradiint in the Drosophila embryo. D euelopment129:563 5-5645. Chopra,V.S., and R. K. Mishra. 2006. "Mir" aclesin hox gene regulation.Bioessays28:445448. Dale, J. K., et al. 2006. Oscillationsof the snail genesin the presomitic mesodermcoordinatesegmentalpatterning and morphogenesisin vertebratesomitogenesis. Deuel. Cell lO:355-366. Davis, G. K., and N. H. Patel.2002. Short, long, and beyond: molecularand embryologicalapproachesto insec segmentafion. Ann. Reu.Entomol. 47 :669-699. Dubrulle, J., and O. Pourquie.2004. Coupling segmenranonro axis formation. Deuelopmentl3L:5783-5793. Ephrussi,A., and D. St. Johnston.2004. Seeingis believing:the . bicoid morphogengradient matures.Cell 116:1,43-1,52. . Freeman,M., and J. B. Gurdon. 2002. Regulatoryprinciplesof developmentalsignaling.Ann. Reu.Cell Deuel.Biol. 18:51.5-539. Fujioka, M., et al. 1999. Analysisof an even-skippedrescue transgenerevealsboth compositeand discreteneuronal and early blastodermenhancers,and multi-stripe positioning by gap gene repressorgradients.Deuelopment126:2527-2538. Gridley,T. The long and short of it: somiteformation in mice. 2006. D euel.Dyn. 235 :2330-2336. Houchmandzadeh,B., E. Wieschaus,and S. Leibler.2002.Establishmentof developmentalprecisionand proportions in the early Drosophila embryo. Nature 415:79 8-802. Johnstone,O., and P. Lasko. 2001. Translationalresulation and RNA localizattonin Drosophila oocytesand embryos.Ann. Reu. Genet. 35:365406. Lemons,D., and \7. McGinnis. 2006. Genomic evolution of Hox geneclusters.Science313:1918-1922. Morgan, R. 2006. Hox genes:a continuation of embryonic patterning?Trends Genet.22:67-69. Sanson,B. 2001. Generatingpatternsfrom fields of cells:examples from Drosophila segmentation.EMBO Rep. 2:1083-1088. Stathopoulos,A., et al. 2002. Whole-genomeanalysisof dorsalventral patterning in the Drosophila embryo. Cell lll:687-70l. Swalla,B. J.2006. Building divergentbody plans with similar genericpathways.Heredity 97(31:235-243. Takeda,K., T. Kaisho, and S. Akira. 2003. Toll-like receDtors. Ann. Reu.Immunol. 2I:33 5-376. _ Yrng, M., and P. $7.Sternberg.2001. Patern formation during C. elegansvulval induction. Curr. TopicsDeuel. Bio. 5l:1,89-220. Wellik,D. M., and M. R. Capecchi.2003. Hox10 and Hox11 genesare requiredto globally pattern the mammalian skeleton. Science30I:363-367.

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Cell-Type Specification in Early Neural Development Colas,J-F.,and G. C. Schoenwolf.2003. Differential expression of two cell adhesionmolecules,Ephrin-A5 and Integrin alpha6, during cranial neurulation in the chick embryo. Deuel. Neurosci. 25:357-365. Cooke, J. E., and C. B. Moens. 2002. Boundary formation in the hindbrain: Eph only it were simple. Trends Neurosci. 25:260-267. Grandbarbe,L., et al. 2003. Delta-Notch signalingcontrols the generationof neurons/gliafrom neural stem cellsin a stepwise process.Deuelopment130:1,39'1,-"1402. Jessell,T. M. 2000. Neuronal specificationin the spinal cord: inductive signalsand transcriptionalcodes.Natwre Reu.Genet. l:20-29. Kriegstein,A. R., and S. C. Noctor. 2004. Patternsof neuronal migration in the embryonic cortex. TrendsNeurosci.2T:392-399. Miao, H., et al. 2001. Activation of EphA receptorryrosine kinase inhibits the Ras/MAPK pathway. Nature Cell Biol. 3:527-530. Santiago,A., and C. A. Erickson.2002. Ephrin-B ligandsplay a dual role in the control of neural crestcell migration. Deuelopment 1293627-3632. Growth and Patterning of Limbs Boulet, A. M., et al. 2004. The rolesof Fgf4 and FgfS in limb bud initiationand outgrowth.Deuel.Biol.273:361-372. Colas,J-F.,and G. C. Schoenwolf.2001. Towards a cellular and molecularunderstandingof neurulation. Deuel. Dyn. 221:117-1,45. Han, M., et al. 2005. Limb regenerationin higher vertebrates: developinga roadmap. Anat. Rec. B Neut Anat.287:1,4-24. Li, C., et al. 2005. FGFR1 funcrion at the earlieststagesof mouselimb developmentplays an indispensablerole in subsequent autopod morphogenesi s. D euelopment 132:475 547 64. Martin, G. 2001. Making a vertebratelimb: new playersenter from the wings. Bioessays23:865-868. Minguillon, C., J. Del Buono, and M. P. Logan. 2005. TbxS and Tbx4 are not sufficientto determinelimb-specificmorphologiesbut have common roles in initiating limb outgrowth. Deuel. Cell 8:75-84. Moon, R. T., et al. 2002.The promise and perils of l(nt signaling through beta-catenin.Science296l.1, 644-1646. Nybakken, K., and N. Perrimon.2002.Hedgehog signaltransduction: recentfindings. Curr. Opin. Genet.Deuel. 12:503-511. Pandur,P.,D. Maurus, and M. Kuhl. 2002. Increasinglycomplex: new playersenter the'Wnt signalingnetwork. Bioessays 24:881.-884. Pires-daSilva, A., and R. J. Sommer.2003. The evolution of signalingpathways in animal development.Natwre Reu.Genet. 4:3949. Rubin, C., et al. 2003. Sprouty fine-tunesEGF signaling through interlinked positive and negativefeedbackloops. Curr. Biol. 13:297-307. Salsi,V., andY. Zappavigna.2006. Hoxdl.3 and Hoxa13 directly control the expressionof the EphAT Ephrin tyrosine kinase receptorin developinglimbs./. Biol. Chem.28l:1992-1999. Tickle, C. 2006. Making digit patternsin the vertebratelimb. Nature Reu.Molec. Cell Biol.7:45-53. Tickle, C. 2003. Patterningsysrems-from one end of the Lim to the other.Deuel. Cell 4:449458. Tickle, C., and A. Munsterberg.2001. Vertebrarelimb development: the early stagesin chick and mouse.Curr. Opin. Genet. Deuel. ll:476-481. Zuniga, A. 2005. Globalizationreachesgeneregulation:the casefor vertebratelimb development.Curr. Opin. Genet.Deuel. 15:403409.

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TO STUDYDEVELOPMENT USINGLETHALMUTATIONS C. Nrlsslein-Volhard and E. Wieschaus,1980, Nature 287:.795

One of the most fascinating questions in developmentalbiology concernsthe proper formation of an embryo. How does a fertilized egg "know" how to form a complex organism? Scientists have puzzled over how to address this question for a long time. In 1980, Christiane Niisslein-Volhard and Eric l7ieschaus first reported studies with the fruit fly Drosophila melanogaster in which a genetic approach was used to addressthis question.

Background To examine complex processessuch as development of an embryo from a fertilized egg, biologists often collect mutant organisms that differ from the normal (wild-type) organism. To apply this genetic approach to developmental biology, geneticists first look for mutant organisms that display an obvious defectin overall formation. Early work uncovereda number of genesinvolved in the development of the fruit fIy Drosophila melanogaster. ln the first genesexamined, the mutations resulted in the birth of flies with obvious physical defects, such as the presence of an extra set of wings. Becausethis approach relied on examining viable flies with physical malformations, it missed many developmentally important genesthat, when mutated, result in the death of the fly embryo. In the late 1.970s.Niisslein-Volhard and Wieschausbegan their pioneering work on the developmentof Drosophila embryos. They sought to identify as many genesin the developmentalprocess as possible by looking for genesthat, when mutated. resulted in the death of the embryonic fly. Their work unveiled severalkey genesactive in the early development of not only Drosophila, but higher organismsas well.

progeny would be heterozygous for any mutations on the chromosomes Geneticists develop systematic methreceived from the father. The hetthey ods, known as genetic screens, to erozygoteswere then bred as separate searchfor mutations that affect biologlines, in essenceisolating eachnew muical processes.Niisslein-Volhard and tation in a separate fly stock. Flies $Tieschaushad to consider severalprewithin each stock then were crossed vious observations on Drosophila dewith each other to generate homozyvelopment when they designed their gous embryos. If the mutation affected screen.First, they knew that genesexa gene needed for embryogenesis,the pressed in the egg, called maternalhomozygous embryos would die but effect genes,as well as genesexpressed could be examined for phenotype. The after fertrlization in the developing emmutant gene, however, would not be bryo, called zygotically actiue genes, lost as it could be recoveredin the hetcontrolled the early developmentof an erozygousflies of that same stock. embryo. They choseto focus on isolatUsing this screen,Niisslein-Volhard ing mutations that affect the zygotiWieschausamasseda large collecand cally active genes.Second,they had to of mutant flies. The next step was tion consider that the Drosophila genome the various mutants to speto assign is diploid, which means that the progbasedon their phenotype. cific classes, eny receive a copy of each gene from focused on the segmentation of They '$Thereas both parents. Scientistshad previously all mutants in the larvae. demonstrated that DrosoPhila renecessarilydisplayed the screen their quired only a single wild-type copy of phenotype of embryonic lethality they most genesin order to develop into a differed greatly in their segmentation viable fly. This made it likely that redefects. To classify these defects, cessivemutations in developmentally Niisslein-Volhard and l7ieschaus exactivegeneswould not resultin embryamined the larvae under the microonic death, the phenotypic screenused scope. They compared the body Patby Ntisslein-Volhardand'$Tieschaus. tern of a wild-type viable larva to those Therefore, they had to breed mutant of the embryonic lethal mutants. By Drosophila to obtain flies that were these patterns, they uncovcomparing homozygous for the mutations of inered three classesof genes that affect terest. segmentation, which they called gap, The overall mutation rate in a natpair-rule, and segment-PolaritY. urally occurring population is quite Gap mutants are missing uP to eight low If a geneticistwere to search for segmentsfrom the overall body which mutants in a natural population, he or results in a smaller body due to the she would have to examine a large death of some of the embryo cells.Three number of individuals. To circumvent mutants-knirps, hunchback, and the this difficulty, Niisslein-Volhard and '!Tieschaus previously characterized Krilppel-felI induced mutations in a into this class,as shown in the accomDrosophila population at the onset of panying photographs. The next class the screen by feeding a mutagenic of mutants, the pair-rule mutants' had chemicalto male flies and mating them deletions of alternating segmentsof to a genetically defined population of body, which caused roughly halfthe wild-type female flies in a process normal-size larval bodies to form. Six known as a geneticcross.The resulting

The Experiment

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< FIGURE 1 A viable, wild-type larva(Normal) iscompared with threelarvaethat havemutationsin the gap genesKr1ppel, hunchback, or knirps. Thoracic segments arelabeled T1-T3,whereas abdominal segments aredesignated A1-A8.Gapmutants are missing entiresegments fromthe bodyplan,asillustrated by the labeled segments on the left.lFrom C Nusslein-Volhard andE Wieschaus, 1980,Nature 287:7951

Normal

Kriippel

hunchback

previously uncharacterizedmutantspaired, euen-skipped, odd-skipped, odd-paired, fushi tarazu, dnd runtwere placed in this class.The final set of mutants, the segment-polarity mutants, had the same overall number of segments as wild-type larvae. But a part of the body partern within each segment was deleted in each mutant. The deleted porrion of the pattern within each segmentwas replaced by a mirror image of the portion that remained. Niisslein-Volhard and rWieschaus'sinitial screen uncovered six mutants of this class, three of which-goo seberry, hedgehog, and patched-had not been previously observed.

Discussion In the first publishedreportof their screen,Niisslein-Volhard and'sfieschaus described 15 mutations that affected segmentation.Of these,only five were

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in previously identified genes. Ifhen they completed the study-often referred to as the Heidelberg screensthey had identified 139 different genes that, when mutated, resultedin embryonic death. These mutants fell into 17 different classes including the ones described here. Together these genes act in a field of equivalent cells and direct each cell to take on a fate appropriate to its position within the field. In this way some cellsform segmentanterior, some make bristles, some make sensory organs, and so forth. Genes that control these sorts of organizing cell-fate decisions are called patternformation genes. As molecular techniques evolved, scientistscloned many of these genesand characterizedtheir gene products. These mutants formed the base for the next quarter century of research into the development of Drosopbila. Moreover, many vertebrate pattern-formation genes are close homologs of the corresponding

T H E M O L E C U L A RC E L LB t O L O G YO F D E V E L O P M E N T

fly genes, and use similar molecular mechanismsto organize cells, tissues, and organs. Thus the work of Niisslein-Volhard and \Tieschaus greatly advancedthe study of development in all vertebrates,including ourselves. The majority of genesidentified by Niisslein-Volhard and Wieschaus encode transcription factors, but their screen also uncovered genesencoding signaling molecules, receptors, enzymes, adhesion molecules,cytoskeleton proteins, and some proteins whose functions remain unknown. Mammalian genes related to some of the Drosophila genes uncovered by Niisslein-Volhard and Wieschaussubsequentlywere found to be important in human diseasessuch as cancer and birth defects.In 1995, the Nobel Foundation awarded its prize for Physiology and Medicine to Niisslein-Volhard and \flieschaus for their pioneering work.

CHAPTER t

t

NERVECELLS

Theconvolutedsurfaceof the cerebrumand(lowerright) Unlimited] the cerebeilum[O RalphHutchingstuisuals

as the pinnahen humanswish to view themselves cle of evolution,the assertionis usuallyrelatedto the brain. since manv of our other abilities fare poorly in comparisonwith other animals.The complexityof the human brain is staggeringand seemsadequateto account for its amazing abilities.In the 1.3-kg adult human brain (78 percentof which is water!), there are about 1011 nervecells,calledneurons.The number of human brain neurons is comparableto the estimated1011starsin our galaxn the Milky Way. The neurons in one human bratn are conthe iunction pointswheretwo nectedby some1014synapses, or more neurons communicate.\X/ith 6.5 x 10o people on r h ee a r t h ,t h e r ea r ea b o u t6 . 5 x 1 0 2 3h u m a ns y n a p s ei sn e x istence,which is also about the total number of starsin the universe. . . so far as we know. Using our neurons,we can keep searchingfor more. Right this moment you are vigorously employing neurons to detectand interpret visual information. Neurons gobble ATP, made exclusively from glucose, at a tremendous rate. Although the brain is only about 2 percent of the body's mass,it usesabout 20 percentof the body's restingenergy. This extensivebrain energyis usedto drive electricalsignaling along neurons, which are often elongated cells, and chemicalsignalingbetweenthem. The electricalpulsesthat travel along neuronsare called action potentials,and information is encodedas the frequencyat which action potentials are fired. Owing to the speedof electricaltransmission, neuronsare champion signaltransducers,much faster than cellsthat secretehormonesor developmentalsignalingproteins. The rapidity of neural signalingmakes it possibleto handle large amounts of information quickly. The amazing

complexity of our neural network makes possiblesophisticated perception, analysis,and response,and forms the cellular machinery underlying instinct, learning, memory' and emoilon. In this chapter we will focus on neurobiology at the cell and molecular level. We will start by looking at the general architectureof neurons and at how they carry signals(Figure 23-1). Next. we will look at ion flow, channelproteins,and membrane properties:how electricalpulses move rapidly along neurons. Third, we will examine communication between neurons: electrical signals traveling along a cell must be translated into a chemical pulse between cells and then back into an electrical signal in the receiving cells. In the

OUTLINE 23Jl

N e u r o n sa n d G l i a :B u i l d i n gB l o c k so f the NervousSYstem

23.2

Voltage-Gatedlon Channelsand the Propagationof Action Potentials i n N e r v eC e l l s

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Communicationat SYnaPses

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73.3

, earing, , e e l i n gH : e e i n gF 2 3 . 4 S e n s a t i o n aCl e l l sS T a s t i n ga, n d S m e l l i n g

1027

ControllingAxon The Pathto Success: Growth and Targeting

1040

23.5

1001

A

)

l!, (f)

(h)

FIGURE 23-1 lllustrationsof the nervoussystemand nerve cellsby Ramony Cajal(1852-1934). (a)Sensory andmotor nervous systems of a worm(A : sensory cellsof the skin,C : motor cellswith crossed processes, G : terminalramifications of a motor neuronon a muscle)(b)Cross sectron of a spinalcord (c)Sectron throughtheopticlobeof a chameleon (numbers indicate layers from d e e pt o s u p e r f i c iAa ,l ;C ,D : S h e p h esr d c r o o kc e l l s () d ,e )C e l l si n the nuclear layerof the kittensuperior (f)Chiasm (crossinq colliculus

place) andcentralprojection (c : crossed of humanvisualpathways bundleof theopticnerve, d : largeuncrossed bundle,Rv: projection of the mentalimage-thearrow-ontovisualareasof the cerebral cortex)(g)Neurons in thefusrform layerof the human motorcortex(A,E : pyramidal cells,a : axon)(h)Motorneurons (a : terminal terminating on rabbitmuscles arborization of an axon, 4 : pointwheremyelinsheathends,n : branchof a nerve).

Neuronsand Glia:BuildingBlocks of the NervousSystem

high-throughput signal processingand analysis.rWerefer to the processingas "thinking," and molecular cell biology is at the heart of it. 1002

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In this sectionwe take an initial look at the structure of neurons and how they propagateelectricaland chemical signals. Neurons are distinguished by their elongated, asymmetric shape,by their highly localized proteins and organelles,and most of all by a set of proteins rhat controls the flow of ions across the plasma membrane. Becauseone neuron can respond to the inputs from multiple neurons,generateelectrical

signals,and transmit the signalsto multiple neurons, a nervous system has considerablepowers of signal analysis' For example, a neuron might pass on a signal only if it receives five simultaneousactivating signalsfrom input neurons.The receiving neuron measuresboth the total amownt of incoming signal and whether the five signalsare roughly synchronous.Input from one neuron to another can be either excitatory-combining with other signals to trigger electrical transduction in the receiving cell-or inhibitory, discouraging such transmission.Thus the properties and connections of individual neurons set the stage for integration and re'$7e will begin by looking at how finement of information. signals are receivedand sent, and in subsequentparts of the chapter we will look at the molecular details of the machinery involved.

I n f o r m a t i o nF l o w sT h r o u g hN e u r o n sf r o m Dendritesto Axons Neurons arise from roughly sphericalnewroblastprecursors. Newly born neurons can migrate long distanceswhile still in the form of simple round cells before growing into dramatically elongatedcells. Fully differentiated neurons take many forms, but generally share certain key features (seeFigure 23-1). The nucleusis found in a rounded part of the cell called the cell body (Figure 23-21. Branching cell processes called dendrites (from the Greek for "treelike") are found at one end, and are the main structure where signals are receivedfrom other neurons via synapses.Incoming signalsare also receivedat synapsesthat form on neuronal cell bodies. Neurons often have extremely long dendrites with complex branches,particularly in the central nervous system.This allows them to form synapseswith, and receivesignalsfrom' a large number of other neurons-up to tens of thousands. Thus the converging dendritic branchesallow signals from many cells to be receivedand integrated by a single neuron.

'When

a neuron is first differentiating' the end of the cell opposite the dendrites undergoesdramatic outgrowth to form a long extended arm called the axon, which is essentially a transmissionwire. The growth of axons must be controlled so that proper connections are formed, a processcalled axon guidancethat is discussedin Section23.5. The diametersof rr"ry from just a micrometer in certain nerves of the hu"*orr, man brain to a millimeter in the giant fiber of the squid' Axons can be metersin length (in giraffe necks,for example),and

next neuron. The asymmetryof the neuron' with dendritesat one end and axon termini at the other, is indicative of the unidirectional flow of information from dendritesto axons'

they have other important roles. Much current work is devoted to learning how glia build the myelin insulators that control neuronal electricaltransmission,provide growth factors and other signals to neurons' receivesignals from neurons, and influence the formation of synapses.

lnformation Moves as Pulsesof lon Flow C a l l e dA c t i o n P o t e n t i a l s Nerve cells are members of a class of excitable cells, which also includesmusclecells,cellsin the pancreas,and someothers. The term indicatesthat the cells can build up a voltage acrosstheir plasma membranes,the membranepotential and

( a )M u l t i p o l ai nr t e r n e u r o n Axon terminal

Dendrite I

I

D i r e c t i o no f a c t i o no o t e n t i a l

hillock Muscle

(b)Motorneuron C e l lb o d y -i

Nodes of Ranvier Dendrite

Myelin sheath

D i r e c t i o no f a c t i o np o t e n t i a l

Axon terminal

23-2 Typicalmorphologyof < FIGURE two typesof mammalianneurons.Action potentials arisein the axonhillockandare towardtheaxonterminus(a)A conducted branched hasprofusely interneuron multipolar at synapses signals whichreceive dendrites, Small otherneurons. hundred with several by inputsin the imparted changes voltage cansumto giveriseto the more dendrites whichstartsin the actionpotential, massive A singlelongaxonthat branches hillock. to signals transmits at itsterminus laterally (b) innervating neuron A motor otherneurons hasa singlelongaxon celltypically a muscle fromthe cellbodyto the effector extending s ,n c e l ll.n m a m m a l i amno t o rn e u r o n a all covers usually myelin of sheath insulating of nodes the at except the axon of oarts andthe axonterminalsThemyelin Ranvier of cellscalledg/r,a sheathiscomposed

OFTHENERVOUSYSTEM A N D G L I A :B U I L D I N GB L O C K S NEURONS

1003

that this voltage can be discharged(allowed to come back to zero voltage or even swing to positive) in various ways and for various purposes (Chapter 11). The voltage in a typical neuron, called the resting potential becauseit is the state when no signalis in transit, is establishedby ion pumps in the plasma membrane. The pumps use energy, in the form of ATP, to move positively charged ions out of the cell. The result is a net negativechargeinside the cell compared with the outside environment. A typical resting potential is -60 mV. Neurons have a languageall their own. The signalstake the form of brief local voltagechanges,from negativeinsideto positive,an eventdesignateddepolarization.A powerful surge of depolarizingvoltage change,moving from one end of the neuron to the other, is called an action potential. "Depolarization" is somewhat of a misnomer,sincethe neuron goes from negative inside to neutral to positive inside, which could be accurately described as depolarization followed by the oppositepolarization(FigureZ:-:1. et the peak of an action potential, the membranepotential can be as much as *50 mV (insidepositive),a net changeof =110 mV. As we shall seein greater detail in Section 23.2, the voltage change (which is eventuallyadded to other voltage changesro creare the action potential) beginsat the dendriteend ofthe cell in responseto inputs from other cellsand movesalong the axon to the axon terminus.Action potentialsmove at speedsup to 100

tentials are all or none. Once the threshold to start one is reached,a full firing occurs.The signal information is therefore carried primarily not by the intensity of the action porentials, but by the timing and frequencyof them. Someexcitablecells are not neurons.Muscle contraction is triggered by motor neurons that synapsedirectly with ex, citablemusclecells(seeFigure23-2b).Insulin secretionfrom the beta cells of the pancreasis triggered by neurons.In both casesthe activating event involves an opening of plasma

Axon of presynaptic cell Exocytosisof neurotransmitter

Synaptic vesicle Synapticcleft Postsynaptic cell Receptorsfor neurotransmitter

Directionof f signaling

Axonterminal of presynaptic c el l

negative-insiderestingpotential (repolarization).Theresrora_ tlon processchasesthe action potential down the axon to the terminus,leavingthe neuron ready to signalagain.Action po-

Synapticvesicles Synapticcleft

A c t i o np o t e n t i a l s Dendriteof postsynaptic cell

+50 mV o c q)

o

q) c o

o)

o N

0.5 prm

E

o

-60 mV

Time ----> EXPERIMENTAL FTGURE 23-3 Recordingof an axonal membrane potential over time revealsthe amplitude and frequency of action poentials. An actionpotentialis a suooen, transientdepolarization of the membrane, followedby reporarization to the restingpotentialof about -60 mV Theaxonalmembranepotential can be measured with a smaljelectrode placedinto it (seeFigure1.1_1g) Thisrecording of the axonalmembranepotentialin this neuronshows that it is generating one actionpotentialaboutevery4 milliseconds 1004

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A FIGURE 23-4 A chemicalsynapse. (a)A narrowregion-the synaptic cleft-separates the plasma membranes of tne presynaprrc andpostsynaptrc cellsArrivalof actionpotentials at a synapse causes release (redcircles) of neurotransmitters bythepresynaptic cell,their diffusion across thesynaptic cleft,andtheirbindingbyspecific receptors on theplasma membrane of the postsynaptic cell.Generally thesesignals depolarize the postsynaptic (making membrane the potential inside lessnegative), tendingto inducean actionpotential in it (b)Electron micrograph showsa dendrite synapsing wtrnan axon terminal filledwithsynaptic vesicles Inthesynaptic region, theplasma membrane of the presynaptic cellisspecialized for vesicle exocytosis; synaptic vesicles containrng a neurotransmitter areclustered in these regionsTheopposing membrane of the postsynaptic cell(inthiscase, a neuron) contains receptors for the neurotransmitter. (b)fromC [part Raineet al , eds , 1981, BasicNeurochemistry3d ed, Little,Brown, p 32 l

membranechannelsthat causeschangesin the transmembrane flow of ions and in the electricalproperties of the regulatedcells.

T h e N e r v o u sS y s t e mU s e sS i g n a l i n gC i r c u i t s C o m p o s e do f M u l t i P l eN e u r o n s

l n f o r m a t i o nF l o w sB e t w e e nN e u r o n s via Synapses What starts an action potential?Axon termini from one neuron are closelyapposedto dendritesof another,at the junction called a synapse(Figure23-4). The axon termini of the presynaptic cell use exocytosisto releasesmall molecules called neurotransmitters.Neurotransmitters, glutamate or acetylcholine,for example,diffuseacrossthe synapsein about 0.5 ms and bind to receptorson the dendrite of the adiacentneuron. Binding of neurotransmittertriggersopeningor closingof specific ion channelsin the plasmamembraneof postsynapticcell dendrites, leading to changesin the membrane potential at this point. The ensuinglocal depolarization,if large enough' triggers an action potential. Transmissionis unidirectional, from the axon termini of the presynapticcell to dendritesof the postsynapticcell. In some synapses,the effect of the neurotransmittersis to hyperpolarizeand thereforelower the likelihood of an action potential in the postsynapticcell. A single axon in the central nervous system can synapsewith many neurons and induce responsesin all of them simultaneously. Conversely,sometimesmultiple neuronsmust act on the postsynaptic cell roughly synchronouslyto have a strong enough impact to trigger an action potential. Neuronal integration of depolarizingand hyperpolarizingsignalsdeterminesthe likelihood of an actionpotential. Thus neurons employ a combination of extremely fast electricaltransmissionalong the axon with rapid chemical

"-

O u a d r i c e p sm u s c l e

communication between cells' Now we will look at how a chain of neurons, a circuit, can achievea useful function'

In complex multicellularanimals,such as insectsand mammals, various types of neurons form signaling circuits. A sensory neulon reports an event that has happened' like the arrival of a flash of light or the movement of a muscle.A motor neuron carries a signal to a muscle to stimulate its contraction (Figure23-5, and seeFigure23-2b). An interneuron bridges other neurons, sometimesallowing integration or divergenceof signals, sometimes extending the reach of a signal. In a simple type of circuit called a reflex arc interneurons connect multiple sensoryand motor neurons' allowing one sensory neuron to affect multiple motor neurons and one motor neuron to be affected by multiple sensory neurons; in this way interneurons integrate and enhancereflexes.For example, the knee-jerk reflex in humans (seeFigure 23-5) involves a complex reflex arc in which one muscle is stimulated to contract while another is inhibited from contracting. The reflex also sends information to the brain to announcewhat happened.Such circuits allow an organism to respond to a sensoryinput by the coordinated action of setsof musclesthat together achievea single purpose. These simple signaling circuits, however, do not directly explain higher-orderbrain functions such as reasoning,compuiation, and memory development.Typical neurons in the brain receive signals from up to a thousand other neurons and, in turn, can direct chemical signalsto many other neurons. The output of the nervous systemdependson its circuit

-*-.*/ --

S e n s o r yn e u r o n Axon carries cell body i n f o r m a t i o nt o b r a i n S p i n a lc o r d

Sensory -J n e ur o n D o r s a l - r o o tJ ganglion

Motor neuron

Biceps muscle (flexor) Motorneuron a x o nt e r m i n a l

S-

K n e ec a p Motor neuron

Inhibitory I n t e r n e ur o n

23-5 The knee-jerkreflex.A tapof thehammer A FIGURE in the electrical activtty muscle, thustriggering thequadriceps stretches in the traveling neuronTheactionpotential, sensory stretchreceptor srgnals to thebrainsowe are of thetop bluearrowsends direction andalsoto two kindsof cellsin thedorsalawareof whatishappening, in the spinalcord Onecell,a motor that islocated rootganglion (red), muscle stimulates backto thequadriceps neuron thatconnects

yourkneeThe sothatyoukickthe personwho hammered contraction interneuron "excites," inhibitory an or activates, connection second by a activity (black)Theinterneuron hasa dampingeffect,blocking activate thatwould,in othercircumstances, flexormotorneuron(green) Inthisway,relaxation thequadriceps thatopposes muscle the hamstring Thisisa quadrrceps the of to contraction iscoupled of thehamstring decision conscious no requires movement reflexbecause

B L O C K SO F T H E N E R V O U S Y S T E M A N D G L I A :B U I L D I N G NEURONS

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properties-the wiring, or interconnections, between neurons and the strength of theseinterconnections.Complex aspects of the nervous system, such as vision and consciousness,cannot be understood at the single-celllevel. but onlv a t t h e l e v e lo f n e t w o r k so f n e r v ec e l l si h a t c a n b e s t u d i e db y techniques of systemsanalysis.The nervous sysremls constantly changing;alterations in the number and nature of the interconnections between individual neurons occur, for example, in the formation of new memones.

N e u r o n sa n d G l i a : B u i l d i n g B l o c k so f t h e Nervous System r Neuron are highly asymmetric cells composed of dendritesat one end, a cell body containingthe nucleus,a long a x o n ,a n d a x o n t e r m i n i . r Neurons carry information from one end to the other using pulses of ion flow across the plasma membrane. Branchedcell processes, dendrites,at one end of the cell receive chemical signals from other neurons, triggering ion flow. The electricalsignal moves rapidly ro axon termini at the other end of the cell (seeFigure23-2). r Glial cellsare ten times as abundant as neurons and serve many purposes such as building the insulation that coats neurons and supporting the formation of new synapses. A resting neuron carrying no signal has protern pumps at move ions across the plasma membrane. The movement of ions such as K+ and Na* and Cl crearesa net negative chargeinside the cell. This voltage is called the resting potentialand usuallyis about -60 mV (seeFigure23-3). a stimuluscauseschannelproteinsto open so that ions flow more freely, a strong pulse of voltage changemay down the neuron from dendritesto axon termini. The cell goesfrom being -60 mV insideto +50 mV inside,relative to the extracellular world. This pulse is called an action potential. r The action potential travels down the neuron becausea change in voltage near the dendrites triggers a change in voltage in the cell body, which in rurn does the sameto the proximal and then distal axon, and so forth. r Neurons connect across small gaps called synapses. Sincean action potential cannot jump the gap, atthe axon termini of the presynaptic cell the signal is converted from electricalto chemical to stimulate the postsynapticcell. r Upon stimulation by an action potential, axon termini release,by exocytosis, small packets of chemicals called neurotransmrtters. Neurotransmitters diffuse across the synapseand bind to receptorson the dendriteson the other side of the synapse.Thesereceptorsinitiate a new axon potential in the postsynapticcell (seeFigure 23-4). Neurons form circuits. They may consist of sensoryneuns, interneurons,and motor neurons,as in the knee-ierk response(seeFigure23-5).

r006

C H A P T E R2 3

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Voltage-Gated lon Channelsand the Propagation of ActionPotentialsin NerveCells Action potentials are propagated becausea change in voltage in one part of the cell triggers the opening of channelsin the next section of the cell. Voltage-gated channels therefore lie at the heart of neural transmission(Chapter 11). In this section,we first introduce some of the key properties of action potentials, which move rapidly along the axon. I7e then describe how the voltage-gatedchannels responsiblefor propagating action potentials in neurons operate.Thus electric signals carry information within a nerve cell, while chemical signals, discussedin the next section, carry information from one neuron to another, or from a neuron to a muscle or other target cell.

The Magnitude of the Action potential ls Close to Ep. Operation of the Na*/K* pn-p generatesa high concentration of K+ and a low concentrationof Na+ in the cytosol,relative to those in the extracellularmedium (Chapter 11). The subsequentoutvvardmovement of K+ ions through nongated K- channels is driven by the K+ concentration gradient (cytosol ) medium), generatingthe restingmembranepotential. The entry of Na* ions into the cytosol from the medium also is thermodynamically favored, driven by the Na+ concentration gradient (medium > cytosol) and the inside-negative membranepotential (seeFigure 11-24). However, mosr Na+ channelsin the plasmamembraneare closedin restingcells,so little inward movement of Na* ions can occur (Figure 23-6a). If enough Na* channelsopen, the resulting influx of Na- ions will more rhan compensatefor the efflux of K+ ions through open resting K+ channels.The result would be a net inward movement of cations, generating an excessof positive charges on the cytosolic face and a corresponding excessof negative charges (owing to the Cl- ions .,left behind" in the extracellular medium after influx of Na* ions) on the extracellular face (Figure 23-6b). In other words, the plasma membrane is depolarized to such an extent that the inside face becomesoositive. The magnitude of the membrane potenrial at the peak of depolarization in an action potential is very closeto the Na+ equilibrium potential Ey^ given by the Nernsr equation (Equation I1.-2), as would be expectedif opening of voltagegated Na* channelsis responsiblefor generatingaction potentials. For example, the measuredpeak value of the action potential for the squid giant axon is 35 mV, which is closeto the calculated value of En1,(55 mV) based on Na+ concentrations of 440 mM outside and 50 mM inside. The relationship befweenthe magnitude of the action potential and the concentrationof Na+ ions inside and outside the cell has been confirmed experimentally.For instance,if the concentration of Na* ions in the solution bathing the squid axon is reduced to one-third of normal, the magnitude of the depolarization is reduced by 40 mV, nearly as predicted.

(a)Restingstate(cytosolicfacenegative) b
++

++

tc 1 2m M

140mM

Mngated Kr channel (partlyopen)

CVtosol

uuu Na*channels (closed)

(b)Depolarized state(cytosolic facepositive) E
K+

1 5 0m M M-

'12 mM CYtosol

Lq\ 140mM

Voltage-gated K+channel (open)

Na*

Na*

M+

M* channels (open)

23-6 Depolarization of the plasmamembranedue a FIGURE neurons, a type to openingof gated Na+channels.(a)In resting ispartially K* channel open,butthe morenumerous of nongated gatedNa* channels areclosedThemovement of K* ionsoutward potential of membrane characteristic the inside-negative establishes permits an influxof of gatedNa* channels mostcells(b)Opening potential Na* ionsto causea reversal of the membrane sufficient openandsubstate,voltage-gated K* channels In the depolarized repolarize the membraneNotethattheflowsof ionsare sequently concentration of either too smallto havemucheffecton the overall or exterior fluid. Na* or K* in thecytosol

S e q u e n t i aO l p e n i n ga n d C l o s i n go f V o l t a g e G a t e dN a + a n d K + C h a n n e l sG e n e r a t eA c t i o n Potentials The cycle of changesin membrane potential and return to the resting value that constitutes an action potential lasts 1-2 millisecondsand can occur hundreds of times a second in a typical neuron (seeFigure23-3). Thesecyclicalchanges in the membrane potential result from the sequentialopening and closing first of uoltage-gatedNa* channelsand then of uohage-gatedK* channels.The role of these channelsin the generation of action potentials was elucidated in classic studiesdone on the giant axon of the squid, in which multiple microelectrodescan be insertedwithout causing damage to the integrity of the plasma membrane.However, the same basicmechanismis usedby all neurons. voltageVoltage-Gated Na+ Channels As just discussed, gated Na+ channelsare closedin resting neurons.A small depolarization of the membrane causesa conformational changein thesechannel proteins that opens a gate on the cytosolic surface of the pore, permitting Na- ions to pass through the pore into the cell. The greater the initial membrane depolarizatron,the more voltage-gatedNa* channels that open and the more Na- ions that enter. As Na* ions flow inward through opened channels,the excesspositive charges on the cytosolic face and negative

chargeson the exoplasmicface diffuse a short distanceaway from the initial site of depolarization.This passiuespread of positive and negative chargesdepolarizes(makes the inside less negative) adjacent segmentsof the plasma membrane' causingopening of additional voltage-gatedNa* channelsin thesesegmentsand an increasein Na* influx. As more Na* ions enter the cell, the inside of the cell membrane becomes more depolarized,causing the opening of yet more voltagegated Na+ channels and even more membrane depolarizaiion, setting into motion an explosiveentry of Na+ ions. For a fraction of a millisecond, the permeability of this region of the membrane to Na* becomesvastly greater than that for K*, and the membrane potential approachesEv" the equilibrium potential for a membrane permeable only to Naions. As the membrane potential approachesE51",however, further net inward movement of Na* ions ceases,since the concentrationgradient of Na* ions (outside > inside) is now offset by the inside-positive membrane potential Ev". The action potential is, at its peak' close to the value of E1q". Figure 23-7 schematicallydepicts the critical structural features of voltage-gatedNa+ channelsand the conformational changes that cause their opening and closing. In the resting state,a segmentof the protein on the cytosolic face-the gateobstructsthe central pore, preventingpassageof ions' A small depolarizationof the membrane triggers movement of positively charged uoltage-sensiago. helicestoward the exoplasmic surface,causinga conformational changein the gate that opens the channel and allows ion flow. After about 1 ms, further Nainflux is preventedby movement of the cytosol-facing channelinactiuating segment into the open channel. As long as the membraneremains depolarized,the channel-inactivatingsegment remains in the channel opening; during this refractory period,thechannel is inactivatedand cannot be reopened.A few milliseconds after the inside-negativeresting potential is reestablished,the channel-inactivating segment swings away from the pore and the channel returns to the closed resting state,once again able to be openedby depolarization. Voltage-Gated K+ Channels The repolarization of the membrane that occurs during the refractory period is due Iargely to opening of voltage-gatedK+ channels'The subsequent increasedefflux of K* from the cytosol removes the excesspositive chargesfrom the cytosolic faceof the plasma membrane (i.e., makes it more negative), thereby restoring the inside-negativeresting potential. ActuallS for a brief instant the membrane becomeshyperpolarized.At the peak of this hyperpolarization, the potential approachesE6' which

ized, and close only when the membrane potential has returned to an inside-negativevalue. Becausethe voltage-gated K* channelsopen slightly after the initial depolarization, at the height of the action potential, they sometimesare called delayedK* channels.Eventually all the voltage-gatedK* and Na+ channelsreturn to their closed resting state. The only

O F A C T I O NP O T E N T I A L ISN N E R V EC E L L S AND THE PROPAGATION V O L T A G E - G A T E IDO N C H A N N E L S

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R e p o l a r i z a t i oonf m e m b r a n e d, i s p l a c e m e not f c h a n n e l i n a c t i v a t i n sge g m e n t ,a n d c l o s u r eo f g a t e ( s l o w ,s e v e r a m l s) Na+ Depolarized membrane

Cytosol Gate

Voltage-

In n e r

C h an n e l - i n a c t i v a t i n g s e n sn rg

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segment s helix ClosedNa* channel

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Open Na+ channel

I n i t i a ld e p o l a r i z a t i o nm, o v e m e n t o f v o l t a g e - s e n s i nag h e l i c e s ,

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channel iioli',?,n it

lnactiveNa* channel (refractory periodl

Returnof voltage-sensing cr helicesto restingposition, i n a c t i v a t i oo nf c h a n n e l (0.5-1.0 ms )

E

A FIGURE23-7 Operational model of the voltage-gated Na+ c h a n n e l .F o u rt r a n s m e m b r a ndeo m a i n si n t h e p r o t e i nc o n t r i b u t e to the centralporethroughwhich ionsmove The criticalcomponents that controlmovementof Na+ ionsare shown here in the cutaway viewsdepictingthree of the four transmembrane domains Il In the closed,restingstate,the voltage-sensing ct helices,which have positively chargedsidechainseverythird residue,are attractedto the negatrvechargeson the cytosolicsideof the restingmembraneThis keepsthe gate segmentin a positionthat blocksthe cnanner, preventingentryof Na* ions I In response to a smail depolarization, the voltage-sensing helicesrotatein a screwlike

mannertoward the outer membranesurface,causingan immediate conformational changein the gate segmentthat opensthe channel helicesrapidlyreturnto the restingposition, fl The voltage-sensing and the channel-inactivating segmentmovesinto the open channel, preventingpassageof furtherions 4 Oncethe membraneis repolarized, the channel-inactivating segmentis displacedfrom the channelopeningand the gate closes;the proteinrevertsto the closed,restingstateand can be openedagainby depolarization 2001,Nature 409:988;M Zhouet al , 2001.Nature [SeeW A Catterall, 411:657;andB A Yi andL Y Jan,2000,Neuron27:4231

open channels in this baseline condition are the non-sated K+ channels that generate the resting membrane porenri;1, which soon returns to its usual value (seeFigure 23-6a). The patch-clamp tracings in Figure 23-8 reveal the essential properties of voltage-gated K+ channels. In this experiment, small segments of a neuronal plasma membrane were

held clamped at different voltages, and the flux of electric chargesthrough the patch due to flow of K* ions through open K+ channelswas measured.At the modestdepolarizing voltage of -10 mV, the channelsin the membrane patch open infrequently and remain open for only a few milliseconds, as judged, respectively,by the number and width of

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2 0 0m s p r o b a b i l i t yo f c h a n n e l EXPERIMENT FA TL G U R2 E 3-8 o p e n i n ga n d c u r r e n tf l u x t h r o u g hi n d i v i d u avl o l t a g e - g a t e d K* channelsincreases with the extent of membrane depolarization. Thesepatch-clamp tracings wereobtained from patches of neuronal plasma membrane clamped at threedifferent p o t e n t i a l+s 5, 0 , + 2 0 ,a n d- 1 0 m V T h eu p w a r d e v i a t i o ni nst h e c u r r e ni tn d i c a tteh eo p e n i n o g f K * c h a n n eal sn dm o v e m e notf 1008

.

cHAprER 23 |

N E R V cEE L L s

K- ionsoutward (cytosolic to exoplasmic face)acrossthe m e m b r a n eI n c r e a s i ntgh e m e m b r a n ed e p o l a r i z a t i o( n i e , the c l a m p i n gv o l t a g ef)r o m - 1 0 m V t o + 5 0 m V i n c r e a s et sh e p r o b a b i l i tay c h a n n ew l i l l o p e n ,t h e t i m e i t s t a y so p e n ,a n d t h e a m o u n to f e l e c t r i c u r r e n t( n u m b e r so f i o n s )t h a t p a s st h r o u g hi t . et a1, 1981,Nature 293:47 [FromB Pallota 1, asmodified by B Hille,1992, lon Channels of Excitable Membranes, 2d ed , Sinauer, p 122l

the upward blips on the tracings. Further, the ion flux through them is rather small, as measuredby the electriccurrent passing through each open channel (the height of the blips). Depolarizing the membrane further to +20 mV causes these channels to open about twice as frequently. Also, more K* ions move through each open channel (the height of the blips is greater) becausethe force driving cytosolic K* ions outward is greater at a membrane potential of +20 mV than at -10 mV. Depolarizingthe membrane further to +50 mV, the value at the peak of an action potential, causesopening of more K- channelsand also increases the flux of K* through them. Thus, by opening during the peak of the action potential, theseK* channelspermit the outward movement of K* ions and repolarization of the membrane potential while the voltage-gatedNa* channels are closed and inactivated. More than 1.00voltage-gatedK* channel proteins have been identified in humans and other vertebrates.As we discuss later, all these channel proteins have a similar overall structure, but they exhibit different voltage dependencies, conductivities, channel kinetics, and other functional properties. Many open only at strongly depolarizing voltages, a property required for generation of the maximal depolarization characteristicof the action potential before repolarization of the membrane besins.

Action PotentialsAre Propagated U n i d i r e c t i o n a l lW y ithout Diminution The generation of an action potential relatesto the changes that occur in a small patch of the neuronal plasma membrane. At the peak of the action potential, passivespread of the membrane depolarization is sufficient to depolarize a neighboringsegmentof membrane.This causesa few voltagegated Na+ channels in this region to open, thereby increasing the extent of depolarization in this region and causing an explosive opening of more Na* channelsand generation of an action potential. This depolarization soon triggers opening of voltage-gatedK* channels and restoration of the resting potential. The action potential thus spreadsas a traveling wave away from its initial site without diminution. As noted earlier, during the refractory period voltagegated Na+ channels are inactivated for several milliseconds. Suchpreviouslyopenedchannelscannot open during this period even if the membrane is depolarizedowing to passivespread.As illustratedin Figure23-9,the inability of Na* channelsto reopen during the refractory period ensures that action potentials are propagated only in one direction, from the axon initial segmentwhere they originate to the axon terminus. This property of the Na* channels also limits the number of action potentialsper secondthat a neuron can conduct. This is important, since it is the frequency of action potentials that carries the information. Reopeningof Na* channelsupstream of an action potential (i.e., closer to the cell body) also is delayed by the membrane hyperpolarizationthat resultsfrom opening of voltagegated K" channels.

Nerve CellsCan ConductMany Action Potentials in the Absenceof ATP The depolarizaion of the membrane during an action potential resultsfrom movement of just a small number of Na* ions into a neuron and doesnot significandy affectthe intracellular Na+ concentration.A typical nerve cell has about 10 voltage-gatedNa+ channelsper squaremicrometer 1pm21of plasmamembrane.Sinceeachchannelpasses=5000-10,000 ions during the millisecond it is open (seeFigure 11-22), a maximum of 10s ions per pm2 of plasma membrane will move inward during each action potential. To assessthe effect of this ion flux on the cytosolic Na* concentration of 10 mM (0.01 moVL), typical of a resting axon, we focus on a segmentof axon 1 micrometer (pm) lon^g and 10 pm in diameter.The volume of this segmentis 78 pmr, 1a liters, and it contains 4.7 x 108 Na+ ions: or 7.8 x 10

ber of Na* ions in this segmentby only one part in about 1.50:(4.7 x 108) + (3.1 x 106).Likewise,the repolarization of the membrane due to the efflux of K* ions through voltage-gated K* channels does not significantly change the intracellularK' concentration. Becauseso few Na* and K* ions move acrossthe plasma membrane during each action potential, the ATP-driven Na*/K* pump that maintains the usual ion gradients plays no direct role in impulse conduction. Since the ion movements during each action potential involve only a minute fraction of the cell's K* and Na* ions, a nerve cell can fire hundreds or even thousandsof times in the absenceof ATP.

All Voltage-Gatedlon ChannelsHave S i m i l a rS t r u c t u r e s Having explained how the action potential is dependenton regulatedopening and closing of voltage-gatedchannels,we turn to a molecular dissectionof theseremarkable proteins. After describingthe basic structure of thesechannels'we focus on three questions: r How do theseproteins sensechangesin membrane potential? r How is this changetransducedinto opening of the channel? 'What causesthesechannelsto becomeinactivatedshortly r after opening? The initial breakthrough in understandingvoltage-gatedion channels came from analysis of fruit flies (Drosopbila melanogaster) carrying the shaker mutation' These flies shake vigorously under ether anesthesia,reflecting a loss of motor control and a defect in certain motor neurons that have an abnormally prolonged action potential. Researchers suspectedthat the shaker mutation causeda defect in channel function. Cloning of the gene involved confirmed that

O F A C T I O NP O T E N T I A L ISN N E R V EC E L L S AND THE PROPAGATION V O L T A G E - G A T E IDO N C H A N N E L S

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> FIGURE23-9 Unidirectionalconduction of an action potential due to transient inactivation of voltage-gated Na* channels.At time 0, an actionpotential(red) i s a t t h e 2 - m m p o s i t i o no n t h e a x o n ;t h e N a + c h a n n e l as t t h i sp o s i t i o na r eo p e na n d N a + ionsare flowing inward The excessNa+ ions diffusein both directionsalongthe insideof t h e m e m b r a n ep, a s s i v e sl yp r e a d i ntgh e depolarizationBecause the Na+ channelsat '1 the -mm positionare stillinactivated (green), they cannotyet be reopenedby the small depolarization causedby passivespread;the N a - c h a n n e las t t h e 3 - m m p o s i t i o ni,n contrast,beginto open Eachregionof the membraneis refractory(inactive) for a few mrlliseconds after an actionpotentialhas p a s s e dT h u s t, h e d e p o l a r i z a t i oant t h e 2 - m m siteat time 0 triggersactionpotentials .l downstreamonly;at ms an actionpotential i sp a s s i n g t h e 3 - m m p o s i t i o na, n d a t 2 m s a n actionpotentialrs passingthe 4-mm position

+

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Eb-50 >6 234 D i s t a n cael o n ga x o n( m m )

the defective protein was a channel. The shaker mutation prevents the mutant channel from opening normally immediately upon depolarization. To test whether the wild-type shakergeneencodeda K* channel,cloned wild-type shaier cDNA was usedas a templateto produceshakermRNA in a cell-free system. Expression of this mRNA in frog oocyres and patch-clamp measurementson the newly synthesized channel protein showed that its functional proDertieswere i d e n t i c a lw i t h t h o s eo f r h e v o l t a g e - g a r eK< J c h a n n e li n r h e neuronal membrane, demonstrating conclusiveiythat the shakergeneencodesthis K+-channelprotein. The ShakerK* channeland most other voltage-gated K* channelsthat have been identified are tetrameric proteins composed of four identical subunits arranged in the membrane around a centralpore. Each subunitis constructedof six membrane-spanning crhelixes,designatedS1-S6,and a p 1010

.

cHAprER 23 |

N E R V cEE L L s

segment(Figure23-10a). The 55 and 56 helicesand the p segmentare structurally and functionally homologous to those in the nongated resting K+ channel discussedearlier (seeFigure 11.-19).The 55 and 56 helicesform the lining of the channel through which the ion travels.The S1-S4 helices act as a voltage sensor(with 54 acting as the primary sensor) and are describedas paddlesowing to the way they protrude from the central complex. The N-terminal "ball" extending into the cytosolfrom S1 is the channel-inactivaring segment. Voltage-gated Na* channels and Ca2* channels are monomericproteinsorganizedinto four homologousdomains, I-IV (Figure 23-10b). Each of these domains is similar to a subunit of a voltage-gatedK* channel.However, in contrast to voltage-gated K+ channels, which have four channel, inactivating segments,the monomeric voltage-gatedchannels have a single channel-inactivatingsegment.Except for this

(a) Voltage-gatedK+channel (tetramer)

Exterior

Cytosol

a/'

lnactivation segment

( b ) V o l t a g e - g a t e dN a * c h a n n e l( m o n o m e r )

minor structural differenceand their varying ion permeabilities, all voltage-gatedion channelsare thought to function in a similar manner and to have evolved from a monomeric ancestral channel protein that contained six transmembranea helices.

V o l t a g e - S e n s i n5g4 c H e l i c e sM o v e i n R e s p o n s e t o M e m b r a n eD e p o l a r i z a t i o n The understandingof channel-proteinbiochemistryis advancing rapidly owing to new crystal structuresfor bacterial and shaker potassiumchannelsand other channels.One method used to obtain crystals of these difficult membrane proteins was to surround them with bound fragmentsof monoclonal antibodies(Fab's;Chapter 24);in other casesthey were crystallized in complexeswith normal protein-bindingpartners. The structuresof the channelsrevealremarkable arrangements of the voltage-sensingdomains, and suggesthow parts of the protein move in order to open the channel. The tetramer has a pore whose walls are formed by helicesS5 and 56 (Figure 23-1la). Outside that core structure four arms, or "paddles," protrude into the surrounding membranel these are the voltage sensors,and they are in minimal contact with the pore. Sensitiveelectric measurementssuggestedthat the opening of a voltage-gatedNa* or K* channel is accompanied by the movement of 12-14 protein-bound positive chargesfrom the cytosolic to the exoplasmic surface of the membrane. The moving part of the protein is composed of helicesS1-S4; 54 accountsfor much of the positivecharge and is therefore the primary voltage sensor,with a positively charged lysine or arginine every third or fourth residue. Arginines in 54 have been measuredmoving as much as 1.5

depictionsof the 23-10 Schematic < FIGURE secondarystructuresof voltage-gatedK+ and (a)Voltage-gated are K* channels Na* channels. eachcontaining subunits, of fouridentical composed andsixmembrane-spanning 600-700aminoacids, of eachsubunit, S1-56.TheN-terminus cthelices, N,formsa globular andlabeled inthecytosol located of the for inactivation ball)essential domain(orange (green) and openchannelTheS5and56 helices (blue)arehomologous to thosein the Psegment but eachsubunit K+ channels, resting nongated o helices. transmembrane fouradditional contains voltageOneof these,54 (red),isthe primary in thisroleby helices o helixandisassisted sensing (b) aremonomers Na* channels Voltage-gated S1-3 s r g a n i z ei nd t o c o n t a i n i n1g8 0 0 - 2 0 0a0m i n oa c i d o (l-lv)thataresimilar domains fourtransmembrane The K* channels. in voltage-gated to the subunits located in the segment, singlechannel-inactivating a lllandlV contains domains cytosol between motif(H;yellow)Voltagehydrophobic conserved overall gatedCa2*channels havea similar also ionchannels structureMostvoltage-gated (F) are not that subunits regulatory contain (a)adapted 1992, fromC Miller, here[Part depicted eral, 1996,Neuron Biol2.573,andH Larsson Curr. 2001, part(b)adapted fromW A Catterall, 16:387; 409:988 Nature l nm as the channelopens,which can be comparedwith the =5nm thicknessof the membrane or the L.2-nm diameterof the cr helix itself. The movement of thesegating charges(or voltage sensors)under the force of the electric field triggersa conformational changein the protein that opensthe channel. Thus the 54 helix is the key part of the voltage sensor,which then moves the S1-S4 helicesacrossmuch of the membrane. The most unusual aspect of the voltage-sensitivechannel structuresis the presenceofcharged groups' e.g.' arginines,in contact with lipid. The location of the voltage sensorhelps to explain earlier experimentswhere a non-voltage-sensitive channel was converted into a voltage-sensingchannel by adding to it voltage-sensingdomains' Such an experiment would seemunlikely to work if the voltage sensorshad to be deeply embeddedin the core stmcture. the imStudieswith mutant ShakerK* channelssupport 'When one or portance of the 54 helix in voltage sensing. more arginine or lysine residuesin the 54 helix of the Shaker K* channel were replaced with neutral or acidic residues' fewer positive chargesthan normal moved acrossthe membrane in responseto a membrane depolarization, indicating that arginine and lysine residuesin the 54 helix do indeed move acrossthe membrane. In other studies,mutant Shaker oroteins in which various 54 residueswere converted to cysteinewere tested for their reactivity with a water-soluble cysteine-modifyingchemical agent that cannot cross the membrane. On the basis of whether the cysteinesreacted with the agent added to one side or the other of the membrane, the results indicated that in the resting state amino acids near the C-terminus of the 54 helix face the cytosol; after the membrane is depolarized,some of these same amino

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A FIGURE 23-11 Molecularstructureof a voltage-sensitive nearthe interiorto the exterior of the membrane in response to potassium channel.Thetwo ribbondiagrams showmodels of the depolarization. Since eachoneisattached to an S4-S5linker, each potassium channel in (a)openand(b)closed statesSince themolecule linkeranditsattached S5helixismoved, in turnmoving56 helices, isa tetramer of thesamesubunit, fourcopies of eachhelixarevisible; whichopenstheporeThestructure of theopenmammalian channel the onesfarthestawayareseenonlydimly.Thebrown(S5)andgreen (a)hasbeendeterminedTheclosed-channel structure in (b)is (56)alphahelices spanthemembrane, withtheinterior of thecellat hypothetical, but is basedon observations of a closedbacterial the bottomandexterior at thetop Thepurplespheres potassium-channel (c)Theball-and-chain represent K+ structure. modelfor ions,whichpassthroughtheopenchannel partof the andoccupy inactivation of voltage-gated K* channels in three-dimensional closed channel withoutpassing throughThe56 (green) helices line cutawayviewof the inactive state In additionto thefour crsubunits the pore Notehowthe helices (tanandgray)thatformthechannel, aretlghtlypackedat the bottomin proteins thesechannel havefour (b),closing thechannel sothatthe K* ioncannotpassthrough (purple) regulatory At theN terminus of eachof the B B subunits thedistances betweenS5helices [compare asshownbythe arrows proteins subunit isa smalldomain(purple"ball"on theendof the below(a)and(b)I The54-55linker,locatedin the cytoplasm, connects purple"chain")thatcontrols theopening pore.Inthis of thecentral the54 helix(notshown) to theS5helix(brown). Forclarity, helices illustration S1 theN terminus of onesubunit hasmovedthrougha lateral through54 havebeenomittedfromthemodel;theywouldnormally (a)and(b)fromS B Long, windowto blockthe pore [Parts E B Campbell, beattached to theendof theS4-S5linkerandprotrude fromthe andR MacKinnon, part(c)adapted 2005,Science 309:903-908; fromR molecule "paddles asthevoltage-sensing " These paddles movefrom Aldrich, 2001,Nature 411:643, andM Zhouetal, 200'1, Nature 4'11:657 l acids becomeexposedto the exoplasmic surfaceof the channel. These experiments directly demonstratedmovement of the 54 helix acrossthe membrane, as schematicallydepicted in Figure23-7 for voltage-gatedNa+ channels. The structure of the open form of a mammalian Shaker K* channel has been contrastedwith the closed structure of a crystallizedbacterial K* channel. The results suggesta model for the closing of the channel in responseto movements of the voltage sensorsacross the membrane (Figure 23-11.a,b). In the model, the voltage sensors,composed of helices S1-S4, move in responseto voltage and exert a

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torque on a linker helix that connects54 to 55. In the openchannel conformation, the position of the S4-S5 linkers alIows the 55 helicesto lie at about a 45" angle to the plane of the membrane(Figure23-11a, brown helices),and the pore 'lfhen insidehas a'1.2-nm opening. the cell is repolarized and the voltage sensor moves toward the intracellular membrane surface,the S4-S5 linkers are twisted down, toward the inside of the cell. The 55 helicesare consequentlymoved more orthogonal to the plane of the membrane(Figure23-11b, brown helices).This position leavesthe 55 and 56 helicesin closer proximiry squeezingthe channelclosed(Figure23-11a, b; the

double headedarrows indicate spacingbetween adjacent 55 helices).Thus the gate probably is composed of the cytosolfacing ends of the 55 and 55 helices,where the pore is narrowest.

M o v e m e n to f t h e C h a n n e l - l n a c t i v a t i nSge g m e n t into the Open PoreBlockslon Flow An important characteristic of most voltage-gatedchannels is inactivation; that is, soon after opening they close spontaneously forming an inactive channelthat will not reopen until the membraneis repolarized.In the resting state,the positively charged globular balls at the N-termini of the four subunitsin a voltage-gatedK* channelare free in the cytosol. Severalmillisecondsafter the channel is opened by depolarization, one ball moves through an opening (lateral window) between two of the subunits and binds in a hydrophobic pocket in the pore's central cavity, blocking the flow of K* ions (Figure 23-L1,c).After a few milliseconds,the ball is displaced from the pore, and the protein reverts to the closed, resting state.The ball-and-chaindomains in K+ channelsare functionally equivalent to the channel-inactivatingsegment in Na- channels. The experimental results shown in Figure 23-12 demonstrate that inactivation of K* channels dependson the ball domains, occurs after channel opening, and doesnot require the ball domains to be covalently linked to the channel protein. In other experiments,mutant K* channelslacking portions of the =40-residuechain connecting the ball to the S1

helix were expressedin frog oocytes. Patch-clampmeasurements of channel activity showed that the shorter the chain, the more rapid the inactivation, as if a ball attached to a shorter chain can move into the open channel more readily. Conversely,addition of random amino acids to lengthen the normal chain slows channel inactivation. The singlechannel-inactivatingsegmentin voltage-gated Na* channelscontains a conservedhydrophobic motif composed of isoleucine,phenylalanine) methionine, and threonine (seeFigure 23-1,0b).Like the longer ball-and-chain domain in K* channels,this segmentfolds into and blocks the Na*-conducting pore until the membrane is repolarized (see Figure23-71.

MyelinationIncreasesthe Velocity o f l m p u l s eC o n d u c t i o n

As we have seen, action potentials can move down an axon without diminution at speedsup to 1 meter per second. But even such fast speedsare insufficient to permit the complex movementstypical of animals.In humans, for instance.the cell bodies of motor neurons innervating leg musclesare located in the spinal cord, and the axons are about a meter in length. The coordinated muscle contractions required for walking, running, and similar movementswould be impossibleif it took 1 secondfor an action potential to move from the spinal cord down the axon of a motor neuron to a leg muscle. The solution is to wrap cells in insulation that increasesthe rate of movement of an action potential. The insulation is called a myelin sheath (seeFigure 23-2b). The presenceof a myelin sheath around an axon increasesthe velocity of impulse conducM u t a n tS h a k e rK +c h a n n e l tion to 10-100 metersper second.As a result, in a typical human motor neuron, an action potential can travel the Wild-typeShakerK' channel length of a 1-meter-longaxon and stimulate a muscle to contract within 0.01 seconds. In nonmyelinated neurons,the conduction velocity of an action potential is roughly proportional to the diameter of the axon, becausea thicker axon will have a greater number of ions that can diffuse. The human brain is packed with relatively small, myelinated neurons' If the neurons in the hu020406080 man brain were not myelinated' their axonal diameters T i m e( m s ) would have to increaseabout 10,000-fold to achievethe FIGURE 23-12Experiments with a mutantK+ EXPERIMENTAL sameconduction velocitiesas myelinated neurons.Thus verchannellackingthe N-terminalglobulardomainssupportthe tebrate brains, with their densely packed neurons, never K' channel could have evolved without myelin. inactivation model.Thewild-type Shaker ball-and-chain theaminoacidscomposing theN-terminal anda mutantformlacking in Xenopus ballwereexpressed oocytesTheactivityof thechannels Action Potentials"Jump" from Node to Node Whenpatches bythepatch-clamp technique thenwasmonitored opened from-0 to +30 mV thewild-type channel weredepolarized in MyelinatedAxons (redcurve)Themutantchannel opened for =5 msandthenclosed The myelin sheathsurrounding an axon is formed from many curve)Whena chemically normally, butcouldnotclose(green glial cells.Each region of myelin formed by an individual glial faceof the patch, synthesized ballpeptidewasaddedto the cytosolic unmyelinated (bluecurve). This cell is separatedfrom the next region by an openednormally andthenclosed the mutantchannel pm called the in length 1 about membrane of axonal area the channelafterit demonstrated thatthe addedpeptideinactivated ax23-2).The Figure (or see node; simplg Ranuier node of openedandthatthe balldoesnot haveto betetheredto the proteinin extracellular with the contact is in direct membrane onal order to function [FromW N Zagottaet al , 1990,Science250:568]

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A FIGURE 23-13 Conductionof actionpotentialsin myelinatedaxons.Because voltage-gated Na+channels are localized to the axonalmembrane at the nodesof Ranvier, the influx of Na* ionsassociated with an actionpotential canoccuronlyat nodesWhenan actionpotential isgenerated at onenode(stepIl), positive the excess ionsin thecytosol, whichcannotmoveoutward across thesheath, diffuserapidly downtheaxon,causing sufficient depolarization at the nextnode(step[) to inducean action potential at thatnode(stepB) Bythismechanism theaction jumpsfromnodeto nodealonqthe axon potential

fluid only at the nodes. Moreover, all the voltage-gatedNa+ channels and all the Na+/K+ pumps, which maintain the ionic gradientsin the axon, are located in the nodes. As a consequenceof this localization, the inward movement of Na* ions that generatesthe action potential can occur only at the myelin-free nodes (Figure 23-13). The excess cytosolic positive ions generatedat a node during the membrane depolarization associated with an action potential spreadpassivelythrough the axonal cytosol to the next node with very little loss or attenuation, since they cannot cross the myelinated axonal membrane. This causesa depolarization at one node to spread rapidly to the next node, permitting the action potential, in effect, to jump from node to node. The transmission is called sahatory conduction. This phenomenon explains why the conduction velocity of myelinated neurons is about the sameas that of much larger diameter unmyelinated neurons. For instance,a 12-p,m-diameter myelinatedvertebrateaxon and a 600-pm-diameter unmyelinated squid axon both conduct impulsesat L2 mls.

G l i a P r o d u c eM y e l i n S h e a t h sa n d S y n a p s e s Of the four types of glia (three of which are shown in Figure 23-14), two produce myelin sheaths: oligodendrocytes make sheathsfor the central nervous system (CNS), and Schwann cells make them for the peripheral nervous system. Astrocyfes, a third type, are necessaryfor neurons to produce synapsesand use them to communicate with other neurons. The fourth type, microglia, produce survival fac1014

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tors for cells and carry out immune functions. These cells participate in inflammatory responsesand constitute a part of the CNS immune system.They can differentiate to form phagocyticcellswith characteristics of macrophages(Chapter 241.Microglia form in the bone marro% are not related by lineageto neurons or to other glia, and will not be discussedfurther. Oligodendrocytes Oligodendrocytes form the spiral myelin sheath around axons of the central nervous system (Figure 23-14c). Each oligodendrocyte provides myelin sheathsto multiple neurons. The major protein constituents are myelin basicprotein (MBP) and proteolipid protein (PLP). MBP, a peripheral membrane protein found in both the CNS and the PNS, has sevenRNA splicing variants that encodedifferent forms of the protein. It is synthesized6y ribosomes locatedin the growing myelin sheath(Figure23-14c),an example of specifictransport of mRNAs to a peripheral cell region. The localizationof MBP mRNA dependson microtubules. Damage to proteins produced by oligodendrocytesunderlies a prevalent human neurological disease,multiple sclerosis (MS). MS is usually characterizedby spasms and weaknessin one or more limbs, bladderdysfunction,local sensorylosses,and visual disturbances.This disorderthe prototype demyelinating disease-is caused by patchy Ioss of myelin in areas of the brain and spinal cord. In MS patients, conduction of action potentials by the demyelinated neurons is slowed, and the Na* channelsspread outward from the nodes, lowering their nodal concentration. The causeof the diseaseis not known but appearsto involve either the body's production of auto-antibodies (antibodies that bind to normal body proteins) that react with MBP or the secretion of proteasesthat destroy myelin proteins. A mouse mutant, shiuerer,has a deletion of much of the MBP gene, Ieading to tremors, convulsions,and early death. Similarly human (Pelizaeus-Merzbacher disease)and mouse mutations in gene the coding for the other major Ui*pylt protein of CNS myelin, PLP, causeloss of oligodendrocytes and inadequatemyelination. I Schwann Cells Schwann cells form myelin sheaths around peripheral nerves.A Schwanncell myelin sheathis a remarkable spiral wrap (seeFigure 23-14b). A long axon can have as many as severalhundred Schwann cells along its length, each contributing insulation to an internode stretch of about 1-1.5 pm of axon. Not all axons are myelinated,for reasonsthat are not known. Mutations in mice that eliminate Schwanncells causethe death of most neurons. In contrast to oligodendrocytes,Schwann cells each dedicate themselvesto one axon. The sheathsare composed of about 70 percent lipid (rich in cholesterol) and 30 percent protein. In the peripheral nervous system the principle protein constituent(-80 percent)of myelin is called protein 0 (Pe),an integral membraneprotein that has immunoglobulin (Ig) domains.MBP is also an abundantcomponent.The extracellular Ig domains bind together the surfaces of

(b) Peripheralnervoussystem neuron

(a) Centralnervoussystem neurons Central nervous system neu rons

Capillary

A$trocyte

Astrocyte

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Oligodendrocyte

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2003,Curr.Biol 13:469,andadaptedfrom D L S h e r m a na n d P B r o p h y , 23-14Threetypesof glia cells.(a)Astrocytes interact A FIGURE withneurons butdo notinsulate them (b)EachSchwann cellinsulates 2005,NatureRev.Neurosci6:683-690,Photos:C o u r t e s yo f V a r s h aS h u k l a f romNIHl andDougField of a singleperipheral nervous system axon (c)A single a section canmyelinate multiple B Stevens, oligodendrocyte CNSaxonslFrom

sequentialwraps around the axon to compact the spiral of myelin sheath (Figure 23-15b). Other proteins play this kind of role in the CNS. In humans, peripheral myelin, like CNS myelin, is a target of autoimmune disease,antibodies forming against P6. The Guillain-Barre syndrome (GBS), also known as acute inflammatory demyelinating polyneuropathy, is one such disease.GBS is the most common causeof rapid-onset paralysis, occurring at a frequency of 10-t. The causeis unknown. The common inherited neurological disorder called Charcot-Marie-Tooth disease,which damagesperipheralmotor and sensorynerve function, is due to overexpressionof the gene that encodesPMP22 protein, another constituentof peripheralnerve myelin. I

Interactions betweenglia and neurons control the placement and spacing of myelin sheaths,and the assemblyof nervetransmission machinery at the nodes of Ranvier. VoltagegatedNa* channelsand Nan/K* pumps' for example,congregate at the nodes of Ranvier through interactions with '$fhile all the details of the node ascytoskeletal proteins. sembly processare not understood, a number of key players have beenidentified. In the PNS, where the processhas been most studied, surface adhesion molecules in the Schwann cell membrane interact with neuronal adhesion molecules. The glial membrane immunoglobulin cell-adhesionrnolecule (lgCAM) called newrofascinl55 contacts two axonal proteins, contactin and contactin-associatedprotein at the edge of the node. Thesecell-cell contact eventscreate boundaries at eachside of the node.

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A FIGURE 23-15 Formationand structureof a myelinsheathin the peripheralnervoussystem.(a) At highmagnification the specialized spiralmyelin(My)membrane appears asa series of layers, or lamellae, of phospholipid bilayers wrapped aroundtheaxon(Ax); (b)Closeup viewof threelayers Mit : mitochondrion of the myelin membrane spiralThetwo mostabundant peripheral membrane myelinproteins, PoandPMP22, areproduced onlyby Schwann cells. Theexoplasmic domainof a Poprotein, whichhasan immunoglobulin fold,associates withsimilar domains emanating frompeproteins in theopposite membrane surface, thereby"zippering" together the exoplasmic membrane surfaces in closeapposition Theseinteractions

arestabilized by bindingof a tryptophan residue on the tip of the e x o p l a s mdi o cmain t o l i p i d si n t h eo p p o s i tm e e m b r a nC e lose apposition of the cytosolic facesof the membrane mayresultfrom bindingof the cytosolic tailof eachP0proteinto phospholipids in the opposite membranePMP22mayalsocontribute to membrane protein,remains compactionMyelinbasicprotein(MBP), a cytosolic betweenthe closely apposed membranes asthe cytosolissqueezed (a)@Science out [Part part(b)adapted VU/CRainel/isuals Unlimited; from

The channel proteins and other moleculesthat will accumulate at the node are initially dispersedthrough the axons. Then axonal proteins, including two IgCAMs called NrCAM and neurofascinlSS,as well as ankyrin G (Chapter 17), accumulatewithin the node. The two IgCAMs bind to a single transmembrane domain protein called gliomedin that is expressedin the glial cell. Experimenrsrhat eliminated gliomedin production showed that without it nodes do not form, so it is a key regulator.Ankyrin in the node contactsBIV spectrin,a major constituentof the cytoskeleton, thus tethering the node's protein complex to the cytoskeleton.Na* channelsbecomeassociatedwith neurofascin186, NTCAM, and ankyrin G, firmly trapping the channel where it is needed.As a result of these multiple protein-protein interactions, the concentration of Na* channels is roughly a hundredfold higher in the nodal membrane of myelinated axons than in the axonal membrane of nonmyelinatedneurons.

Astrocytes are critical regulators of the formation of the blood-brain barrier. A mass of blood vesselsin the brain suppliesoxygen and removesC02, and deliversglucose and amino acids, with capillaries within a few micrometers of every cell. These capillaries form the bloodbrain barrier, which prevents, for example, blood-borne circulating neurotransmittersand some drugs from entering the brain. The barrier consistsof a set of tight junctions (Chapter 19) made by the endothelialcells that form the walls of capillaries.Astrocytespromote specialization of theseendothelialcells, making them less permeable ( F i g u r e2 3 - 1 , 6 ) . Many synapsesand dendritesare also surrounded by astrocyte processes.Astrocytesproduce abundant extracellular matrix proteins, some of which are used as guidancecuesby migrating neurons,and a host of growth factors that carry a variety of types of information to neurons. The Ca2*, K*. Na-, and Cl channels,among others, found in the plasma membranesof astrocytesinfluence the concentration of free ions in the extracellular space,thus affecting the membrane potentialsof neurons and of the astrocytesthemselves.Astrocytes also take up glutamate,a neurotransmitter,from extra'When cellular spacesand turn it into glutamine. nearby neurons have fired, glutamate binds to glutamate receprors on astrocytes, enabling the astrocytes to sensethe event. Astrocytes are joined to each other by gap junctions, so changesin ionic composition in any of them are communicated to others, over distancesof hundreds of mrcrons.

Astrocytes The third type of glial cell is the astrocyte, named for its starlike shape (seeFigure 23-14a). These can constitutemore than a third of brain mass and half of the brain's cells. There are two kinds. Proloplasmic astrocytes are in the gray matter (the areasrich in cell bodies);fibrous astrocytes are in the white matter (the areas composed mainly of axonsl it is the myelin that makes it look white). The astrocytesmake long thin processesthat envelop all the brain'sblood vessels. 1016

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L S h a p i r oe t a l , 1 9 9 6 , N e u r o n1 7 i 4 3 5 ,a n d E J A r r o y oa n d S S S c h e r e r , 2000, Histochem Cell Biol 113:1 l

< FIGURE 23-16 Astrocytesinteractwith endothelialcellsat the barrieristo of the blood-brain blood-brainbarrier.Thepurpose into cantravelout of the bloodstream controlwhattypesof molecules of thebanierbythe thebrainandviceversaTheformation thebrainis wallsentering cellsthatmakeup bloodvessel endothelial inthe brainareformed astrocytes Capillaries directed bysurrounding to most thatareimpermeable cellswithtightjunctions byendothelial soonlysmallmolecules cellsisblocked, Transport between molecules throughcellscan transported specifically or substances thatcandiffuse andpermeability Theendothelial cellshavetransporters cross the barrier. prevent but selectively thatallowoxygenandC02across characteristics the bloodvessels, surround fromcrossingAstrocytes othersubstances protein signals andsendsecreted cells, withtheendothelial in contact barrier. Unless cellsto producea selective to inducethe endothelial standthebest molecules involved, lipid-soluble carrier thereisa specific thoughtheymaytravelpoorlyin the blood. chanceof gettingacross, by specific channel likeNa+andCl-, aremovedacross Electrolytes, proteins. cellshavelessvesicle endothelial Thebrain's andtransport presumably is sincetransport cells, transport thanmostendothelial bya areensheathed cells(burgundy) Theendothelial moreselective (orange) by on theoutside andcontacted layerof basallamina (tan).Pericytes cellsthat provide processes aremesenchymal astrocyte L R6nnbdck, andE Hansson, N J Abbott, to thecapillaries. support [From 7:41-53]l Neurosci Rev. 2006,Nature

r As the action potential reachesits peak, opening of voltagegated K+ channelspermits efflux of K* ions, which repolarizes and then hyperpolarizes the membrane. As these channelsclose, the membrane returns to its resting potential (seeFigure23-3). r The excesscytosolic cations associatedwith an action potential generatedat one point on an axon spread passivelyto the adjacentsegment,triggeringopeningof voltagegated Na+ channelsand movement of the action potential along the axon.

Voltage-Gated lon Channelsand the Propagation of Action Potentials in Nerve Cells r Action potentials are sudden membrane depolarizations followed by a rapid repolarization. They originate at the axon initial segmentand move down the axon toward the axon terminals, where the electric impulse is transmitted to other cellsvia a synapse(seeFigures23-3 and23-6). r An action potential resultsfrom the sequentialopening and closingof voltage-gatedNa* and K* channelsin the plasma membraneof neuronsand musclecells(seeFigure23-9). r Opening of voltage-gatedNat channels permits influx of Na* ions for about 1 ms, causing a sudden large depolarization of a segmentof the membrane.The channels then closeand becomeunableto open (refractory)for several milliseconds,preventingfurther Na* flow (seeFigure23-7).

r Becauseof the absolute refractory period of the voltagegated Na* channelsand the brief hyperpolarization resulting from K* efflux, the action potential is propagated in one direction only, toward the axon terminus' ' r Voltage-gatedNa and Ca2" channelsare monomeric proteins containing four domains that are structurally and functionally similar to each of the subunits in the t tetramericvoltage-gatedK channels' r Each domain or subunit in voltage-gatedcation channels contains six transmembranea helicesand a nonhelical P segment that forms the ion-selectivirypore (seeFigure23-10). r Opening of voltage-gatedchannelsresults from movement of the positively charged 54 ct helicestoward the extracellular side of the membrane in responseto a depolarization of sufficient magnitude. r Closing and inactivation of voltage-gatedcation channels result from movement of a cytosolic segmentinto the open pore (seeFigure23-11c). r Myelination, which increasesthe rate of impulse conduction up to a hundredfold, permits the close packing of neurons characteristicof vertebrate brains.

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r In myelinated neurons, voltage-gatedNa* channels are concentrated at the nodes of Ranvier. Depolarization at one node spreadsrapidly with little attenuation to the next node, so that the action potential jumps from node to node (seeFigure 23-13). r Myelin sheathsare produced by glial cells that wrap themselves in spirals around neurons. Oligodendrocytes produce myelin for the CNS; Schwann cells, for the PNS (seeFigure 23-14). r Astrocytes,a third type of glial cell, wrap their processes around synapsesand blood vessels.Astrocytes secreteproteins that stimulate synapseformation, and also induce the endothelial cells of blood vesselsto oroduce a blood-brain barrier that limits transepithelial flow of subsranceslsee Figure 23-16).

had been stored and upon the frequency of action potentials arriving at the synapse.The duration of signalalso dep e n d s o n h o w r a p i d l y a n y r e m a i n i n gn e u r o t r a n s m i t t e ri s retrieved by the presynaptic cell. Presynaptic cell plasma membranes,as well as glia, contain transporter proteins that pump neurotransmittersacrossthe plasma membrane back into the cell, thus keepingthe extracellularconcentrations of transmitter low. Here we focus first on how synapsesform and how they control the regulated secretion of neurotransmitters in the context of the basic principles of vesicular trafficking outlined in Chapter 14. Next we look at the mechanismsthat limit the duration of the synaptic signal, and how neurotransmirters are receivedand interpretedby the postsynapticcell.

Formationof SynapsesRequiresAssemblyof Presynapticand PostsynapticStructures

Communication at Synapses As we have discussed,electricalpulsestransmit signalsalong neurons, but signals are transmitted between neurons and other excitable cells by chemical signals. Synapsesare the junctions where presynaptic neurons releasethese chemical signals,or neurotransmitters,that act on a postsynaptic target cell (Figure 23-4), which can be another neuron or a muscle or gland cell. Neurotransmitters are small, water-soluble molecules(e.g.,acetylcholine,dopamine).The cell-cell communication at chemical synapsesgoes in one direction: pre- to postsynapticcell. Arrival of an action potential at an axon terminal leads to opening of voltage-sensitiveCa2* channels and an influx of Ca2*, causing a localized rise in the cytosolic Ca2* concentration in the axon terminus. The rise in Caz* in turn rriggers fusion of small (40-50-nm) synaptic vesicles containing neurotransmitters with the plasma membrane, releasingneurotransmittersfrom this presynapticcell into the synaptic cleft, the narrow spaceseparating it from posrsynapticcells. The membrane of the postsynaptic cell is located within approximarely 50 nm of the presynapticmembrane. Neurotransmitter receptors fall into two broad classes: ligand-gated ion channels, which open immediately upon neurotransmitter binding, and G protein-coupled receptors. Neurotransmitter binding to a G protein-coupled receptor inducesthe opening or closing of a separateion-channelprotein over a period of secondsto minutes. These "slow" neurotransmitter receptors are discussedin Chapter 15 along with G protein-coupled receptors that bind different types of ligands and modulate the activity of cytosolic proteins other than ion channels.Here we examine the structure and operation of the nicotinic acetylcholine receptor found at many nerve-musclesynapses.It was the first ligand-gated ion channel to be purified, cloned, and characteized at the molecular level, and provides a paradigm for other neurotransmitter-gatedion channels. The duration of the neurotransmittersignal dependson the amount of transmitter releasedby the presynaptic cell, which in turn depends on the amount of transmitter that 1018

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Axons extend from the cell body during development,guided by signalsfrom other cellsalong the way so that the axon rermini will reach the correct location (seeSection23.5). As axons grow, they come into contact with the dendritesof other neurons, and often at such sites synapsesform. In the CNS, synapseswith presynapticspecializationsoccur frequently all along an axon, in contrast to motor neurons, which form synapseswith muscle cellsonly at the axon termini. Neurons cultured in isolation will not lorm synapses very efficiently, but when glia are added, the rate of synapse formation increasessubstantially. Astrocytes and Schwann cells send signals to neurons to stimulate the formation of synapsesand then help to preservethem (Figure 23-17).To

tlcm I EXPERIMENTAL FIGURE 23-17 Signalsfrom astrocyteshave beenshown to inducesynapseformation.lmmunostaining for the presynaptic (red)andthe postsynaptic proteinsynaptotagmin protein (yellow) yieldsfew measured puncta(dotsof stain)in control PSD-95 retinalganglioncellsculturedin the absence (/eft). of astrocytes However, a 3-5-foldincrease in punctaoccurswhenthesecellsare culturedin the presence proteinproduct of astrocytes or theastrocyte : the recombinant (right)(rTSP2 thrombospondin thrombospondin 2 thatwasused).Astrocytes secrete thrombospondin, whichby itselfhas muchthe sameeffecton synapse formationasastrocytes themselves. Scale baris30 pm. [Reproduced withpermission fromK S Christopherson et al . 2005. Cell 12O:421-433 |

discover the signals involved, culture medium in which glia had been incubated was added to neuron cultures, and synapse formation was stimulated. By purifying different substancesfrom that medium it was possibleto identify the signal. Thrombospondin (TSP)protein, a component of extracellular matrix, was found to be the active agent. Confirmation came from mice lacking two tbrombospondin geflesi the mice had only 70 percent of the normal number of synapsesin their brains. TSP probably does not work alone, as it is not as potent in inducing synapsesas are whole glia. Another molecule that appears to account for some of the synapse-inducingactivity of glia is cholesterol, and direct contact betweenglia and neurons may contribute as well. Mutual communication between neurons and the glia that surround them is frequent and complex. The signalsand information they carry is an area of active research.There is even evidencethat neurons form synapseson glia. \fhile glia do not have action potentials, they do have complex arrays of channelsand ion fluxes. At the site of a synapse,the presynapticcell has hundreds to thousands of synaptic vesicles,some docked at the membrane and others waiting in reserve. The releaseinto the synaptic cleft occurs in the actiue zone, a specializedregion of the plasma membrane containing a remarkable assemblage of proteins whose functions include modifying the propertiesof the synapticvesiclesand bringing them into position for docking and fusing with the plasma membrane. Mewed by electron microscopg the active zone has electrondensematerial and fine cytoskeletalfilaments (Figure 23-18). The active zone is assembledgradually, with synaptic vesicles accumulating first, then cytoskeletalelements,and then other proteins. A similarly denseregion of specializedstructures is seenacrossthe synapsein the postsynapticcell, the postsynaptic density (PSD). Cell-adhesion molecules that connect pre- and postsynapticcells keep the active zone and PSD aligned. After releaseof synaptic vesiclesin responsero an action potential,the presynapticneuron retrievessynaptic vesicle membrane proteins by endocytosis both within and outside the active zone. The induction of PSD assemblyhas been extensively studied at the neuromuscular iunction (NMJ). At these synapsesacetylcholineis the neurotransmitter produced by motor neurons, and its receptor,AChR, is produced by the postsynapticcell, which is a muscle cell. Muscle cell precursors,myoblasts,put into culture will spontaneouslyfuse into multinucleate myotubes that look similar to normal muscle cells (Chapter 21.).As myotubes form, AChR is produced and inserted into the plasma membrane of the myotubes, reaching a density of about 1000 receptors/pm2.The AChR is dispersedthrough the membrane, but if neurons are added to the culture, the AChR starts to concentrate at points of contact with the neurons. The neurons cause movement of preexisting AChR and also induce the myotubes to produce additional AChR. The density of receptors in a mature synapsereachesabout 10,000-20,000/p"m2,while elsewhere in the plasma membrane the density is <10/pm2. These observationsled to an investigation of the mutual signaling by neurons and myotubes. The conclusion from

t

Schwann celI

Axon terminal Synaptic vesicles B a s al a m i n a in synaptic cleft Muscle prasma memorane Muscle cell

Muscle contractile proteins

' 0 . 1p m ' 23-18 Synapticvesiclesin the axon terminalnear A FIGURE the regionwhere neurotransmitteris released.Inthislongitudinal junction, the basallaminaliesin the section througha neuromuscular membrane, the neuronfromthe muscle synaptic cleftseparating foldedAcetylcholine receptors areconcentrated whichisextensively membrane at thetop andpartwaydown in the postsynaptic muscle A Schwann cellsurrounds thesidesof thefoldsin the membrane. andT.Reese, 197],inE R Kandel, J E Heuser theaxonterminal[From vol 1,Handbook of Physiology, Williams and System, ed, IheNervous p 266l Wilkins,

this work is that muscle cells begin to organize their postsynaptic structures before there is any discernibleinfluence of motor axons. The arrival of an axon stabilizesand further modifies the structuresthat have formed.

NeurotransmittersAre Transportedinto SynapticVesiclesby H*-LinkedAntiport Proteins In this section,we focus on how neurotransmittersare packaged in membrane-bound synaptic uesiclesin the axon terminus. Numerous small molecules function as neurotransmitters at various synapses.\fith the exception of acetylcholine,the neurotransmitters shown in Figure 23-19 are amino acids or derivatives of amino acids. Nucleotides such as ATP and the corresponding nucleosides,which lack phosphategroups, also function as neurotransmitters.Each neuron generallyproducesjust one type of neurotransmitter. All the "classic" neurotransmittersare synthesizedin the cytosol and imported into membrane-bound synaptic vesicles within axon terminals, where they are stored. These vesiclesare 40-50 nm in diameter,and their lumen has a low pH, generatedby operation of a V-classproton pump in the vesiclemembrane.Similar to the accumulationof metabolites AT SYNAPSES COMMUNICATION

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o cH3-c-o-cH2-cH2-N+-(cHs)g Acetylcholine

o tl

that < FIGURE 23-19 Structures of severalsmallmolecules allthese function as neurotransmitters.Exceptfor acetylcholine, fromthe indicated or derived andglutamate) areaminoacids(glycine fromtyrosine, which synthesized aminoacidsThethreetransmitters arereferred to as moiety(bluehighlight), containthe catechol catecholamines

H3N+-CH2-C-O-

in plant vacuoles (seeFigure 1,1,-28),this proton concentration gradient (vesiclelumen > cytosol) powers neurotransmitter import by ligand-specificH*-linked antiporters in the vesiclemembrane. For example, acetylcholine is synthesized from acetyl coenzymeA (acetyl CoA), an intermediate in the degradation of glucose andfatty acids, and choline in a reaction catalyzed by choline acetyltransferase:

Glycine

oC:O O C n r C H r [ O H3N+-CH Glutamate

o tl

cH2-cH2-

NH3r

CH"

CH3-C-S-CoA

o

Choline

?r.

CH3-C-O-CH2-CH2-N*-CHs Acetylcholine

Norepinephrine (derivedfrom tyrosine)

cH-cH2-NH2+-CH3 OH

C h o iln e acetyltransferase

+ HO-CH2-CH2-N+-CH3

Acetyl CoA Dopamine (derivedfrom tyrosine)

t-

CHs

+ CoA-SH

CH:

Synapticvesiclestake up and concentrateacetylcholinefrom the cytosol against a steepconcentration gradient, using an Ht/acetylcholine antiporter in the vesiclemembrane. Curiously, the geneencoding this antiporter is contained entirely within the first intron of the gene encoding choline acetyltransferase,a mechanism conserved throughout evolution for ensuring coordinate expression of these two proteins. Different H+/neurotransmitter antiport proteins are usedfor import of other neurotransmittersinto synaptic vesicles.

Epinephrine (derivedfrom tyrosine)

Ho;Z+cH2-cH2-NH3*

\r1-^,/ H Serotonin, o r S-hydroxytryptamine (derivedfrom tryptophan) HC:C-CHz-CH2-NH3+ N\,/NH

'cH

Histamine (derivedfrom histidine)

o tl

H3N+-CH2-CH2-CH2-C-Oy-Aminobutyric acid, or GABA (derivedfrom glutamate)

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SynapticVesiclesLoadedwith Neurotransmitter Are Localizednear the PlasmaMembrane Neurotransmitters are synthesizedby enzymesin the cytosol and then transportedinto synapticvesiclesby transporterproteinsdedicatedto the task. For exampleglutamateis imported into synaptic vesicles by proteins called uesicular glutamate transporters (VGLUTs). VGLUTs are highly specific for glutamate but have rather low substrateaffinity (K- : 1-3mM). The transporters are antiporters, moving glutamate into synaptic vesicleswhile protons move in the other direction. The membrane-potentialgradient that drives the transport processis establishedby a vacuolar-typeMPase (Chapter11). The exocytosisof neurotransmitters from synaptic vesicles involves targeting and fusion events similar to those that lead to releaseof secretedproteins in the secretorypathway (Figure 23-20). However, severalunique featurespermit the very rapid releaseof neurotransmitters in responseto arrival of an action potential at the presynaptic axon terminal. For example, in resting neurons some neurotransmitter-filled synaptic vesicles are "docked" at the plasma membranel others are in reservein the active zone near the plasma membrane at the synaptic cleft.

H+-linked a ntiporter

@ I

Synaptotagmin

Neurotransmitter/ transporter Voltage-gated

U C a 2 +c h a n n e l

I

lmport of neurotransmitter

N a+-neurotransmitter symport protern

VAMP

V-classH* pump

ADP+ P; Movementof vesicle to active zone a

a

.

Cytosol of presynaptic cell

l.

+

-ql

[

S

coated vesicle o

Na*

Plasma membrane Dynamin

g SNARE I comolex

clathrin

\,, Clathrin-

t.'

Synaptic cleft Botulinum -1 toxin

,lt .

Exocytosisof neurotransmitter triggeredby influx ol Ca2+

T

Shibire mutation

'-'E a

Reuptakeof neurotransmitter

23-20 Cyclingof neurotransmitters A FIGURE and of synaptic vesiclesin axon terminals.Mostsynaptic vesicles areformedby endocytic recycling asdepicted hereTheentirecycletypically takes Step[:The uncoated about60 seconds. vesicles employa varietyof (blue)andothertransport (green) proteins antiporters to import (reddots)fromthe cytosol. neurotransmitters StepZ: Synaptic vesicles loadedwith neurotransmitter moveto the activezone StepB: Vesicles dockat definedsiteson the plasmamembrane of prevents a presynaptic cell.Synaptotagmin membrane fusionand release of neurotransmitter Botulinum toxinprevents exocytosis by proteolytically VAMP, cleaving thev-SNARE on vesicles. Synaptotagmin in steps4-E or [, thoughit isstillpresent. doesnot participate For it isnot shown.StepZl: In response simplicity, to a nerveimpulse (action potential), voltage-gated in the plasma Ca2*channels membrane open,allowing an influxof Ca2*fromtheextracellular

in change conformational Ca2*-induced Theresulting medium. with the plasma leadsto fusionof dockedvesicles synaptotagmin cleft intothe synaptic of neurotransmitters andrelease membrane v-SNARE and vesicles containing StepE: Afterclathrin/AP proteins bud inwardandarepinchedoff transporter neurotransmitter process, theylosetheircoatproteinsDynamin in a dynamin-mediated of blockthe re-formation suchasshlbrrein Drosophila mutations At the sametime,Na- symporter leadingto paralysis vesicles, synaptic cleft,whichlimits proteins fromthe synaptic takeup neurotransmitter recharges the cellwith andpartially the durationof the actionpotential creating by endocytosis, are recovered Step6: Vesicles transmitter. vesicles, readyto be refilledandbeginthe cycleanew Unlike uncoated Seethetextfor isnot recycled. acetylcholine mostneurotransmitters, VMurthyandC 133:1237; etal,1996,JCellBiol. details[SeeKTakei andR Jahnetal, 2003,Cell112:519 392:497; I 1998,Nature Stevens,

In addition, the membrane of synaptic vesiclescontains a specialized Caz*-binding protein that sensesthe rise in cytosolic Ca2* after arrival of an action potential, triggering rapid fusion of docked vesicleswith the presynaptic membrane. A highly organized arrangementof cytoskeletalfibers in the axon terminal helps localize synaptic vesiclesin the active zone. The vesiclesthemselvesare linked together by synapsin, a fibrous phosphoprotein associatedwith the cytosolic surface of all synaptic-vesiclemembranes.Filaments of synapsin also radiate from the plasma membrane and bind to vesicleassociatedsynapsin.Theseinteractionsprobably keep synap-

tic vesiclescloseto the part of the plasmamembranefacingthe synapse.Indeed,synapsinknockout mice, although viable,are prone to seizures;during repetitive stimulation of many neurons in such mice, the number of synaptic vesiclesthat fuse with the plasmamembraneis greatly reduced.Thus synapsins are thought to recruit synaptic vesiclesto the active zone' Rab3A, a GTP-binding protein located in the membrane of synaptic vesicles,is also required for targeting of neurotransmitter-filled vesiclesto the active zone of presynaptic cells facing the synaptic cleft. Rab3A knockout mice, like synapsin-deficientmice, exhibit a reduced number of synaptic AT SYNAPSES COMMUNICATION

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vesiclesable to fuse with the plasma membrane after repetitive stimulation. The neuron-specificRab3 is similar in sequence and function to other Rab proteins that participate in docking vesicleson particular target membranes in the secretorypathway.

chinery of endocytosis and exocytosis is highly conserved, and is explained extensivelyin Chapter 14.

Influx of Ca2+TriggersRelease of Neurotransmitters

Fusion of synaptic vesicleswith the plasma membrane of axon terminals dependson SNAREs, the same type of proteins that mediatemembrane fusion of other regulatedsecretory vesicles (Figure 23-20). The principal v-SNARE in synaptic vesicles(VAMP) tightly binds syntaxin and SNAP25,the principal I-SNAREs in the plasma membraneof axon terminals, to form four-helix SNARE complexes. After fusion, SNAP proteins and NSF within the axon terminal promote disassociationof VAMP from I-SNAREs, as in the fusion of secretoryvesiclesdepictedpreviously (Figure 14-10).

The exocytosis of neurotransmitters from synaptic vesicles involves vesicle-targetingand fusion events similar to those that occur during the intracellular transport of secretedand plasma-membrane proteins (Chapter 13). Two featurescritical to synapse function differ from other secretory pathways: (1) secretionis tightly coupled to arrival of an action potential at the axon terminus, and (2) synaptic vesiclesare recycled locally to the axon terminus after fusion with the plasma membrane. Figure 23-20 shows the entire cycle whereby synaptic vesiclesare filled with neurotransmitter, releasetheir contents, and are recycled. Depolarization of the plasma membrane cannot, by irself, cause synaptic vesiclesto fuse with the plasma membrane. In order to trigger vesiclefusion, an action potential must be converted into a chemical signal-namely, a localized rise in the cytosolic Ca2+ concentration. The transducers of the electric signals areuoltage-gatedCa2* channelslocalizedto the region of the plasma membrane adjacentto the synaptic vesicles.The membrane depolarization due to arrival of an action potential opens thesechannels,permitting an influx of Ca2* ions from the extracellular medium into the axon terminal. A simple experiment demonstrates the importance of voltage-gatedCa2* channelsin releaseof neurotransmrtters. A preparation of neurons in a Ca2*-containing medium is treated with tetrodotoxin, a drug that blocks voltage-gated Na- channelsand thus preventsconduction of action potentials. As expected,no neurotransmirtersare secretedinto the culture medium. If the axonal membrane then is artificially depolarized by making the medium =100 mM KCI in the presence of extracellular Ca2* , neurotransmitters are releasedfrom the cells becauseof the influx of Ca2* through open voltage-gatedCaz* channels.Indeed, patch-clampiig experiments show that voltage-gatedCa2*channels,like voltage-gatedNa* channels,open transiently upon depolarization of the membrane. Two pools of neurotransmitter-filledsynaptic vesiclesare presentin axon terminals: those docked at the plasma membrane, which can be readily exocytosed,and those ln reserve in the active zone near the plasma membrane. Each rise in Ca2* triggers exocytosisof about 10 percent of the docked vesicles.Membrane proteins unique to synaptic vesiclesthen are specifically internalized by endocytosis,usually via the same types of clathrin-coated vesiclesused to recover other plasma-membraneproteins by other types of cells. After the endocytosedvesicleslose their clathrin coat, they are rapidly refilled with neurotransmitter.The ability of many neurons to fire 50 times a secondis clear evidencethat the recycling of vesiclemembrane proteins occurs quite rapidly. The ma-

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A C a l c i u m - B i n d i nPgr o t e i nR e g u l a t e sF u s i o no f SynapticVesicleswith the PlasmaMembrane

Strong evidencefor the role of VAMP in neurotransmitter exocytosisis provided by the mechanism of action of botulinum toxin, a bacterial protein that can cause the paralysis and death characteristicof botulism. a type of food poisoning. The toxin is composedof two polypeptides: One binds to motor neurons that releaseacetylcholine at synapseswith muscle cells, facilitating entry of the other polypeptide, a protease,into the cytosol of the axon terminal. The only protein this proteasecleavesis VAMP (seeFigure 23-201.After the botulinum proteaseentersan axon terminal, synaptic vesiclesthat are not already docked rapidly lose their ability to fuse with the plasma membrane because cleavageof VAMP preventsassemblyof SNARE complexes. The resulting block in acetylcholinereleaseat neuromuscular synapsescausesparalysis. However, vesiclesthat are already docked exhibit remarkable resistanceto the toxin, indicating that SNARE complexes may aheady be in a partially assembled,protease-resistant state when vesicles are docked on the presynapticmembrane. I The signal that triggers exocytosis of docked synaptic vesicles is a rise in the Ca"* concentration in the cytosol near vesiclesfrom <0.1 pM, characteristicof resting cells, to 1-100 pM following arrival of an action potential in stimulated cells. The speedwith which synaptic vesiclesfuse with the presynapticmembrane after a rise in cytosolic Ca2* (less than 1 ms) indicatesthat the fusion machinery is entirely assembledin the resting state and can rapidly undergo a conformational change leading to exocytosisof neurotransmitter. A Ca2*-binding protein calledsynaptotagmin,located in the membrane of synaptic vesicles,is thought to be a key component of the vesicle-fusionmachinery that triggers exocytosis in responseto Ca'- (seeFigure 23-20). Severallines of evidencesupport a role for synaptotagmin as the Ca2+ sensorfor exocytosisof neurotransmitters.Mutant embryos of Drosophila and C. elegansthat completely lack synaptotagminfail to hatch and exhibit very reduced,uncoordinated muscle contractions. Laruae with partial loss-offunction mutations of synaptotagminsurvive,but their neurons are defectivein Ca2+-stimulatedvesicleexocytosis.Moreover, in mice, mutations in synaptotagmin that decreaseits affinity

for Cazt causea corresponding increasein the amount of cytosolic Ca2* neededto trigger rapid exocytosis.The precise mechanismof synaptotagminfunction is still unresolved.

S i g n a l i n ga t S y n a p s e lss T e r m i n a t e db y Degradationor Reuptakeof Neurotransmitters Following their releasefrom a presynaptic cell, neurotransmitters must be removed or destroyedto prevent continued stimulation of the postsynaptic cell. Signaling can be terminated by diffusion of a transmitter away from the synaptic cleft, but this is a slow process.Instead, one of two more rapid mechanismsterminates the action of neurotransmrtters at most synapses. Signaling by acetylcholine is terminated when it is hydrolyzed to acetate and choline by acetylcbolinesterase,an enzymelocalizedto the synapticcleft. Choline releasedin this reaction is transported back into the presynapticaxon terminal by a Na*/choline symporterand usedin synthesisof more acetylcholine.The operation of this transporter is similar to that of the Na+-linked symportersused to transport glucose into cellsagainsta concentrationgradient (seeFigure 11-25). \fith the exception of acetylcholine,all the neurotransmitters shown in Figure 23-19 are removedfrom the synaptic cleft by transport into the axon terminals that releasedthem. Thus thesetransmittersare recycledintact, as depictedin Fig:ure23-20 (step p ). Transportersfor GABA, norepinephrine, dopamine,and serotoninwere the first to be cloned and studied. These four transport proteins are all Na*-linked symporters. They are 60-70 percentidentical in their amino acid sequences, and eachis thought to contain 12 transmembrane ct helices.As with other Na+ symporters, the movement of Na* into the cell down its electrochemicalgradient provides the energy for uptake of the neurotransmitter.To maintain electroneutrality,Cl often is transported via an ion channel along with the Na- and neurotransmitter. Neurotransmitters and their transporters are targets ffi of a variety of powerful and sometimesdevastating fl drugs. Cocaine inhibits the transporters for norepinephl rine, serotonin, and dopamine. Binding of cocaine to the dopamine transporter inhibits reuptake of dopamine,thus prolonging signaling at key brain synapses;indeed, the dopaminetransporteris the principal brain "cocainereceptor." Therapeutic agents such as the antidepressantdrugs fluoxetine (Prozac) and imipramine block serotonin uptake, and the tricyclic antidepressantdesipramineblocks norepinephrineuptake. I

Fly Mutants LackingDynaminCannotRecycle SynapticVesicles Synapticvesiclesare formed primarily by endocytic budding from the plasma membrane of axon terminals. Endocytosis usually involves clathrin-coated pits and is quite specific,in that severalmembraneproteinsunique to the synapticvesicles (e.g.,neurotransmitter transporters)are specificallyincorporated into the endocytosed vesicles.In this way, synaptic-

vesicle membrane proteins can be reused and the recycled vesiclesrefilled with neurotransmitter (seeFigure 23-20). As in the formation of other clathrin/AP-coatedvesicles, pinching off of endocytosed synaptic vesiclesrequires the GTP-binding protein dynamin (see Figure 1,4-1,9).Indeed, Drosophila mutant called analysisof a temperature-sensitive shibire (shi), which encodesthe fly dynamin protein, provided early evidencefor the role of dynamin in endocytosis. At the permissivetemperatureof 20'C, the mutant flies are normal, but at the nonpermissivetemperatureof 30 "C, they are paralyzed (shibire, "paralyzed," in Japanese)because pinching off of clathrin-coated pits in neurons and other cellsis blocked. When viewed in the electronmicroscope,the sEl neurons at 30 "C show abundant clathrin-coatedpits with long necks but few clathrin-coated vesicles.The appearanceof nerve terminals in sbl mutants at the nonpermissive temperature is similar to that of terminals from normal neurons incubated in the presenceof a nonhydrolyzableanalog of GTP (seeFigure 14-20). Becauseof their inability to pinch off new synaptic vesicles,the neurons in slrl mutants eventually become depleted of synaptic vesicleswhen flies are shifted to the nonpermissivetemperature, leading to a cessationof synaptic signaling and to paralysis.

Cation Openingof Acetylcholine-Gated ChannelsLeadsto MuscleContraction In this section we look at how binding of neurotransmitters by receptors on postsynaptic cells leads to changesin their membranepotential, using the communication betweenmotor neurons and musclesas an example. At these synapses, often called neuromuscular junctions, acetylcholine is the neurotransmitter. A single axon terminus of a frog motor neuron may contain a million or more synapticvesicles,each containing 1000-10,000 molecules of acetylcholine; these vesiclesoften accumulatein rows in the active zone (seeFigure 23-18). Such a neuron can form synapseswith a single skeletalmuscle cell at severalhundred points. The nicotinic acetylcholinereceptor,which is expressed in muscle cells, is a ligand-gated channel that admits both K* and Na*. Thesereceptorsare also produced in the brain and are important in learning and memory; acetylcholinereceptor loss is observed in schizophrenia,epilepsy,drug addiction, and Alzheimer's disease.Antibodies against acetylcholine receptorsconstitute a major paft of the autoimmune reactivity in the diseasemyastheniagravis. The receptor is so named becauseit is bound by nicotine; it has beenimplicated in addiction to nicotine by tobacco smokers.The receptor is also the target of potent neurotoxins, such as the conotoxins produced by certain Pacific Ocean snails. There are at least 14 different isoforms of the receptor, which assembleinto homo- and heteropentamerswith varied properties. The effect of acetylcholineon this receptor can be determined by patch-clampingstudies on isolated outside-out patchesof muscle plasma membranes(seeFigure 1'1'-21,c)' Such measurementshave shown that acetylcholine causes opening of a cation channel in the receptor capable of transmitting 15,000-30,000 Na* or K* ions per millisecond. AT SYNAPSES COMMUNICATION

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Voltage-gated C a 2 *c h a n n e l

,r, Motor :: neuron Acetylcholine Voltage-gated N a *c h a n n e l

Nicotinic acetylchol i ne receptor

Na*

Na* f' Musclecell

Sarcoplasmic r e t i c ul u m

Ca2*-release c h a nn e l

A FIGURE 23-21 Sequentialactivationof gated ion channelsat a junction,Arrival neuromuscular of anactionpotential at theterminus of a presynaptic motorneuroninduces openingof voltage-gated (steptr) andsubsequent Ca2*channels release of acetylcholine, whichtriggers opening of theligand-gated acetylcholine receptors in (stepZ) Theopenchannel plasma themuscle membrane allows an influxof Na*andaneffluxof K* TheNa+influxproduces a localized depolarization of themembrane, leading to opening of voltage-gated (stepp) When Na- channels andgeneration of anactionpotential thespreading depolarization reaches T tubules, it issensed byvoltagegatedCa2+channels in theplasma membrane Through an unknown (indicated mechanism as?)thesechannels remain closed butinfluence (anetwork Ca2*channels in thesarcoplasmic reticulum membrane of membrane-bound compartments in muscle), releasing storedCa2*into (step4) Theresulting thecytosol risein cytosolic Ca2*causes muscle contraction by mechanisms discussed in Chapter 17 However, since the resting potential of the muscle plasma membrane is near Es, the potassium equilibrium potenrial, openingof acetylcholinereceptorchannelscauseslittle increase in the efflux of K* ions;Na* ions, on the orher hand, flow into the musclecell, driven by the Na+ electrochemicalgradient. The simultaneousincreasein permeabilityto Na+ and K* ions following binding of acetylcholineproducesa net depolarization to about -15 mV from the muscle resting potential of -85 to -90 mV. As shown in Figure 23-21, this localized depolarizationof the muscleplasma membranetriggersopening of voltage-gatedNa* channels,leading ro generationand conduction of an action potential in the muscle cell surface membrane by the same mechanismsdescribedpreviously for neurons. When the membrane depolarization reachesT tubules,specializedinvaginationsof the plasma membrane,it affects Ca2* channels in the plasma mimbrane apparently without causingthem to open. Somehowthis causesopening of adjacentCa2*-releasechannelsin the sarcoplasmicieticul lum membrane.The subsequentflow of storedCa2* ions from the^sarcoplasmic reticulum into the cytosol raisesthe cytosolic Ca'- concentrationsufficientlyto inducemusclecontraction. Careful monitoring of the membranepotential of the muscle membrane at a synapsewirh a cholinergic motor neuron

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has demonstratedspontaneous,intermittent, and random =2ms depolarizationsof about 0.5-1.0 mV in the absenceof stimulation of the motor neuron. Each of these depolarizations is caused by the spontaneousreleaseof acetylcholine from a singlesynaptic vesicle.Indeed, demonstrationof such spontaneoussmall depolarizationsled to the notion of the quantal releaseof acetylcholine(later applied to other neurotransmitters)and therebyled to the hypothesisof vesicleexocytosis at synapses.The releaseof one acetylcholine-containing synaptic vesicleresultsin the opening of about 3000 ion channelsin the postsynapticmembrane,far short of the number neededto reach the threshold depolarizationthat induces an action potential. Clearly,stimulation of musclecontraction by a motor neuron requiresthe nearly simultaneousreleaseof acetylcholinefrom numeroussynapticvesicles.

A l l F i v eS u b u n i t si n t h e N i c o t i n i cA c e t y l c h o l i n e ReceptorContributeto the lon Channel The acetylcholinereceptorfrom skeletalmuscleis a pentameric protein with a subunit compositionof o2B16.The cr, B, ^y,and 6 subunitshave considerablesequencehomology; on average, about 35-40 percent of the residuesin any two subunits are similar. The complete receptor has fivefold symmerry, and the actualcation channelis a taperedcentralpore lined by homologoussegmentsfrom eachof the five subunits(Figure23-22). The channelopenswhen the receptorcooperativelybinds two acetylcholinemoleculesto siteslocatedat the interfacesof the oE and e1 subunits.Once acetylcholineis bound to a receptor,the channelis openedwithin a few microseconds.Studies measuringthe permeabilityof different small cations suggest that the open ion channel is, at its narrowest, about 0.65-0.80 nm in diameter,in agreementwith estimatesfrom electron micrographs. This would be sufficient to allow passageof both Na* and K* ions with their shellof bound water molecules.Thus the acetylcholinereceptorprobably transports hydratedions, unlike Na* and K* channels,both of which allow passageonly of nonhydratedions (seeFigure 11-20). The central ion channelis lined by five homologoustransmembrane M2 a helices,one from each of the five subunits (seeFigure 23-22a). The M2 helicesare composedlargely of hydrophobic or unchargedpolar amino acids,but negatively charged aspartateor glutamate residuesare located at each end, near the membranefaces,and severalserineor threonine residuesare near the middle. Mutant acetylcholinereceptors in which a singlenegativelychargedglutamateor asparrarein one M2 helix is replacedby a positively charged lysine have beenexpressedin frog oocytes.Patch-clampingmeasurements indicate that such altered proteins can function as channels, but the number of ions that passthrough during the open state is reduced.The greaterthe number of glutamate or aspartate residuesmutated (in one or multiple M2 helices),the greater the reduction in ion conductivity.Thesefindings suggestthat aspartate and glutamate residues form a ring of negative chargeson the external surfaceof the pore that help to screen out anions and attract Na* or K* ions as they enter the channel. A similar ring of negativechargeslining the cytosolicpore surfacealso helpsselectcationsfor passage(seeFigure 23-22).

(a)

T I

I 6nm (b) Synaptic spaGe

Acetylcholine b i n d i n gs i t e

3nm

xI

structureof < FIGURE 23-22Three-dimensional the nicotinicacetylcholinereceptor.(a)Schematic receptor in the modelof the pentameric cutaway for clarity, the B subunitisnot shownEach membrane; an M2 crhelix(red)thatfacesthe subunitcontains at sidechains andglutamate centralporeAspartate formtwo ringsof negative bothendsof the M2 helices anionsfromandattract charges thathelpexclude Thegate,whichisopenedby to thechannel. cations lieswithrnthe pore (b)Cross bindingof acetylcholine, showing faceof the receptor section of theexoplasmic aroundthe centralpore of subunits thearrangement about3 bindingsitesarelocated Thetwo acetylcholine surface nm fromthe membrane

z l'-

I

,2nm,

l<-

I nm ----->l

The two acetylcholine binding sitesin the extracellular domain of the receptor lie =4 to 5 nm from the center of the pore. Binding of acetylcholine thus must trigger conformational changesin the receptorsubunitsthat can causechannelopening at some distancefrom the binding sites.Receprorsin isolated postsynapticmembranescan be trapped in the open or closedstateby rapid freezingin liquid nitrogen.Imagesof such preparationssuggestthat the five M2 helicesrotate relativeto the vertical axis of the channelduring openingand closing. 'We have discussedthe neuromusculariunctron as an excellent example of how neurotransmitters and their receptors work. Similar ideas apply to glutamate and GABA, the two principal neurotransmitters in vertebrate brain. They use ligand-gatedchannelsthat work along the same principles as AchR.

N e r v eC e l l sM a k e a n A l l - o r - N o n eD e c i s i o nt o Generatean Action Potential At the neuromuscular junction, virtuaily every action potential in the presynapticmotor neuron triggersan action potential in the postsynapticmusclecell that propagatesalong the muscle fiber. The situation at synapsesbetween neurons, especially those in the brain, is much more complex becausethe postsynaptic neuron commonly receivessignalsfrom many presynaptic neurons. The neurotransmitters releasedfrom presynaptic neurons may bind to an excitatory receptor on the postsynaptic neuron, thereby opening a channel that admits Na- ions or both Na* and K* ions. The acetylcholinereceptor just discussedis one of many excitatory receptors,and opening of such ion channelsleadsto depolarizationof the postsynapticplasma membrane, promoting generation of an action potential. In contrast,binding of a neurotransmitterto an inhibitory receptor on the postsynapticcell causesopeningof K* or Cl- channels,leading to an efflux of additional K* ions from the cytosol or an influx of CI ions. In either case,the ion flow tends to hyperpolarize the plasma membrane,which inhibits generationof an action potential in the postsynapticcell. A singleneuron can be affectedsimultaneouslyby signals receivedat multiple excitatory and inhibitory synapses.The

neuron continuously integratesthese signals and determines whether or not to generatean action potential. In this process, the various small depolarizationsand hyperpolarizationsgeneratedat synapsesmove along the plasmamembranefrom the dendritesto the cell body and then to the axon initial segment, where they are summedtogether.An action potential is generated wheneverthe membraneat the axon initial segmentbecomes depolarizedto a certain voltage called the threshold potential (Figure 23-23). Thus an action potential is generated in an all-or-nothingfashion:depolarizationto the thresholdalways leadsto an action potential,whereasany depolarization that doesnot reachthe thresholdpotential neverinducesit. Whether a neuron generatesan action potential in the axon initial segmentdependson the balance of the timing, amplitude, and localization of all the various inputs it receiveslthis signal computation differs for each type of neuron. In a sense,each neuron is a tiny computer that averages all the receptor activations and electric disturbanceson its membraneand makes a decisionwhether to trigger an action potential and conduct it down the axon. An action potential will always have the samemagnitude in any particular neuron. As we have noted, the freqwencywith which action potentials are generatedin a particular neuron is the important parameter in its ability to signal other cells.

G a p J u n c t i o n sA l s o A l l o w N e u r o n st o Communicate Chemical synapsesemploying neurotransmitters allow oneway communication at reasonably high speed. However sometimessignalsgo from cell to cell electrically,without the intervention of chemical synapses.Electrical synapses depend on gap junction channels that link two or more cells (Chapter 19). The effectof gap junction connectionsis to perfectly coordinate the activities of joined cells. An electrical synapse also is bidirectional; either neuron can excite the other.In the neocortex and thalamus and some other parts of the brain, electricalsynapsesare common. The key feature of electricalsynapsesis their speed.\Whileit takesabout 0.5-5 ms for a signal to cross a chemical synapse,transmissionacross AT SYNAPSES COMMUNICATION

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> EXPERIMENTAL FIGURE 23-23Incoming signalsmust reachthe thresholdpotential to triggeran actionpotentialin a postsynaptic cell.In thisexample, the presynaptic neuronisgenerating aboutone actionpotential every4 milliseconds Arrivalof eachactionpotential at thesynapse causes a smallchangein the membrane potential at the axonhillockof the postsynaptic cell,in this example a depolarization of =5 mV When m u l t i p lset i m u cl ia u s teh em e m b r a noef t h i s postsynaptic cellto become depolarized to the -40 potential, threshold hereapproximately mV an actionpotential isinduced in it

D i r e c t i o no f a c t i o np o t e n t i a l

Dendrite

Cell body

- -S

Axon hillock

\

Postsynaptic \ cell

Electrodeto measure electricpotential

Membranepotentialin the postsynapticcell

-40 mV

Threshold potential

-60 mV

an electricalsynapseis almost instantaneous,on the order of a fraction of a millisecond. The cytoplasm is conrinuous between the cells. In addition, the presynaptic cell (the one sending the signal) does not have to reach a threshold at which it can causean action potential in the postsynapticcell. Instead,any electricalcurrent continuesinto the next cell and causesdepolarizationin proportion to the current. An electricalsynapsemay contain thousandsof gap channels, each composed of two hemichannels,one in each apposed cell. Gap junction channelshave a structure similar to conventional gap junctions (Chapter 19). Each hemichannel is an assemblyof six copies of rhe connexin protein. Since there are about 20 mammalian connexin genes,diversity in channel structure and function can arise from the different protein components. The 1.6-2.}-nm channel itself allows the diffusion of moleculesup to about 1000 Da in sizeand has no trouble at all accommodatingions.

Communication at Synapses r Synapsesare the junctions between a presynapticcell and a postsynapticcell, and consist of small gaps. r Neurotransmitters are released by the presynaptic cell using exocytosis.They diffuse acrossrhe synapseand bind to receptors on the postsynaptic cell, which can be a neuron or a muscle. r Chemical synapsesof this sort are unidirectional (see Figure23-4). r Neurotransmitters (seeFigure 23-19) are stored in hundreds to thousandsof synaptic vesiclesin the axon termini 'S7hen of the presynaptic cell (seeFigure 23-1,8). an action potential arrives there, voltage-sensitiveCa2* channels open and the calcium causessynaptic vesiclesto fuse with

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the plasma membrane, releasingneurotransmittermolecules into the synapse(seeFigure 23-20). r Communication between presynaptic and posrsynaptic cellsis abundant as a synapseis being formed. Cell-adhesion moleculeskeepthe cellsaligned.Neurons inducethe accumulation of acetylcholine receptor, for example, in the postsynaptic muscleplasmamembranein the vicinity of a synapse. r Synapticvesiclesfuse to the plasma membrane using cellular machinery that is standard issue for exocytosis, including SNAREs, syntaxin, and SNAP proteins. Synaptotagmin protein is the calcium sensorthat detectsthe action potential-stimulated rise in calcium that leads to synaptic vesiclemembrane fusion (seeFigure 23-20). r Dynamin, an endocytosisprotein, is critical for the formation of new synaptic vesicles,probably to "pinch off" inbound vesicles. r Neurotransmitter receptorsfall into two classes:ligandgated ion channels,which permit ion passagewhen open, and G protein-coupled receptors, which are linked to a separateion channel. r At synapsesimpulses are transmitted by neurotransmitters releasedfrom the axon terminal of the presynapticcell and subsequentlybound to specific receptors on the postsynaptic cell (seeFigure 23-4). r Low-molecular-weightneurotransmitters(e.g., acetylcholine, dopamine, epinephrine) are imported from the cytosol into synaptic vesiclesby H*-linked antiporters. V-class proton pumps maintain the low intravesicular p H t h a t d r i v e s n e u r o r r a n s m i t t e ri m p o r t a g a i n s t a c o n centration gradient. r Arrival of an action potential at a presynaptic axon terminal opensvoltage-gatedCa2* channels,leadingto a localized rise in the cytosolic Ca'* level that triggers exocytosisof

synapticvesicles.Following neurotransmitterrelease,vesicles are formed by endocytosisand recycled (seeFigure 23-20). r Coordinated operation of four gated ion channelsat the synapseof a motor neuron and striatedmusclecell leadsto releaseof acetylcholine from the axon terminal, depolarization of the muscle membrane, generation of an action potential, and then contraction (seeFigure 23-21). r The nicotinic acetylcholine receptor, a ligand-gated cation channel, contains five subunits, each of which has a transmembrane ct helix (M2) that lines the channel (see F i g u r e2 3 - 2 2 ) . r A postsynapticneuron generatesan action potential only when the plasma membrane at the axon hillock is depolarized to the threshold potential by the summation of small depolarizations and hyperpolarizations caused by activation of multiple neuronal receptors(seeFigure 23-23). r Electrical synapsesare direct, gap junction connections between neurons. Electrical synapses,unlike chemical synapsesthat employ neufotfansmitter systems,are extremely fast in signal transmissionand are bidirectional.

Cells:Seeing,Feeling, Sensational and Smelling Hearing,Tasting, Dramatic progress has been made in understandinghow our sensesrecord impressionsof the outside world, and how that information is processedby the brain. In this section we discusscellular and molecular mechanismsand specializednerve cells underlying vision' touch, hearing, taste, and olfaction.

The Eye FeaturesLight-SensitiveNerve Cells Vhile owls would favor hearing, and dogs smell, most humans would choose vision as the sensethat provides the most effective window on the world. Light is fast, about 300,000 km/s, and moves in straight lines, so it is excellent for information transfer. The human eye is a complex structure that gathers light from the environment and focuses it on specializedlight-sensitive nerve cells, which send signalsto the brain, where they are translatedinto an i m a g e( F i g u r e2 3 - 2 4 a ) .

(a)

C i l i a r ym u s c l e Zonular Anterior chamber----rCornea .....-

eupit/,

Vitreous numor

Optic disk

Ir i s

(b)

Interneurons

Horizontal Amacrine cell c el l Axons of g a n g l i o n c e l l s G a n g l i o n In n e r Bipolar Outer plexiform plexiform cell to optic nerve cell layer layer

Light

Photoreceptors Cone

Rod

23-24 Structureof the < FIGURE human eye and three classesof neuronsin the retina.(a)Themain lightpasses of the eye Incoming tissues bythe isfocused throughthe cornea, cells light-sensing lens,andactivates the Theirisrestricts in the retina. located the lensThe amountof lightentering fibers, bythezonular lensissupported andmovedbytheciliarymuscleTheeye cushioning, isfilledwith transparent, fluid Thefoveaisthe location vitreous of cells,and density of the highest the highestsenses consequently imageThereisa blindspot resolution (opticdisc)wherethe opticnerveleaves of cells organization theeye (b)Detailed light in the retinaNotethat incoming hasto passthroughmultiplelayers the of neuronsbeforereaching photoreceptor cells,the rodsandcones cells; includehorizontal Theinterneurons bipolarcells,of whichthereareabouta cells,of dozentypes;andamacrine whichtherearemorethan20 tYPes. ganglion cellscarrythesignal Retinal The to theopticnerve. information arewheremost layers two plexiform (a)adapted aremade.lPart connections andK French, W Burggren, fromD Randall, 5thed, W H 2002,EckertAnimalPhysrology, p 259;part(b)from Freeman andCompany, 2006,An B KolbandI Q Whishaw, lntroductionto Brainand Behaviot2d ed , Worth,p 278l

S E N S A T I O N ACLE L L SS: E E I N GF, E E L I N GH, E A R I N GT, A S T I N GA, N D S M E L L I N G

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The human retina (Figure 23-24b) is about 200 pm thick. As we learnedin Chapter 15, it containstwo types of photoreceptors,rods and cones, which are the primary recipientsof visual stimulation.Conesare involved in color vision, while rods are stimulatedby weak light, like moonlight, over a rangeof wavelengths.In contrastto many other sensoryneurons,stimulatedphotoreceptorcellsbecomebyperpolarized, not depolarized. The photoreceptors synapse on layer upon layer of interneuronsthat are innervatedby different combinations of photoreceptor cells. All these signals are processedand interpretedby the part of the brain called the uisual cortex. In each eye we have about 6 million conesand 120 million rods, connectingto 540 million visual cortex cells, so a substantialpart of our nervous systemis devotedto detectingand interpretinglight. Rods detect faint light, as low as a single photon, owing to their sensitivevisual pigment and their ability to amplify a

Pigment epithelium

Free-floating d isks

"" .?%",

Rod outer segment Inner segment

weak signal.Rods have a singlepigment that allows an equal responseto a broad spectrum of wavelengths.Cones (Figure 23-25) come in the three types: red, green, and blue. The brain deducescolor information by comparing the signals from a trio of cone cells,one of eachkind, that sharethe same receptiuefield.The receptivefield of each cell is measuredas an angle with its vertex at the cell. If cells are denselypacked and eachhas a small receptivefield, highly detailedvisual information is collected. If cells are less dense,have large receptive fields, or both, the image will have lower resolution. Rods and conesare packedwith light-absorbingpigments consistingof an opsin protein covalently bound to a small light-sensitivemolecule called 11-cis-retinal.These pigments are arranged in flattened membrane disks in the outer segmentsof rods and cones(Figure23-25; also seeFigure 15-16). Opsin-containingdisks are continuouslyreplaced,completely turning over about every 12 days. Opsins are G protein-
EyesReflectEvolutionaryHistory The eye structuresof organismsreflect their different evolutionary histories.The eyesof some simpler animals, such as planarians,consistof a light-sensitiveoptic nerveaccompanied by pigment cells, without lensesor orher means to obtain a FIGURE 23-25 Rodsand Cones.Vertebrates havetwo typesof sharp image. In contrast, the compound eyesof insectshave photoreceptors, rodsandcones, thatdiffermorphologically and hundreds of lenses,one for each facet of the eye (seeFigure functionally Conesdetectcolor;rodsdetectlightintensity but not 16-21). Yet, some of the proteins that regulate eye developcolorandaremoresensitive thanconesto low lightlevelsThe ment play the same role in a vast array of animals that have prgments thatabsorblightarepackedintoflattened disksin the outersegments strikingly different eyes.One of the most peculiar aspectsof the of rodsandconesNotethatthe outersegments containing the pigments human retina, arelocated causedby the way our eyesevolvedfrom more on the innermost sideof the retina, so lightmusttraverse layers of cellsbeforereaching thesensory primitive light-sensingstructures,is that the photosensitive o r g a n e l l e s [ A d a p t e df r o m f r o m D R a n d a l lW , B u r g g r e na, n d K F r e n c h , cells lie behind the mass of neural connections.Light must 2002, EckeftAnimalPhysiology,5thed, W H Freemanand Company,p 261 passthrough the lens,vitreousfluid, and axons and dendrites l

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and interneuronsbeforereachingthe photoreceptors(seeFigure23-24b; note direction of incoming light). Comparedwith optimal designof a light detector,the eye is backwards.The arrangementis also the reason we have a blind spot.'Where the optic nerveconnects,no light can be sensed.

o

Podcast:Vision-Detecting and Recogniaing Patterns

I n t e g r a t e dI n f o r m a t i o nf r o m M u l t i p l eG a n g l i o n C e l l sF o r m sl m a g e so f t h e W o r l d If eachphotoreceptor cell respondsto a tiny point of light, how The massof neurons is a larger imageof the world assembled? in the visual systemmakes this problem seeminsurmountable. Fortunately the cells are organizedin a rather simple hierarchical fashion that has allowed considerableprogressto be made. The first stageof information processingis done in the retina, immediately after light is received by interneurons (seeFigure 23-24b).In fact, processingof visual information beginsat the very first synapseswhere the photoreceptor cell connectsto interneurons. Interneurons allow signalsfrom multiple photoreceptor cellsto be combinedand compared.By the time signals leave the eye via the axons of retinal ganglion cells that constitute the optic nerve, eachsignal conveysnot a point of light but a pattern of light. Let us start by looking at what sorts of pattern information emergesfrom the retina. The experimentalapproach to the problem useselectrical

Excitatory center

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ticularcellinto which theelectrodeis inserted.Next, variations in thesize,position,andshapeof thelight spotaretested.

(bI Off-centerfield

(a) On-centerfield

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and the retinal ganglioncellsto which they connect(Figure2324b, shown in yellow) are sensitiveto particularpatternsof retinalillumination.This is possiblebecauseeachphotoreceptor (rod or cone)cell simultaneouslyreceiveslight and, a signal from nearby photoreceptorcells.Theselateral signalsare carried by horizontal cells,a type of interneuron (Figure23-24b, eachphotoreceptorcell compares shown in green).In essence, what it seesto what it learnsits tr.ighbort aie seeing. The o] su.h Let'slook at the consequences "n ".r".rg!-.nt. receptivefield of a singlebipolar cell is approximaielycircular, and its information is p"r..d on to a ..tin"l g".gfi." cell thai thereforehasthe samefield. A singlebipolar.-.ll ,"...iuesi.rpots from a group of photoreceptorcellscovering,h; ;;;i;;;: .r cle. Each bipolar cell receivessignalsfrom a d,ii.i#.oa.

to a specificpart of the field that corresponds .ii-fu,. receptive patternsand illumination eyesto different retina.Byexposing ganglion neurons, retinal individual recording simultaneously to an annulus that eachneuronresponds discovered researchers (doughnut)-shaped field,a patterndescribedas centersurround Somecellsfire trainsof actionpotentialswhen the field"); is light("off-center centeris darkandthe periphery (a) ("on-center field") pattern opposite to the respond some An on-centerfield Spofon center:a light spotfocusedon the centerof the cell'sfield triggersa series,or "train"' of action potentialsSpoton surround:alight spotfocusedon the peripheralregionof the samecell'sf ield inhibitsaction potentials'Centerilluminated;illuminationof the entirecenter ti.:i."t the fieldtriggersa rapidburstof actionpotentials'

i,,.,"F1,;;;; i:::',ii":;T:f,ni rlrdp"tt.,n rhisiyp.orreceptive ceus. !,lff:ni,iii?:iJ;ill,ll##,i,1"n'"' surround (Figure 23-26). There is a critical added feature: a bipolar cell respondsto light shining in the centerof its receptive field-the group of photoreceptorcellsnear the centerof the circle-by producing a set of action potentials.However' action potentials are inhibited if light strikes photoreceptor cellssurroundingthe centralpart of the circle(Figure23-26a). This type of cell is an on-centercellbecauselight directedat the middle of the field turns the cell on. Some bipolar cells, and thereforetheir correspondingretinal ganglion cells,have just

illuminationof the cellsentirereceptive illumination:diffuse i.e.,a dampingof the response field causesa weak response, seenif onlythe centeris illuminated.(b) An "off-center"field of a cellthat hasthe oppositepropertyof the cellin (a). llluminationof a spot in the centerof the off-centercell'sfield whilea spotof lightin the periphery inhibitsactionpotentials, 1998, fromF.Delcomyn, actionpotentials[Adapted stimulates p 265) andcompany, w H Freeman of Neurobiology, Foundations

S E N S A T I O N A L C E L L S : S E E I N G , F E E L I N G , H E A R I N G , T A S T I N G , A N D S M E L1029 LING

the opposite response:light in the center reducesthe frequency of action potentials, while light in the surround stimulates more frequentaction potentials(Figure23-26b).This is an offcenter ceIl.The two types of cells, on-center and off-center,are presentin roughly equal numbers.Both typesof cellssensethe relative light intensity in the center versusthe surround, not the absoluteamount of light in either place.Bipolar and retinal ganglion cellsare therefore contrast detectors. How do bipolar cells integrate photoreceptor cell information to detect center-surround patterns? To answer this

> FfGURE23-27 fhe influenceof light and dark on on-centerconecellsin image (a)In the dark,a conecellin interpretation. the centerof the receptive fieldisdepolarized, resulting in the release of glutamate G l u t a m a it ne h i b i ttsh eb i p o l acre l lc, a u s i nigt to hyperpolarize andthuspreventing allbut rareactionpotentials Notethatbipolaroffcentercellshavetheopposite response to glutamate(b)Whenlightstrikes the conecell, it becomes hyperpolarized, with a resultant dropin glutamate secretion Freeof the inhibitory effectof glutamate, the bipolarcell depolarizes, andfrequent actionpotentials result(c)Theactivity of a conecellisaffected by surrounding conecellsthroughhorizontal cellsthatconnect cellslaterally lf onlythe centercellisilluminated, the cellwillstimulate t h eb i p o l aar n dc o n s e q u e ntthl yeg a n g l i o n cellslf bothcenterandsurround cellsare i l l u m i n a t et d h ,ec e n t ecr e l l ss i g n at lo t h e bipolarcellwill be inhibitedThesystem is therefore a contrast detector, lookingfor light patterns that illuminate a smallcenterspotbut not thesurrounding retinaHerearethe steps involved: Lightstriking theconecellin the surround of a receptive fieldhyperpolarizes it Il, whichresults in a reduction in the release of glutamate in E This,in turn,results hyperpolarization of the horizontal cell, causing it to release lessinhibitory transmitter to thecenterconecellB Thecentercone cell,in theabsence of inhibition fromsurround c e l l si ,sd e p o l a r i z 4e ,d d e s e n s i t i z i tntgo l i g h t andinducing an increase of glutamate release to the on-center bipolarcell,asin (a) The bipolarcellistherefore g, hyperpolarized resulting in suppression of actionpotentials @ T h es c h e m a st ah o w na r eh i g h l ys i m p l i f i e d , sinceallthe cellscanbe connected to more thanonecellat eachstageof signal transm ission

(a)

we will look at the connectionsof cone photoreceptor cells to bipolar and horizontal interneurons (seeFigure 23-24b). On-center and off-center bipolar cells differ in the types of channel proteins they use, giving opposite responsesto the same glutamate neurotransmitter. For simplicity, here we will focus on bipolar on-centercells. Bipolar and horizontal interneuron cells,like photoreceptor cells, lack voltage-gatedNa* channels,so none of them generateactionpotentials.Instead,the secretionof neurotransmitters from the cells' synaptic termini is controlled by the

(b) Cone cell in center of receptive field

Cone cell in center of receptive field Depolarized-40 mV

Hyperpolarized-65 mV

Inhibitory glutamate released On-center bipolar celI

Glutamate secretion inhibited

Hyperpolarized

Depolarized

Frequent action potentials

Rareaction potentials G a n gl i o n c e l l

----> Optic nerve

@ (c) Cone cell in "surround"of receptive field

Cone cell in "center" of receptive field

E

E

Cone cell is hyperpolarized-65 mV

Center cone cell is depolarized-40 mV

p Glutamate secreilon inhibited

HorizontaI inhibited

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H o r i z o n t acl e l l E On-center bypolar cell is hyperpolarized

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Cone cell in "surround" of receptive field

degreeof membranepolarization.In the dark, cone cellshave a membranepotential of about -40 mV, which opensvoltagegated Ca2+ channelsand causesthe continuousreleaseof the neurotransminerglutamate (Figure 23-27a). This glutamate, coming from cone cells in the center of a receptivefield, hyperpolarizesthe bipolar neuron that coversthat field, suppressing action potentials. Light striking the center field cone cells hyan inward perpolarizesthem to about -65 mV by sup^pressing flow of Na* and other ions, closingthe Ca'* channelsand reducing emissionof glutamate(Figure23-27b).This depolarizes the bipolar neuron, which in turn depolarizes ganglion cells and triggersaction potentialsto be sentto the brain. On-centerbipolar cellsare most stimulatedif the conecells in the center of the receptivefield are illuminated and the surround cone cells are in the dark. How do bipolar cells detect the light condition in the surround part of the receptive field? The surround input is mediated by horizontal cell interneurons (seeFigure23-24b, greencells).If therels light on a conecell in the surround region of the field, the horizontal cell that is connected to that cone cell becomeshyperpolarized which means it reducesinhibitory transmitterreleaseonto the conecell in the center of the receptivefield. The central cone cell becomesdepolarized,as though it was in the dark, and consequentlythe bipolar cell in the centerof the receptorfield is hyperpolarized (seeFigure23-27c). Ganglion cell action potentialsin the center of the field are suppresseddespite the light falling on the centralconecells.Thus light in the surroundinhibits sensingof the light in the center,a contrast detector. The retina'sprocessingof visual information is just the beginning of a hierarchicalchain of pattern representationand interpretation events.The refined processingof visual information occurs, it must be remembered,by reading trains of action potentials coming from the eye. In the visual cortex, cells are found that are specifically sensitiveto bars of light and dark, and eachcell prefersbars at a certain angle.It is easyto seehow the information that passesthrough retinal ganglion cells, the center-surroundinformation, can be usedto detecta bar of light or dark. If a visual cortex cell is stimulatedby ganglion cells whose visual fields are arrangedin a line, the integratedpattern would be a bar that passesthrough the centersof the individual ganglion cells' center-surroundpatterns (Figure 23-28). Further combinations can lead to recognition of more complex patterns by single cells. Some cells respond to a change of light-on to off or off to on. Others respondto edges,moving spots,or moving bars. Somecellsin the higher levelsof the visual cortex have even been found to recognizea certain face. The discovery of cells that integrate spatial information from cells that have simple receptivefields was describedby Nobel prizewinner David Hubel, who made the discovery with his collaboratorTorstenWiesel: Our first real discoverycameabout as a surprise.For three or four hours, we got absolutelynowhere.Then gradually we beganto elicit some vague and inconsistentresponses by stimulating somewhere in the midperiphery of the 'We retina. were insertingthe glassslidewith its black spot into the slot of the ophthalmoscopewhen suddenly,over the audio monitot the cell went off like a machine gun.

C i r c luar rrouno center-su field receptive

Corticalcell detects the verticalbar

Other cortical c e l l sc a n respondto different patterns

cell A cortical 23-28 Complexpatternrecognition. FIGURE thus ganglion cell"centers," to the sumof multiple responds to cellsrespond barshownOthercortical thevertical detecting of fieldsor combinations receptor of ganglion combinations different patterns. more complex recognize neuronfieldsto cortical After some fussing and fiddling, we found out what was happening.The responsehad nothing to do with the black dot. As the glass slide was inserted,its edge was casting onto the retina a faint but sharp shadow, a straight dark line on a light background.That was what the cell wanted' and it wanted, moreover,in just one narrow range of orientations.This was unheard of. It is hard now to think back and realize iust how free we were from any idea of what cortical cellsmight be doing in an animal'sdaily life'

CellsDetectPain,Heat, Cold, Mechanosensory Touch,and Pressure Our skin, especiallythe skin of our fingers, is expert at collecting sensory information. Our whole body, in fact, has numerous mechanosensorsembedded in its various tissues' These sensorsfrequently make us aware of touch, the positions and movementsof our limbs or head (proprioception), pain, and temperature,though we often go through periods where we ignore the inputs. Mammals use one set of receptor cells to report on touch, and other sets of receptorsfor temperature,heat, and pain' Pain receptors' called nociceptors, respond to mechanicalchange, heat' and certain toxic chemicals (e.g., hot pepper). Genetic insensitivity to pain is

o S E N S A T I o N AcLE L L S 5: E E I N GF, E E L | N GH, E A R I N GT, A S T | N GA, N D S M E L L I N G

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Sensoryhomunculus

Motor homunculus

F I G U R2E3 - 2 9H o m u n c u l u T s .h eh o m u n c u l u i ssa m a po f t h e regions of the brain's cortexthatarededicated to parttcular f u n c t i o nS s e n s oa r yn dm o t o rh o m u n c ual ri es h o w nT h ef a c ea n d often due to mutations in a gene,trkA, that encodesa receptor for nervegrowth factor (NGF), a protein mostly studied in different contexts. NGF and other neurotrophins have now been implicated as signalsof pain. Thermal receptors detecttemperaturechanges.Thesecellssteadilysendaction potentials (2-5h) that indicate the currenr remDerature. Each temperaturerange has receptorstuned to rt, so which cellsare firing conveysthe temperature. Connecting from the skin's sensory cells to the brain does not take many synapses.Mechanosensorsin the skin, for example, connect to the medulla, where they pass the signal along to neurons that go to the thalamus. A third neuron goes from there to the sensorycortex. A mere trio of neurons connectsthe periphery to the brain centers.In the cortex the sensory inputs are combined, through interneurons,with proprioceptiveinputs that report the positions of musclesand joints. Presumablythis makesit possible to know what you are feeling and where it must be if that armin that position is feelingit. proprioceprionreceptors take multiple forms. Some of the most studied are m u s c l e s p i n d l e s ,s e n s o r ya s s e m b l a g etsh a t a r e b u r i e d i n musclesto report on how much that muscle is extended. Suchstretchreceptorsare crucial to smooth movementand well-timed responses. Remarkably,the organizationof the body is reflectedin a rrap, or more accuratelyseveralmaps, in the brain. The organization of the cortical neurons that respond to sensory signalsis physicallyrelatedto the spatial origins of the signals.In the brain the sensoryneurons are laid out in a distorted map of the body. The motor neurons are also 1032

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arranged in a map that can be aligned with the muscles they control. The maps are called the sensoryhomuncwlus and the motor homwncwlus(Figure 23-29). A bomunculus is a "little human," an image of us. The map dimensions are not proportional to the body's dimensions,becausethe homunculi reflect the number of sensory or motor cells rather than the area of the body. The hands and feet are highly representedand take up much more space in the sensorymap.

I n n e r E a rC e l l sD e t e c tS o u n da n d M o t i o n The outer ear captures sound, which moves three tiny bones (ossicles)in the middle ear, which in turn transmit soundinduced motions to the inner ear, or cochlea (Figure 23-30a). The cochlea is shapedlike a snail, with nearly three turns, and indeed the name derives from the Greek word for ,,snail" (cochlos). The cochlea housesthe organ of Corti, the sensory part of the inner ear, which transducesmechanical movement into electricalimpulses.The human organ has about 16,000 hair cellsarrangedin four rows (Figure23-30b, c), attachedto about 30,000 afferent neurons that carry any signalsto the brain. Hair cells produce stereocilia,which are moved by vibrations induced by sound. The oscillatingvibrations alternately bend the stereociliaone way or the other, triggering depolarization eventscalled receptor potentialsin the 10 or so axons associatedwith each hair cell. Theserecepror potentials, which are milder than full action porentials,range up to 25 mV. Hair cells and the neurons they influence are responsive to different sound frequencies.There is a gradient acrossthe

(a)

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23-30 Structuresof the ear.(a)Soundenterstheouter a FIGURE to the middleear,wherethreetinybones(themalleus, earandtravels in thetympanic vibration sound-rnduced transfer andstapes) incus, innerear,where in the cochlea the ear to middle the across membrane Thecochlea, signals. to electrical istransduced vibration mechanical of theorganof wouldbe 33 mm long (b)Theinnersurface unwound, electronmicroscopy' asviewedby scanning Corti,foundin the cochlea, (white)wouldbe in contactwith the Thetopsof allthe stereocilia 23-31)Thestereocilia ear(seeFigure the intact in membrane tectorial in a line,whilethethree of the innerhaircells(leftrow)arearranged in V shapes(c).Higher rowsof outerhaircellshavestereocilia Thehaircellsare of outerhaircells. of thestereocilia magnification cellsarecovered support sunounding while cilia, for the smoothexcept K andFrench, Burggren, (a) W Randall, from D adapted microvilli by IPart ed , W H Freemanand Company,p 243 2OO2,EckertAnimal Physiology,5th Instituteof Healthl Parts(b) and (c):Courtesyof BecharaKachar/National

cochleaof frequency sensitivity so that the spatial locations of the cellsthat are stimulatedreflect the frequencycomposition of a sound. Hair cells and neurons at one end of the cochleahear low-frequencysoundsand at the other end high frequencies.This is not becauseof a differencein either hairs o. ,r.uront. The graded sensitivityis due to a tapering tissue called the basilar membrane (Figure 23-31')that respondsto low frequenciesat one end and high frequenciesat the other' Each friquency excites motion in a particular region of the 33-mmlong basilar membrane, which is then locally transferred to nearby hair cells. The orientation of the hair cells, and in particular of their bundles of stereocilia,with respect to the basilar membrane allows sensitivedetection of deflections causedby sound' The polarity of the hair cellsand their cytoskeletonsare key to proper transduction of sound into electricalsignals. Some of the proteins that control the structure of hair cells and stereocilia have been identified through human genetics,tracking genesresponsiblefor deafness'Five g..r., h"u. been implicated in Usher type 1 syndrome, the . S E N S A T I O N AC L E L L SS E E I N GF, E E L I N GH, E A R I N GT, A S T I N GA, N D S M E L L I N G

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bent stereocilia

!

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most frequent causeof hereditary deafnessand blindnessin humans. The genesencodemyosin VIIa, cadherin 23, protocadherin 15, aPDZ domain protein calledharmonin, and a putative scaffolding protein called Sans. All these proteins are localized within stereociliain auditory hair bundles. Harmonin associateswith both F-actin and with the cadherins implicated in the disease,while myosin VIIa and Sans help to Iocahze harmonin in stereocilia. These discoveries have emergedfrom medical geneticscreeningof patients and are revealingthe key molecular underpinnings of stereocilia and auditory sensing.

Five PrimaryTastesAre Sensedby Subsetsof Cellsin EachTasteBud (b)

T e c t o r i a lm e m b r a n e

(c)

FIGURE23-31 Sterociliamovement. The stereocilia of the innerand outer hair cells(purple)are stimulatedby a sideways movementwith respectto the overhangingtectorialmembrane, which in turn is influencedby oscillating fluid pressure changesin the organof Corti.The fluid pressure in the organoscillates at the frequencyof the incomingsound.(a)As vibrationbegins,the basilar membrane(pink)is forcedupward(indicatedby the arrow)by fluid pressure changes,which translates into a leftwardmovementwlth respectto the tectorialmembrane,thus bendingthe stereocilia to the right (b) At the midpointof the oscillation, theitereocitiarelax (c) When the oscillationgoesthe other way and the basilarmembrane movesdown (indicatedby the arrow),the hair bundlesare movedin the oppositedirectionby the shearingeffectof the tecrorrar membraneThe motionsare repeatedwith eachwave [Adapted from D Randall, W Burggren, and K French, 2002,Eckert Animatphysiology, 5th ed , W H Freeman andCompany, p 24gl 1034

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Taste buds are located in bumps called papillae, and each bud has a pore through which fluid carries solutesinside. About 50-100 tastecellsare locatedin eachtaste bud (Figure 23-32a, b). Cells in the tongue and other parts of the mouth are subjectedto a lot of wear and tear,and tastebud cells are continuously replacedby cell divisions in the underlying epithelium. (A taste bud cell in rars has a lifetime of 10 days.) Tastecellsare epithelial cells that show some of the functions of neurons. Reception of a taste signal causescell depolarization and receptor potentials that trigger action potentials;these,in turn causeCa2+ uptake through voltagedependent Ca2* channels and releaseof neurotransmrtters at synapses.Taste cells do not grow axons, instead signaling over short distancesto other neurons. In conrrast to most other sensory systems,there is, as yet, no known topographic representationat any level of the brain thar correspondsto the different rasres. .$7e taste certain chemicals, all hydrophilic, nonvolatile moleculesfloating in saliva. Although all tastesare sensed on all areas of the tongue and there is no topographical taste map of the tongue, selectivecells do respond preferentially to certain tastes. Taste is less demanding of the nervous systemthan olfaction becausefewer types of moleculesare monitored. \il/hat is impressiveis the sensitivity of taste; bitter moleculescan be detectedat concentrations as low as 10 r2 M. There are receptors for salt, sweet, sour, umami (e.g., monosodium glutamate and other amino acids),and bitter (Figure23-32c, d, e, f) in all parts of the tongue.The receptorsare of two different ,,flavors": channel proteins for salt and sour tastesand seven-transmembrane-domainproteins (G protein-coupled receptors) for sweetness,umami. and bitterness. Salt is probably sensedby members of a family of Na* channels called ENaC channels,though definitive proof is lacking; other membersof the family have diversefunctions including neural memory. The influx of Na+ through a channel depolarizesrhe cell. The role of ENaC channelsas salt sensorsis old, since ENaC proteins clearly detect salt in insects. In Drosophila, taste sensors are located in multiple placesincluding the legs,so when the fly stepson somerhing tasty, the proboscis extends to explore it further. However, the ENaC studieswere done using fly Iarvae,which can respond

23-32 A mammaliantaste bud and its receptors. V FfGURE cells (a)Thepinkcellsarethetastecells.These receptor epithelial arriveat the signals contactthe nervecells(yellow)Thechemical of a pairof tastebuds, seenat thetop (b)Photograph microvilli visible in the areclearly cellsThemicrovilli showingthe receptor tastebudon the /eft (c-f)Typesof tastereceptors[Part(a)adapted andBehavtor, to Brain An lntroductton 2006, fromB KolbandI Q Whrshaw Arnoldl 2ded. Worth,p 400:Part(b)fromEdReschke/Peter

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to salt if they have either of their two ENaC proteins.Sour reception is the detectionof H* ions, which ."n -ou. through the samechannelsas Na*. H* may also be senseddue to its interferencewith K* channelsand consequentincreasein intracellular positive charge(i.e.,a depolarizingeffect). Bitter tastes are more diverse than salt and have been found to depend on a diverse family of about 25 genesencoding various T2Rs, taste-receptorproteins with seven

T1R3 fail to detect sugar; it is thought that the actual receptor is a heterodimerof the two. T1R3 appearsto be r...p" tor for both sweet tastesand umami, and that is because it detectssweetswhen combined with T1R2 and umami when it combines with T1R1. Accordingln taste cells express T1R1 or T1R2 but not borh, as otherwisethey would send an ambiguous messageto the brain.

A Plethoraof ReceptorsDetectOdors

The first member of the T2R family to be identified came from human genetics studies that showed an important bitterness-detecrion gene on chromosome5. Multiple T2R types can be expressedin the same taste cell, and about 15 percentof all tastecellsexpressT2Rs. Bitter tastemole_ culesare quite distinctin structure,which probably accounts for the need for the diverse family of T2Rs. Mice that have five amino acid changesin the receptorT2R5 are unable to taste the bitter taste of cycloheximide(a protein synthesis inhibitor, Chapter 8). A dramatic gene regulation swap experiment was done to demonstrate the role of T2R proteins. Mice were engineered to express a bitter taste receptor, a T2R protein, in cells that normally detect sweet tastesthat attract mice. The mice developeda strong attraction for bitter tastes.evidentlv becausethe cellscontinuedto senda ,,goand eat this.' signal even though they were detecting bitter taste. This exferiment demonsrratesthat the specificity of taste cells is diter_ mined within the cells themselves,and that the signals they send are interpreted according to the neural connections made by that classof cells.This in turn implies a highly regulated systemconnecting the different classesof trrle r..eptor cells to specifichigher regions of the brain. One bitter taste is especiallyfamous becauseit is often used in geneticsclassesto teach about human variation. The chemical phenylthiocarbamide (pTC) tastes exceedingly bitter to many people but is tastelessto others. Human sensitivity to PTC can differ by a factor of 16. The inability to detectPTC is inherited as a recessivetrait, meaning that rast_ ing is dominant over nonrasrrng. Sweetand umami tastesare detectedby a protein family relatedto the T2Rs, called T1Rs. When T1R proteins bind an appropriate tastant, they act through G proteins to rrig_ ger the releaseof calcium inside the cell. The three mam_ malian TlRs differ from one another in a small number of

The perceptionof volatile airborne chemicalsimposesdifferent demands than the perceprion of light, sound, touch, or taste. Light is sensedby only four molecules,tuned to different wavelengths.Sound is detectedby mechanicaleffects through hairs that are tuned to different wavelengths.Touch requiresa small number of types of transducers.The senseof taste measuresa small number of substancesdissolvedin water. In contrast to all theseother senses,olfactory systems can discriminateberweenmany hundredsof volatile molecules moving through air. Discriminarion between a large number of chemicals is useful in finding food or a mate, sensingpheromones,and avoiding predators, toxins, and fires. Olfactory receptorscan work with enormous sensitivity. Male moths, for example, can detect single moleculesof the signalssent drifting through the air by females. In order to cope with so many signals,the olfactory system employs a large family of olfactory recepror proteins. Humans have about a thousand olfactory receptor genes,of which about a third are functional (the rest are unproductive pseudogenes), a remarkably largeproportion of the estimated 25,000 human genes.Mice are more efficient, with 1300 genes,of which about 1000 are functional. That means3 percent of the mouse genomeis composedof olfactory receptor genes.Drosophila has about 60 olfactory receptor genes.In this sectionwe will examinehow olfactory receprorgenesare

FIGURE23-33 Sequenceorganization in olfactory receptors. Olfactoryreceptorsare seven-transmembrane-domain G protein_ coupledreceptorproteins.The cyllndersindicatethe extentof alpha helicesthat crossthe membrane.Residues in blackare highlyvariable, and someof thesedifferences accountfor specificinteractions with odorants [FromL BuckandR Axel.j991,Celt65:1j51

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employed, and how the brain can recognizewhich odor has beensensed-the initial stagesof interpretation of our chemical world. Odor moleculesare calledodorants.They have diversechemicalstructures,so olfactory receptorsface some of the same challengesfaced by antibodies-the need to bind and distinguishmany variants of relatively small molecules. Olfactory receptors are seven-transmembrane-domain proteins(Figure23-33).In mammals,olfactoryreceptorsare produced by cells of the nasal epithelium. Thesecells' called olfactory receptor neurons (ORNs), transducethe chemical signal into action potentials(Figure 23-34).ln Drosophila, ORNs are located in the antennae.The ORNs project their axons to the next higher level of the nervous system,which in mammals is located in the olfactory bulb of the brain. The ORN axons synapsewith dendritesfrom proiectton neurons in insects(calledmitral neurons in mammals);thesesynapses occur in the clustersof synaptic structures calledglomerwli. The projection neurons connect to higher olfactory centers in the brain (Figure 23-35). Each ORN producesonly a singletype of odorant receptor. Any electricalsignalfrom that cell will conveyto the brain a simple message:"my odor is binding to my receptors."Receptorsare not always completelymonospecificfor odorants. Somereceptorscan bind more than one kind of molecule,but the moleculesdetectedusually are closelyrelatedin structure. Conversely,some odorants bind to multiple receptors. There are about a million ORNs in the mouse;so on averageeach of the thousand or so olfactory receptor genesis active in a thousand cells.There are about 2000 glomeruli (2 for eachgene),so on averagethe axons from 500 ORNs convergeon eachglomerulus.From there the axons of about 50,000 mitral neurons,about 25 per glomerulus,connectto higher brain centers.Note that in contrast to the visual system, very little signal interpretation and refinement occurs in the sensoryepithelium or even the projection neurons. The initial sensory information is carried to higher parts of the brain without processing,a simple report of what has been detectedwith no further analysisor commentary. The one neuron-one receptorrule extendsto Drosophila. Detailed studieshave been done in larvae,where a simple olfactory systemwith only 21 ORNs usesabout 10-20 olfactory receptorgenes.It appearsthat a uniquereceptoris expressedin one ORN, which sends its proiections to one glomerulus.

pounds or aromatic compounds.The arrangementmay reflect errolutionof new receptorsconcomitantwith a processof subdivision of the olfactory part of the brain. The simple system of having each cell make only one receptortypi also has some impressivedifficulties:(1) Each ....ptot must be able to distinguish a type of odorant molecule or a set of molecules with specificity adequate to the needsof the organism. A receptor stimulated too frequently will probably not be too useful. (2) Each cell must produce one and only one receptor. All the other genes must be turned off. At the same time the collective efforts of all the cells in the nasal epithelium must allow the production of enough different receptorsto give the animal adequatesensory versatility.It doeslittle good to have hundreds of receptors if most of them are never used' but it is a regulatory

cell is receivingwhich odorant so that electricalsignalsfrom the nose can be interpreted. It is often the case that a responseto a particular odor is programmed in the genes,like b.h"uio."l responseto a pheromone. In such casesthe " brain must know which cells are detecting that pheromone' Otherwise the animal might be feeling romantic when it should be running away as fast as possible. The solution to the first problem is the great variability of the olfactory receptor proteins, both within and between

Nematode

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ORNs can send either excitatory or inhibitory signalsfrom their axon termini, probably in order to distinguishattractive versusrepulsiveodors. The ORNs project to glomeruli in the antennallobe of the larval brain. The researchbeganwith tests of which odorants bind to which receptors(Figure 23-36a)' Someodorants are detectedby a singlereceptor'some by sev-

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23-34 Structuresof olfactory < FIGURE receptorneurons.Acrossa vastspanof insect, distance-vertebrate, evolutionary neuronshave actoryreceptor nematode-olf exposed forms Eachhasfineprocesses srmilar t o v o l a t i loed o r a n tdsi s s o l v ei ndf l u i d H i g h l y (notshown)in the receptors olfactory specific The the odorants cellsshownare cellssense fromF not drawnto the samescale[Adapted Delcomyn,1998, Foundationsof Neurobiology, W H F r e e m a na n d C o m P a n YP, 3 2 7 l

S E N S A T I O N ACLE L L SS: E E I N GF, E E L I N GH, E A R I N GT, A S T I N GA, N D S M E L L I N G

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A FIGURE 23-35 Theanatomyof olfactionin mouseand fly. ln boththe mouse(a)andthefly (b),olfactory (ORNs) receptor neurons thatexpress a singletypeof receptor sendtheiraxonsto thesame glomerulus Inthlsfigurethe redandbluecolorsrepresent the neural connections for two distinct expressed receptors Inthe mousethe glomeruli areIocated in theolfactory bulb;in thefly theyarein the

brain ln the glomeruli, the ORNssynapse withprojectionneuronsin the fly,or mitralneuronsin mammalsEachprojection neuron(or mitralneuron) hasitsdendrites in a singleglomerulus, thuscarrying to highercenters of the braininformation abouta particular odorant. T.Komiyama andL Luo,2005,CurrOpinNeurobiol. [From 16:67-73]

species(seeFigure 23-33; the black residuesare highly vari_ able). The solution to the secondproblem, the expressionof a single olfactory receptor gene, has been expltred using transgenicmice, but the mechanism is still not understood. rX/henan engineered olfactory receptor gene is used to pro_ duce an olfactory receptor,other genesare turned off tian_

receptor send their axons to the same glomerulus. Thus all cells that respond to the same odorant send processesto the same destination. This convergenceprocesscould be due to (1) an attractive signal that somehow is specificfor a certain olfactory receptor or (2) to mutual recognition, and subsequent coordinate growth, of axons that have the samereceptor on their surfaceor (3) to a pruning processin which many connectionsare made but only those that share the same olfactory receptorpersist,possiblyregulatedby neuronal activity. Developmentalanalysesshow that ORN axons do not arrive at a glomerular "blank slate." Rather the glomerulushas organizedits projection neurons prior to the arrival of ORN axons. The systemis to some degreehardwired. In mice a crucial clue about the patterning of the olfactory systemcame from the discoverythat olfactory receptorsplay

The third problem, how the systemis wired so the brain can understand which odor has been detected,has been partly answered.First, ORNs that have expressedthe same 1038

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23-39 Antibodylabelingreveals FIGURE A EXPERIMENTAL cytoskeletoncomponentsinsideculturedhippocampalgrowth cones.A singlegrowthconeisshownthreetimes,in eachcase tyrosinylated F-actin, with an antibodyfor a differentstructure: labeled (ace-MTs)' The (tyr-MTs), microtubules acetylated and microtubules of the otherthree Notethe relative fourthpanelshowsa composite andthe edgesandperiphery, at the leading lackof mrcrotubules in areconcentrated of actinthereThemicrotubules concentration actin with (although colocalize tyr-MTs some region the central so regionThegrowthconeis paused, in the peripheral bundlespikes F B Gertler,2003,Neuron E W Bentand loop [From the microtubules 40:209-227 l

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23-38 Theadvanceof the growth cone.During a FIGURE from protrusion, extendunderpressure filopodia andlamellipodia (ribs)During bundles F-actin meshworks andelongated intracellular andcarry intotheprotrusions microtubules areelongated engorgement, depolyintothem.Duringconsolidation, organelles membrane-bound neckisfollowedbynanowingof merization of actinin thegrowth-cone bundleto formtheaxonshaftA new thecellaroundthe mrcrotubule protrusion axonshaft off thesideof a cylindrical canformby branching z10:209-227 2003,Neuron E W BentandF.B Gertler, | lFrom appearanceof extra outgrowths of lamellipodia and filopodia along the axon shaft, implying that microtubules may normally prevent the assemblyof microfilaments in the axon. The concentration of actin in the periphery and leading edge of the growth cone, and of microtubules in the more central and lagging regions,reflectsthe different roles played by the two (Figure 23-39; see also Chapter 17). Actin is

assembledinto filaments in the leading cone, the filamentous mesh of actin flows backward as the cone advances,and actin is disassembledas the cone transforms into an axon' The transport of actin toward the rear occurs in the filopodia and lamellipodia and is driven by a myosin motor' Note that this is completely different from treadmilling (Chapter 17). Movement of the actin mesh with respectto the growth 'l'-7 cone involves movement of the entire filament at pm,/min, attachedto myosin motors. For a filopodium to advance, the rate of actin polymerization at the leading edge must exceedthe rate of retrograde flow. Actin filaments have beenviewed as major determinantsin turning growth cones'a processthat is crucial for the neuron to respond to guiding signals' Microtubules are also involved, sincemicrotubule-inhibiting drugs prevent turning as well' Microtubules are assembledat the neuron'scentrosomeand transthe advancing growth cone' ported by dynein motors toward 'sfhile traveling' the microtubules with the plus end leading.

C:O N T R O L L I N A GX O N G R O W T HA N D T A R G E T I N G T H E P A T HT O S U C C E S S

o

1041

> FIGURE 23-40 The retinotectalmaps. (a)Thedorsalretinaisconnected to the lateral tectumon theopposite sideof thebrarn, andthe ventralretinaisconnected to medialtectumon theopposite sideSimilarly thetemporal-nasal (T-N) axisof theeyeisreflected in the rostral(R-C) caudal mapof thetectum(b)Themaps of thevisual worldon theretina andthe corresponding tectumareturned90 degrees but otherwise arein register. Thearrowshowshowa patternof lighton the retinaisreproduced asa setof retinal ganglion cellconnections in the tectumThetectumin mammals isreferred to as (SC)[G Lemke lhe superiorcolliculus andM Reber,

(b)

Medialr

2005,Ann Reu CellDev.Eloi 21:551-580l

can undergopolymerizationand depolymerization.A tyrosinylated form of tubulin is preferentiallypresentin more advanced parts of the growth cone, while acetylatedtubulin is enriched in central and lagging parts and in the axon itself (seeFigve 23-39). The roles of such post-translationalmodifications are describedin Chapter 2. An ordered processof tubule assemblyand modification underliesgrowrh-coneadvancement. As we have seen (Chapter !7), actin polymerization is controlled by a startlingly large set of regulatory proteins. More than 20 actin-binding proteins have been found in growth cones,most of which control nucleation or polymerization of actin filaments, or tether the filaments to the membrane. Many of these actin-binding proteins are targets of signal transduction events triggered by axon guidance signals, as we shall seein the next section.

The RetinotectalMap Revealedan Ordered Systemof Axon Connections Researcherslong debatedtwo generalideas about how neurons might get wired. One, the "resonancehypothesis," proposedthat cellsextend axons along pathwaysthat are defined by mechanicalforces.After many paths are taken and connections formed, the ones that work are preservedwhile others are removed.The secondidea, a "tprri1i, pathways hypothesls," suggestedthat axons choosetheir path bv chemicalaffinitg molecules on the growing axons contacting molecules along the way thar provide guidepostsor signals. In 1963 Roger Sperryproposeda version of the specificparhwaysidea called the "chemoaffinity hypothesir.'tH. suggesredthat growth coneswould find their way following molecular cues that form a gradient from start to desdnation,a seminalproposal that could not be properly testedfor decades.Sperry's proposalwas basedon his studiesof how the axons of the retinal ganglion cells, which form the optic nerve, are arranged when they arrive at the optic tectum. The optic tectum is locatedin the roof of the midbrain and is the destinationof retinal ganglion cell axons that grow from the retina. The incoming retinal neuronsform a map on the tectum (the retinotectal map) that reflectsthe arrangementof rods and conesin the retina, and indeedthe visual world outside(Figure23-40).The spatialmap on the retina is in essence copied into the brain. 1042

.

c H A p r E R2 3

|

NERVE cELLs

Sperryperformed experimentswith the frog eye and brain to distinguish the two models, "resonance" versus "chemoaffinity," that describe how axons may accomplish this mapping (Figure 23-47). The frog optic nerve will regenerate if it is severed,and the pattern of regeneration-the route-finding by axons-is revealing about how axons are guided.In the normal arrangement,retinal ganglioncell axons from the ventral part of the eye connectto the medial part of the tectum, while axons from the dorsal part of the eye connect to the lateraltectum (seeFigure 23-41a).For eacheye,the connectionsare made on the opposite side of the brain, left eyeto right brain and right eyeto left brain. Sperrynext added a secondsurgeryto the experiment,rotating one eye 180 degrees,so that ventral and dorsal are reversed(Figure 23-41b). If the resonancehypothesisis correct, i.e., mechanicalforces followed by a functional sorting-out processwere governing regeneration,the visual systemshould end up functioning normally, since the proper connectionswill be establishedand maintained (Figure 23-41c, left). lf there is a chemoaffinity guidance system,then vision should be inverted becausedespite the rotation the ventral axons would find their way to the lateral tectum and the dorsal ones to the medial tectum (Figure23-41c, right).ln this casethe invertedeyewould lead to the frog's having inverted vision: it would see something above and would think it was below (Figure 23-4Id). The results were clear: after regenerationthe frog respondedto a fly passingabove by shooting its tongue down. The axons were originating from abnormal locations yet finding their way to the right connections,so the inverted eye was tricking the frog's brain. The chemoaffinityhypothesiswas affirmed. It seemsa necessary conclusion...thatthe cellsand fibers of the brain and cord must carry some kind of individual identification tags, presumably cytochemical in nature, by which they are distinguished one from another almost, in many regions, to the level of the single neuron. -Roger Sperry, 1963 There is now substantialevidencethat Sperry'sgeneralideas were correct. Nonethelessmuch remainsto be learnedabout how the enormous complexity of the neural circuitry is successfullyassembled.The relativeimportanceof local versus

23-41 Eyerotation experimentstest < EXPERf MENTALFIGURE of the propertiesof axon pathfinding'(a)Normalprojection (L)tectumandventral(V)eyeto fromdorsal(D)eyeto lateral nerves, of two sequential representation medial(M)tectum.(b)Schematic (2) a 180' rotation (1 nerve and of the optic a severing operations,) of Regeneration of the eye(orno rotationin thecontrolexperiment) to occurNotethatin the rotatedleft wasthenallowed the nerves (D),nevertheless still dorsally half,noworiented eye,the dark-shaded (a). half of the light Likewise the in as seen theventralretina, contains ventrally thedorsalretina,isnoworiented eye,whichencompasses is on whichhypothesis (V) (c)Twopossible dependinq outcomes, predicts X: visionisrestored hypothesis correctTheresonance axon Thespecific properconnections functionselects because retinal dorsal inverted because predicts is Y: vision pathway hypothesis affinityspecified, of chemical tectumbecause axonsstillgo to lateral in theventralposition eventhoughthedorsalretinaisnow located (d)Theresults Y; thefrogs visionisinverted supporthypothesis

(a)

Tectum

(b)

T h e r eA r e F o u r F a m i l i e so f A x o n G u i d a n c e Molecules

Optic nerve is severed andeyeis rotated 180'

Specificaxon pathway hypothesis

Resonance hypothesis

X

(d)

Regeneration

Y

Flv

long-rangesignaling,the roles of glia, the influencesof neural electrical activity, and the signal transduction and cytoskeletal changesthat form the responseto signalsare important areas of current research.The lure of this field is substantial,since understandinghow neuronsare wired to one anotherunderlies the working of the brain and at the sametime is important for learninghow to stimulaterepair of damagedneural circuits.

For many years, attempts were made to identify key axon guidance molecules.Approaches included making panels of antibodies against surfacemolecules,culturing neurons and testing extracts for their ability to make growth conesturn, and using geneticsto identify mutants that fail to properly wire the nervous system.All these attempts worked to a degree, but geneticswas the most powerful approach, since it identified previously unknown moleculeswhile at the same time convincingly demonstrating their importance in vivo. 'We can set a high standard for what constitutesa proper guidancemolecule:it must be produced by cellsthat actually guide neurons in vivo, it must be necessaryfor guidance' the guided cells must have sensorsand signal-transductionmaihitl.ry for responding,and the mislocalization of the signal must causecells to turn the wrong way. 1Wewill discusshere four families of proteins (Figure 23-42) that fulfill our criteria and, with their receptors,provide crucial information to growing axons: Ephrins, Semaphorins, Netrins, and two related proteins called Robo and Slit. These proteins have both attractive and repulsive effectson growing axons. Sincethe growth cone is an active sender of signals,as well, the communication is mutual. After the initial connectionsform, the wiring is refined by preservingconnections that work and discarding those that do not contribute to neural function. Many cellsthat fail to make useful connectionsdie bY aPoPtosis. Ephrins The retinotectalmap describedabove is a striking example of the simplicity of the nervous system' which belies the seeminglychaotic mass of neurons in the visual part of the brain. This rather amazingphenomenonis due in part to a remarkable signaling system involving the ephrins, a family of cell-surface signaling proteins, and their receptors' the Ephs' (The word "Eph" comes from the erythropoietin-producing Eepatocellularcarcinomacell line wherethe proteinswere originally found.) Ephs constitute the largest family of receptor tyrosinekinases(RTKs; Chapter 16), with 14 Ephsand 8 ephrins in mice. Although Ephs usually serve as receptors for ephrins,

C:O N T R O L L I N A GX O N G R O W T HA N D T A R G E T I N G T H E P A T HT O S U C C E S S

O

1043

> FIGURE 23-42Families of guidance ( a ) 4 c l a s s e so f l i g a n d s molecules. Fourmajorfamilies of signaling Netrin Slit proteins provide crucial information to direct growingaxonsTheligands (a)areNetrins, Robo/Slit, Semaphorin, andEphrin proteins (b)areasfollows:Netrininteracts Thereceptors Cytosol with itsreceptor DCCin vertebrates; the corresponding but differentreceptor in nematodes iscalledUnc5Bothcontain (lg)domains immunoglobulin (bluecrescents) anda variety of otherdomains asshownThe Slitligandinteracts withthe Roboreceptor, whichalsohaslg domainsTheSemaphorins interact with diverse receptors, generically calledPlexins Someof theinteractions are through"sema"domains (redbars), present in bothligandandreceptor, othersrequire lg domainsEphrin ligands interact with Eph Exterior receptors, although signaling appears to go in bothdirections andin somecases the Ephrins actmorelikereceptors All of the receptors havesingle transmembrane domainsThe NetrinandSlitligands aresecreted andnot membrane associated TheSemaphorin ligands canassociate with membranes to varying degrees-some notat all TheEphrins are membrane-associated Seethetextfor a detailed discussion of thesefourfamiliesln addition to thesefourfamilies of proteins a few others, including thedevelopmental regulators Wnt andShh,contribute guidance additional information Rp Kruget J Aurandt, [From andK-L Cytosol Guan, 2005,Nature Rev. Mol CellBiol6:789-800,

Semaphorins Invertebrate

Ephrins

Vertebrate

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membranesby GPI links. Ephrin Bs, the other classof ephrins, are transmembraneproteins (seeFigure 23-42). The ephrin proteins affect cell migration, axon guidance,synapsedevelopment, and vascular development,but we will focus on their rolesin guidanceand formation of the retinotectalmap. Ephs and ephrins are distributed in gradients so that advancing axons can recognizeand grow toward appropriate targets. Antibodies against ephrin A5 show a gradient of protein with the highest levels in the anterior. Mice lacking ephrin A5 have guidance defects; axons rhat should have been targetinganterior tectum grow into posterior regions. Further investigationsshowed that cells expressEph receptor proteins in two orthogonal gradients, to control axon 1044

.

C H A P T E2R3

I

NERVE cELLs

Plexins

Eph

guidance along each of the two axes (Figure 23-43).In each axis, the graded amount of receptor in retinal ganglion cells confers differential sensitivity to specific ephrins emitted from the tectum targets.The Ephlt's and ephrin lfs controlling one axis do not cross-reactwith the EphB's and ephrin B's for the other axis. Therefore, axons can "learn" their positions on an XY coordinate system by reading levels of iigand for which they have receptors.To give one example, axons that have EphB2,3, and 4 receptorsare attracted to places where there are high levels of ephrin 81 ligands, so axons originating in ventral retina tend to go to medial tectum. The full situation with all thesegradients is more complex and not yet fully understood.Eph tyrosine kinase signal transduction influencesthe small GTPasesRho. Cdc42. and Rac, thus regulating assemblyof the actin cytoskeleton and controlling guidance of the growth cone. The activation of an Eph receptor may cause arrraction or repulsion of a growth cone, dependingon the cell.

Retina gradients

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EohrinA2lA5 23-43 Gradientsof Ephproteinsform two A FIGURE gradients in the retina orthogonalsignalingsystems.Ephreceptor in the superior colliculus areshownon the /eft;ephringradients (retina) (tectum), on the nght Gradients alongthe nasal-temporal (tectum) gradients in along axes are shown blue; androstral-caudal (retina) (tectum) axesareshown andlateral-medial the dorsal-ventral in red Themorecolor,the moreproteinpresentThewhitearrows (seeFigure 23howthe mapsarerelated indicate andarrowoutlines so for EphA-expressing axons, 40) Ephrin A'sarechemorepellent EphA receptors tendto go fromtheir retinalganglion cellscarrying in the rostral sideof the retinato destinations originsat thetemporal of ephrrnA'sislower tectum,wherethe concentration Semaphorins Semaphorinsare a diversefamily (seeFigure 23-42\. and much remainsto be learnedabout all their effects. They were named for the alphabeticsignalingsystemof flags that was usedto communicateover long distances.Semaphore signalscan spell out any message,but in the nervous system go away.They are semaphorinslargely caffy a singlemessage: potent repellents.The family of two invertebrateand five vertebratesemaphoringlycoproteins(Figure23-42) includessome that are secretedand somethat are membrane-bound.This impliesthat someof them act on adjacentcells,while othershave a longer reach. Motor, sensory,olfactory, and hippocampal neuronscan be repulsedby semaphorinsignals.Semaphorins transmembind to receptorscalledplexins that are single-pass proteins capapicture is receptor proteins. The overall of brane ble of acting as scaffolds both inside the cell and outside, assemblingprotein complexesand modifying their activities. How exactlythis modifiesgrowth-coneadvancementis an area of current research;at leastpart of the answeris differentialadhesionto cellson one sideof the srowth cone versusthe other. Netrins Netrins are secretedproteinsrelatedto laminin (see Figure 23-42). They were discoveredin geneticscreenslook-

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23-44 Evolutionaryconservationof netrin signaling. FIGURE with cellbodiesin neurons spinalcord,commissural Inthevertebrate by attracted growaxonstowardtheventralmtdline, dorsalregions followa similar neurons sensory C. elegans' Intherirrorm netrinsignals. followthe whilemotorneurons pathtowardventralUnc6/Netrin, axonsarealso vertebrate Certain pathawayfromUnc6/Netrin opposite that in wormsviamutations werediscovered by netrinsNetrins repelled as neurons, andin vertebrates bysensory alteredthe pathfinding pathfinding of commissural thatcouldinfluence molecules secreted cordexPlants in spinal neurons ing for misrouting of neurons in C. elegans,the nematode worm for which every cell and every neuron has been identified. About 30 geneswere found, three of which affecteddorsal-ventralrouting of the sensoryand muscleneurons (Figure 23-44\: unc-6, which encodesa netrin protein; wnc-40, which encodesthe netrin receptor (called DCC in mammals); and wnc-S,which encodesa secondtype of netrin receptor.Unc6lNetrin mutations affected both dorsally directed and ventrally directedaxon extensions.Vertebratenetrins were found in studiesof how commissural (crossing)neurons find their way through the spinalcord (seeFigute23-44). Theseneurons emerge from dorsal regions of the spinal cord and extend around the periphery of the cord toward the ventral midline. To test for the presenceof guidance molecules,parts of the spinal cord were cultured either separatelyor together. When the most dorsal part was cultured near a ventral part' axons grew out toward the ventral tissues.No axon outgrowth was observedwhen the two parts were cultured separately.Extracts from the ventral part had the same activitg stimulating axon outgrowth from dorsal tissue. A heroic protein purification, starting with about 20,000 embryonic chick brains, succeededin identifying two proteins that were potent chemoattractant signals.Both were netrins. Netrin 1 is highly expressedin the spinal cord floor plate' the most ventral part. Further proof of the role of netrins came from mouse gene knockouts, in which the commissural neurons were unable to properly find their way (Figure 23-45). Netrins guide axons to the ventral midline in nematodes, flies, and vertebrates,an example of evolutionary conservation of protein function over more than half a billion years. A puzzle remained.The worm version of the protein appearedto have two functions: attracting the axons of sensory neurons to grow toward the ventral midline and driving the axons of motor neurons ,Luay from the ventral midline' The simplest possibility was that netrin is attractive to some axons and repellent to others.Indeed evidencequickly emerged

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EXPERIMENTAL FTGURE 23-45 Mouse netrin-/- mutants havecommissural neuronguidancedefects.(a)Wild-type tracing (red)thatoriginate of commissural neurons (top)andgrow dorsally towardandacross (greenarrowheads) theventralmidline underthe influence of netrinproduced (floorplate)cells(b) by ventralmidline Homozygous netrin-/- mousemutantManycommissural neurons wanderoff track(greenarrows) beforereaching theventralregions, andothers(greenarrowheads) turn instead of crossing theventral midline. of Marc Tessier-Lavigne, [Courtesy Genentech Inc] that certain vertebrate neurons found netrin repulsive. Genetic analysesin worms showed that Unc-40 receptor is required for attraction by netrin, while Unc-5 receptor in combination with Unc-40 is required for repulsion by netrin. Another puzzleremained.If the axonsof commissuralneurons are attracted by netrin coming from the ventral midline, how do the axons continue to grow after they have crossedthe midline? One would expecrthem to turn around and go right back. The solution to this puzzle awaited the discovery of still other key playersin axon guidance,Slit, Robo, and Comm. Robo and Slit Guidance Molecules The path of growing axons in the insect nerve cord is reminiscent of a subway map (Figure 23-46a). Geneticsallowed the discoveryof guidance genesand proteins that affect the pathfinding process,changing the map. A large set of random mutations was introduced into the Drosophila genome to generatelethal mutations. The mutationscould be carriedin heterozygotes, and when the heterozygotesof eachline were crossed,a quarter of their progeny were homozygous for the newly induced mutation. These progeny were stained to show the embryonic nerve cord, the equivalent of our spinal cord, which in the wild type looks like a ladder (Figure23-46bl.In lines of flies where the mutarion was in a genenecessary for axon guidance,defectsin the nerve cord could be seen.Among the genesidentifiedin this manner were three, slit, roundabout (robo), and commisswreless (comm). They defined yet another set of critically important and evolutionarilyconservedaxon guidancemolecules. Slit is a secretedprotein (seeFigure 23-42) made by midline glia. The Slit receptor is Robo, a single-passtransmembrane protein with only a short sequencein the cytoplasm and fibronectin and immunoglobulin domains on the outside of the plasma membrane (see Figure 23-42). The Robo/Slit complex is a chemorepellentinteraction. The presence of Slit in the midline servesto repel axons containing Robo receptors,thereby ensuring that ipsilateral (same-side) neurons do not crossto the opposite side. In loss-of-function 1046

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s/it mutants, or in double mutants that lack two Drosophila robo genes,axons go to the midline but can never leave (Figure 23-46c, d). This can be explained by the fact that, in the absenceof Robo receptor or its ligand, the chemorepellent Robo/Slit interaction at the midline does not occur. This raisesthe question of how an axon that needsto cross the midline is first attracted to it and then repelled from it. An axon that will cross the midline does produce Robo protein and should be repelled by Slit, but the axon is initially refractory to the Slit signal becausethe Robo protein is trapped in the Golgi network by a Golgi protein called Commissureless (Comm) and neverreachesthe cell membrane.Once the axon reaches the midline, Comm becomes inactive and Robo reachesthe surfaceagain. The newly accessiblereceptor allows a responseto midline Slit, and the axon grows away from the midline on the far side.Loss of comm function allows excessive Robo to reach the surface,so no axons cross the midline (Figure 23-46e).The expressionof comm is normally regulatedso that it is "off" in cellswhose axons are supposedto remain in only the left or right longitudinal axon tract, and is "on" in cellswhose axons must crossto the other side. None of these protein guidance systems is dedicated solely to the nervoussystem;in fact, all of them are employed in other tissuesfor various purposes.Indeed most of the special attributes of neural cells appear to be more or lessexaggerated versions of processescommon to many or all cells. That is apparent ( 1 ) in the polarization of neurons from dendrite to axon, which employs cell-asymmetryproteins, (2) in the neuronal intracellular organelle-transportsystems,which depend on variations of endo- and exocytosis, (3) in outgrowths of axons and dendrites, which have features of chemotaxis,and (4) in the use of channelproteins to control ion flow. Neurons are a variation on familiar cell biology themes,a variation with enormous functional Dower.

DevelopmentalRegulatorsAlso GuideAxons In Chapter 22 we becamefamiliar with a set of secretedsignaling proteins that control cell fates and, in some cases,cell division. Sincethesewere discoveredfor their roles in development and differentiation, they were not initially suspects in the hunt for axon guidancemolecules.Yet it turns out that at least three cell fate regulators, Hedgehog (specifically Sonic hedgehog,Shh), BMP, and Wnt proteins, can also be axon guidancemolecules.This is in a sensereassuring,as the sum total of complexity of the other guidancemoleculesstill seemsrather low compared with the challenge of routing millions of axons on complex paths. In the developingspinal cord (seeFigure 23-44), even in the absenceof Netrin 1 some commissuralaxons still extend toward the ventral midline. Another protein, Shh, made in the floor plate, accountsfor that remaining guidanceactivity. Proof of its role came from the discovery that (1) cultured cellsthat secreteShh reorient commissuralaxons in tissueexplants, (2) isolatedneuronsin culture turn toward a sourceof pure Shh protein, and (3) mutants affecting Shh signal transduction interfere with axon pathfinding. Thus commissural axons are guided toward the ventral midline by both Netrin

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for sweetness,bitterness,and umami; and odor receptor molecules. 15. How did Roger Sperry'sexperimentsin frogs distinguish betweenthe resonancehypothesisand the chemoaffinity hypothesisfor how neurons becomewired?

Analyze the Data Olfaction occurs when volatile compounds bind to specific odorant receptors. In mammals, each olfactory receptor neuron in the olfactory nasal epithelium expressesa single type of odorant receptor.These odorant receptorsconstitute a large multigene family (>1000 members)of related proteins. Binding of odorant induces a signaling cascade that is mediatedvia a G protein, Goo15. Recentstudiessuggestthat there are a small number of olfactory sensoryneurons in the nasal epithelium that expressmembers of the trace-amineassociatedreceptor (TAAR) family, chemoreceptorsthat are G protein-coupled receptors(GPCRs)but are unrelated to classicalodorant receptors (see Liberles and Buck, 2006, Natwre 442:645-650). The mouse genomeencodes15 TAAR geneswhile the human genome encodes6. a. In order to examine the expressionpattern of different TAARs in the olfactory nasal epithelium, researchers localized TAAR RNA by in situ hybridization in pairwise combinations. All possiblepairwise combinations of the 15 mouse TAARs were examined. A typical example of the results obtained is shown in the top set of panels in the figure below in which TAAR6 and TAART have been localized with fluorescent probes in the nasal epithelium of mouse. The Taar6 probe was labeledwith a green fluor, the TAART probe with a red fluor. The lower set of panels shows localization of mouse odorant receptor 28 (MOR28; green), a classical odorant receptor, and TAAR6 (red). Each stained patch in the images is the staining pattern of an individual olfactory neuron. The "merge" panels show the two other imagessuperimposed.\What do thesedata suggestabout expressionpatterns of the TAARs?

phosphatase (SEAP) under control of a cAMP-responsive element. The cells are then exposed to various amines, as shown in the following figure, and SEAP activity in the medium is determined.The figure shows data for some representativeTAARs (m : mouse,h : human). What do these data reveal about TAARs? lfhat does the SEAP activity assay reveal about the signaling pathway utilized by chemoreception involving TAARs? 400

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References Neurons and Glia: Building Blocks of the Nervous System Allen, N. J., and B. A. Barres.2005. Signalingbetweenglia and neurons:focus on synapticplasticity.Curr. Opin. Neurobiol. 15:542-548. Eshed,Y., et al. 2005. Giiomedin mediaresSchwanncell-asn interaction and the molecularassemblyof the nodesof Ranvier. Neuron 47:215-229. Jessen,K. R., and R. Mirsky. 2005. The origin and development of glial cellsin peripheralnerves.Nature Reu.Neurosci.6(91:671-682. Parker,R. J., and V. J. Auld. 2005. Rolesof glia in the Drosophila nervoussystem.Semin. Cell Deu. Biol. 17(1):66-77. Ram6n y Cajal, S. 1911. Histology of the NeruowsSystemof Man and Vertebrates(trans.N. Swansonand L. \7. Swanson.1995, Oxford UniversityPress). Salzer,J. L. 2003. Polarizeddomains of myelinatedaxons. Neuron. 402297-31.8. Seifert,G., K. Schilling,and C. Steinhauser. 2005. Asrrocyte dysfunctionin neurologicaldisorders:a molecularperspective. N ature Reu.N ewrosci. 7 (31:194-20 6. Sherman,D. L., and P.J. Brophy.2005. Mechanrsmsof axon ensheathmentand myelin growrh. Nature Reu.Neurosci. 619),:683-690. Stevens,B. 2003. Glia: much more than the neuron'sside-kick. Curr. B iol. 13:R4 69 -R47 2. Voltage-Gated lon Channels and the Propagation of Action Potentials in Nerve Cells Aldrich, R. W. 2001. Fifty yearsof inactivation.Nature 4lt:643-644. Armstrong, C., and B. Hille. 1998. Voltage-gatedion channels and electricalexcitability.N euron 20:371,-380. Brunger,A. T. 2005. Srructureand function of SNARE and SNARE-interactingproteins.Quart. Reu.B iophys. 38(1):147 . Cannon, S. C. 2006. Pathomechanisms in channelopathiesof skeletalmuscleand brain. Ann. Reu.Neurosci.29z387-415. Catterall,\f. A. 2000. From ionic currentsto molecularmechanisms:the structureand function of voltage-gatedsodium channels. Neuron 26:1.3-25. Catterall,\7. A. 2000. Strucrureand regulation of voltage-gated Car" * channels. Ann. Reu.Cell Deu. Biol. 16:521.-555 . Catterall,W. A. 2001. A 3D view of sodium channels.Nature 409988-989. Clapham,D.1999. Unlockingfamily secrets: K+ channeltransmembranedomains. Cell 97:,547-5 50.

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Brejc,K., et al. 2001. Crystal structureof an ACh-binding protein revealsthe ligand-bindingdomain of nicotinic receptors.Nature 4ll:269-276. Fatt, P.,and B. Katz 1952. Spontaneoussubthresholdactivity at motor nerveendings.J Physiol. 117:709-128. Fernandez,J. M. 1,997.Cellular and molecularmechanicsby atomic force microscopy:capturing the exocytoticfusion pore in vivo? Proc. Nat'l Acad. Scl. USA 94:9-10. Ikeda, S. R. 2001. Signaltransduction.Calcium channels-link locally,act globally.Science294:31,8-319. Jan, L. Y., and C. F. Stevens.2000. Signalingmechanisms:a decadeof signaling.Curr. Opin. Neurobiol. 10':625-630. Karlin, A. 2002. Emergingstructureof the nicotinic acetylcholine receptors.Nature Reu.Neurosci. 3:1.02-1.1.4. Kavanaugh,M. P. 1998. Neurotransmittertransport: modelsin flux. Proc. Nat'l Acad. Sci.USA 95:1,2737-1,2738. Klann. E.. and T. E. Dever.2004. Biochemicalmechanismsfor translationalregulationin synapticplasticity.Nature Reu.Neurosci. 5(1.2\:931.-942. Kummer, T. T., T. Misgeld, and J. R. Sanes.2005. Assemblyof the postsynapticmembraneat the neuromuscularjunction: paradigm lost. Cwrr.Opin. Neurobiol. 16(1,\:74-82. Lin, R. C., and R. H. Scheller.2000. Mechanismsof synaptic vesicleexocytosis.Ann. Reu.Cell Deu. Biol. 16':1,949. Neher,E. 1998.Vesiclepools and Ca2' microdomains:new tools for understandingtheir roles in neurotransmitterrelease. Neuron 20:389-399. Reith, M., ed. 1997. NeurotransmitterTransporters:Structure, Function, and Regulatioz.Humana Press. Sakmann.B. 1992.Elementarystepsin synaptictransmission revealedby currentsthrough singleion channels.Nobel Lecture reprinted in EMBO J. 17:2002-20L6 and Science256:503-51,2. Sosinsky,G. E., and B. J. Nicholson.2005. Structuralorganization of gap junction channels.Biochim.Biophys.Acta 17II(2\:99-1,25. Sudhof,T. C. 1995. The synapticvesiclecycle:a cascadeof protein-proteininteractions.Nature 37 5:645-653. Ule, J., and R. B. Darnell. 2005. RNA binding proteins and the regulationof neuronal synapticplasticity.Curr. Opin. Neurobiol. 1 6 (1 ) : 1 0 2 - 1 0 . Usdin, T. B., et al. 1995. Molecular biology of the vesicularACh ff ansporter. Trends Neurosci. 18:21,8-224. White-Grindley,E., and K. Si. 2005. RISC-y memories.Cel/ 124(1\':23-60. Ziv. N. E.. and C. C. Garner.2004. Cellularand molecularmechanismsof presynapticassembly.Nature Reu.Neurosci.5(5):385-399. Sensational Cells: Seeing, Feeling, Hearing, Tasting, and 5melling Buck, L., and R. Axel. 1991. A novel multigenefamily may encodeodorant receptors:a molecular basisfor odor recognition. 5-1.87. Cell 65(1.)21.7 Cohen-Cory,S., and B. Lom. 2004. Neurotrophic regulation of retinal ganglion cell synapticconnectivity:from axons and dendrites to synapses.lnt'l J. Deu. Biol.48(8-91:947-956. Eatock, R. A., and K. M. Hurley. 2003. Functional development of hair cells. Cun Top. Deu. Biol. 57:389448. Esteve,P.,and P. Bovolenta.2006.Secretedinducersin vertebrate eye development:more functions for old morphogens.Curr. Opin. -1.9. Neur o bi ol. 76(1.\21.3 Gillespie,P. G., and R. G. Walker. 2001. Molecular basisof mechanosensory fransduction.Natwre 413(68 52):194-202.

Hoon, M. A., et al. 1,999.Putativemammalian tastereceptors:a class of taste-specificGPCRs with distinct topographic selectivity. Cell 96(41:541-551. Hudspeth,A. J. 2005. How the ear'sworks work: mechanoelectrical transductionand amplification by hair cells. ComptesRendus 5 5-162. Biol. 328(2\21. Lin, S. Y., and D. P. Corey.2005. TRP channelsin mechanosensation. Cur r. O p in. N euro bi o l. 15 (3):3 5 0-3 57 . Marin, E. C., et al. 2002. Representationof the glomerular olfactory map in the Drosophila brain. Cell 109(21243-255 ' McKemy, D. D,'W M. Neuhausser,and D. Julius. 2002. Identification of a cold receptorrevealsa generalrole for TRP channelsin n. Natur e 476252-5 8. thermosensatio Mombaerts, P. 1999. Molecular biology of odorant receptorsin vertebrates.Ann. Reu.Neurosci.22:487-509. Nelson, G., et al. 2001. Mammalian sweettastereceptors.Cel/ 106(3):381-390. Zhang, X, and S. Firestein.2002.The olfactory receptorgene superfamilyof the mouse.Nature Neurosci. 5(21:124-733. The Path to Success:Controlling Axon Growth and Targeting Chilton, J. K. 2005. Molecular mechanismsof axon guidance. Deu. Biol. 292(11:13-24. Dickson,B.J.and G.F.Gilestro.2005. Regulationof commissural axon pathfinding by slit and its robo receptors.Ann. Reu.Cell Deu. Biol.22:65L-75. Gallo, G., and P. C. Letourneau.2004. Regulationof growth cone actin filaments by guidancecues.J. Neurobiol. 58(I):92-102Gomez,T.,M. and J. Q. Zheng. 2006.The molecular basisfor calcium-dependentaxon pathfinding. Nature Reu.Neurosct.

7(21:1.1.5-r25. Hedgecock,E. M., J. G. Culotti, and D. H. Hall' 1990. The unc-5, unc-6, and unc-40 genesguide circumferentialmigrations of pioneeraxons and mesodermalcellson the epidermisin C. elegans. Neuron 4(1):61-85. Hilliard, M. A., and C.I. Bargmann.2006. I(nt signalsand frizzled activity orient anterior-posterior axon outgrowth in C. elegans. D eu. Cell 10(3\:379-390. Hindges,R, et al. 2002. EphB forward signalingcontrols directional branch extensionand arborization required for dorsal-ventral retinotopic mapping. Neuron 35(31:475487 . Jin, M., et al. 2005. Ca2*-dependentregulationof rho GTPases triggersturning of nervegrowth cones./. Neurosci.25(9):2338-2347. Kidd, T., K. S. Bland, and C. S. Goodman. 1999' Slit is the midline repeflent for the robo receptor in Drosophild- Cell 96(61:785-794. Kruger,R. P.,J. Aurandt, and K.-L. Guan. 2005. Semaphorins command cellsto move. Nature Reu.Mol. Cell Biol.6(10):789-800' Lemke, G., and M. Reber.2005. Retinotectal mapping: new insights from molecular genetics.Ann. Reu.Cell Deu' Biol. 21:551-580. Li, W., et al. 2004. Activation of FAK and Src are receptorproximal eventsrequiredfor netrin signaling.Nature Neurosci. 7 (11\:1213-7221. Marquardt, T., et al. 2005. CoexpressedEphA receptorsand ephrin-A ligandsmediateopposingactionson growth cone navigation from distinct membranedomains. Cell l2t(11:127-139. Mclaughlin, T., and D. D. O'Leary' 2005. Molecular gradientsand developmentof retinotopic maps.Ann. Reu.Newrosci.28327-355. Pan, C. L., et al. 2006. Multiple Wnts and frizzled receptors regulateanteriorly directedcell and growth cone migrations in -377 . Caenorhabditiselegans.D eu.Cell. lO(3)2367

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Schmitt,A.M., et al.2006.I7ncRyk signallingmediatesmediallateral retinotectal topographic mapping. Nature 439(70721:31-37. SerafiniT., et al. 1996. Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cel/ 87(6\:1.00L-1.014. Sperry,R. \/. 1963. Chemoaffinity in the orderly growth of nerve fiber patternsand connections.Proc. Nat'l Acad. Sci. IISA 5O:703-710. Tessier-Lavigne, M., et al. 1988. Chemotropicguidanceof developing axons in the mammalian central nervous system.Nature. 3366201.\2775-778.

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Walter,J., et al. 1987. Recognitionof position-specificproperties of tectal cell membranes by retinal axons in vitro. Deuel. 10l(4):685-696. 'Wen,Z., and J. Q. Zheng.2006. Directional guidance of nerve growth cones.Curr. Opin. Neurobiol. 16(1):52-58. Xie, Y., et al. 2005. Phosphatidylinositol transfer protein-alpha in netrin-1-induced PLC signalling and neurite outgrowth. Nature Cell BioL 7 (ll):1124-1132. 2o1,Y.2006. Navigating the anterior-posterioraxis with Wnts. Neuron 49:787-789.

CHAPTER

IMMUNOLOGY

Dendritic cellsin the skinhaveclassll MHC molecules on their Thoseshownherewereengineered surface. to express a classll green [Courtesy MHC-GFP fusionprotein,whichfluoresces M BoesandH L Ploeoh I

I mmunity is a stateof protection againstthe harmful effects I of exposureto pathogens.Host defensecan take many difI ferent forms, and all successfulpathogens have found ways to disarm the immune systemor manipulate it to their own advantage. Host-pathogen interactions are therefore an evolutionary work in progress.This explains why we continue to be assaultedby pathogenicviruses,bacteria,and parasites.The prevalenceof infectious diseasesillustratesthe imperfectionsof host defense.But killing its host is not necessarilyadvantageousto a pathogen becausecomplete elimination of the host would immediately remove the reservoir in which the pathogenreplicatesor survives.An immune system that could produce perfect sterilizing immunity would yield a world without pathogens,an outcome clearly at variance with life as we know it. Rather, the co-evolution of pathogensand their hosts allows pathogens,which have relatively short generationtimes, to continue to evolve sophisticated countermeasures,againstwhich the host must respond by adjusting,if not improving, its defenses.Sophisticateddefensecomesat a price: An immune systemcapableof dealing with a massively diverse collection of rapidly evolving pathogensmay mount an attack on the host organism'sown cells and tissues,a phenomenoncalled autoimmunity. In this chapter we deal mostly with the vertebrate immune system,with particular emphasison those molecules, cell types, and pathways that uniquely distinguish the immune system from other types of cell and tissues. Host defense comprises three layers: (1) mechanical/chemical defenses,(2) innate immunity, and (3) adaptive immunity (Figure 24-1). Mechanical and chemical defensesoperate continuously. Innate immune responses,which involve cells

and molecules present at all times, are rapidly activated (minutes to hours), but their ability to distinguish among many different pathogensis somewhat limited. In contrast, adaptive immune responsestake several days to develop fully and are highly specific;that is, they can distinguish between closelyrelated pathogensbasedon very small molecular differencesin structure. The manner in which antigens-any material that can evoke an immune response-are recognizedand how these and cell foreign materials are eliminated involve molecular'We begin biological principles unique to the immune system. this chapter with a brief sketch of the organization of the

OUTLINE 24.'l

Overview of Host Defenses

and Function Structure 24.2 lmmunoglobulins:

1057 1063

Generationof Antibody Diversity l evelopment a n d B - C e lD

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24.4

The MHC and Antigen Presentation

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24.5

T Cells,T-CellReceptors,and T-Cell DeveloPment

24.6

Cells Collaborationof lmmune-System in the Adaptive ResPonse

24.3

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Chemical defenses

M i n u t e st o h o u r s

Adaptive immunity

Innate immunity

lnnate immunity

Adaptive immunity

Adaptive immunity

A FIGURE 24-1 Thethree layersof vertebrateimmune defenses. Left;Mechanical defenses consist of epithelia andskin. Chemical defenses include the low pHof the gastric environment andantibacterial enzymes in tearfluid.Thesebarriers provide protection contrnuous against invadersPathogens mustphysically ([) to infectthe host Middle.Pathogens breachthesedefenses that havebreached (Z) are the mechanical andchemical defenses handled by cellsandmolecules (blue), of the innateimmunesystem w h i c hi n c l u d epsh a g o c y tci ce l l s( n e u t r o p h i dl se, n d r i t icce l l s , macrophages), naturalkiller(NK)cells,complement proretns, ano (lL-1,lL-6).Innatedefenses certainrnterleukins areactivated within

minutes to hoursof infection. Rrght:Pathogens thatarenotcleared bythe innateimmunesystem aredealtwith by theadaptive immune (B), in particular system B andT lymphocytes Fullactivation of adaptive immunity requires daysTheproducts of an innateresponse (4). Likewise, maypotentiate an ensuing adaptive response the products of an adaptive immuneresponse, including antibodies (Y-shaped icons), mayfacilitate functioning of the innateimmune (S) Several system products celltypesandsecreted straddle the fencebetween the innateandadaptive immunesystems, andserve to connectthesetwo layersof hostdefense

mammalian immune system,introducing the essentialplayers of innate and adaptive immunity and describing inflammation, a localized response ro injury or infection that leadsto the activation of immune-systemcellsand their recruitment to the affected site. In the next two secrions,we discuss the structure and function of antibody (or immunoglobulin) molecules,which bind to specificmolecular features on antigens, and how variability in antibody structure contributes to specific recognition of antigens. The enormous diversity of antigensthat can be recognized by the immune system finds its explanation in unique rearrangementsof the genetic material in B and T lymphocytes, commonly called B cells and T cells, which are the white blood cells that carry out anrigen-specificrecognition. These gene rearrangements not only control the specificity of antigen receptors on lymphocytes but also determine cell-fate decisionsin the course of lymphocyte development.

Although the mechanismsthat give rise to antigen-specific receptors on B and T cells are very similar, the manner in which these receptors recognize antigen is very different. The receptors on B cells can interact with intact antigens directly, but the receptors on T cells cannot. Instead, as describedin Section24.4,the receptorson T cells recognize cleaved (processed)forms of antigen, presentedon the surface of target cells by glycoproteins encoded by the major histocompatibility complex (MHC). How MHC-encoded glycoproteins display these processedantigens is important for our understanding of how immune responsesare initiated. MHC-encoded glycoproteins also help determine the developmentalfate of T cells so that an organism'sown cells and tissues(self antigens)normally do not evoke an immune response,whereasforeign antigensdo. We concludethe chapter with an integrated view of the immune responseto a pathogen, highlighting the collaboration between different immune-systemcellsthat is requiredfor an effectiveresponse.

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cHAprER 24 |

tMMUNolocy

Overviewof HostDefenses Becausethe immune systemevolved to deal with pathogens, we begin our overview of host defensesby examining where rypical pathogensare found and where they replicate.Then we introduce basic concepts of innate and adaptive immunity, including some of the key cellular and molecular players.

PathogensEnterthe Body Through Different Routesand Replicateat Different Sites Pathogensaffect alI life forms capable of independentreplication. Two general classesof pathogen, viruses and bacteria, have fundamentally different modes of propagation. 'S(ith the exception of polymerases involved in copying geneticmaterial, virusesgenerallylack the necessarymachinery to synthesizetheir component parts and therefore are completely dependenton host cells for their propagation. In contrast, most bacteria are metabolically autonomous and do not rely on their host for replication, which allows them to be grown in the laboratory in the appropriate culture media. (An exception is those bacteriathat can replicateonly within a mammalian host cell.) Bacteria can cause disease becausethey possessvirulence factors that act on the host's metabolism and physiology. Parasitic organisms also can causedisease.With increasinglycomplex life cyclessuch as those of the protozoa that cause sleeping sickness (trypanosomes)or malaria (Plasmodiumspecies)(seeFigure 1-4), the pathogen'scountermeasuresalso become increasingly complex. Bacteria,protozoa, and fungi-especially thosethat can causediseasein animals-often are called microbes. Exposure to pathogensoccurs via different routes. The skin itself has a surface areaof =20 sq. ft.; the epithelial surfacesthat line the airways, gastrointestinaltract, and genital tract present an evenmore formidablesurfaceareaof =4000 sq. ft. All these surfacesare continuously exposedto viruses and bacteria in the environment. Foodborne pathogens and sexually transmitted agentstarget the epithelia to which they are exposed. The sneezeof a flu-infected individual releasesmillions of virus particles in aerosolizedform, ready for inhalation by the next person to be infected. Rupture of the skin, even if only by minor abrasions, or of the epithelial barriers that protect the underlying tissues,provides an easy route of entry for pathogens,which then gain accessto a rich source of nutrients (for bacteria) and to the cells required for their replication(viruses). Replicationof virusesis strictly confined to the cytoplasm or nucleusof host cells, where protein synthesisand replication of the viral geneticmaterial occur.Virusesspreadto other cells either as free virus particles (virions) or by cell-to-cell spread.Many bacteriacan replicatein the intercellularspace, but someare specializedto invadehost cellsand survivethere. Such intracellular bacteria reside either in membranedelimited vesiclesthrough which they enter cells by endocytosis or phagocytosisor in the cpoplasm if they escapefrom these vesicles.An effective host defensesystem, therefore, needsto be capableof eliminatingnot only cell-freevirusesand free-living bacteria but also cells that harbor thesepathogens.

LeukocytesCirculateThroughoutthe Body and TakeUp Residencein Tissuesand Lymph Nodes 'With

the exception of erythrocytes,few cells in the courseof their assignedfunction cover such distancesas do the cells that provide immunity. The mammalian circulation servesas the necessarytransport vehicle for erythrocytes,Ieukocytes, and platelets.Although erythrocytesnever leave the circulation (their oxygen-carrying function does not require it), leukocytes(white blood cells) use the circulation exclusively for transport and may leave and re-enter the circulation in the course of their tasks. The immune system is an interconnectedsystem of vessels,organs, and cells, divided into primary and secondary lymphoid structures(Figure 24-21.Primary lymphoid organs -the sites at which lymphocytes (the subset of leukocytes that includes B and T cells) are generatedand acquire their Lymph nodes (filteringof lymphand maturationof white blood cells) Thoracic duct (discharges lymph into blood)

Lymphvessels (conveylymph) Thymus (T-cell maturation) Spleen (lymphocyte maturation and filtering of lymph)

Bone marrow (B-cell development, T-cell precursors)

24-2 The circulatoryand lymphaticsystems.Positive A FfGURE for lossof exertedbythe pumpingheartisresponsible arterialpressure (red)intothe interstitial spaces of thetissues, liouidfromthecirculation of andcandispose to nutrients sothat allcellsof the bodyhaveaccess threetimesthat fluid,whosevolumeisroughly waste.Thisinterstitial in theform to thecirculation isreturned of allbloodin thecirculation, structures anatomical throughspecialized of lymph,whichpasses wherelymphocytes organs, calledlymphnodes.Theprimarylymphoid (B precursors) andthe T-cell cells, marrow generated, the bone are are involves the of an immuneresponse thymus(Tcells)Theinitiation (lymphnodes, spleen) organs lymphoid secondary O V E R V I E WO F H O S TD E F E N S E S

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functional properties-include the thymus, where T cells are generated,and the bone marrow, where B cellsare generated. Adaptive immune responses,which require functionally competent lymphocytes are initiated in secondary lymphoid orgaas including lymph nodes and the spleen.All of the lymphoid organs are populated by cells of hematopoieticorigin (seeFigure 21-15), generatedin the fetal liver and throughout life in the bone marrow. The total number of lymphocytes in a young adult male is estimatedto be 500 x 10v, roughly 15 percent of which are found in the spleen,40 percent in the other secondarylymphoid organs (tonsils,lymph nodes), 10 percent in the thymus, and 10 percenr in the bone marrow; the remainder are circulating in the bloodstream. Leukocytesmust leavethe bloodstreamand enrertissuesto perform their functions.Vertebrateblood vesselsallow the escape of fluid from the circulation, driven by the positive arrerial pressureexertedby the pumping heart.This fluid contains not only nutrients but also proteins that carry out defensive functions. To maintain homeostasis,the fluid that leavesthe circulation must ultimately return and does so in the form of lymph, via lymphatic vessels.The total volume of lymph is up

to three times the total blood volume. At their most distal ends, Iymphatic vesselsare open to collect the interstitial fluid that bathes the cells in tissues.The lymphatic vesselsmerge into larger collecting vessels,which deliver lymph to lymph nodes. A lymph node consistsof a capsule,organized into areas that are defined by the cell types that inhabit them. Blood vessels entering a lymph node deliver B and T cells to it. The lymph that arrives in a lymph node carries cellsthat have encountered ("sampled") antigen,as well as soluble antigens,from the tissue drained by that particular afferent lymphatic vessel.In the lymph node, the cells and moleculesrequired for the adaptive immune responseinteract, respond to the newly acquired antigenic information, and then execute the necessaryeffector functionsto rid the body of the pathogen(Figure24-3). Lymph nodes can be thought of as filters in which antigenic information gathered from distal sitesthroughout the body is collected and displayed to the immune system in a form suitable to evoke an appropriate response.All the relevant steps that lead to lymphocyte activation take place in Iymphoid organs.Cells that have receivedproper insrructions to becomefunctionally active leavethe lymph node via efferent

Antigen-laden d e n d r i t i cc e l l I

B c e l l b i n d ss o l u b l ea n t i g e n and movesto follicle

B - c e l fl o l l i c l e s Afferent lymphatic vessel

E

M a t u r e Ta n d B c e l l s are deliveredvia the c i r c u l a t i o na n d t a k e u o r e s i d e n c ei n lymphnodes

!

Activationof T cell by antigenladen,activated d e n d r i t i cc e l l ; activatedTcells may re-enter circulation

Blood vessels @ Efferent lymphatic vessel FIGURE24-3 Initiation of the adaptive immune responsein lymph nodes. Recognition of antigenby B and T cells(lymphocytes) locatedin lymph nodesinitiatesan adaptiveimmuneresponse Lymphocytes leavethe circulationand take up residence in lymph nodes(Il) Lymphcarriesantigenin two forms-soluble antigenand antigen-ladendendriticcells;both are deliveredto lvmph nodesvia a f f e r e n tl y m p h a t i c(s4 , B ) . S o l u b l ea n t i g e ni s r e c o g n i z e b dy B c e l l s

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IMMUNOLOGY

ActivatedTcells interact w i t h B c e l l s ,l e a d i n gt o B-celldifferentiationand antibody production

( 4 ) , a n da n t i g e n - l a d de en n d r i t icce l l sp r e s e natn t i g e n to T cells (E) Productive interactions betweenT andB cells(El) allowB cells to moveintofollicles anddifferentiate intoplasma cells,which p r o d u c lea r g ea m o u n tosf s e c r e t ei d m m u n o g l o b u l(i a nn s tibodies) Efferent lymphatic vessels returnlymphfromthe lymphnodeto the circulation

lymphatic vesselsthat ultimately drain into the circulation. Such activated cells recirculate through the bloodstream and-now ready for action-may reach a location where they again leave the circulation, move into tissues,and seek out pathogenicinvadersor destroy virus-infectedcells. The exit of lymphocytes and other leukocytes from the circulation, recruitment of these cells to sites of infection, processingof antigenic information, and return of immunesystem cells to the circulation are all carefully regulated processes that involve specificcell-adhesionevents,chemotactic cues,and the traversalof endothelialbarriers,as we discuss Iater.

M e c h a n i c aal n d C h e m i c aB l o u n d a r i e sF o r m a FirstLayerof DefenseAgainst Pathogens As noted already,mechanicaland chemicaldefensesform the first line of host defenseagainstpathogens(seeFigure 24-1). Mechanical defensesinclude the skin, epithelia, and arthropod exoskeleton,which are barriers that can be breachedonly by mechanicaldamage or through specificchemo-enzymatic attack. Chemicaldefensesinclude not only the low pH found in gastric secretionsbut also enzymessuch as lysozyme, found in tear fluid, which can attack microbes directly. The importance of mechanical defenses,which operate continuously,are immediatelyobvious in the caseof burn victims: When the integrity of the epidermisand dermis is compromised,the rich sourceof nutrients in the underlyingtissues is exposed,and airborne bacteria or otherwise harmlessbacteria found on the skin can multiply unchecked,ultimately overwhelming the host. Viruses and bacteria have also evolved strategiesto breach the integrity of these physical barriers. Enveloped viruses such as HIV, rabies virus, and influenza virus possessmembrane proteins endowed with fusogenicproperties.Following adhesionof a virion to the surface of the cell to be infected, direct fusion of the viral envelope with the host cell's membrane results in delivery of the viral genetic material into the host cytoplasm, where it is now available for transcription, translation, and replication (see Figures4-47 and 4-49). Certain pathogenicbacteria (e.9., S. aureus)secretecollagenasesthat compromisethe integrity of connectivetissueand so facilitate entry of the bacteria.

I n n a t el m m u n i t y P r o v i d e sa S e c o n dL i n e of DefenseAfter Mechanicaland Chemical BarriersAre Crossed The innate immune system is activated once the mechanical and chemical defenseshave failed, and the presenceof an invader is sensed(seeFigure24-1,).Theinnate immune system comprises cells and molecules that are immediately available for responding to pathogens.Phagorytes,cells that ingest and destroy pathogens, are widespread throughout tissues and epithelia and can be recruited to sitesof infection. Severalsoluble proteins presentconstitutively in the blood, or produced in responseto infection or inflammation, also contribute to innate defense.Animals that lack an adaptiveimmune system,suchas insects.rely exclusivelvon innate defensesto combat infections.

Phagocytes and Antigen'Presenting Cells The innate immune systemincludesmacrophages'neutrophils, and dendritic cells. All of these cells are phagocytic and come equipped with Toll-like receptors (TLRs). Members of this family of cell-surface proteins detect broad patterns of pathogen-specificmarkers and thus are key sensorsfor detecting the presenceof viral or bacterial invaders. Engagement of Toll-like receptors is important in eliciting effector molecules,including antimicrobial peptides. Dendritic cells and macrophageswhose Toll-like receptors have detected pathogens also function as antigen-presentingcells (APCs) by displaying processedforeign materials to antigen-specific T cells.The structure and function of Toll-like receptorsand their role in activating dendritic cells are describedin detail in Section24.6. Complement System Another important component of the innate immune system is complement, a collection of constitutive serum proteins that can bind directly to microbial or fungal surfaces.This binding activatesa proteolytic cascadethat culminates in formation of pore-forming proteins constituting the membrane attack complex, which is capable of permeabilizing the pathogen's protective membrane (Figure 24-4). The complement cascadeis conceptually similar to the blood-clotting cascade,with amplification of the reaction at each successivestageof activation' At least three distinct pathways can activate complement. The classical pathluay requiresthe presenceof antibodies produced in the course of an adaptive responseand bound to the surface of the microbe. Many microbial surfacesdirectly activate complement via the abernatiuepathway. Finally, pathogensthat contain mannose-rich cell walls activate complement through the mannose-binding lectin pathway. The bound lectin then triggers activation of two mannose-binding lectin-associatedproteases,MASP-1 and MASP-2, which allow activation of the downstream componentsof the complementcascade. In the course of complement activation, the C3 and C4 complement proteins occupy a specialrole. These abundant serum proteins are synthesizedas precursorsthat contain an internal, strained thioester linkage between a cysteineand a glutamate residue in close proximity' This thioester linkage becomeshighly reactive upon proteolytic activation of C3 and C4 by their respectiveupstream partners. The activated thioester bond can react with primary amines or hydroxyls in close proximitS yielding a covalent bond linking C3 or C4 with a protein or carbohydrate close-by.If no such reactants are available, the thioester bond is simply hydrolyzed. This mode of action ensuresthat C3 and C4 fragments will be covalently depositedonly on antigen-antibody complexes in close proximity. Regardlessof the pathway of complement activation engaged,activated C3 unleashesthe terminal components of the complement cascade,C5 through C9, culminating in formation of the membrane attack complex, which inserts itself into most biological membranes and renders them small permeable. The resulting loss of electrolytes and 'Whenever cell. solutes leads to lysis and death of the target O V E R V I EO WF H O S TD E F E N S E S.

1059

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complement is activated, the membrane attack complex is formed and results in death of the cell onto which the complex is deposited. The direct microbicidal effect of a fully activated complement cascadeis an important protective function. All three complement activation pathways also generate the C3a and 5a cleavagefragmenrs,which bind to G protein-coupled receptorsand function as chemoattractants for neutrophils and other cells involved in inflammation (see below). All three pathways also result in the covalent decoration of the structures targeted by complement activation with fragments of C3. Phagocytic cells make use of these C3-derived tags to recognize,ingest, and destroy the decorated particles,a processtermed opsonization.

MASP2

Natural Killer (NK) Cells In addition to bacterialinvaders, the innate immune system also defends against viruses. \(hen the presenceof a virus-infected cell is detected, yet other cell types of the innate immune system becomeactive, seek out the virus-infected targets, and kill them. For instance,many virus-infected cells produce type I interferons, which are good at activating natural killer (NK) cells. Acti, vated NK cells not only afford direct protection by eliminating the factory of new virus particles, but they also secreteinterferon "y (IFN-1), which is essentialfor orchestrating many aspectsof anti-viral defenses(Figure 24-5). Recognition by NK cellsinvolves severalclassesof receptors, capable of delivering either stimulatory (promoting cell killing) or inhibitory signals. The interferons are classified as cytokines, small, secretedproteins that help regulate im'We mune responsesin a variety of ways. will encounter

t*

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Surfaceof targetcell (pathogen or antibodydecorated hostcell) FIGURE 24-4 Threepathwaysof complementactivation. Theclassical pathwayinvolves theformation of antibody-antigen complexes, whichrecruit thecomplement component C1q,leading to activation of C1r andC1s.Thiscomplex, in turnactivates C4andC2, whichthenconvert C3to itsactive form Inthe mannose-binding lectinpathway, mannose-rich structures foundon thesurface of many pathogens arerecognized by mannose-binding lectin, an interaction that results in activation of two serineproteases, MASP-1 andMASp-2 Thealternative pathway requires deposition of a special formof the serumproteinC3,a majorcomplement component, ontoa microbial surfaceSubsequent activation of C3 involves factorsB,D andB foundin serum.Eachof theactivation pathways isorganized asa cascade of proteases in whichthedownstream component isitselfa protease Amplification of activity occurs with eachsuccessive step All threepathways converge on C3,whichtriggers formation of the membrane attackcomplex, leading to destruction of targetcellsThe smallfragments of C3andC5 generated in thecourse of complement activation attractneutrophils, phagocytic cellsthatcankillbacteria at shortrangeor uponingestion 1060

.

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@ Killing (perforin/ granzyme)

FIGURE 24-5 Naturalkillercells.Natural killer(NK)cellsare an important source of the cytokine interferon f (lFN-r)andcankill virus-infected andcancerous cellsby meansof perforins. Theseporeformingproteins allowaccess of serineproteases calledgranzymes to thecytoplasm of the cellaboutto be killed.Granzymes alsocan inrtiate (Chapter apoptosis throughactrvation of caspases 21)

Bacterium

other cytokines and discusssome of their receptorsas the chapterprogresses.

I n f l a m m a t i o nl s a C o m p l e xR e s p o n s teo I n j u r y Both Innate and Adaptive That Encompasses lmmunity

Dendriticcell

\Vhen a vascularizedtissueis injured, the stereotypicalresponse that follows is inflammation. Damage may be a simple paper cut or result from infection with a pathogen. Inflammation, or the inflammatory response,is characterized 6y four classical signs:redness,swelling, heat, and pain. These signs are caused by increasedleakinessof blood vessels(vasodilation),the attraction of cells to the site of damage, and the production of soluble mediators responsiblefor the sensationof heat and pain. Inflammation has immediate protective value through the activation of the cell types and soluble products that together mount the innate immune response.Further, inflammation createsa local environment conduciveto the initiation of the adaptive immune response.However, if not properly controlled, inflammation can also be a major causeof tissuedamage. Figure24-6 depictsthe key playersin the inflammatory responseto bacterialpathogensand the subsequentinitiation of an adaptive immune response.Tissue-residentdendritic cells sensethe presenceof pathogensvia their Toll-like receptors (TLRs) and respond to them by releasingsoluble mediators such as cytokines and chemokines;the latter act as chemoattractants for immune-system cells. Neutrophils, a second important cell type in the inflammatory response,leave the circulation and migrate to wherevertissueinjury or infection has occurred in responseto various soluble mediators produced upon tissue damage. Neutrophils, which constitute almost half of all circulating leukocytes,are phagocytic, directly ingestingand destroyingpathogenicbacteria.They also can interact with a wide variety of pathogen-derivedmacromolecules via their Toll-like receptors. Activation of these receptors allows neutrophils to produce cytokines and chemokines; the latter can attract more leukocytesneutrophils,macrophages,and ultimately lymphocytes(T and B cells)-to the area.Activatedneutrophilscan releasebacteriadestroyingenzymes(e.g.,lysozymeand proteases)as well as small peptides with microbicidal activity, collectively called defensins.Activated neutrophilsalso turn on the enzymesthat generatesuperoxideanion and other reactiveoxygen species (seeChapter 12, p. 502), which can kill microbes at short range.Another cell type contributing to the inflammatory reent mast cells.When activatedby a varisponseis tissue-resid ety of physical or chemical stimuli, mast cells releasehistamine, a mediator that increasesvascular permeability and thereby facilitatesaccessto the site of plasma proteins (e.g., complement)that can act againstthe invading pathogen. A very important early responseto infection or injury is activation of a variety of plasma proteases,including the proteins of the complement cascadediscussedabove (seeFigure 24-4l.The peptidesproduced during activation of theseproteasespossesschemoattractant activity, responsible for attracting neutrophils to the site of tissuedamage.They further induce production of proinflammatory cytokines such as

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24-6 Interplayof innateand adaptiveimmune FIGURE breaches againsta bacterialpathogen.Oncea bacterium responses is exposed the bacterium defenses, andchemical thehostsmechanical aswellasto cellsthat cascade, of thecomplement to components (E) Various protection, suchasneutrophils conferimmediate to a contribute induced by tissuedamage mediators inflammatory of the bacterium Localdestruction response. inflammatory localized viathe whicharedelivered antigens, of bacterial in the release results cells lymphnode(E). Dendritic to thedraining lymphatics afferent in response to becomemigratory antigenat thesiteof infection, acquire products, andmoveto the lymphnode,wheretheyactivate microbial and T cellsproliferate T cells(B) Inthelymphnode,antigen-stimulated (4), cells help B to the ability including functions, effector acquire their someof whichmaymoveto the bonemarrowandcomplete of the immune intoplasma cellsthere(E). In laterstages differentiation to anttgenassistance additional T cellsprovrde activated response, antigen-specific plasma secrete yield that cells B cells to experienced produced asa at a highrate(step6) Antibodies antibodies with actin synergy to bacteria of the initialexposure consequence (Z), shouldit persist, or afford the infection to eliminate complement to thesamepathogen in thecaseof re-exposure rapidprotection interleukin 1. and 6 (IL-1 and IL-6). The recruitment of neutrophils also dependson an increasein vascularpermeability, controlled in part by lipid mediators (e.g.,prostaglandinsand leukotrienes)that are derived from phospholipids and fatty O F H O S TD E F E N S E S . OVERVIEW

1061

acids. All of theseeventsoccur rapidl5 starting within minutes of injury. A failure to resolvethe causeof this immediate responsemay result in chronic inflammation, in which cells of the adaptive immune systemplay an important role. 'When the pathogen burden at the site of tissuedamageis high, it may exceed the capacity of innate defensemechanisms to deal with them. Moreover, some pathogens have acquired,in the courseof evolution, tools to disableor bypass innate immune defenses.In such situations, the adaptive immune responseis required to control the infection. This adaptive responsedependson specializedcells that straddle the interface between adaptive and innate immunity, including antigen-presentingcells such as macrophagesand dendritic cells,which are capableof acquiring intact pathogensand of killing them upon ingestion.Theseantigen-presenting cells,in particular dendritic cells, can initiate an adaprive immune responseby deliveringnewly acquiredpathogen-derivedantigensto secondarylymphoid organs (seeFigure 24-6).

Adaptive lmmunity,the Third Line of Defense, ExhibitsSpecificity Lymphocytes bearing antigen-specificreceptors are the key cells responsiblefor adaptive immuniry. An early indication of the specificnature of adaptiveresponses camewith the discovery of antibodies, key effector moleculesof adaptive immuniry, by Emil von Behringand ShibasaburoKitasato in 1905. They observedthat when serum (the straw colored liquid that separates from cellular debris upon completion of the blood clotting process) from guinea pigs immunized with a sublethal dose of the deadly diphtheria toxin was transferred to animals never before exposed to the bacterium, the recipient animals were protected against a lethal dose of the same bacterium, which kills its host by production of a toxin (Figure 24-7). Transfer of serum from animals never exposedto diphtheria toxin failed to protect, and protection was limited to the microbe used as the sourceof toxin with which the animal that

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EXPERIMENTAL FTGURE 24-7 The existenceof antibody in serumfrom infectedanimalswas demonstratedby von Behringand Kitasato.Exposure of animals to a sublethal dose of diphtheria toxin(orthe bacteria thatproduce it)elicitsin their seruma substance that protects against a subsequent challenge with a lethaldoseof thetoxin(orthe bacteria thatproduce it) The protective effectof thisserumsubstance canbe transferred from an animalthat has beenexposedto the pathogento a naive (nonexposed) animal.When the serumrecipientis subsequently

1062

CHAPTER 24

I

IMMUNOLOGY

exposed to a lethaldoseof the bacteria, theanimalsurvives. This effectisspecific for the pathogen usedto elicitthe response. Serum (antibody) thuscontains a transferable substance thatprotects against the harmfuleffects pathogenSerumharvested of a virulent fromtheseanimals, saidto be immune, displays bactericidal activity rnvitro.Heating of immuneserumdestroys itsbactericidal actrvrty Additionof freshnonheated serumfroma naiveanimalrestores the bactericidal activity of heatedimmuneserum.Serumthuscontains anothersubstance thatcomplements the activitv of antibodies.

servedas the serum donor was immunized. This experiment demonstratesspecificity-that is, the ability to distinguish between two closely related substancesof the same class. Such specificity is a hallmark of the adaptive immune system. Even proteins that differ by a single amino acid may be distinguishedby immunological means. From these experiments, von Behring inferred the existenceof corpuscles( "Antikorper" ), or antibodies,as the factor responsiblefor protection. The antibody-containing (immune) sera not only afforded protection in vivo, they also killed microbesin the test tube. Heating the immune serato 56" C destroyed this killing activity, but it was restored by the addition of unheatedfresh serumfrom naive animals (i.e.,animals never exposed to the microbe). This finding suggestedthat a second factor, now called complement, acts in synergy with antibodies to kill bacteria. We now know that von Behring's antibodies are serum proteins referred to as immunoglobulins and that complementis actually a seriesof proteases(seeFigure 24-4l.Immunoglobulins can neutralizenot only bacterial toxins, but also harmful agents such as viruses, by directly binding to them in a manner that prevents the virus from attaching itself to host cells.In the samevein, antibodiesraised against snake venoms can be administeredto the victims of snakebites to protect them from intoxication: The anti-snake venom antibodies bind to the venom, keep it from binding to its targets in the host, and in so doing neutralize it. Antibodies can thus have immediate orotective effects.

Structure lmmunoglobulins: and Function Immunoglobulins, produced by B cells,are the best-understood molecules that confer adaptive immunity. In this section we describe the overall structural organization of immunoglobulins, their structural diversiry and how they bind to antigens.

l m m u n o g l o b u l i n sH a v ea C o n s e r v e dS t r u c t u r e Consistingof Heavyand Light Chains Like complement, immunoglobulins are abundant serum proteins that can be classifiedin terms of their structural and functional properties. Fractionation of antisera, basedon their functional activity (e.g.,killing of microbes,binding of antigen), led to the identification of the immunoglobulins as the classof serum proteins responsiblefor antibody activity. Immunoglobulins are composed of two identical heauy (H) chains, covalently attached to two identical light (L) chains (Figute 24-8). Structure of an antibodY molecule

L i g h tc h a i n

Light chain

Disulfidebonds Heavychain

Overview of Host Defenses r Mechanical and chemical defensesprovide protection against most pathogens.This protection is immediate and little specificity.Innate and adaptive continuous, yet possesses immunity provide defensesagainstpathogensthat breach the boundaries(seeFigure24-1). body'smechanicaVchemical

vy chain

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F(ab): monovalent

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r The circulatory and lymphatic systems distribute the molecular and cellular playersin innate and adaptiveimmunity throughout the body (seeFigure24-2). r Innate immunity is mediated by the complement system (seeFigure 24-4) and severaltypes of leukocytes,the most important of which are neutrophils and other phagocytic cells such as macrophagesand dendritic cells.The cells and molecules of innate immunity are deployed rapidly (minutes to hours). Molecular patterns diagnostic of the presenceof pathogenscan be recognizedby Toll-like receptors' but the specificity of recognition is modest. r Adaptive immunity is mediated by T and B lymphocytes. Thesecells require days for full activation and deployment, but they can distinguish between closely related antigens. This specificity of antigen recognition is the key distinguishingfeatureof adaptiveimmunity. r Innate and adaptive immunity act in a mutually synergistic fashion. Inflammation, an early responseto tissueinjury or infection, involves a seriesof eventsthat combines elements of innate and adaptive immunity (seeFigure 24-6).

Fc

24-8 The basicstructureof an immunoglobulin A FIGURE alsoknownas areserumproteins molecule,Antibodies structures Theyaretwo-foldsymmetrical immunoglobulins light heavychainsandtwo identical of two identical composed yields fragments with proteases of antibodies Fragmentation chains. papainyields Theprotease capacity. thatretainantigen-binding yieldsbivalent pepsin protease the and fragments, F(ab) monovalent butthis TheFcfragmentis unableto bindantigen, fragments. F(ab')z properties hasotherfunctional portionof the intactmolecule S:T R U C T U RAEN D F U N C T I O N IMMUNOGLOBULINS

1063

PentamericlgM is stabilizedby an additional polypeptide,the J chain

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J chain

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< FIGURE 24-9 lmmunoglobulin isotypes. Thedifferent classes of immunoglobulins, called isotypes, maybedistinguished biochemically andby immunological techniques In mouse andhumans therearetwo light-chain isotypes (r
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The typical immunoglobulin therefore has a two-fold sym, metrical structure, described asH2L2. An exception to this basic H2L2 architecture occurs in the camelids (camels.llamas, vicunas).Theseanimalscan make some immunoglobulins that are heavy-chain dimers (H2) and lack light chains. 1064

.

C H A P T E2R4

I

IMMUNoLoGY

A biochemicalapproach was usedto answer the question of how antibodies manage to distinguish between related antigens. Proteolytic enzymes were used to fragment immunoglobulins, which are rather large proteins, to identify the regions directly involved in antigen binding (seeFigure 24-8). The protease papain yields monovalent fragments,

calledP(ab), that can bind a singleantigenmolecule,whereas the proteasepepsin yields bivalent fragments,referred to as F(ab')2 (F : fragment; af : antibody). These enzymesare commonly used to convert intact immunoglobulin molecules into monovalent or bivalent reagents.Although F(ab) fragments are incapableof cross-linking,F(ab')2 fragmentscan do so, a property frequently usedto cross-linkand so activate surface receptors.The portion releasedupon papain digestion and incapableof antigen binding is called Fc, becauseof its easeof crystallization (F : fragment; c : crystallizable). This biochemical approach using proteaseswas followed by peptide mapping and sequencingstrategiesto determine the primary structure of the immunoglobulins.

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M u l t i p l e l m m u n o g l o b u l i nl s o t y p e sE x i s t , Eachwith Different Functions Basedon their distinct biochemicalproperties,immunoglobulins are divided into different classes,or isotypes.There are two light-chain isotypes, r and tr. The heavy chains show more variation:In mammals,the major heavy-chainisotypes are p, E, 1, c, and e. Theseheavychainscan associatewith either r or \ light chains. Depending on the vertebrate species,further subdivisions occur for the ct and 1 chains, and fish possessan isotype not found in mammals. The fully assembledimmunoglobulin (Ig) derives its name from the heavy chain: p chains yield IgM; cr chains, IgA; r chains, IgG, E chains,IgD; and e chains,IgE. The generalstructures of the major Ig isotypes are depicted in Figure 24-9. By means of their unique structural features,each of the different Ig isotypescarriesout specializedfunctions. The IgM moleculeis secretedas a pentamer,stabilizedby disulfide bonds and an additional chain, the J chain. In its 10 identicalantigen-binding pentamericform, IgM possesses sites, which allow high-avidity interactionswith surfaces that displaythe corresponding(cognate)antigen.Upon deposition of IgM onto a surface that carries the antigen, the pentamericIgM molecule assumesa conformation highly conduciveto activationof the complementcascade,an effective means of damagingthe membraneonto which IgM is adsorbed and onto which complement proteins are depositedas a consequence. The IgA moleculealso interactswith the J chain, forming a dimeric structure.Dimeric IgA can bind to the polymeric IgA receptoron the basolateralside of epithelialcells, endocywhere its engagementresultsin receptor-mediated dimeric is and IgA receptor cleaved the tosis. Subsequently, IgA with the proteolytic receptorfragment (secretorypiece) still attachedis releasedfrom the apicalsideof the epithelial cell. This process,called transcytosis,is an effective means of deliveringimmunoglobulinsfrom the basolateralside of an epitheliumto the apical side (Figure24-1'0a\.Tear fluid and other secretionsare rich in IgA and so provide protection againstenvironmentalpathogens. The IgG isotype is important for neutralization of virus particles.This isotype also helps prepareparticulate antigens for acquisition by cells equipped with receptorsspecific for the Fc portion of IgG molecules(seebelow).

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D i m e r i cl g A b i n d st o polymeric lg Receptor( p l s R )

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of l9A and lgc. (a)lgA,foundin 24-10Transcytosis FIGURE the across transport requires mucosae, the different of secretions andisendocytosed. l9Areceptor epitheliumlgAbindsto the polymeric a portionof the monolayer, theepithelial across Afterbeingtransported atthe apicalsidetogether andthe lgAisreleased iscleaved, receptor rodents piece.(b)Suckling thesecretory with a portionof the receptor, apical possesses at the newborn milk The mother's lg from acquire (FcRn), Fcreceptor the neonatal epithelium of itsintestinal surface (seeFigure I MHCmolecules thatof class resembles whosestructure bindsto the Fcportionof lgG,transcytosis 24-21)Aflerthisreceptor sideof theepitheliumIn thebasolateral lgG to movestheacquired FcRn andso expresses in the placenta trophoblast thesyncytial humans, anddelivery circulation of lgGfromthe maternal acquisition mediates transport) to thefetus(transplacental The immune systemof the newborn is immature, and in rodentsprotectiveantibodiesare transferredfrom the mother to the fetus via the mother's milk. The receptor responsible for caplrring maternal IgG is the neonatal Fc receptor,which ' I M M U N O G L O B U L I NSST: R U C T U RAEN D F U N C T I O N

1065

is presenton intestinal epithelial cells in rodents. By transcytosis, IgG captured on the luminal side of the newborn's intestinal tract is delivered across the gut epithelium and so makesmaternal antibodiesin the milk availablefor passive protection of the infant rodent (Figure 24-l0b).In humans, the Fc receptor is found on fetal cells that contact the maternal circulation. Transcytosisof IgG antibodies from the maternal circulation across the placenta delivers maternal antibodiesto the fetus.Thesematernal antibodieswill protect the newborn until its own immune systemis sufficiently mature to produceantibodiesunder its own steam.

their discoverers,are readily purified and afforded the first target for a protein chemical analysis. Two key observationsemergedform this work: (1) no two tumors produced light chains of the identical biochemical properties,suggestingthat they were all unique in sequence and (2) the differencesin amino acid sequencethat distinguish one light chain from anotherare nor randomly distributedbut occur clusteredin a domain referred to as rhe uariable region of the light chain, or V1. This domain comprisesthe N-terminal

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E a c hB C e l lP r o d u c e a s U n i q u e ,C l o n a l l y D i s t r i b u t e dl m m u n o g l o b u l i n The clonal selectiontheory stipulatesthat each lymphocyte carriesan antigen-bindingreceptorof unique specificity.\When a lymphocyte encountersthe antigen for which it is specific, clonal expansion (or multiplication) occurs and so allows an amplification of the response,culminating in clearing of the antigen(Figure24-Il). B-cell tumors, which representmalignant clonal expansions of individual lymphocytes, enabled the first molecular analysisof the processesrhat underlie the generationof antibody diversity.A key observationwas thar tumors derived from lymphocyres may produce large quantities of secreted immunoglobulins. Some of the light chains of the immunoglobulins are secretedin the urine of tumor-bearing patients.Theselight chains, calledBence-lonesDroteins after

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A FfGURE 24-12 Hypervariable regionsand the immunoglobulin fold. (a)Varration in aminoacidvariability position with residue in lg lightchainsThepercentage of variable-region sequences with variantaminoacidsisplottedfor eachposition in thesequence P o s i t i o nf osr w h i c hm a n yd i f f e r e natm i n oa c i ds i d ec n a t n as r e p r e s e ni n t t h e d a t as e ta r ea s s i g n eadh i g hv a r i a b i l iitnyd e xt;h o s e t h a ta r ei n v a r i a natm o n gt h es e q u e n c ce os m p a r eadr ea s s i g n ead valueof 0 Thisanalysis reveals threeregions of increased variability, h y p e r v a r i a b i(lH i t yV )r e g i o n1s , 2 , a n d3 ; t h e s ea r ea l s oc a l l e d complementarity-determining (CDRs) (b)Volume-rendered regions depiction of F(ab')z fragment(ight) andribbondiagram of a typical19 light-chain variable domain(Vr)withthe positions of the hypervariable regions indicated in red(left)Ihe hypervariable regions arefoundin the loopsthatconnect theB strands andmakecontact withantigenTheB (rendered strands asarrows) makeup two B sheets andconstitute the framework regionNotethateachvanable andconstant domainhasa characteristic three-dimensional structure, called theimmunoglobulin fold L : lightchain;H : heavy chain; Vs : heavy-charn variable domain; VL: light-chain variable domain; CH1 , CH2,Cs3: heavychainconstant domains; CL: ljght-chain constant domain

ftPP?fiR?PgHR A FIGURE24-11 Clonal selection.The clonalselectiontheory proposesthe existence of a largeset of lymphocytes, eachequipped with its own uniqueantigen-specific receptor(indicatedby different colors)The antigenthat showsa fit with the receptorcarriedby a particularlymphocyteallowsthat lymphocyteto expandclonally Froma modestnumberof antigen-specific cells,a largenumberof cellsof the desiredspecificity (and largeamountsof their secreted outputs)may be generated

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110 amino acidsor so. The remainderof the sequenceis identical for the different light chains (provided they derive from the identical isotype, rcor ),) and is therefore referred to as the constant region, or Cr. From the serum of tumor-bearingindividuals, immunoglobulins unique to that individual patient subsequentlywere purified. Sequencingof the heavy chains from these preparations revealed that the variable residues that distinguishone heavychain from anotherwere againconcentrated in a well-demarcated domain, referred to as the uariable region of the heauy chain, or Vs. An alignment of sequencesobtained from different homogeneouslight-chain preparations showed a non-random pattern of regions of variability, revealing three hyperuari' able regions-Hv1, HVz, and HV3-which are sandwiched betweenwhat are called framework regions (Figure 24-12a). (Similar alignments for the immunoglobulin heavy-chain sequencesalso yield hypervariable regions.) In the properly folded three-dimensionalstructure of immunoglobulins, these hypervariable regions are in close proximity (Figure 24-12b) and make contact with antigen. Thus that portion of an Ig moleculecontainingthe hypervariableregionsconstitutes the antigen-bindingsite. For this reason,hypervariable regions are also referred to as complementarity-determining regions (CDRs). The difficulty of encoding in the germ line all of the information necessaryto generatethis enormously diverse antibody repertoire led to suggestionsof unique genetic mechanismsto account for this diversity.

l m m u n o g l o b u l i nD o m a i n sH a v ea C h a r a c t e r i s t i c Fold Composedof Two p SheetsStabilized b y a D i s u l f i d eB o n d Both the variable and constant domains of immunoglobulins fold into a compact three-dimensionalstructure composed

exclusively of B sheets(seeFigure 24-1'2b). A typical Ig domain contains two P sheets(one with three strands and one with four strands) held together by a disulfide bond. The residuesthat point inwards are mostly hydrophobic and help stabilize this sandwich structure. Solvent-exposedresidues show a gteater frequency of polar and charged side chains. The spacing of the cysteineresiduesthat make up the disulfide bond and a small number of strongly conservedresidues characterize this evolutionarily ancient structural motif, termed the immunoglobulin fold. The basicimmunoglobulin fold is found in numerous eukaryotic proteins that are not directly involved in antigen-specificrecognition, including the Ig superfamily of cell-adhesionmolecules' or IgCAMs ( C h a p t e r1 9 ) .

Structureof Antibody The Three-Dimensional MoleculesAccountsfor Their Exquisite Specificity The three-dimensional structure of immunoglobulins has been solved and the details of how an antibody interacts with an antigenare known at atomic resolution (Figure24-1'3; see also Figure 3-1'9b).The contact area between an antibody and a protein antigen is on the order of 20 x 30 A and involves mostly interactionsthat require perfect complementarity. Hydrogen bonds and van der'sfaals interactionsmake important contributions to antigen-antibody binding. (For more discussion of the role of molecular complementarity in protein binding and function, seeChapter 2, p. 39.) Antibodies can be elicited not only against proteins' but also against modifications carried by some proteins (e.g., attached oligosaccharidechains or phosphategroups) or even small organic molecules that do not occur in nature. For reasonsto be describedin Section24.4,the production of

L i g h tc h a i n

Thismodelshows structure. 24-13lmmunoglobulin A FIGURE with complexed structure of an immunoglobulin thethree-dimensional

byx-ray (a proteinantigen) asdetermined henegg-whitelysozyme 5938 PNAS 86: 1989 al Padlan et ] E A on , crystallography. lBased S :T R U C T U RAEN D F U N C T I O N IMMUNOGLOBULINS

'

1067

antibodies specific for small nonprotein antigens requires conjugation of the antigen to carrier proteins, but the antibodies themselvescan recognize these smaller antigens as such. The smaller the antigen, often the deeperit lies buried in the antigen-binding site of the antibody. The hypervariable regions of the heavy and light chains make the most extenslve contacts with the antigen to which the antibody binds, with the third hypervariable regions making particuIarly significant contributions. The region on an antigen where it makes contact with the corresponding antibody is called an epitope. A protein antigen usually contains multiple epitopes,which often are exposedloops or surfaceson the protein and thus accessible to antibody molecules.Each homogeneousantibody preparation, derived from a clonal population of B cells, recognizes a single molecularly defined epitope on the corresponding antlgen. In order ro solve the structure of an antibody complexed to its cognate epitope on an antigen, it is important to have a source of homogeneousimmunoglobulin and the antigen in pure form. Homogeneous immunoglobulins can be obtained from B-cell tumors (malignant monoclonal expansions of immunoglobulin-secreting B cells),but in that case the antigen for which the antibody is specific is not known. The breakthrough essentialfor generatinghomogeneousantibody preparations suitable for structural analysis was the development of techniquesto obtain monoclonal antibody produced by hybridomas by use of a special selection medium (seeChapter 9, pp. 400402).

A n l m m u n o g l o b u l i n 'C s o n s t a n tR e g i o n D e t e r m i n e sl t s F u n c t i o n apl r o p e r t i e s Antibodies recognizeantigen via their variable regions, but their constant regions determine many of the functional properties of antibodies. An importanr functional property of antibodiesis their neutralizing capacity.By binding to epitopes on the surfaceof virus particles or bacteria,antibodies may block a productive interaction between a pathogen and receptorson host cells, thereby inhibiting (neutralizing) infection. Antibodies attached to a virus or microbial surface can be recognizeddirectly by cells that expressreceptorsspecific for the Fc portion of immunoglobulins. These Fc reieptors (FcRs),which are specific for individual classesand subclassesof immunoglobulins, display considerablestructural and functional heterogeneity.By means of FcR-dependent events, specializedphagocytic cells such as dendritic cells and macrophagescan engageantibody-decoratedparticles, then ingest and destroy them in the processof opsonization. FcR-dependenteventsalso allow some immune-systemcells (e.g., monocytes and natural killer cells) to directly engage target cells that display viral or other antigens to which a n t i b o d i e sa r e a r t a c h e d T . h i s e n g a g . - . n r - 1 y i n d u c et h e immune-systemcells to releasetoxic small molecules(e.g., oxygen radicals) or the contents of cytotoxic granules, including perforins and granzymes.These proteins can attach themselvesto the surface of rhe ..rg"g.J target cell, inflict 1058

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c H A p r E2R4 | I M M U N o L o G y

membrane damage, and so kill the targer (seeFigure 24-5). This process,called antibody-dependentcell-rnediatedcytotoxicity, illustrates how cells of the innate immune system interact with, and benefit from, the products of the adaptive lmmune response. Depending on the immunoglobulin isotype, anrigenantibody (immune) complexescan initiate the classicalpathway of complement activation (seeFigure 24-4).lgM and IgG3 are particularly good at complement activation, but all IgG classescan in principle activate complement, whereas IgA and IgE are unable to do so.

lmmunoglobulins: Structure and Function r Most immunoglobulins (antibodies)are composed of two identical heavy (H) chains and two light (L) chains, with each chain containing a variable (V) region and constant (C) region. Proteolytic fragmentation yields monovalent F(ab) and bivalent F(ab')2 fragments, which contain variable-region domains and retain antigen-binding capa, bility (seeFigure 24-8). The Fc portion contains constantregion domains and determineseffector functions. r Immunoglobulins are divided into classesbased on the constant regions of the heavy chains they carry (seeFigure 24-9).ln mammalsthere are five major classes:IgM, IgD, IgG, IgA, IgE; the corresponding heavy chains are referred to as p, 6 T, o, and e. There are two major classesof light chain, r and }', again characterized by the attributes of their constant regions. r Each individual B lymphocyte expresses an rmmunoglobulin of unique sequenceand is therefore uniquely specificfor a particular antigen. Upon recognition of antigen, only a B lymphocyte rhat bearsa receptor specificfor it will be activated and expand clonally (clonal selection) (seeFigure 24-11). r The antigen specificityof antibodiesis conferred by their variable domains, which contain regions of high variabilit5 called hypervariable or complementarity-determining regions (seeFigure 24-l2a). These hypervariableregions are positioned at the tip of the variable domain, where they can make specific contacts with the antigen for which a particular antibody is specific. r The repeating domains that make up immunoglobulin moleculeshave a characteristicthree-dimensionalstructure, the immunoglobulin fold: It consistsof two B pleated sheetsheld together by a disulfide bond (see Figure 2412b). The immunoglobulin fold is widespreadin evolution and is found in many proteins other than antibodies. including an important classof cell,adhesionmolecules. r The constant regions endow antibodies with unique functional properties or effector functions, such as ihe capacity to bind complement, the ability to be transported across epithelia, or the ability to interact with receptors specificfor the Fc portion of immunoglobulins.

(Figure 24-14). Although the rearrangementof heavy-chain genesprecedesthe rearrangement of light-chain genes,we discusslight-chain genesfirst becauseof their less complex organrzatlon. The immunoglobulin light chains are encodedby clusters of V gene segments,followed at some distancedownstream by a singleC segment.Each V genesegmentcarries its own promoter sequenceand encodesthe bulk of the light-chain variable region, although a small piece of the nucleotide sequenceencoding the light-chain variable region is missingfrom the V genesegment.This missingportion is provided by one of the multiple J segmentslocated between the V segmentsand the single C segmentin the unrearranged r light-chain locus (seeFigure 24-14a). ln the course of B-cell development'commitment to a partlcular V gene segment-a random process-results in its juxtaposition with one of the J segments,again a random choice, forming an exon encoding the entire light-chain variable region (V1). The act of recombination not only generatesan intact and functional light-chain gene, but also placesthe promoter sequenceof the rearrangedgene within controlling distanceof enhancerelementsrequired for its transcription. Only a rearrangedlight-chain gene is transcribed.

Ji,tFll Generationof Antibody Diversity and B-CellDevelopment Pathogenshave short replicationtimes, are quite diversein their genetic makeup, and evolve quickly, generating even more antigenic variation. An adequate defense must thus be capableof mounting an equally diverseresponse.Antibodies fulfill this role. B cells, which are responsiblefor antibody production, make use of a unique mechanismby which the genetic information required for synthesisof immunoglobulin heavy and light chainsis stitchedtogether from separate DNA sequenceelements, or Ig gene segments, to create a functional transcriptional unit. The act of recombination that combines Ig gene segmentsitself dramatically expands the variability in sequenceprecisely where these genetic elements are joined together. This mechanism of generatinga diverse array of antibodies is fundamentally different from meiotic recombination, which occurs only in germ cells, and from alternative splicing of exons (Chapter 8). Becausethis recombination mechanismoccurs in somatic cells but not in germ cells, it is known as somatic gene rearrangement or somatic recombination.

G e n eR e q u i r e s A F u n c t i o n aLl i g h t - C h a i n A s s e m b l yo f V a n d J G e n eS e g m e n t s

Recombination Signal Sequences Detailed sequence analysis of the light-chain and heavy-chain loci revealed a conservedsequenceelementat the 3' end of eachV genesegment. This conservedelement,called a recombination signal sequence/RSS/, is composed of heptamer and nonamer sequencesseparatedby a23-bp spacer.At the 5' end of each

Immunoglobulin genesencoding intact immunoglobulins do not exist already assembledin the genome,ready for expression. Instead, the required gene segmentsare brought together and assembledin the course of B-cell development

( a ) K a p p a( r ) l i g h tc h a i n V

s' G e r m - l i n eD N A

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24-14 Overviewof somaticgene rearrangement A FIGURE in immunoglobulinDNA.Thestemcellsthatgiveriseto B cells portions of immunoglobulin genesegments encoding multiple contain of a B cell,somatic heavyandlightchainsDuringdevelopment yields light-chain functional of thesegenesegments recombination carries genes(b) EachV genesegment genes(a)andheavy-chain closeenough bringsan enhancer itsown promoterRearrangement Thelighttranscription VJsequence to activate to the combined

bytwo joinedgenesegments, region(Vi isencoded chainvariable (Vs)is encodedby three region variable andthe heavy-chain t h e c h r o m o s o mraelg i o nesn c o d i n g j o i n e ds e g m e n t N t h a t o t e s. s an i m m u n o g l o b u l ci nosn t a i nm a n ym o r eV D , a n dJ s e g m e n t h n c u sc o n t a i nas s i n g l ec o n s t a n( tC ) s h o w nA , l s o t, h e x l i g h t - c h a li o several distinct locuscontains asshown,butthe heavy-chain segment, immunoglobulin to the (not corresponding C segments shown) isotvpes.

A N D B - C E L LD E V E L O P M E N T O F A N T I B O D YD I V E R S I T Y GENERATION

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1059

Recombination s i g n a ls e q u e n c e( R S S )

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Recombination s i g n a ls e q u e n c e( R S S )

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J element,there is a similarly conservedRSSthat contains a 12-bp spacer (Figure 24-l5a). The 12- and 23-bp spacers separatethe conservedheptamer and nonamer sequencesby one and two turns of the DNA helix, respectively. 1070

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Somatic recombinarion is catalyzed by the RAG1 and RAG2 recombinases,which are expressedonly in lymphocytes.Juxtaposition of the two genesegmentsto be joined is stabilized by the RAG1/RAG2 complex (Figure 24-1Sb).

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deoxynucleotidyltransferase(TdT) may add nucleotides to free 3' OH ends of DNA in a template-independentfashion. Up to a dozen or so nucleotides,called the N-region, may be added, generatingadditional sequencediversity at the junctions wheneverD-J and V-DJ rearrangementsoccur (seeFigure 24-1,5, step 6 ). Only one in three rearrangementsyields the proper reading frame for the rearrangedVDJ sequence.If the rearrangementyields a sequenceencoding a functional protein, it is called productive. Although the heavy-chainIocus is present on two homologous chromosomes,only one productive rearrangementis permitted, as discussedbelow. An enhancerlocated downstream of the cluster of J segments and upstream of the p, constant-regionsegmentactivatestranscription from the promoter at the 5' end of the rearranged VDJ sequence(seeFigure 24-1.6). Splicing of the primary transcript produced from the rearranged heavychain gene generatesa functional mRNA encoding the p heavy chain. For both immunoglobulin heavy- and lightchain genes, somatic recombination places the promoters upstream of the V segmentswithin functional reach of the enhancersnecessaryto allow transcription, so that only rearranged VJ and VDJ sequences,and not the V segments that remain in the germ-line configuration, are transcribed.

S o m a t i cH y p e r m u t a t i o nA l l o w s t h e G e n e r a t i o n and Selectionof Antibodieswith lmproved Aff inities In addition to the diversity createdby somatic recombination and junctional imprecision,antigen-activatedB cells can undergo somatic hypermutation. Upon receipt of proper additional signals,most of which are provided by T cells,expression of activation-induceddeaminase(AID) is turned on. This enzymedeaminatescytosineresiduesto uracil. lfhen a B cell that carriesthis lesion replicates,it may place an adenineon the complementarystrand, thus generatinga G-to-A transition (seeFigure 4-35). Alternatively,the uracil may be excised by DNA glycosylaseto yield an abasicsite.Theseabasicsites, when copied,give rise to possibletransitionsaswell as a transversion,unlessthe nucleotideoppositethe gap is chosento be the original G that paired with the cytosinetarget.Mutations thus accumulatewith every successiveround of B-cell division, yielding numerous mutations in the rearrangedVJ and VDJ segments.Many of these mutations are deleterious,in that they reducethe affinity of the encodedantibody for antigen, but some improve the encoded antibody's affinity for antigen. B cells carrying affinity-increasingmutations have a selectiveadvantagewhen they competefor the limited amount of antigenthat evokesclonal selection(seeFigure 24-11').The net result is generationof a B-cell population whose antibodies, as a rule, show a higher affinity for the antigen. In the course of an immune responseor upon repeated immunization, the antibody responseexhibits affinity maturation, an increasein the averageaffinity of antibodies for antigen, as the result of somatic hypermutation. Antibodies produced during this phase of the immune responsedisplay affinities for antigen in the nanomolar (or better) range. For reasonsthat are not understood. the activity of activation-

induced deaminaseis focused mostly on rearrangedVJ and VDJ segments,and this targetingmay thereforerequire active transcription. The entire processof somatic hypermutation is strictly antigen dependent and shows an absolute requirement for interactionsbetweenthe B cell and certain T cells.

B-CellDevelopmentRequiresInput from a Pre-BCell Receptor As we have seen,B cells destinedto make immunoglobulins must rearrange the necessarygene segmentsto assemblea functional heavy-and light-chain gene.Theserearrangements occur in a carefully ordered sequenceduring developmentof a B cell, startingwith heavy-chainrearrangements.Moreover, the rearrangedheavy-chainis first usedto build a membranebound receptor that executesa cell fate decisionnecessaryto drive further B-cell development(and antibody synthesis). Successfulrearrangementof V, D, and J segmentsin the heavy-chain locus allows synthesisof a p chain. B cells at this stageof developmentare calledpre-B cells,as they have not yet completed assemblyof a functional light-chain gene and therefore cannot engagein antigen recognition. The newly rearrangedheavy-chaingene encodesthe p polypeptide, which becomespart of a signaling receptor whose expressionis essentialfor B-cell developmentto proceed in orderly fashion. The p chain made at this stage of B cell development is a membrane-bound version. Following engagementwith antigen, the B cell switchesto producing soluble, secretedimmunoglobulins from the sametranscription unit, as we describebelow. In pre-B cells,newly made p chains form a complex with so-calledsurrogate light chains, composed of two subunits, no \5 and VpreB (Figure24-1'7).The p chain itself possesses cytoplasmic tail and is therefore incapable of recruiting cytoplasmic components for the purpose of signal transduction. Instead, early B cells expresstwo auxiliary transmembrane proteins, called Igct and IgB, each of which carries in its cytoplasmic tail an immunoreceptor tyrosine-basedactivation motif, or ITAM. The entire complex including Igct and IgB constitutes the pre-B cell receptor (pre-BCR). Engagement of this receptor by suitable signals results in recruitment and activation of a Src family tyrosine kinase, which phosphorylates tyrosine residues in the ITAMs. In their phosphorylated form, ITAMs recruit other molecules essentialfor signal transduction (seebelow). Becauseno functional light chains are yet part of this receptor,it is incapable of antigenrecognition' The pre-B cell receptor has severalimportant functions. First, it shuts off expressionof the RAG recombinases,so rearrangement of the other (allelic) heavy-chainlocus cannot occur. This phenomenon, called allelic exclusion, ensures that only one of the two available copies of the heavy-chain locus will be rearrangedand thus expressed.Second,because of the associationof the pre-B cell receptor with Igct and IgB, the receptor becomesa functional signal-transductionunit. The pre-BCR also initiates cell proliferation and so expands the numbers of those B cellsthat have undergoneproductive D-J and V-DJ recombination.

O G E N E R A T I OO NF A N T I B O D YD I V E R S I TAYN D B - C E L LD E V E L O P M E N T

1073

,_

Pre_BCR______]

],.5

'gro,tsl Exterior

E Expressionof VpreB and 1,5 turned off

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t,a

E Light-chain rearrangement

Cytosol

E

Assembly with surrogate l i g h tc h a i n s

A FIGURE 24-17 Structureof the pre-Bcell receptorandits role in B-celldevelopment.Successful rearrangement of V,D, and j heavy-chain genesegments allowssynthesis of membrane-bound p heavychainsin the endoplasmic (ER) reticulum of a pre-Bcell At this stage,no light-chain generearrangement hasoccurred. Newlymade p chains assemble with surrogate lightchains, composed of L5 and VpreB, to yieldthe pre-Bcellreceptor, (tr) Thisreceptor pre-BCR proliferation drives of thoseB cellsthatcarryit lt alsosuppresses rearrangement of the heavy-chain locuson theotherchromosome

andso mediates allelic exclusion. In thecourse of proliferation, the synthesis of L5 andVpreBisshutoff ([), resulting in "dilution"of theavailable surrogate lightchains andreduced expression of the pre-BCR As a result,rearrangement of the light-chain locican (E) lf thisrearrangement proceed isproductive, the B cellcan synthesize lightchains andcomplete assembly of the B-cellreceptor (BCR), comprising a membrane-bound lgMandassociated lgo and lgB TheB cellisnow responsive to antigen-specific stimulation.

In the course of this expansion, expressionof the surrogate light-chain subunits, VpreB and },5, is turned off. The progressive dilution of VpreB and \5 with every successivecell division allows reinitiation of expressionof the RAG enzymes,which now rarget the rcor L light-chain locus for recombination. A productive V-J rearrangemenr also shuts off rearrangementof the allelic locus (allelic exclusion). Upon completion of a successfulV-J light-chain rearrangement,the B cell can make both p, heavy chains and r or I light chains, and assemblethem into a functional B-cell receptor (BCR), which can recognizeanrigen (seeFigure 24-17). Once a B cell expressesa complete BCR on its cell surface, alI subsequentstepsin B-cell activation and differentiation involve recognition of the antigen for which that BCR is specific.The BCR not only plays a role in driving B-cell proliferation upon successfulencounter with antigen, but also functions as a device for ingesting antigen, an essentialstep that allows the B cell to process the acquired antigen and convert it into a signal that sendsout a call for assistanceby T lymphocytes.This antigen-presentationfunction of B cells is describedin later sections.

During an Adaptive Response,B CellsSwitch f r o m M a k i n g M e m b r a n e - B o u nldg t o M a k i n g Secretedlg

1074

.

C H A P T E2R4 I

IMMUNoLoGY

As just described, theB-cellrecepror(BCR),a membranebound IgM, provides a B cell with the ability to recognizea particular antigen, an event that triggersclonal selectionand proliferation of that B cell, thus increasingthe number of B cells specific for the antigen (see Figure 24-11). However, key functions of immunoglobulins, such as neutralization of antigen or killing of bacteria, require that theseproducts be releasedby the B cell, so that they may accumulatein the extracellular environment and act at a distance from the site where they were produced. The choice between the synthesisof membrane-bound versus secretedimmunoglobulin is made during processing of the heavy-chain primary transcript. As shown in Figure 24-18, the p locus conrainstwo exons (TM1 and TM2) that together encode a C-terminal domain that anchors IgM in the plasma membrane. One polyadenylation site is found upstream of these exons; a second polyadenylation site is presentdownstream.If the downstreampoly(A) siteis chosen,

.}l Primary transcript

TM1 TM2

Cp4 lts

5

I Enhancer

Poly (A) sites

( a )P o l y a d e n y l a t i o n at upstreamsite

mRNA

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i I

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AAAAAAA

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J SecretedlgM

J

M e m b r a nleg M A FIGURE 24-18 Synthesis of secretedand membranelgM. The primary organization of the p heavy-chain isshownat the transcript top:C,,4istheexonencoding thefourthp constant-region domain, p, isa codingsequence uniquefor secreted lgM;TM1andTM2are exonsthatspecify the transmembrane domainof the p chain Whethersecreted or membrane-bound lgM ismadedepends on whichpoly(A) siteisselected duringprocessing of the primary

(a)lf the upstream poly(A) mRNA siteisused,the resulting transcript formof the p thesecreted includes the entireC.4 exonandspecifies poly(A) siteisused,a splicedonorsitein chain(b)lf the downstream yielding exons, to the transmembrane splicing the Cp4 exonallows formof the p chain. the membrane-bound a mRNAthatencodes forms generate andmembrane-bound secreted mechanisms Similar SS: signalsequence of otherlg isotypes

then further processingyields a mRNA encoding the membrane-bound form of p. (As describedabove, this choice is necessaryfor formation of the B-cell receptor,which includesmembrane-boundIgM.) If the upstreampoly(A) site is chosen, processing yields the secretedversion of the p chain. Similar arrangementsare found for the other Ig constant-regiongene segments,each of which can specify either a membrane-boundor a secretedheavy chain. In the courseof B-cell differentiation, the B cell acquiresthe capacity to switch from the synthesisof exclusivelymembrane immunoglobulin to the synthesisof secretedimmunoglobulin. Terminally differentiated B cells, called plasma cells, are devoted almost exclusivelyto the synthesisof secretedantibodies (seeFigure 24-6). Plasma cells synthesizeand secreteseveral thousand antibody moleculesper second.It is this ramped up production of secretedantibodies that underlies the effective-

nessof the adaptive immune responsein eliminating pathogens. The protective value of antibodies is proportional to the concentration at which they are present in the circulation' Indeed' circulating antibody levelsare often used as the key parameter to determinewhether vaccination againsta particular pathogen has beensuccessful.The ability of plasma cellsto establishadequate antibody levelsis a function of their ability to secretelarge amounts of immunoglobulins and so requiresa massiveexpansion of the endoplasmicreticulum, a hallmark of plasma cells.

P

VDJ

6

Y3

Factorsrequired for classswitching:

VDJ

p

B CellsCan Switch the lsotyPeof l m m u n o g l o b u l i nT h e y M a k e In the immunoglobulin heavy-chainlocus, the exons that encode the p, chain lie immediately downstream of the rearranged VDJ exon (Figure 24-19, /op). This is followed by

Yl

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t

ct

C D 4 Tc e l l s tL-4 AID

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- b l o o di m m u n o g l o b u l i n lgG12 24-19 Classswitchrecombination in the A FIGURE locus.Class switchrecombinatron immunoglobulinheavy-chain (colored involves switchsites,whicharerepetitive sequences circles) genesRecombination of the heavy-chain constant-region upstream (AlD),assistance byT cells, requires activation-induced deaminase (e q., lL-4)produced T cellsRecombination andcytokines by certain

lgA - secretionacrossepithelia

of theswitchsiteupstream of DNAbetween thesegment eliminates Class occurs switching to which region constant and the exons [.i, for with thesamespecificity generates molecules antibody switching the original mounted B cell that lgM-bearing antigenasthatof the and constant-regions heavy-chain butwith different response, thereforedifferenteffectorf unctions

Y N D B - C E L LD E V E L O P M E N T O F A N T I B O D YD I V E R S I T A GENERATION

1075

exons that specify the 6 chain. Transcription of a newly rearranged immunoglobulin heavy-chainlocus yields a single primary transcript that includes the p and 6 constant regions. Splicing of this large transcript determineswhether a p chain or a 6 chain will be produced.Downstream of the p/E combination are the exons that together encodeall of the other heavy-chainisotypes.Upstream of each cluster of exons (with the exception of the 6 locus) encodingthe different isotypes are repetitive sequences(switch sites) that are recombination prone, presumablybecauseof their repetitive nature. Becauseeach B cell necessarilystarts out with surface IgM, recombination involving thesesites,if it occurs, results in a classswitch from IgM to one of the other isotypes located downstream in the array of constant-regiongenes(see Figure 24-19). The intervening DNA is deleted. In the course of its differentiation, a B cell can switch sequentially. Importantly, the light chain is not affectedby this process,nor is the rearrangedVDJ segmentwith which the B cell started out on this pathway. Classswitch recombination thus generatesantibodies with different constant regions, but of identical antigenic specificity.Each immunoglobulin isotype is characterizedby its own unique consrantregion. As discussedpreviously,these constant regions determine the functional properties of the various isotypes. Class switch recombination is absolutely dependenton rhe activity of activation-induceddeaminase(AID) and the presenceof antigen and T cells. Somatic hypermutation and classswitch recombination occur concurrently,and their combined effect allows fine tuning of the adaptive immune responsewith respect to the affinity of the antibodies produced and the effector functions called for.

Generation of Antibody Diversity and B-Cell Development r Functional antibody-encodinggenesare generatedby somatic rearrangementof multiple DNA segmentsat the heavy-chainand light-chain loci. Theserearrangemenrslnvolve V and J segmentsfor immunoglobulin light chains, and V, D, and J segmentsfor immunoglobulin heavy chains (seeFigure 24-14). r Rearrangementof the V and J, as well as of the V, D, and J gene segmentsis controlled by conservedrecombination signalsequences (RSSs),composedof heptamersand nonamers separatedby 12- or 23-bp spacers(seeFigure 2415). Only those segmentsthar have spacersof different length can rearrangesuccessfully. r The molecular machinery that carries out the rearrangement processincludes recombinases(RAG1 and RAG2) made only by lymphocytes and numerous other proteins that participate in nonhomologous end yoining of DNA moleculesin other cell types as well. r Antibody diversity is created by the random selectionof Ig genesegmentsto be recombined (combinatorial yoining) and by the ability of the heavy and light chains produced from rearranged Ig genesto associatewith many different 1076

CHAPTER 24

I

IMMUNOLOGY

light chains and heavy chains, respectively(combinatorial association). r Junctional imprecision generatesadditional antibody diversity at the joints of the gene segmentsjoined during somatic recombination. r Further antibody diversity arisesafter B cells encounter antigen as a consequenceof somatic hypermutation, which can lead to the selection and proliferation of B cells producing high-affinity antibodies, a process termed affinity maturatlon. r During B-cell development,heavy-chaingenesare rearrangedfirst, leading to expressionof the pre-B cell receptor. Subsequentrearrangementof light-chain genesresults in assembly of an IgM membrane-bound B-cell receptor (seeFigure24-17). r Only one of the allelic copies of the heavy-chain locus and of the light-chain locus is rearranged(allelic exclusion), ensuring that a B cell expressesIg with a single antigenic specificity. r Polyadenylationof different poly(A) sitesin an Ig primary transcript determineswhether the membrane-boundor secretedform of an antibody is produced (seeFigure 24-18). r During an immune response,classswitchingallows B cells to adjust the effectorfunctions of the immunoglobulinsproduced but retain their specificityfor antigen (Figure 24-19).

W

rhe MHCand Antigenpresentation

Antibodies can recognizeantigenwithout the involvement of any third-party molecules;the presenceof antigen and antibody is sufficient for their interaction. Although antibodies contribute to the elimination of bacterial and viral pathogens,it is often necessaryto destroy also the infected cellsthat serveas a sourceof new virus particles.This task is carried out by T cellswith cytotoxic activity. Thesecytotoxic T cells make use of antigen-specificreceptors whose genes are generatedby mechanismsanalogous to those used by B cells to generateimmunoglobulin genes.However, antigen recognition is accomplishedvery differently by T cells than by B cells.The antigen-specificreceptorson T cells rccognize short snippets of protein antigens,presentedto thesereceptors by membrane glycoproteins encoded by the maior histocompatibility complex (MHC). Various anrigen-presenting cells, in the course of their normal activity, digest pathogen-derived(and self) proteins and then "post" these protein snippets (peptides)to their cell surfacein a physical complex with an MHC protein. T cells can inspect these complexes, and if they detect a pathogen-derivedpeptide, the T cells take appropriate action, which may include killing the cell that carries the MHC-peptide complex. MHC proteins, which commonly are called MHC molecules,also facilitate communication between T cells and B cells.B cellsdo not usuallyengagein production of secreted antibodies unless they receive assistancefrom another subset of T cells, called helper T cells. These T cells also use

antigen-specificreceptors to recognize MHC-peptide complexes.In this section,we describethe MHC and the proteins it encodes,and then examine how theseMHC moleculesare involvedin antigenrecognition.

The MHC Determinesthe Ability of Two U n r e l a t e dI n d i v i d u a l so f t h e S a m eS p e c i e s to Accept or RejectGrafts The major histocompatibility complex was discovered,as its name implies, as the genetic locus that controls acceptance or rejection of grafts. At a time when tissueculture had not yet been developed to the stage where tumor-derived cell lines could be propagated in the laboratorS investigatorsrelied on serial passagein vivo of tumor tissue.It was quickly observed that a tumor that arose spontaneously in one

inbred strain of mice could be propagated successfullyin the strain in which it arose, but not in a geneticallydistinct line of mice. Geneticanalysissoon showed that a singlemajor locus was responsiblefor this behavior. Similarly, transplantation of healthy skin was feasible within the same strain of mice, but not when the recipient was of a geneticallydistinct background. Genetic analysis of transplant rejection likewise identified a single major locus that controlled acceptance or rejection, which is an immune reaction. As we now knoq all vertebratesthat possessan adaptive immune system have a geneticregion that correspondsto the major histocompatibility complex as originally defined in the mouse' An important step in discovering the functions of the MHC was development of mice strains congenic for the MHC. Congenic strains are genetically identical except for the locus or geneticregion of interest. Figure 24-20 outlines

With eachsuccessivebackcross to StrainA, the geneticcontribution of StrainA is increased.

At eachsuccessivegenerationafter the Fr, checkwhether skin from offspiing shows rapid rejectionby a o u r e S t r a i nA a n i m a l . lf yes, offspringpossessesan MHC that is non-A,thereforeit must be MHCB.

By continuingto selectfor the desiredtrait (MHC B, allotypic m a r k e r )a . c o n g e n i cl i n e o f m i c ei s o b t a i n e d .

+ + 20 Generations +

EXPERIMENTAL FIGURE 24-20 Micecongenicfor the major histocompatibility(MHC)are generatedby crossingtwo histo-incompatible strains.Strain A andstrainB,whichrejecteach grafts,aresaidto be histo-incompatible other's anddifferat their MHC.The(A x B)F1progeny acceptgraftsfromeitherparental (e.g, strain.Bybackcrossing F1miceto oneof the parental strains strainA) for manygenerations, thecontribution of strainA to the geneticmaterial of the resulting offspring will increase Breeding is

Breedto homozygosity for MHC B bv brother/sister m a t i n g .S t r a i ni s g e n e t i c a l l y 'A' with the exceptionof MHC which is "Bl'

the MHC thathaveretained performed suchthatonlythoseanimals of the strainB MHCis of strainB areusedfor breedingThepresence Onlyif a skingraftontoa strainA recipient. by performing assessed will inbredmiceof strainA rejectthe the B-typeMHCispresent for the B-typeMHC andassays graft Byperforming suchbackcrosses thatis a strainof miceisobtained for 20 or moregenerations, the MHCof strainB A in itsgeneticmakeup,yetretains essentially for the MHC. Thesemicearesaidto beconqenic THE MHC AND ANTIGENPRESENTATION

1077

( a ) M o u s eM H C ( H - 2c o m p l e x ) H-2K

l-A

t-E

H-2D

L

F3qP ( b ) H u m a nM H C ( H L Ac o m p l e x ) HLA-DO

HLA-DR

HLA-B

HLA-C HLA-A

*fl ep

organization and gene content show considerablevariation betweenspecies. The human fetus may also be considereda graft: The fetus sharesonly half of its genetic material with the mother, the other half being contributed by the father. Antigens encoded by this paternal contribution may differ sufficiently from their maternal counterparts to elicit an immune responsein the mother. In the course of pregnancy,fetal cells that slough off into the maternal circulation stimulate the maternal immune system to mount an antibody response against these paternal antigens. The antibodies recognize structures encoded by the human MHC. The fetus itself is spared rejection becauseof the specialized organtzation of the placenta, which prevents initiation of an immune responseby the mother againstfetal tissue.

The Killing Activity of CytotoxicT Cellsls Antigen Specificand MHC Restricted

Clearly the function of MHC moleculesis not to prevent the exchangeof surgical grafts. MHC moleculesplay an essenprotein protein il tial role in the recognition ofvirus-infected cells by cytotoxic T cells; these cells also are called cytotoxic T lymphocytes A FfGURE 24-21 Organizationof the major histocompatibitity (CTLs). In virus-infectedcells,MHC moleculesinteract with complexin miceand humans.Themajorlociaredepicted with protein fragments derived from viral pathogensand display schematic diagrams proteins of theirencoded belowClassI MHC these on the cell surface where cytotoxic T cells, charged proteins arecomposed of a MHC-encoded single-pass transmembrane with eliminating the infection, can recognizethem. glycoprotein in noncovalent association with a smallsubunit, called Mice that have recoveredfrom a particular virus infecB 2 - m i c r o g l o b uwl ihni ,c hi sn o te n c o d eidn t h eM H Ca n di sn o t tion are a ready sourceof cytotoxic T cellsthat can recognize membrane bound Classll MHCproteins consist of two nonidentical and kill target cells infected with the same virus. The rasingle-pass transmembrane glycoproteins, bothof whichareencoded dioactivechromium (51Cr)releaseassaycan be used to deby the MHC tect the presenceof cytotoxic T cells in single-cell suspensions prepared from the spleenof an animal that has cleared how mice strains congenic for the MHC can be generated. an infection (Figure24-22a).If T cells are obtained from a Congenic strains are essentialtools for assigning complex mouse that successfullycleared an infection with influenza immunological functions to a particular locus, such as the virus, cytotoxic activity is observedagainstinfluenza-infected MHC. As long as it is possibleto selectfor a particularphetarget cells,but not againstuninfectedcontrols (Figure24-22b1. notypic trait in the form of an allelic marker (e.g.,graft reMoreover, the influenza-specificcytotoxic T cells will not jection in the case of the MHC), congenic strains may be kill target cells infected with a different virus, such as vesicproducedfor other loci. ular stomatitis virus. Cytotoxic T cells can even discriminate In the mouse, the genetic region that encodesthe antibetweencloselyrelated strains of influenza virus, and can do gensresponsiblefor a strong graft rejection is called the H-2 so with pinpoint precision: Differences of a single amino complex (Figure 24-21a). The initial characterizationof the acid in the viral antigen may suffice to avoid recognition and MHC was followed by an appreciation of the genetic comkilling by cytotoxic T cells. These experiments show that plexity of this region. After coarsemapping by standard gecytotoxic T cells are truly antigen-specificand do not simply netic means (recombination within the MHC), the complete recognizesome attribute that is shared by all virus-infected nucleotidesequenceof the entire MHC was determined.The cells, regardlessof the identity of the virus. typical mammalian MHC contains dozens of genes,many In this example, it is assumedthat the T cells harvested encoding proteins of immunological relevance. from an influenza-immunemouse are assayedon influenzaIn humans, the discoveryof the MHC relied on the charinfected target cells derived from the identical strain of acterizationof antiseraproduced in patients who underwent mouse (strain a). However, if target cells from a completely multiple blood transfusions:Antigens expressedon the surunrelated strain of mouse (strain b) are infected with the face of the geneticallynonidentical donor cells provoked an same strain of influenza and used as targets,the cytotoxic T immune responsein the recipient. The predominant target cells from the strain a mouse are unable to kill the infected antigens recognized by these antisera are encoded by the strain b target cells (seeFigure 24-22b, E vs. E). It is therehuman MHC, a genetic region also referred to as the HLA fore not sufficient that the antigen (an influenza-derivedprocomplex (Figure 24-Z1b). All vertebrateMHCs encode a tein) is present;recognition by cytotoxic T cells is restricted highly homologous set of proteins, although the details of by strain-specificelements.By making use of MHC congenic

Class I

Class ll frp

MHC

10 7 8

CHAPTER 24

MHC

I

*4tt.,

IMMUNOLOGY

(b)

Target c el l

I Spleen

lnfect mouseawith virus X +

X XX

- K i l l e r Tc e l l s .--.-',' Single-cell -r' Labeled suspension target cell

J

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HarvestTcells

Virus-infected targetcell Control K i l l e r Tc e l l

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rnfectedwith virus X

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s

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t

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Infectedwith virus X '

TargetDX

M e a s u r e5 1 c r .i n the supernatant

A EXPERIMENTAL FIGURE 24-22 Chromium(slCr)releaseassay allows the direct demonstrationof the cytotoxicityand specificityof cytotoxicT cellsin a heterogeneouspopulation (killer) of spleen cytotoxic of cells.(a)A suspension cells,containing prepared particular from T cellsis micethat havebeenexposed to a virus(eg , influenza virus)andhavecleared the infection. Target cells fromthe samestrainareinfected with the identical virusor obtained proteins Afterinfection, leftuninfected cellular arelabeled nonspecifically by incubation of thetarget-cell suspension with slCr of radiolabeled targetcellswiththe suspension of T Uponincubation in release of the slCr-labeled cells,killingof targetcellsresults proteinsUninfected targetcellsarenot killedandretaintheir

T cellscanthereforebe of cellsby cytotoxic radioactive contentsLysis the radioactivity by measuring andquantitated detected readily (CTLs) (b)Cytotoxic T lymphocytes intothesupernatant released X can be with virus infected have been frommicethat harvested of CTLthe specificity various targetcellsto determine testedagainst targetcells virusX-infected of lysing killing.CTLscapable mediated with a different (Z) cannotkilluninfected cells(tr) or cellsinfected targets virus,Y (B). WhentheseCTLsaretestedon virusX-infected MHCtype(b),again different an altogether froma strainthatcarries (@).Cytotoxic isthusvirusspecific T-cell activity no killingisobserved bythe MHC. andrestricted

strains, the genesthat encodetheserestricting elementswere mapped to the MHC. Thus cytotoxic T cellsfrom one mouse strain immune to influenza will kill influenza-infectedtarget cells from another strain only if the two strains match at the MHC for the relevant MHC molecules.This phenomenonis therefore known as MHC restriction.

T Cellswith Different FunctionalProperties Are Guidedby Two DistinctClassesof MHC Molecules The MHC encodestwo types of glycoproteins essentialfor immune recognition, commonly called class I and class II THE MHC AND ANTIGENPRESENTATION

1079

( a ) C l a s sI M H C m o l e c u l e

End view

< FIGURE 2tt-23Three-dimensional structureof classI and class (a)Shownhereisthestructure ll MHCmolecules. of a classI MHC molecule with boundpeptideasdetermined byx-raycrystallography. Theportionof a classI MHCmolecule thatbindspeptideconsists of a B sheetcomposed of eightB strands andflanked bytwo cthelices Thepeptide-binding deftisformedentirely fromtheMHC-encoded large subunit, whichassociates noncovalently with the smallsubunit(82(b)Class microglobulin) encoded elsewhere. ll MHCmolecules are structurally similar to classI molecules, butwith several important distinctions Boththeo andB subunits of class ll MHCmolecules are MHCencoded andcontribute to formationof the peptide-binding cleft Thepeptide-binding cleftof classll MHCmolecules accommodates a (a)based widerrangeof peptidesizes thanthatof classI molecules [Part (b)based onD N Garboczi, 1996Nature384:134, Part onJ Hennecke etal, 2000,EMBO 19:561'1l the complementcascade.ClassI and ClassII MHC molecules are recognized by different populations of immune system cells and therefore servedifferent functions. As should be clear from the experimentsoutlined in Figure 24-22, cytotoxic T cells are guided in the recognition of their targets by MHC molecules.These T cells mostly use classI MHC moleculesas their restriction elements,and also are characterizedby the presenceof the CD8 glycoprotein marker on their surface.Most, if not all, nucleatedcellsconstitutively expressclass I MHC molecules and can support replication of viruses. Cytotoxic T cells recognize and kill the infected targetsvia the expressedclassI MHC molecules that display virus-derived antigen. As mentioned previously, B cells do not undergo final differentiation into antibody-secretingplasma cells without assistance from another subset of T cells, the helper T cells (or T helper cells). Helper T cells expresson their surface the CD4 glycoprotein marker and use class II MHC molecules as restriction elements.The constitutive expressionof classII MHC moleculesis confined to so-calledprofessional antigen-presenting cells, including B cells, dendritic cells, and macrophages. (Severalother cell types, some in epithelia, can be induced to expressclassII MHC molecules,but we will not discussthese.) The two major groups of functionally distinct T lymphocytes-cytotoxic T cells and helper T cells-can thus be distinguishedbasedon the unique profile of membraneproteins displayedat the cell surfaceand by the MHC moleculesused as restriction elements:

Side view Top view

( b ) C l a s sl l M H C m o l e c u l e

Top view

Cytotoxic T cells: CD8 marker; classI MHC restricted Helper T cells: CD4 marker; classII MHC restricted

MHC molecules.A comparison of the genetic maps of the mouse and human MHCs shows the Dresenceof severalclass I MHC genesand severalclass II MHC genes,even though their arrangement shows variation between the different species(seeFigure24-21.).In addition to the classI and classII MHC molecules,the MHC encodeskey components of the antigen-processingand presentation machinery. Finally the typical vertebrate MHC also encodes key components of

1080

CHAPTER 24

|

TMMUNOLOGY

Both CD4 and CD8 belong to the immunoglobulin (Ig) superfamily of proteins, which all include one or more Ig domains. The B-cell and T-cell receptors,polymeric IgA receptor, and many cell-adhesionmolecules(Chapter 19) also belong to the Ig superfamily.The molecular basis for the strict correlation between expression of CD8 and utilization of class I MHC molecules or between expressionof CD4 and utilization of class II MHC moleculesas the restriction element will becomeevident once the structure and mode of action of MHC moleculeshas been described.

r80r

N O I I - V I N ] S 3 UNd] 9 I T N V C N V ) H I A 3 H T

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Plasma membrane

Itr

High error rate in translation targets misfits for Ub addition and proteolysis

Cytosol

I

G o l g ic o m p l e x

VesiculartransDort

Aminopeptidases Calreticulin

Peptide epitope

I /

I

Peptides

,

t- l

Tapasin

complex Peptide-loading

A FIGURE 24-25 ClassI MHCpathwayof antigenprocessing and presentation. Step[: Acquisition of antigenissynonymous with the production of proteins with errors(premature termination, misincorporation) proteins StepE: Misfolded aretargeted for degradation throughconjugation with ubiquitinStepf,l: Proteolysis iscarried out bythe proteasome. In cellsexposed to interferon 1, the catalytically activeB subunits of the proteasome arereplaced by

are interferon-induced immune-specific B subunitsStep4: Peptides (ER) reticulum viathe delivered to the interiorof the endoplasmic is loadedonto StepE: Peptide dimeric TAPpeptide transporter withinthe peptide-loading newlymadeclassI MHCmolecules complex classI MHC-peptide complex. Step@: Thefullyassembled pathway. Seetext istransported to the cellsurfaceviathe secretory for details

cytoplasm or engagepartner proteins in nonproductive interactions.The rate of cytosolic proteolysis must be matched to the rate at which mistakesin orotein svnthesis occur. Theserapidly degradedproreins are an important source of antigen peptidesdestinedfor presentationby class I MHC molecules.\fith the exception of cross-presenration (discussed below), the classI MHC pathway resultsin the formation of peptide-MHC complexesin which the peptides are derived from proteins synthesizedby the classI MHC-bearing cell itself.

B Proteolysis:Ubiquitin-conjugatedproteins are destroyed by proteasomalproteolysis.The proteasomeis a highly processiveproteasethat engagesits substratesand, without the releaseof intermediates,yields final digestion products, peptidesin the size range of 3-20 amino acids (seeFigure 3-29). During the course of an inflammatory responseand in responseto interferon ^y,the three cata l y t i c a l l ya c t i v eB s u b u n i t s( 9 1 , P 2 , 9 5 ) o f t h e p r o t e a some can be replacedby three immune-specificsubunits: B 1 i , B 2 i a n d B 5 i . T h e B 1 i , B 2 i a n d B 5 i s u b u n i t sa r e e n coded in the MHC. The net result of this replacementis the generationof the immunoproteasorne,the output of which is matched to the requirementsfor peptide binding by classI MHC molecules.The immunoproteasomeadjusts the averagelength of the peptidesproduced, as well

Z Tagging the Antigen for Destruction: For the mosr part, the ubiquitin conjugation systemis responsiblefor tagging a protein for destruction(seeChapter 3, p. 88). Ubiquitin conjugation is tightly regulated.

THE MHC AND ANTIGENPRESENTATION

1083

as the sitesat which cleavageoccurs. Given the central role of the proteasomein the generationof peptidespresentedby classI MHC molecules,proteasomeinhibitors potently interfere with antigen processingvia the classI MHC pathway. 4 Deliuery of Peptidesto ClassI Molecules: Protein synthesis,ubiquitin conjugation, and proteasomal proteolysis all occur in the cytoplasm, whereaspeptide binding by class I MHC moleculesoccurs in the lumen of the endoplasmic reticulum (ER). Thus peptidesmust cross the ER membrane to gain accessto classI molecules,a processmediatedby the heterodimericTAP complex, a member of the ABC superfamily of ATP-poweredpumps (seeFigure 11-14).The TAP complex binds peptides on the cytoplasmic face and, in a cyclethat includesAIP binding and hydrolysis,peptides are translocatedinto the ER. The specificity of the TAP complex is such that it can transport only a subsetof all cytosolic peptides,primarily those in the length range of 5-10 amino acids. The mouse TAP complex shows a pronounced preferencefor peptidesthat terminate in leucine, valine, isoleucine,or methionineresidues,which match the binding preferenceof the classI MHC moleculesservedby the TAP complex. The genesencoding the TAP1 and TAP2 subunits composing the TAP complex are located in the MHC. E Binding of Peptidesto ClassI Molecules:Within the ER, newly synthesizedclassI MHC moleculesare part of a multiprotein complex referred to as the peptide-loading complex. This complex includestwo chaperones(calnexin and calreticulin) and the oxidoreductaseErp57. Another chaperone(tapasin)inreractswith both the TAP complex and the classI MHC molecule about to receive peptide. The physical proximity of TAP and the classI MHC molecule is maintained by tapasin. Once peptide loading has occurred, a conformational changereleases the loaded classI MHC molecule from the oeotide-loading complex. 6 Display of Class I MHC-Peptide Complexes at the Cell Swrface:Once peptide loading is complete, the classI MHC-peptide complex is releasedfrom the peptide-loading complex and entersthe constitutive secretorypathway (see Figure 14-1). ClassI MHC molecules,dependingon the speciesand allelic identity, contain between one and three N-linked oligosaccharides,which receiveextensivemodifications in the Golgi complex. Transfer from the Golgi to the cell surfaceis rapid and completesthe biosynthetic pathway of a classI MHC-peptide complex. The entire sequenceof eventsin the classI pathway occurs constitutively in all nucleated cells, which express classI MHC moleculesand the other required proteins or can be induced to do so. In the absenceof a virus infection. protein synthesisand proteolysis continuously generatea stream of peptidesthat are loaded onto classI MHC molecules.Healthy, normal cells therefore display on their surface a representativeselection of peptides derived from 1084

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host proteins. There may be several thousand distinct MHC-peptide combinations displayed at the surface of a typical classI MHC positive cell. DevelopingT cells in the thymus calibrate their antigen-specificreceptorson these sets of MHC-peptide complexes, and learn to recognize self-MHC products as the "restriction elements"on which they must henceforth rely for antigen recognition. At the same time, the display of self-peptidesby self-MHC molecules enablesthe developingT cell to learn which peptideMHC combinationsare self-derivedand must thereforebe ignored to avoid a self-destructiveautoimmune reaction. It is not until a virus makes its appearancethat virus-derived peptides begin to make a contribution to the display of peptide-MHC complexes. The overall efficiency of this pathway is such that approximately 4000 moleculesof a given protein must be destroyed to generatea single MHCpeptide complex carrying a peptide from that particular polypeptide. An unusual mode of antigen presentation that is n o n e t h e l e s sc r u c i a l i n t h e d e v e l o p m e n t o f c y t o t o x i c T cells is cross-presentation.This term refers to the acquisition by dendritic cells of apoptotic cell remnants, immune complexes, and possibly other forms of antigen by phagocytosis. By a pathway that has yet to be understood, these materials escapefrom phagosomal/endosomal compartments into the cytosol, where they are then h a n d l e d a c c o r d i n g t o t h e s t e p s d e s c r i b e da b o v e . O n l y dendritic cells are capable of cross-presentation,and so allow the loading of class I MHC molecules complexed with peptides that derive from cells other than the antigen-presentingcell itself.

Classll MHC PathwayPresentsAntigens Deliveredto the EndocyticPathway Although classI MHC and classII MHC moleculesshow a striking structural resemblance,the manner in which the two classesacquire peptide and their function in immune recognition differ greatly. Whereas the primary function of classI MHC moleculesis to guide CD8-bearing cytotoxic T cells to their target cells, classII MHC moleculesserveto guide CD4-bearing helper T cells to the cells with which they interact, primarily professional antigen-presenting cells. As noted previously, class II MHC molecules are expressed primarily by professional antigen-presentingcells: dendritic cells and macrophages,which are phagocytic, and B cells, which are not. Hence, the classII MHC pathway of antigen processingand presentationgenerallyoccurs only in these cells. The stepsin this pathway are depicted in Figure 24-26 and describedbelow: A Acqwisition of Antigen:Inthe classII MHC pathway, antigen is acquired by pinocytosis,phagocytosis,or receptor-mediated endocytosis.Pinocytosis,which is rather nonspecific,involves the delivery by a processof membrane invagination and fission, of a volume of extracellular fluid and the moleculesdissolvedtherein. Phagocytosis,the

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across the peptide-binding portion of the MHC-peptide complex. As a result, the T-cell receptor makes extensive contacts with the peptide cargo as well as with the cr helicesof the MHC molecule to which it binds. The positions at which allelic MHC molecules differ from one another frequently involve residuesthat directly contact the T-cell receptor, thus precluding tight binding of the "wrong" allele. Amino acid differencesthat distinguish one MHC allele from an other also affect the architecture of the peptidebinding cleft. Even if the MHC residuesthat interact directly with the T-cell receptor were shared by two MHC allelic molecules,their peptide-binding specificity is likely to differ becauseof amino acid differencesin the peptide-binding cleft. Consequently,the TCR contact residuesprovided by bound peptide and essentialfor stable interaction with a T-cell receptor would be absent from the "wrong" MHCpeptidecombination. A productive interactionwith the T cell receptor is then unlikely to occur.

S i g n a l i n gv i a A n t i g e n - S p e c i f iRce c e p t o r s TriggersProliferationand Differentiation ofTandBCells Both B-cell and T-cell receptorsfor antigen transducesignals by means of proteins associatedwith the antigen-specific portions of the receptor (i.e., Ig heavy and light chains for the BCR; c and B chains for the TCR). The cytosolic portions of the antigen-specificreceptors themselvesare very short, do not protrude much beyond the cytosolic leaflet of the plasma membrane, and are incapable of recruitment of downstream signaling molecules. Instead, as discussed previously,the antigen-specificreceptorson T and B cells associatewith auxiliary subunits that contain ITAMs (immunoreceptor tyrosine based activation motifs). Engagement of antigen-specificreceptorsby ligand initiates a series of receptor-proximal events:kinase activation, modification of ITAMs, and subsequentrecruitment of adapter molecules that serve as scaffolds for recruitment of yet other downstream signaling molecules. As outlined in Figure 24-31, engagementof antigenspecific receptors activatesSrc family tyrosine kinases (e.g., Lck in CD4 T cells;Lyn and Fyn in B cells).Thesekinasesare found in closeproximity to or physically associatedwith the antigen receptor. The active Src kinases phosphorylate the ITAMs in the antigen receptors' auxiliary subunits. In their phosphorylated forms, these ITAMs recruit and activate non-Src family tyrosine kinases(ZAP-70 in T cells, Syk in B cells) as well as other adapter molecules.Such recruitment and activation involves phosphoinositide-specificphospholipase C" and PI-3 kinases. Subsequentdownstream events parallel those discussedin Chapter 1.6 for signaling from receptor tyrosine kinases.Ultimately signaling via antigenspecificreceptorsinitiates transcription programs that determine the fate of the activated lymphocyte: proliferation and differentiation.

T cells depend critically on the cytokine interleukin 2 (lL-2) for clonal expansion. Following antigen stimulation of a T cell, one of the first genesto be turned on is that for IL-2.The T cell respondsto its own initial burst of IL-2 and proceedsto make more IL-2, an example of autocrine stimulation and part of a positive feedback loop. An important transcription factor required for the induction of IL-2 synthesis is the NF-AT protein (nuclear factor of activated T cells). This protein is sequesteredin the cytoplasm in phosphorylated form and cannot enter the nucleus unless it is dephosphorylatedfirst. The phosphataseresponsibleis calcineurin, a Ca2* -activated enzyme. The rise in cytosolic Ca2* leading to activation of calcineurin results from mobilization of ER-resident Caz+ stores triggered by hydrolysis of PIP2 and the concomitant generation of IP3 (seeFigure 1 5 - 3 0 ,s t e p sZ - 4 ) . The immunosuppressant drug cyclosporine inhibits calcineurin activity through formation of a cyclosporinecyclophilin complex, which binds and inhibits calcineurin. If dephosphorylation of NF-AT is suppressed,NF-AT cannot enter the nucleus and participate in the up-regulation of transcription of the lL-2 gene. This precludesexpansion of antigen-stimulatedT cells and so leads to immunosuppression, arguably the single most important intervention that contributes to successfulorgan transplantation. Although the successof transplantation varies with the organ used, the availability of strong immunosuppressantssuch as cyclosporine has expanded enormously the possibilities of clinical transplantation. I

M H CM o l e c u l e s T C e l l sC a p a b l eo f R e c o g n i z i n g of Positive a Process DevelopThrough and NegativeSelection The rearrangementof the gene segmentsthat are assembled into a functional T-cell receptor is a stochastic event' completed on the part of the T cell without any prior knowledge of the MHC molecules with which these T-cell receptors must ultimately interact. Similar to somatic recombination of Ig heavy-chainloci in B cells,the first TCR gene segmentsto rearrange are the TCRP D and J elements, followed by joining of a V segment to the newly recombined DJ. At this stage of T-cell development' productive rearrangement allows the synthesis of the TCR B chain, which is incorporated into the pre-TCR through association with the pre-T cr subunit. This pre-TCR fulfills a function strictly analogous to that of the pre-BCR in B-cell development: It allows expansion of pre-T cells that successfully underwent rearrangement, and it imposes allelic exclusion to ensure that, as a rule, a single functional TCRB subunit is generatedfor a given T cell and its descendants.After the expansion phase of pre-T cells is complete' rearrangement of the TCRcr locus is initiated, ultimately leading to the generation of T cells with a fully assembledTCR ctB receptor.

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Pseq

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AUVSSO'19

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uolue

and other organized groups of cells (e.g., muscle), separating them from connective tissue or other cells. (Figures 19-19 andtg-201 base Any compound, often containing nitrogen, that can accept a proton (H-) from an acid. Also, commonly used to denote the purines and pyrimidines in DNA and RNA. base pair Association of two complementary nucleotides in a DNA or RNA molecule stabilized by hydrogen bonding berween their base components. Adenine pairs with thymine or uracil (A.T, A.U) and guanine pairs with cytosine(G.C). (Figure4-3b) basichelix-loop-helix

Seehelix-loop-helix,basic.

Calvin cycle The major metabolic pathway that fixes CO2 into carbohydrates during photosynthesis;also called carbon fixation. It is indirectly dependent on light but can occur both in the dark and light. (Figre 12-44) cancer General term denoting any of various malignant tumors, whose cells grow and divide more rapidly than normal, invade surrounding tissue,and sometimesspread (metastasize)to other sites. capsid The outer proteinaceouscoat of a virus, formed by multiple copies of one or more protein subunits and enclosing the viral nucleic acid.

basolateral Referring to the base (basal) and side (lateral) of a polarized cell, organ, or other body structure. In the case of epithelial cells, the basolateral surface abuts adjacent cells and the underlying basal lamina. (Figure 19-8)

carbohydrate General term for certain polyhydroxyaldehydes, polyhydroxyketones, or compounds derived from these usually having the formula (CH2O)". Primary type of compound used for storing and supplying energy in animal cells. (Figure 2-18)

B cell A lymphocyte that matures in the bone marrow and expressesantigen-specificreceptors (membrane-bound immunoglobulin). After interacting with antigen, a B cell proliferates and differentiates into antibody-secreringplasma cells.

carbon fixation

SeeCalvin cycle.

carcinogen Any chemical or physical agent that can cause cancer when cells or organisms are exposed to it.

B-cell receptor Complex composed of an antigen-specific membrane-boundimmunoglobulin molecule and associatedsignaltransducing Igct and IgB chains. (Figure24-171

caretaker gene Any gene whose encoded protein helps protect the integrity of the genome by participating in the repair of damaged DNA. Loss of function of a caretaker gene leads to increased mutation ratesand promotes carcinogenesis.

benign Referring to a tumor containing cells that closely resemble normal cells. Benign tumors stay in the tissue where they originate but can be harmful due to continued growth. Seealso malignant.

caspases A class of vertebrate protein-degrading enzymes (proteases)that function in apoptosis and work in a cascadewith each type activating the next. (Figures 21.-37 and 2l-38)

beta (B) sheet A flat secondary structure in proteins that is created by hydrogen bonding between the backbone atoms in two different polypeptide chains or segmenrs of a single folded chain. (Figure 3-5)

catabolism Cellular degradation of complex molecules to simpler ones usually accompaniedby the releaseof energy.Anabolism is the reverse process in which energy is used to synthesize complex moleculesfrom simpler ones.

beta (B) turn (Figure3-6)

catalyst A substancethat increasesthe rate of a chemical reaction without undergoing a permanent change in its structure. Enzymes are proteins with catalytic activity, and ribozymes are RNAs that can function as catalysts. (Figure 3-20)

A short U-shaped secondary structure in proteins.

BLAST A widely used computer program for comparing the amino acid sequenceof a protein with the sequencesof known proteins stored in databases.BLAST searchescan provide clues about the structure, function, and evolution of newly discorreredproteins. blastocyst Stageof mammalian embryo composed of :64 cells that have separated into two cell types-rrophectoderm, which will form extra-embryonic tissues,and the inner cell mass,which gives rise to the embryo proper; stagethat implants in the uterine wall and corresponds to the blastula of other animal embryos. (Figure 22-1) buffer A solution of the acid (HA) and base (A ) form of a compound that undergoeslittle change in pH when small quantities of strong acid or base are added at pH values near the compound's pK".

cadherins A family of dimeric cell-adhesion molecules that agg^regatein adherens junctions and desmosomes and mediate Ca"--dependent cell-cell homophilic interactions. (Figure 19-2) calmodulin A sma^llcytosolic regulatory protein that binds four Ca" ions. The Ca'*/calmodulin complex binds ro many proteins, thereby activating or inhibiting them. (Figure 3-31) calorie A unit of heat (thermal energy). One calorie is the amount of heat neededto raise the temperature of 1 gram of water by 1 'C. The kilocalorie (kcal) commonly is used to indicate the energy content of foods and changes in the free energy of a system.

cation

A positively charged ion.

cDNA (complementary DNA) DNA molecule copied from an mRNA molecule by reverse transcriptase and therefore lacking the introns present in the DNA of the genome. cell-adhesion molecules (CAMs) Proteins in the plasma membrane of cells that bind similar proteins on orher cells, thereby mediating cell-cell adhesion. Four major classesof CAMs include the cadherins, IgCAMs, integrins, and selectins. (Figures l9-l and 19-2\ cell cycle Ordered sequenceof events in which a eukaryotic cell duplicates its chromosomes and divides into rwo. The cell cycle normally consists of four phases: G1 before DNA synthesis occurs; S when DNA replication occurs; G2 after DNA synthesis; and M when cell division occurs, yielding two daughter cells. Under certain conditions, cells exit the cell cycle during G1 and remain in the Ge state as nondividing cells. (Figures1-t7 and20-l) cell division Separation of a cell into two daughter cells. In higher eukaryotes, it involves division of the nucleus (mitosis) and of the cytoplasm (cytokinesis); mitosis often is used to refer to both nuclear and cytoplasmic division. cell junctions Specialized regions on the cell surface through which cells are joined to each other or to the extracellular matrix. (Figure 19-9 ; Table 19-2) GLOSSARY

G-3

cell line A population of cultured cells, of plant or animal origin, that has undergone a genetic change allowing the cells to grow indefinitely. (Figure 9-31b) cell strain A population of cultured cells, of plant or animal origin, that has a finite life span and eventually dies, commonly after 25-50 generations.(Figure9-31a) cellulose A structural polysaccharide made of glucose units linked together by B(1 -+ 4) glycosidic bonds. It forms long microfibrils, which are the major component of the cell wall in plants. cell wall A specialized,rigid extracellular matrix that lies next to the plasma membrane, protecting a cell and maintaining its shape; prominent in most fungi, plants, and prokaryotes, but absent in most multicellular animals. (Figure 19-37) centriole Either of nvo cylindrical structures within the centrosome of animal cells and containing nine setsof triplet microtubules; structurally similar to a basal body. (Figure 18-6) centromere DNA sequencerequired for proper segregation of chromosomes during mitosis and meiosis; the region of mitotic chromosomes where the kinetochore forms and that appearsconstricted. (Figures 6-40 and 5-46b) centrosome (cell center) Structure located near the nucleus of animal cells that is the primary microtubule-organizing center (MTOC); it contains a pair of centrioles embedded in a protein matrix and duplicates before mitosis, with each centrosome becoming a spindlepole. (Figures18-6 and 18-35) chaperone Collective term for two types of proteins-rn olecular chaperones and chaperonins-that prevent misfolding of a target protein or actively facilitate proper folding of an incompletely folded target protein, respectively.(Figures 3-t6 and 3-l7l chaperonin

Seechaperone.

checkpoint Any of several points in the eukaryotic cell cycle at which progression of a cell to the next stage can be halted until conditions are suitable. (Figure 20-35) chemical equilibrium The state of a chemical reaction in which the concentration of all products and reactantsis constant because the rates of the forward and reversereactions are equal. chemical potential energy The energy stored in the bonds connecting atoms in molecules. chemiosmosis Processwhereby an electrochemicalproton gradient (pH plus electric potential) acrossa membrane is used to drive an energy-requiring process such as AIP synthesis; also called chemiosmotic coupling. Seeproton-motive force. (Figure 1'2-21 chemokine Any of numerous small, secretedproteins that function as chemotatic cues for leukocytes. chemotaxis Movement of a cell or organism toward or away from certain chemicals. chimera (1) An animal or tissue composed of elements derived {rom genetically distinct individuals; a hybrid. (2) A protein molecule containing segmentsderived from different proteins.

containing membranes (thylakoids) where the light-absorbing reactions of photosynthesisoccur. (Figlue 12-29) cholesterol A lipid containing the four-ring steroid structure with a hydroxyl group on one ring; a component of many eukaryotic membranes and the precursor of steroid hormones, bile acids, and vitamin D. (Figure 10-5c) chromatid One copy of a replicated chromosome, formed during the S phase of the cell cycle, that is joined at the centromere to the other copy; also called sister chromatid. During mitosis, the two chromatids separate,each becoming a chromosome of one of the two daughter cells. (Figure 6-40) chromatin Complex of DNA, histones,and nonhistone proteins from which eukaryotic chromosomes are formed. Condensation of chromatin during mitosis yields the visible metaphasechromosomes. (Figures 6-28 and 6-30) chromatography, liquid Group of biochemical techniques for separating mixtures of molecules (e'g., different proteins) based on their mass (gel fihration chromatography), charge (ionexchangechromatography), or ability to bind specificallyto other molecules (affinity chromatography). (Figure 3-37) chromosome In eukaryotes, the structural unit of the genetic material consisting of a single, linear double-stranded DNA molecule and associatedproteins. In most prokaryotes, a single, circular double-stranded DNA molecule constitutes the bulk of the genetic material. Seealso chromatin and karyotype. cilium (pl. cilia) Short, membrane-enclosedstructure extending from the surface of eukaryotic cells and containing a core bundle of microtubules. Cilia usually occur in groups and beat rhythmically to move a cell (e.g., single-celledorganism) or to move small particles or fluid along a surface (e.g., trachea cells). Seealso axoneme and flagellum. cisterna (pl. cisternae) Flattened membrane-bounded compartment, as found in the Golgi complex and endoplasmic reticulum. citric acid cycle A set of nine coupled reactions occurring in the matrix of the mitochondrion in which acetyl groups are oxidized, generating CO2 and reduced intermediates used to produce ATP; also called Krebs cycle and tricarboxylic acid (TCA) cycle. (Figrre 12:l'01 clathrin A fibrous protein that with the aid of assemblyproteins polymerizes into a lattice-like network at specific regions on the cytosolic side of a membrane, thereby forming a clathrin-coated pit that buds off to form a vesicle. (Figure 14-18; Table 14-18) cleavage In embryogenesis, the series of rapid cell divisions that occurs following fertilization and with little cell growth' producing progressively smaller cells; culminates in formation of the blastocyst in mammals or blastula in other animals. Also used as a synonym for the hydrolysis of molecules. (Figures 22-l and22-8) Large, multiprotein comcleavage/polyadenylation complex pre-mRNA at a 3' poly(A) site of plex that catalyzesthe cleavage (A) to form the residues adenylate of addition initial and the poly(A) tail. (Figure 8-15)

chlorophylls A group of light-absorbing porphyrin pigments that are critical in photosynthesis. (Figure 12-31)

clone (1) A population of genetically identical cells, viruses, or organisms descendedfrom a common ancestor.(2) Multiple identical copies of a gene or DNA fragment generatedand maintained via DNA cloning.

chloroplast A specialized organelle in plant cells that is surrounded by a double membrane and contains internal chlorophyll-

cochlea Snail-shaped structure containing the organ of Corti, the sound-sensingpart of the inner ear. (Figure 23-30)

G-4

GL O S S A R Y

S-9

.

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rcr-91pue g-91 sarn8rg)'sde.vrqredSurleu8rsreln]]ar€rlul Suttetttut dqaraql toldarar eqt qrl^\ palElf,ossesoseull ;y[ oqosotdc Jo uoIle I]rP 'euotu or sppol Surpurq pue8rl 'suoJetJetul pue 'sutlnelrolut -roq qfruor8 'urtetodo.rqldJe JoJ osoql Surpnpur sroldecar 3ur -leu8rs ace;rns-llal to sselcroleu Jo rrqru€W roldooer eurlot,{c 'uorlPJeJrlororo uoIlelluereJJIPJIeqr re88r.rt ot s11actuals.(s-eunurrulpue poo]q uo sroldacar JJeJJns 'suore;retut 'SSC-C 'utlatodorgl -ller ol pulq leqt (sur>1na1.retur -.{ra ''3'a) surato.rdpataJJas'11etussnolatunu ;o duy aur4olb pue uorler (eVyZt a:n8rg) 'srseqtudsotoqd -rdser re1n11ac Sutrnp srorrreJ uoJpalt se uollf,un; I{JIrl^{ Jo auos souorqrol,(c surato.rd Surutetuor-aureq 'paroloc yo dnor8 y (79-97 arnBtg) 'suralo.rd ta8ret rr;nads Surrel,troqdsoqd dg apdc 11erctlodre>1 -ne eql;o se8erstuara;Jlp q8norqr uotsserSordraSStrl saxalduror snolre1 'ur1cb e ol punog ueqm dluo e,rrlre .,t11ert ;q3-urlcdr -ldlelel sr l€ql espull urelord y (yq3) eseurl luopuedap-uqc,b 'serudzua eseqt to dtror;neds eterlsgns aql SuruturaloP Pu€ 3utle.,rtlre ,{qa.raqt 'saseur>1luapuadep-urlcdo qrrm sexaldruor uroJ sullr -d3 'e1cdc11arrrtodrelne eqt Jo esrnor aql Surrnp IIeJ Pue eslr suoneJruaf,uoJesoq^\ sutalo.rdpJtElel leJe^Js;o ,(uy urp,tr (tg-St pue '8I-SI '6-5I sarn8rg)'sller rerllo Pue rlf,snluqloorus JEInf,seAur C eseul4 utatord satelllf,E Pue sl]ef, Por uI slauueqf, uonec suado teql roEuassau Puoros V (aWCr) 4y15 crlrIc (Z-St olqet:6-91 arn8rg) 'V eseuDluralord sale^Ilf,eleql 'sroldecer paldnoc-uratord g utelroc to uoIlEIntulls l?uourrorl ol asuodse:ur pernpord 'lotuassaut Puof,esV (aWVr) 4ytgy rqcdc 'seruosoruol{J (61-g ern8tg) 'uolleulquoter osl€ aes peurqruoJeJornpord ot slsoloru Surrnp sprleruorqc leurared pue turssorc Ieuleteru uaellloq Ielraleu rttauo8 1o aSuegcxg rorro (E1-g ern8rg) 'turcqds VNU tJartotr arnsse pue salodre4ne raq8rq ;o sygaur-ard eql ul suoxa elee -ur1epsdlaq reqr sluauodruof,reqto pue surelord g5 Sutputq-y519 xalduor uorlrutocar uoxe-ssorJ Surpnlcur dlquasse a8rel 'uorlf,erelu luel?aof,uou osle ees 9-Z pue 7-7 sarn8tg) 'suortf,ala srred erour ro euo yo Surreqs dg raqraSor selnJo]otu ;o ur surolE aql sPloq leql errot lPlllueql alqers Puoq luaP^otr

'g-11 arn8tg) 'uolrcerlP (I-tt alqeJ.:[C 'gg] (uodnue) elrsoddo ro (rrodurds) oruesoql ut luarper8 uollertuof,uof, str u^\op elnlelotu puotes e to lueuelour ot Suqdnoc ,(q ue,rtrp tuarper8 uolleJluaf,uof e lsuteSe eueJgruetu E ssorf,E alnJelou IIEurs Jo uor uE Jo tueruolotu PelelPau-ulaloJd lrodsuerloc (9-E1 ern8rg) 'pareSuolaSutaq pue etuosoglr eql ol Punoq IIIrs sI urelord luefseu egl se urnlnf,Iler cruseldopua oql olul utelord drol -erf,esp 1o lrodsuerl snoeu€llnuls uon€tolsueJl leuonelsueJlot (t-tt tlqet) 'r31o3 aqr ol unlnf,Ilrr rruseldopua 'de.Lrqreddrolerl aqt ruort suralord alolu seltlsel PerPol-IIdOf, -es aqt ur sa]tlsel trodsuerl lPoJ leql sutalord Jo ssell V IIdOf, ( t-tt etqet)'eeurelslt r31og roqree ot ret€l ruo{ Pue tunlnrllar cruseldopua aqr or r31o9 'deaaqted drolarc aqt tuo{ sutetord elotu selrlsal PalPol-IdOJ -es eqt uI selrlsel trodsuerl leor 1€I{1sutalord Jo sselr Y IaIOf, '(s11acSurpr,trp ur.Sutt apLcal|uot) fuaw -elorrr IIef,rc (s,taEJssa4s ''B'e) uoISaqPEIIaf,uI uollrun; r€ql sllel elf,snuuou ur ursodru pue ultce Jo solPung tr]0""0 elllJerluol 'sl€uErs leuJolxe Jo I?u -ralur.{q pareln8ar tou sI leql (uorle.rrasolllnlllsuof t'8'e) ssarord J?lnlleJ e;o uotterado snonulluof, JI{l Jo olnf,eloru Jelnllef, € Jo ,fur,lrtre ro uortcnpord snonulluof, egl ot Surrre;ag elllnlllsuot (g 1-51 a.rn8rg)'srlerqaur^ ut suotllun( sulxauuoJ deE rurol teqt suralord aueJqrueusuerr 1o dpure; y (3-E ern8tg) 'elnralotu eql uI stuole 0I{l to uoneJol lerreds aqr uror; Surtlnsar suolsuaulP aorql ur elnlaloru -oJf,eul Jaqlo Jo uralord e ;o adeqs asnerd aq1 uorleluJoJuoJ 'euPrqluetu rP]nllef,€ Jo saPlslueroJJIPuo ro od.rque Jo IIel e ;o suotSer lueJeJtIPuI ef,uelsgnse Jo uollerluaJ -uof, aqt uI erueraulP e ASolorq 11acu1 luarpert uoperluoJuof,

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'uorqse; da>1-pue-po1e ur raqlaSol rr; reql (orerrsqnssll pue eurdzua ue ''3'e) sa]nJalou Suttce;elut o- al uo suot8er Surgrrcs -aq (Z) :raqlo qf,eo qtr-aTsned eseq rce;red luroJ uef, leql spuerls .ro saouanbaspIJe Jlelrnu o.t\l ol turrra;ag (1) ibeluatualdruor ' @-p7 unfu 7l' xaldwoc 4)oilP auotquaut crldloldr el{l Jo uolleruJo, uI saleululnJ leql oPEJseJ3tld1oal -ord e Sutle.ltrce ,(qoreqr 'sare;rns 1e8unl ro lelqorf,Iru or dllf,erlP luarualduroc purq tpqt suralord Iunres elllnlllsuoJ Jo dnort y 'aletrosse doqr qrqr'r qrtm ft-6l elqel!77-51 anfu7) surato.rdaf,pJrns-lleJpue slueuoduroJ relnllaf,Erlxa eql Pue uollng -rrtsrp enssll JIeI{]ul ragrp sad.{rgnssnorelunu eqJ'senssIl elllf,eu -uoJ pue xrJleru Jelnllef,eJlxaaqt;o tueuodtuoc roleu e sI t€t{l ueteloc auqord pue aun.,{13ur gctr uralo.rdocdlSpcrlaq-eldrrr y (e6-g arn8rg)'srolf,EJuotldt.rlsuB.rl urelJar pue sutetord snoJqIJuI Punot dluoruuror isutelord uI seJnl uEJ leql suorSorlecqaq -Jnrts aIrTpoJ'elqets uro; ot aleIf,osse-JIes m crgredrqdure dq pa4reru Jllou lernlrnrls utelord y IIoJ Palrot -uds oullue 1o uolleulruret asnef, Pue sPrJe 'suopor dots e:e earql 'suopor olgrssod t9 osle fsrseqlu.(sutelord Surrnp prce oulrue terlt VNUtu ro VNq uI sePlloelf,nu oarql

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AUVSSO'r9

9-9

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"r'l'ilrl:1t1 "llleturou p Jo euprgrueueurseldaql ssorf,eslsrxa teql lerluelod J]rllale e,rrle8euare;-rqosot.(oaqt ur osEerf,eq uorlezrrel0dep 'VN61 aes prtrerrelf,nuoqrr,(xoop $-y7 enfug).slleJI ro uortelrtre olerlrurpue sepouqdru,(1 ol elet8ru daql 'uorlcayurro drnlur enssr],o elrs E lp ua8rlue Surzrleureturretyy .srotdocera{rIJJoI rrar{lerAsre{retu ua8oqred ;o suraled peorq tlelep ueJ pue sonssrlsnorJel ur aprserlerll sylartunuasard-uetrlue leuorsse;ordcrrdco8eq4 sllal Jrlrrpuap .suorneu rer{to ft-97 anfug) Jo suoxeruory sleu8rssalref,erpue per{luerq ,(11ecrddr pue trorls d1a,tr1e1ar sr teqt uornaue;o dpoq IIat eqt uor; Surpuetxessef,ord alrrpuop 'uortrunJ 1ecr3010rq Jo ssolur sllnserlllensn :s]eJrwaqJurEtJOJ ol ernsodxaro Surteoq.,tqpesnecsuorlJeJol -ur snorJ€ ;o uorldnrsrpol enp prf,ef,re]JnuJo urel luele^oJuou -ord e ;o uorlpruJoJuof,aql ur uorteJellef,rlseJ6l uorluJnleuep '(3 ,z-0I x 99'l) urole uaSorp,{qe;o sseu eqt ol lenballaleurrxorddesseturelnf,elourJolrufl uorlep '1oracd1t1,{rerpea5 )Vq1

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UVSSO'19

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'seteJlsqns patzlar Jo regunu ]letus Jo eleJlsqns rr;nads e 8ur,r1o,rut erur(zua uortf,par lef,rurer{J relncrlred e sazf,1efit reql uratord y 'adola,tua lprrl Jo ado;e,rua JeolJnu eas adola.lua 'reprosrp aqt ratear8 eqt 'ddortuo aqr reg8rq aql fuels,{s e ur sseuuopuer ro raprosrp 1o aar8ep eql to ernseeurV (5) i(dorlua 'ser8rauapuoq ]erot rrer{l ot lenbe sr stcnpord ro sluelJeer aqr ;o ddleqrua egt 'uortceer IEf,IureLIf,e ur ftea11 f1) ddpqtue (69-4 arn8rg) 'suratord Surpuaq-y51q Jo aruel -srsse er{l qtr.la JaJueque ue ur salrs Surpurq Jlaqt ol ,{1a.,rtte.redooc purq daqt se (srossa.rdar pue sJotelrtre) srorre; uotldtrcsuerl uorl euosef,uerluo selquesse tegt xalduroo uralordoapnu e8rel $1-7antug\ 'eue8 patercoss€eqt Jo uorldrrcsuert Jo eteJ ar{l sotelnporu JeJupl{ -ue ue ot suralord cryrcads;oSurpurg 'aruenbesSurpoc oqt ulqtl/r\ uele Jo slorluoJ lr eua8 eqt tuo{ eJuelsrp leat8 e re perplol eq roJueque ,{eru reqr y51q crrodrelna ur aruenbes droteln8er y 'trrureqloxo;o etrsoddo lpaacord ot JePro ur teeq qrosqe tsnru snqt pue 'HV ',(dpqrua ut a8ueqr a.ttltsod e a^eq leqt sasseco.rdpue suorlJ€er ol Sur.rra;aa tlruJoqlopue (97-9 arn8tg)'stuorqtudsop -ua srseldorolqc Eupuor{rollur qroq 'srsaqtod.{q ruort pa^lo^e puE 'drqsraulred tuorqtudsopuo oqt ot Surproccy I€If,Itouagdllenlnut e ur 11accrlodrelne p eprsur seprsorterlt tunlJetf,Eg tuorqrudsopua

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'setuososdy ot suralo;d 1-71 sa.rn8rg) ;o Surtros uI uollf,un; pue 'srsot 'setuosopue awl 11d leuratur JrprJe ue a^eq r{Jrr{.^a Pue -dropua pet€rperu-JotdacarSutrnp au€rqruetu eurseld eql uroJ; '(saprso,r crtdropua ro) seruosopua Qna :sluaru Jto pnq qrrq.la. -lredtuoc pepunoq-euerqueu;o sed& o.41u auosoPut Jo euo

(1-5 arn8rg) 'srseqtu.(spldl ul suortf,un; 'seu;osoqtr srye1 r{rlrl^A 'Ug qpoLus aqt lsutalord euprgruaru pue pale.ltas 1o 3ur -ssacordpue srsaqludsoqt ur suoDf,un;'setuosoqrrqll^\ Petelf,osse

sr qrrrl^\ 'gg qBnot ag1 'adola,tuar€elrnu relno eql qltrrt snon8tl -uoo sllef, crlo.{re4na;o urseldoldc eqt ulqll..l\ seJnlJnJlssnoueJq -rueru patf,euuoJrelul Jo lro. ataN (ag) urnpcrrar erurseldopua 'IurePosatu Pue ruroPope ees Oy-ZZ pue g-17 se;n8tg) 'lrerl drolerrdseJetll lo.{rqrualeru Jo tsotu Pue 1nBaqr ol astr sa,tr8 -ru€ eql;o sredel 11acdreurud eerlp eql Jo lsolurauul luroPoPue 'srsot^{courdpue'srsolboteqd'srsoilcopua Pel?IPeu -rolderar sJpnltul lauerqruau eurseld eql ,o uorleur8e.tur ,(q Ierroteru relnllef,prtxa ;o alerdn roJ rurel IEraurC srsoldropua (67-y1 enfug) 'drnrtce rtaqt eteln8e.l-urrtop ol de.tr P se af,eJJnsIIel erll uror; suralord roldar -eJ elorueJ 01 pue suletord uodsuerl eueJqluetu dq porrodur eq leqt srcolbopua ot a3re1ool sIEIJetEIrrJelnlleJeJxa sJZITeuJeluI palerparu-rotdacarSurtlo,rut derrrqredrelnlle3 dea,r'qtedcrlbopua '(puep pror,(qr ro dreltnttd ''3'a) puep e ur luesard dllensn s11erdrolarcas pazqercedsluelslP ,{q poolq oqr orul paspelerouoruroq e ot puodsar PUBPuIq sllol la8Jpt qrlqa uI luslueqf,eu Surleu8rs ol Surrra;aa ourJJoPue 'cruotroxa ;o elrsoddo !peerord ol Jepro ur dtrauo eary;o lndur ue artnber snql Pue 9y e,rrtrsod e elpq leqt sassef,oJdpue suotlrea.r ol Surrre;ea cruotrapuo (;17en?r.gl 'od.rque tsor{ E olul uorlresuler reqe ro oJIIA uI reqrre seddr 11ar so.(rgtue dlrea dre.t ;o a8ue.r epl^a € otul e]€IlueraulP uEJ teqt ruorJ pa^rrep sller Perntlnl to euII V s11ac(59) uals rruodrqrue '(eto8tz) 33a pazqtr srsouotofuqua

-reJ e ruorj IEnpI^IpuI ue ;o tuaudole.tap dlreg

( g7-y ernSrg)'uotteItIuI 8utmo11o; (srsaqtuls uralord) YNUIu Jo uollelsuerl panulluof, ro1 parrnbe.r (gg) roroel uorletuola dnor8 e Jo euo surelord letuosoglruou;o (gg-g arn8rg) 'PIaIJllrllela Suorls e ol Pelrol -qns runrpeu reglo ro 1eBe ur uotler8tur rlaql uo Peseqselntre]ou -orJeru Surleredasro; senbtuqral IEraAes;oduy slsaroqdorlcale (91-71 arn8tg) 'sreurec uorlcelo Punoq arolu ro euo suleluoJ ureql ''3'a) srouop uorlrele eqt Jo raquaur I{reg 'zO or (Hq1VN PernPer uror] .{\olJ suorlre]e qrrq.v' qSnorgt } aurdzuaoc PUE , eruortlJol -dc a1qrsry;rp snld auerqruaru lelrPuollf,olrru reuul aql ur sexeld -ruoc uretordrtyntu e8rel Jno, to los ureqc lrodsueJl uoJlJele 'srseldorolqctueld;o auerquelu (Og-Zt pue 91-71 sern8rg) PIo>lEI -.(qt aqr ul *dCVN or OzH luorJ ro 'auerqtueru IPIrPuoIIf,olIIu rau -ur eql ur zO ot (HCVN ''3'a) srouop uorltrale parnPer Iuort srelrretr uorlf,ele Jo sarres E eI suorlf,ela Jo .^aol{ ilodsuerl uoJlJole ur ft-Zt elqetl'suollreer uollrnPer Pue uollePtxo peldnor se]nJelou roldecce ol ruaql sJaJsueJlPue selnJalou JouoP ruor1 raIJJ€J uoJlf,ele suortra]a slderce l?rp tuote ro alncalour duy 'prluolod auerqluoru egr Pue euprqrueur aqt ssoJf,eluarpur8 uorleJluetuoJ sGuoIatll Jo eJuenltur peurqruof,aql stueserdarlI'euerqluou P ssorJe (alnca1oupa8reqc .ro) uor ue ;o lrodsu€rl Jo uollf,erlP alqero^et .(llecrta8reue oqt sourruralap r€IJr elro; Sul,rrrp eg1 luarpert ptluaqtorlrele 'sller ssorre ]le dpeau;o euerqruorueurseld eql 'sa8.reqre,rtle8eupue a.ltltsod peuretul€ru sr lerlualod f,lllf,ale uy ,t8raua eq1 prluelod JrJlJele ;o uotteredes aqr qtIA\ PetelJosse (q6-g arn8rg) 'uqnPouler se gf,ns sulelord 3u1pu1g-*.e3 ,(ueur ut srnf,f,o lEtlt Jrlou lPrnlJnrls xITaI{-dool-xIT0I{Jo eo^r V PuPq {g

e ur (sluetf,Eer)selnle]oru Surtrets pue selnreloru lcnpord eqr;o lSroua aer; aqt ur eluoreurp er1J (gy) atueqr d8reua-aar; Ietor '(g) ,tdorrua pue (H) ,(dleqrua eql jo uortrunJ e sr r{JnI.&\ 'uels.{s e yo d8raua lertualod eqt Jo arnseeurV (9) d8roua aarl ddorsorcrur eJuoJseJonltdg eldures eql Surlresgo pue lsaralur;o tueuoduroc e or dlyecryrceds spurg reqr (fpoqrrue ,.3'o) rueSe peloqel-adp tueJseronlt p qlr.ry\ sonssrt ro sllef, Surleerl dq stuauoduor JEInlleJ Surzrlensraro; anbruqcal IeraueC 8ururels lueJsaJong ddorsonru eJuetseJonl; .(g saldues egt Sur,rrasqopue tserelur ;o ecuenbaseql ol ozrprrq -lq regr saqord luof,saron]Jqtr.tr saldues SurtBa.rtlq sonssrlpue sllar ur saruanbesVNU ro y51q rr;nads Surtralap .ro; sanbruqrel potpler lereles;o duy (1151g)uorrezlppqlq nlrs ur oruerserong (97-6 ern8rg) 'eruarseronlJrraql ur satueraJJrpuo paseq rueqt lros PUP sller lar{to to spuesnoql luo{ sller .t\aj e ro euo trelep uef, ter{t lueunrtsur uV (SCVg) relros IIef, pale^llJp-eruersoronlJ (9p-11 arn8rg)ra,{e1 -rq prdrloqdsoqd e ;o ral;pel rrr{to eqt ot lageeJ ouo ruor; sprdrl aueJqueu Jo tuerueloru eqt satelrlrf,E terlt uretoJd aseddrg 'CIv{ aas

aPlloaltnulP euruoPEUIAPU

(gE-g1 ernSrg) .unqrc pu€ euauoxe osle ees .sarnl -cnr1s raldurrs qrnu pue rallerus are ella8eg .runrperu lerretleg pmp e q8norqr IIer orlr sladord Surpueq alrldrq.tr asoq.u ,(urrads ''3'e) s11ac crtodrelne etuosto arpJrnseqt ruorJ Surpuetxa (]1acrad auo dllensn) erntrnrls drotourorol 3uo1 (eloEeg .1d) runlateg 'uopezrprrq,(q nlrs ur ef,ueJseronlJeas HSIC (96-51 arn8rg) 'sroldarer uorser{pe ul:3otur ol xrrteru pup rplnl -laf,prlxe aqt .saddr 1o slueuoduroJ rer{to .{ueu spurg IIec snorJpl ur'Surcqds olrleuJellp dq pererauaB.su.ro;osr snoJerunuur sJnf,Jo leqt urolord xrJleru e^rseqperllnu tuepunqe uv urlJeuoJqrJ 'oJnllnf, enssrt ur pue Suqeaq puno.lr Surrnp sete.ra;qordpue sater8rurixuleur rcFlleJerlxe aqt;o stueuodruoJ reqto pue ua8eloo seteJf,ester{l II0l enssrt alrtf,euuor yo adlr uouruoJ V lselqoJqrl .srarsa $-Z elqe1-:17-7 anfug) ld.rersayoqr 'sprdrloqdsoqd;o pue 'seprracdlSr.rr srsoqtu.{sro; rosrncard e pue usrloqetaru Surrnp d8raua Jo ef,rnos roleu e fpua auo te dnor8 p seq leql urer{r uoqrprorpdq Buol duy proe ,(rtey ldxoqrec (q6E-7 arn8rg) 'uorlnlos eqt urort +H oml pup elnreloru rouop E luo{ suorlf,ele o^{,t Surtdeore dg rarrrer uoJFale ue sE suorlJunJ tpl{l elnlolou crue8ro JlerusV (aprloopnurp eururpe uneg) qyg 'ralros IIeJ pale^llr"_ef,uef,seronu ees sf,vc (t-f f alqet) 'uotsnJJrypawilpo] pe;;1e) osle fuorsngrp aldurrs lq peurerqo ter{t u€qt rateer8 efet e fe ruerper8 uorlerluef,uof,slr u.taopeuerqrueu IIeJ e ssoJJeelnJeloru Iletus ro uor ue Jo uodsuerl popre-urotord lJodsuerl pelplrlrJel 'aseqluds

dIV ees

xaldurot rgog

UVSSO'19

8-9

(ZE-Spue 1g-9 sern8rg)aua8 pauolJ str uor; uralord € Jo stunoue a8rel acnpord ol Jo tseJelur ;o aua8 e ro; drerqrl VNq E ueerrs ol pasn lurelord paporue eqt Jo srsaqludsslrerrp areql pue IIel lsoq elqelrns p otur VNCf, ro aua8 e serrrpf, teqt snrrl ro prruseld perJrpour V ropa,r uorssardxe (99-91 orn8tg) 'urlrodrur osle aog 'tuseld -ordc aql or xalduroo arod reelcnu e q8norqr o8rec aqt slrodsuerl (dpure;redns ospdlg eql to roqureu e) uea Jo pre aqr qlr-^apue sna]rnu eql ur urelo.rd ,,oBteJ,, p spurq teqt uratord urgodxo 'rrrureglopue 1o alrsoddo !paecord daqt se teaq oseeler snr{t pue 'gy 1(dpqruo ur e8ueqr e,rrleSou ? aleq ter{t sassaJordpue suorlreor ol Surrra;eg JnuJeqloxe (1-g arnSrg) 'ruseldoldr eql ur slrer (y)d1od peuouoqs qrr^asVNUur ro snelf,nu eqt ur svNgur-ard pessacord dlrodordrur pue suorlur pef,rlds lno sepe;3ap teqt xalduoc Surureluoc-esealcnuoxaa8rel eruosoxa le,ne porcarrp euerqueu

(g-91 arn8rg) .losordr eqr uro{ IIel e to alpJ er.{J oce; rruseydoxe

.slueuele GL-9 pue g p-9 sarn8rg) vNC elrqou ;o uorrrsod -suert .,{qro seueSaleredas ol!\.1 suoJtur uee./r\tequolteurgruoJ ,o ,.a.r) -er .{g sauo Surtsrxaerduror; (suoxo Jo suorleurquoc mau sauaS .Lrau Surtean ro; ssacord dreuortnlorrE Eulg;nqs uoxa 'uorlur os]E ees 'Olnrelol'u vNul ro rVNUr 'VNU* arnlpru e Jo lred se urseldoldc eql sorlf,Eerter1l (tdrnsuerl dreurrd srr yo ro) eue8crlodrelna e;o luau8a5 uoxe 'llar e Jo euergruerueuseld eql qtr^ elf,rse^oql;o uorsn; dq elJrsa^ PoPunoq-euerqruarue urqtr.ry\peureluoJ (surelord xrJlBru 'seuourroq ''3'a) salnraloru relnllaJeJlur aseeleU srsoldcoxe to 'rruotrapue ;o atrsoddo lpeaoord degr se r(Erauaearl es€olorsnr{rpue CV e^rte -8au e a,req leqt sessecordpup suorlf,paror Surr.re;eg cruo8raxa 'slaf,uEf urelJaf, JoJ {srJ pespaJf,urr{lr.^apalerf,ossE sr ssol rreql pue dlrlepg ;o oar8ep q8rg e grr.lr aterado .(lerurou srualsdsrredor aseql 'suaEourcreool arnsodxo Jo uorleurueop ro uorleurrndep snoeueluods ot anp o8erupp y11q Burrreder .ruelsds rredar-uorsllxo ro1 srusrueqJar.uIEJeAesJo ouo VNq (E-1 arn8lg) 'selo^re{ord pup sasnrra tdacxa susrueS.ro11esapnlcul'oKn4na peller os]E !sursrueSrodep -uJepotu to so8eauqlreuortnlole tJunsrp Jarql oqr Jo euo salntrtsuof, (sol]eue8ro pup snelJnu pasolJua-eueJqueu e Surureluoc leql sllal eJor.uJo euo to pesoduroc ,stusrue8loto ssel3 solodJe{ne (eEg-9 ernSrg) 'urleuorqroreleq osle ae5 'suor8ar e,rrlrz,{lleuorldrrcsuerl tsoru sapnlf,ur !saruosoruorqceseqdralur ur tuasard uneruoJqJ;o suortrod pasuapuof,ssJ-I urleruoJqJna (S-IZ pup 9-91 sarn8rg)'lrorreru euoq oqr ur s11ec rolrueSord prorqr -d.re;o uorlerluererlrp pup uorleraJrlord aql Sunnpur dq s11oc poolq per Jo uortf,npo.rd sraSSrrtreqr aurlordr y (odg) urrarodorqilra

ru'rsuof,uorlprJossrp eqrpue'y slenbagxl r""ir/.LtJ"ffi:T] (I-6I AIqET) 'sllel Jo suorlrunJlelruer{rorq pue lueudola^ep rr{t tf,erreuer ur troddnslerntf,nJlssepr,rord sereds tJ 'ureqfueerrqeq PUEsenssrt eqt otur slor dq peterressaprrerlcresdlod pue suralord;o 1ro.u -qsau Surlelr8rprerur xalduoc y (WCS) xrrleru relnllererlxe

'uorlJeeJ p ro; sluels -se eqt 'gV g y'uortcear p Surpurg E roC + -uotr eler asreler pue pre^\roJ (y) fuefsuot unrrqtlrnbe Jo orre;

dlrcrlrcodsruoroJ;rpJo serpoq -rtue ot purq teql sadolrda aldrrlnur ssessoddllensn sua8rlue ural -o.rde8rel dpoqlrue ot ro sl]af, ro g uo roldecar rrynads-ua8rlue I ue ot spurq terlt elnrelou ua8rlue ue to rred er{J adolrda

6-9

AUVSSO'19

eJEuauo senprsaldueru qcrq,la,ur saprrer{tJesrpSurleodarE ro Jeru -.{1odpa8reqr dlq8rq 'reourT'3uo1 y (5y5) ueefltouruesor,(18 'suratordoc.,{13 are surelord aupJqruaru .,{ueu pue suralord pelerf,estsol4l 'pa{url .{1tue1e.toc are sureqf, epr.reqcreso8rloaJoruJo auo r.{Jrr{.r\ o1 uralord.(uy urelordocr(1t (E-71 arn8rg) AJV Jo uonrnp -ord aqr qrr.tr 1osol.(c oqt ur ate,rnr.(d ro atetrel ol dlleJrgoreeup peper8ap are sre8ns qrrq^^ ur derrrqred trloqerory srsdyoodl8 'auerqrueruetuseld eql ur puno; dluouruoc :poluq dltuele,tor sr urer{r oterpdr{oqrer uoqs e qrrq/!{ or prdrl duy prdqoc,{16 'slleJ ellsnru pue ralrl ur dlrreurrrd puno; lsleurrue ur alerp -lqoqrec e8erols ,(reurrd aql sr lerll 'strun asocnlS ;o lle,rrsnlc -xe pasodtuor 'eprregcres,{1odpaqrue.rq '3uo1 dro,r y uo8ocdlE (g-11 ern8rg)'tuarper8 uorleJluef,uof, slr u.41\oP soueJqrueu IIeJ ssoJf,E(sreSnsJar{]o .,\{aJe pue) asocnlSuodsuert terll'serrlaq n Suruueds-auerqurau71 8ur -uretuoJ 'suratold eueJqueusuerr dpure; y suralord I11C ;o 'dleartoedser 's11erlueld pup sllef, 'qcrels pue l€rurue ur d8reue oJols 01 pesn eJ€ ue3ocdl8 'srarudlod asocnlSa8rel eq1 'sller tsoru ur ]en; rrlogEteru dreurrd Jr{t sr l€rlt (re8ns) eprJerllJesouou uoqJef,-xrs esoonlE 's1a,ra1 asoonlSPoolq lortuoJ 01 uqnsur qlr.r\ slre :re^rl eqt dq esolnlS ol ua3ocd13Jo uorsraluor eqt sraSSrrtteqt srelsr crteercued1o sller eqr ur pacnpord euouroq apudad y uoE€rnlt (y1-97 arn8rg) 'sesuodsorounrururur alros pue srolJel rrqdorl e;4er;.rptlSoDtu pue fuorgeuroy asdeuds ur uorlJunj salKcotlso tsqreoqsuqedru acnpord satCcotpuapo8rlo pue silal uuomq)S (sad[J rno; eqt lO 'slln pu8 payet osle lsaslndurr lerrrlrele lrnpuor rou op 'suornau olrlun 'teql anssrt snolrou Jo sllal Suruoddns elt 'llof, rure6 oas 1lar euq-urat 'sataue8 aqt q8no.rqrtxau eql ot uortEreuaSeuo ruoJ, Pelt[usuEJl ]ErretErucrlaueSaql
'usrue8roJo p.{g parrrecuollpluJotulJIleuaSleloJ etuouat IIaf, 'Peseq-vNC'sro{relurElnrrlolu osle ees 'lenpl^Ipul ue .ro '110ce 'etuosotuorqre 'eua3 pe4ur1 e rot tleles ro dJrluePro1.{lleluaurrrodxepesnore leql addlouaqd elqetratepdpseaue qll.ry\pelerrossesele]lv sJo{rPIuf,Ilauat

eqrJouorleul*rrrro't?lrtJf;il:tJJ; e^Ir€rer seuo'Jouorlrsod (4-g arn8rg)'soua8 tueJaJtrpJo erueserll uI ore ad&ouogdluetnu orueseql qll^ sluelnlu o.t\.l uI suollelnlu e IssoJaJJI eurlgJoleP uEf, srs,tlPuBuoll -erueualdruo3 '.,(errtqredleJlluaqf,olq erues aqt ro; perrnbar st ural -o.ld paporue esoql\ euoS luaJaulp e uI uollelnlu e saIJreJ I{f,II{.{\ 's11eoproldeq ruor; Pat€reue8 s11orsnoSdzoraleqploldlP Jo r{lpe uolleluauraldruoJ f,Ilaua8 ur uorlf,unJ ad-{r-p1rlr e ro uollerolseu (t-l atqet) 'sutato.rduI sPIf,eoulrue lJlf,eds VNU ro VNq1 ul ePoc crleuo8 (suopoo) sleldrr aprroepnu dqareq^{ selnr to ros aql $7- 9 en&g\'aouanbas epltoelrnu eql ur sa8ueqr llelus ot anp eJuoBre^IPluanbesgnspue eua8 leJlsolue uoruruof, e;o uorlecqdnp dq esore 1€gr souoSto res -{pue1 aue6 '(urelord e Jo uollf,nPoJo Aluolllluof, rsotu) eddrouaqd alqe,trosqouE otul Pelre^uor st eua8 E uI PaPor -ue uorleruJoJur eql I{JrI{.A dg ssecord IIPJO^O uorssardxo euat 'sau0b eruos to uorsserdxe Iorluof, dlaq sy|trau 'o uollelsuerl PUP 'uortezrlrqels'Sutssacord aql Sulluengul suslueqf,eur g8noqr -1e 'uorldlrcsueJl to uorleln8er sr uoluluof, lsoy,q 'uorssardxa euat Surteln8er uI peAIo^uI srusluel{tretuaql Jo IIV lorluoc auat 'lrun uondrrcsuerl osle aas 'VNd ro oPlldeddlod leuortcunl E Jo uortJnpo.rd ro; dressalou-suor8ar lorluoo-uorldrrlsuerl pue 'suorlur tsuoxa SurpnlJul-aJuenbes VNC eJllue eql sI tI 'sural relnrelou uI 'lxeu oql ot uolleJoua8 ouo IUory uorl€uro; -ur sarrref,qrrq.tr ltlrparal{ Jo llun leuolllunJ pue leersdq4 euoE (1 1-77 enfu g)' urapopue pue'uraposatu'turapolra 'sra,te1urat aorl{l eql o1 estr Sur,rr8 'eleur8e,tut lsdJorselq oql uorlelrulset Jo sllal qJrr{.lrtuI So,tJgtue ]eturue dlreo ur sseJord 'sllaf,aql uao.,taleq solnr (gp-61 ern8rg) 'eretuseporuseldosle ees -elou llerus Pue suol ;o a8esseds.4.olleleql s]leJ leruluP lurf,E[PB uolpunl det ;o suseldordr eqt Sullteuuof, Ieuueql Paurl-ulslord (q'eLZ-ZZarn8rg) 'srxe rorratsodolretue oqt Suole Sutureued ,(1.reo ur uolllunt IEIJI sJotJet uorldrJf,suerl epoJuo ye lalo?'lz eql uI sVNUru leurol -eu ruo{ pacnpord srotreJ uottdrrcsuerl dq odrqua dpea oql ut 'ofqdoso'tQ rrl seuat deE pel€^Ilf,E ar€ leql sauaS;o dnor8 e 'lenpr^Ipul 1\,\aue B uolun 'uotlcnp to tuaudolJ^eP eql selelllur 334 ue pue rurads Jo -ordar ruJat rosrnf,erd;o srsoratu.(q pecnpord (33e lenxes ur !s11eo proldeq pezrlercad5 oleruet ue .ro rurods e ral{tla slelulue ur) 11ac 'a1ldc z9'rD'oJ 1ar aa5 eseqd

(g1-5 arn8rg) 'luelnru relncrlred e ut euo8 e^llte1ePe egr dlrruept e1 drerqt1 to uourunJ eql sarolsrr leqr eua8 addr-p1r.u VNC E Suruaercslo1 JJnpoJoJd uotleluarualduoJ IPuoIlJunJ 'lnJJo Suo.rlse seq (sseJoJdlaqlo Jo) uollJeJJ .,{ruapuet o1 'uollf,Eer E l€qt salerlPul cv Jo enlE^ e^IleSeu o3re1y IBf,IIUeI{r

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a.tne8au eJour e ur 8ut11nsar'lsel te I]aJ e to aueJqueu euseld aLIl ssorf,e slsrxa dlletuJou leql Iellualod rrrlcale e^IleSeu ef,eJ -crloso/c eqt Jo epnrlu8eu eqt uI eseerf,ul uoltezlrelodreddq (1 1-7 a.rn8rg)'puoq n uotpotalut tqoqdolpCq e pe11erdluoru -ruor :Jelp,^Arllr,,!r suoltf,EJelul lJeJrp Jlaql ezlrulullll ol se os uoll -n1ossnoanbe ur Joqlo r{Jea qll.4aelelf,oss€01 selnJelou Jo sued Jo selnrelour .reloduou yo ,{ruapuar aq1 rooylo rtqoqdorpdq reloduou osle aos ret€,4,ruI elgnlosul ro elqnlos dlrood 'leraua8 ur .'.raternqrr.u .(la.trtcaga Suttceralut loN crgoqdorpr(q :elod osle eas rale.^Aqrrrn dla.trrcege 3ur1cera1u1 lrpqdorpfq (g-7 ern8rg) 'spuerts pIf,Prlelf,nu uoo.4uaqsrred aseq;o uonerurot ur pue suletoJd;o uorteurotuoJ el{t Surzqtqelsut ]ue1 -.rodu1 'a8.reqr a,rtltsod lzryed e Sutdr.rec tuole uaSorPlq P PUE aS.reqcalrleBeu lerlred e Sutd.rrer(ueSortruJo uo8dxo dluotutuoc) ruote ue uea.^ataquollJ€Jalur lualeloJuou V puog uatorpdq 'sruote uaSorp -dq pue uoqrpJ dluo Sututetuor Punoduol duY uoqreJorP^rl (5g-6 arnSrg) '11areruoladu E I{tI.Aa IIar g Sunnpord-lpoqtlue IeruJou e Jo uolsn1 dq peurro; ldpoqrlue leuolJouoru ecnpord pue leuounul ere leql sl1ac pr.rqdq Jo auolJ V europtrqdq 'seruenbas VNU ro y51q cr;tcedsrcarap 'Puerls ot sde.la,snolrel ut .{lleluaurtrodxa pesn VNU euo PUE

'sulelord ureUef, 'urTedruo8urqdslna dueru ur uouruoJ Jrloru lsrarulporateq ro -ouoq rr;rcads urroJ reql sef,rleg D o^.\l Jo pesodruor ytoru lernlrnrls lror-palror ;o ed,{l y raddrz aunnol 'nlls ur ueql lratap ot slua8eor se ro suralo.rdocdpd;rrnd ot .,{qderSoteurorqodlrurge ur pesn aq upf, pue .runlncrla.rcruseld -opua aqt ur suratordordlS auros ;o 8urp1o; redord or{t ur tsrsse sultre.I 'sreSnsrr;rcads or lpq8rr spurq teqr uralo.rd duy urlral (gg-y arn8rg) 'puerls tu€Ee1 osle ees 'IroJ uorterrlda.r eqr Jo luauoloru se alues eql sr srseqlu.{spuerls-SurpeelJo uorlf,arrp eqI 'uorl -Jarlp e ,t /S eqt ur srsaqtu,(ssnonurluof, , q 4roy uorlerrlder e l€ peturo; sPuertsy51q rerq8nep o.,v\teqr Jo euo puerrs tulppel (1p-77 atnfug) 'sere1ruaragrp Sununsse s11ec lualz,rrnbe-reouJo tuele,rrnbaluace(pe ur sllnseJtegl ssooord letueudole,r.ap peterpeu-leu8rs 1ue1;odu1 uorlrqrqul leralel 'JPralelosEqaes IererEI 'adola,rueJeelf,nu egl Jo eJEJJnsrouur eqt uo ,eurruel JpelJnu eql ({ro.{\teu snoJqrJ e urroJ teql surelord lueuelrJ olerperurolur;o dnor8 y suluel (17-61 ern8rg).euruel pspq II? ur punot sr leql uralord xrJleru aArseqperllnu crreurrlo.rateq a8.rel ururuel (gg-y a;n8rg) 'puerls turpeal osle ras .paurof rarel pue uortf,aJrp ,€ <- ,S eqt ur pezrsoqtu.,ts are qcrq.vr,(stuau8e;; ,t:oqs qezelg) sluau8es snonurluoJsrp >poJ se uorlerrldor e tE peturot spuerls y51q rarq8nep o^ r erlt Jo euo puerrs turtEey

esr.4arer{ro ueurerele(pardn;srp) I'uorrunJuouTllt"fft,ffi; -er dq aue8 crlrcads p Jo uortplrlJeur a^rlf,eles

auot ,lno>1oour1

'sluelnIII uorlJun, -Jo-ssol SurleyosrJoJ spoqlau rneua8 IEJrsselJot alqeuarue lou ale teqt srusrueSrour llrelnortred ,urelord e;o d1r,rr1ce eqr Burcnp -rr roJ InJasn JVNUIs ;o :sn dq VNUIU rr;nads E ,o uorlelsuerl Surlrqrgur,{lleruerurradxe roy enbruqcal VNUIS .u,r.op1oou4

/{.uvSso'19

zr-9.

(77-E arn8rg) 'pue -3q str ro; rolderar e 1o trtruqle ar{t ro elnf,alou pegodsuerl aql ro; uralord l;odsuert e p trlruye eqt seqrJlsep .raletuered relrurs 'iuoisuo) stlaoq)rw erll pellpJ osle :elEJuorlf,?Jr V leurrxEur-JlEr{ oqt sp1er,(leql uortPrtueJuof, aleJtsqns aqt slenba Pue elpJlsqns -) str :o; arudzuaue;o dtrur;;e eqt saqrrrsepleqt relourered y (69-gp arn8rg)'eseqdeue Surrnp salod aqr pJen,\ol seuosoruorr{f, Jo luorualoru ur alor elrlf,e ue sdeld !1ee aqr 1o salod oypurdsoqt prelr^otpuetxe selnqnl -oJf,rur rlf,rq^ , ruoJ, aruosouoJqJ f,rlolru rlJEa ,o alauoJlual arll Jpou ro te pa]?rol eJntJnJls uratord releplnur y aloqJoleur{ 'selnt0loru Jo uorlou

aqt sE qJns 'tuarua,roru ;o lSreug

i(8raue JrleuDI

'fi2-st q8norqr 51-91 sarn8rg) 'srsolrur Surrnp tuarualoru euosoruorr{J ur elor e deld pue sallaue8ro pue selJrsel trodsuerl ueJ surseur) 'olnqnlortrru € Jo pue (+) eqr pre.,taot a,rour ol srs.,(1orp.(q41y dq peseela; .(8reua esn leql surelord Joloru Jo ssEIJV sursauDl (Eg-E ern8rg) 'soseleqdsoqdosle aa5 'suratord rElnller dueur;o dtr,rrlre oqr Surleln8ar ur alor IeJ -rlrr e deld 'senprseraursor.(l ro 'ouruoa.rqt'aurres rrlrcads aleydr -oqdsoqd qrrg,u'seseur{ uretord 'etertsqns e ol dIV ruor; dnor8 areqdsoqd (,{")leururar eql sratsu€rl teql aru.{zua uy eseuDl 'arroler oes

(pc1) euopropl

(9y-91 ernSrg) 'stuaruelrJ crraudlodoraleq otur olquessp teqt sllel lerlaqrrde ur punoJ suralord luouelrJ alerpauJelur ;o dnor8 y surleJe{ (arnBr; Suruedo 9 ;atdeq3) '11accrtodre4ne e Jo souosouorqo aseqdeteu 'sezrs taqurng edr(lodre4 ,o tas errtue er{t Jo sedeqs pue 'snJIJnuJqr Jo tno ;o ur Sur,rou surelord o8rec ur aruanbes leu8ls cr;rcadse ol spurq urreqdolre4 qJeE 'rpoq dlleuorsecco .ro 'urgodxe 'urilodurr uE sE suorlJunJ teqt suretord lrodsuert reel)nu;o dlrue; E to euo urreqdodre4

'sllef, lno Jo ur Jalp^{ to Jo luerue^oru leu ou sssnpJ1r leql qtns sr uorleJluef,uoJ etnlos esoq.l, uoDn]os E ol SurJJeJeU truolosr '8urcrlds e^rleuJalle seo8rapun ldrrcsuerl d.returrd asoq.u eue8 o13urse dg ro saue8 tuarottrp dg paporua eq deu srurotosl relr -rurs ere sertrlrlJp pup dpq8rls ragrp socuanbaspne lereue8 esoq,4a ourue esoq.[ urelord eur€seqt to sruJoJIeJeAesto euo urolosr (9E-g arn8rg) 'plerJ JrJlJela u? ur eloru lou saop eJoJeleqt pue onz;o elreqr tau p sEq e]nraloru pa8reqc dllerruarod rar{to ro urelord pe,rlos -sIP E I{rII{.{{ le uollnlos e 1o gd eq1 (1d) lurod rulrolaosr 'aleqdsoqdsrrl.s't'I €dI lollsoulaes 'puoq ?tuot pe11erdluoruuor !(uorue) uor dlarrrte8eu pa8reqc (uorlec) pue uor pe8reqc dlelrl -rsod e uee.{\leq uorlf,EJelur luoploJuou V uorlJeJalur Jruor

uEursrnrroreqlssacord ro uo'r'ar ,"Tiil'jt"i:tlXi ]f,Elur "

'ursrueSro ue ur esoql ruoryernllnr ur 3ur -.,u,or3 s11ac :tJertxeaeJJ-]letpJtEI ot pasnserurleuros qsrn8urtsrp -osrue ur areld 3urlel ssecordro uortceere Surlouaq oJlla ur

€r - 9

AUVSSO''ID

ur alor tueuodrur ue .,{€ldstuelllelrJorf,rw'ruawou utpp pelle) osle:ullte (g) re1nqo13 rlreruouoruto uorlezrrarullod,{q peuroJ sI leqt (releuerp ur tuu :eqry tuouelrJorf,rru 1:l lereleqsord3 '-) oes luelsuoJ srIeEqJrW

Surlel,fuotldsoqddq sasuodserrelnllor ar€rparu teqr pup srolreJ qluor8 ruoreJ;rp dueu dg uouelnturts 11ecot esuodser ur pele^ -rlf,e erp teqt soseurl uralord;o .{purey e;o duy aseuDl dVW 'u6rueq osle eas 'srselseleruo8rapun rolpue enssrt

Surpunorrns

leruJou (c9-91arn8rg)'uorrnlos epe^ur uef, rErlr sller Jorunl Jo Jorunl e ol SuuJOJOu lueuSqeru snoanbeur dlsnoeuetuods ru;o1terlt selnlalorurrqtedrqdueraqto ro sprdqoqdsoqd;oare8arSSe elqnlos-rele.a\ lecr.reqds V allarrru

wz-vTPue

g7-y7 san8r.g)'s1yeo Surruaserd-ue8rtueleuorsse;o.rd.,{q .seynra -loru 1I sszle !s11ac peteelrnu 11edlreau.(g .(1ar'rlnlrtsuocpassardxa ore selnralotu sspl)'sllao I ol uou€tuasard ueSrtue ro; parrnb I ,suretord (y1aspue) u8raro; uor; -er ere pue sller to ateJrns aqt uo pe^rrep 'saprlded deldsrp teqr surelordordlg solnroloru CHW 'xaldruor drilqped-orolsrq

roleru aa5

f,HI I

'qlrrrorS drepuores Jo seorpJo lueuqsrlqElsa pue ur8rro Jo elrs Jrar{t urog s]lel Jef,uet Jo peerd5 srselsuleru (yg-gp ernSrg) 'se1odelpurds eql pre.r\ot ele3ar8es ol pelrets lad tou e^eq tnq 'alpurds rrtotlru ap yo salod eqt uea^4,leq tuelsrprnbe pauSqe ere saruosoruoJrltpasuopuof,gJlym re srsolrru;o e8et5 eseqdeteu 'VNUtu eas

(17-y7 antug) 'suerunq u xalduoc V'IH eql pue errru w xaldwot Z,-H eqt paylec lsurelord 'uorteluaserd ua8rtue ro; parrnbar tuauelduoo eruos se IIaA, se suralord rer{to pue solnJeloruf,HW II ss€1f,pup I ssep epof,uoleql saua8ruarefpe yo le5 (CffW) xelduor trrruqFedruorolsrqroleu 'seuDloldr

Jo aJrnos ro[Bu e erc pve s11arturluasord-uotrlue leuorsse;ordse uonlun; laql'sroldaoar a{rlJIoI Er^ srelrpru uaSoqredJo surel -ted peorq tratep uef, l€ql sellf,olnal ctrdco8erld sateqdorceru 'suotleP Puesnoqt .ry\e;e ueqt .ralee.r3 sseu relnJaloru E qlr-la (aprreqroesdlod 'prJe JralJnu 'uratord e ''B'a) e]nre]our crraurdlod ,(11ensn'o8re1 duy olnJoloruorJpru 'a1cdr

y51g ro8uesseur

'senssrl fiyZZpve g-17 sa;n8rg) Jeqlo pue 'poolq 'alosnu (enssue^rttreuuoo'p:oqrolou eql ot asrr sa,rr8lruropopue pue ruJapolf,o erll uee^{teq 3urd1,odrquta lerurue eqr Jo srad€l 11ec.(:eurrd eorqt eql Jo elppru eql ruraposoru 'sl€Iurue ur ruJopolro Jo ruJsposolu 0q1 rar{treruo{ pa^rrop 's11arpaqteue ,'(1asoo1 pue pazrueSrodlesool;o pesodruoc'onssrl errrlJauuoccruor(rqua aJnlel'ul'ul arulqcuoseur 'suolsrJaruruort esrJesaJnlJnJls tlnpe Jr{t 11y .stueld uI slooJ pue sloor{s Surrrnor8;o sdrf Oqt tp peur€turErueJe tEql sl1acSurpr.,lrp'patertuora;;rpun ;o dno.r8 pezrueSr6 rualsrraru (g-11 arn8rg) 'usruprlteu lrodsue.n er{t Jo ssel -pre8a: oueJqrueru relnllel e ssoJJE selnJelou Ilprus Jo suor JrJrJ -ads arour Jo euo sel€rpeu luelualoru legt urato.rdaueJqruaru Jo urelord ilodsuerl auprqrueru ler8erur due ro; rurel e^rtJello] (8t-tt pue 11-11sa-tn8rg) rel{to aql uo (suorue) suor e.rrle8au pup eprs euo uo (suorrer) suor alrlrsod Jo sseJxerqSqs aqt ol onp aueJqruerue ssorJe ,st1o,r ur pessardxo 'aouaregrp lertualod JrJlJelg lerluolod au?Jqrueu (E-9 arn8rg) '1ec proldrp lertrur ue uo.r; (salerueE)s11ec ptoldeq tuele.a,rnbeuoudllecrreue8rno; to uortrnpo;d ur stlnsag 'uottectldar VNC Jo punor euo lluo qrrrrr suorsrlrp relnllat pue JpelJnuo,rrssaJJns o,r1 sesr.rdruoc !s11eru;e3;o uouernleru Surrnp srnf,Joterll uorsrlrp 11ar;o adl.r lercedse 'selod;elne u1 srsoraru 'uorldr.rcsuer 8ur1e1nrur1s ur rolelrtf,e ftV-Lpue 1g-4 sarn8rg) -oJ e sE suonf,unJ lrarourord p tE punog aseraudlod 1 VNU ot pue Jaf,upqueue ot punoq srot€ rtJe leuorldrrcsuert uee.,lrlega8prrq re1 -nf,alou e srxroJ tpql xo]druof, urelordrllnu e3.re1dra,r y Jolerpau 'e;nleredual pue 'ured 'peeq pue sqtuq eqt to sluaue^ou pue suortrsodoqt'qcnot ot puodsar puE senssrlsnorrel ur pappeqrueeJe IEI{I soJnFnjls drosuas;o sedft lere^esJo duv rosuasoupqf,oru '*'-,1 aes d1rcolo,r Ierurxeru

Uz-9t puegz-gt

sarn8rg) 'suratord tagJet Jer.ltopue sJotJEJuorldr;csue.n crFads

11area5

aseqd (crrorrur) 141

(7-6 arn8rg)'.(Seqdorne ur slueuoduror relnller to pue srsoldoopua dq pozrleurorur s]Err 'saur.(zuecrrdlorpdq surer -eleru Jo uorleper3ap ur suortJunJ pue -uor'g-y;o ue seq reqr alleue8ro 11eur5 euososdl 11d leuratur 'llel eql yo srsdlSursner dllenrua,ra 'salcrtred IeJrA^aeuto uorteruJot ol speol uortelllf,E luenbasqns 'passardxa tou sr lnq VN( Iprrerrpq oqt qtr.e\ 3uo1e palerqdo: pue atuoua8 ller-tsoq oqt otur pelerodrocur sr (e8eqdor.rerceg) ,(ue6os,{1 snrr^ Ieuetf,€g e Jo vNC eql qlnl^ ur uouououorld 'slueluof, ar{t asEeleJ Jo PUP arnldnr lq 1ec e Jo uollrnrlse6l stsdl ou€rqruaru euseld oqr to 'sller raruer pue 's11ac u8raro; 's1yorpatce;ut-errelreg pue -sn.rr.r. rot elqrsuodsarare (s11er1) sor.{ooqdurll 1 fsor 3ur-{olsop -poqnu? g) serdcoqdudl ;o uorlrnpo.ld .ro; alqrsuodsarare (s11ec g 'sesuodsereunrulur elprpeu pue (suo8rlue) salnrelour u8tero; sofrboqdudl azruaotet uef, teqt sllel Poolq etltl.v\ to soss€lJo^\I 'Jlel E urr..lll^\lusrul.red -ruof, pepunoq-eueJqrueure Jo orunlol rolrelul aql ro (euttsalut eql Jo lessa^e ''3'e) ernlcnrls JPInqnl e urqlr^{ ereds eq1 uounl (17-91 en?r.g) re^rt eqr ol .(llErJedso'senssrtueelueq sralsaldratsalor1JJo r.uJoteqt uI IoJel -selorlr;o rouodsuerl dreurrd p sr teql '991-g uratordodqode 8ur -uretuof, 'uralordodrl to sselr V (1q1) urarordodq dtrsuap-,tol 'suosodsuEJloJloJ IeJI Pue vNq lerr,rorlar polerS (srlpd -atur eseq 009 ol dn 8uI Jo uor8ar Surpoc aqr {uelJ lpr{t -ureluof,'sacuanbasleedar lcerrq (sUtf) sleedor punural Euol 'snrol ,{dnlf,o aueS.relnrrilede;o so1e1peql IIV 'eruosolu eruEs el{l (sf,Iteua8 -orqf, E uo aueSe uI (rcol '1d) sncol to etrs cryrcadseql (c9-91 arnSrg) 'suralord euerqruau ureluof, deur pue sprdqoqdsoqd ruoJt oJtrl ur struoJ leqt JoIJetuI snoanbe uE qlr.{r erntrnrts ra,(e1rq prdqoqdsoqd lerrreqds IeIf,IJIITV auosodq '(fqf ) urelordodq dtrsuap-no1osle aa5 dpoq eqr rnog8norgr sprdq Jo re;suert sseruuI suollrunJ teql duy urelordodl xolduroc prdrl pue uretord alqnlos-rale.4a'a3re1

AUVSSO'19

vt-9

'sd'I{U sopnlJurlsetpntse8e1uqJIteueBuI Intasnerepue sercadserueser{t 1o (srustqd,tou.tt4od VNAI sl?npl^IpulSuoure dre,r teqt saouanbasy51q peseq-VNq 'srr{rptu rplnrelotu

g7-71antuglr 'sluetllellJ urqr (urtce) Pup stueluElll (ursoitu) >prqr ;o pesod -ruoJ soJoruoJres;o derre Surleadar reln8ar e Jo Surlsrsuocsllar olf,snu;o urseldor.(curqlr.^ sernlJnrls repuels (3uo'I lrrqgodru

(gE-6ernSrg)'euoprrq,(qe;o asndq dlleruerurradxa pacnpordaq uel tI'(adorrde)uoSlluealSuls E snql pue 11arg a13uts e sazruSora.r tegl urotord snoaueSoruoq e;o dueSordeqr dq pernpo:d dpoqrruy dpoqtrueleuolrouou

(97-71 atnfi g)'uortecolsuert a]f,rsel elerpeu osl€ pue srseur{otdJ pue uortJeJtuof, elJsnru Surrnp sluourelloJf,rru urlre 3uo1e e^nou surso,(141d1r,rr1ceasedIV pele]nurts-urlf,e eleq leqt surolord rolou to sselJ V surso,{tu

(91-E7 ern8rg) 'uorrcnpuor aslndrur1o peads eqr sespaJJurpup suoxe el€rqaual punore redel Surtelnsur up sruroJlpqt euerquaur IIef, pezrlerradspalcer5 qreaqs urladu 'lonpord aue8 egr Jo uorlf,unt eql ur uorleretle ue sasneodluoru -ruor leua8 e18urse ur dllensn 'ouosoruo:ql e yo ocuenbaseprloall -nu eql ur e8ueqJ alqetrJJrl'luauerurad e 'scrlaua8uy uorlElnru 'suorlElnrx seJnpurteqt lue8e lecrsdqd ro Ief,rrueqf,v sureqc aprtdad.(1odlerairesSururetuor 'sureto.rdrog

uaSelnru

'(slrunqnsro) f,rJeruDlnru

'senssrt rurlJouoJqrJ 'eurwel duBtu ur pue luasard lpseq eqr Jo ruou -oduroc roleur e 'ururruel epnlf,ur saldruexg 'euerqueru IIel eql ot stuauodurof,xrJtpru Sur4uqssorcdgJreql 'sroldelar eleJrns-lleJ ot pu? xrJleru JelnllaJerlxo eqt;o sruauodruof,ror{to or purq reql sutetord elqlxelJ Suoy 1o dno.r3 surelord xrrleru e^rseqperlFru (g-91 arn8rg) 'sller ur selngnlorrru sezr -ue8.rotegr (,{poq pseq 'a1odalpurds 'eruosorluer ''3'a) arnlrnrls due ro; ruret IErauaC (ratuer Surzruetro-alnqnlorf,nu) f,OIW (77-g e.rn8rg)'xeydu-rooarod reallnu eqt ur surrodorlf,nu qlr.r\ dlluarsue.rlSurtrBretur ,{q ulseldor.(r ot snalJnu aqt tuory uodxe rIel{t slrerlp pue (s451gur)selcrtred uretordoapnuoqrr Sururetuoc -vNgur ot spurq leql urarord f,rrerurporelaq V rauodxe-dN1ru (91-y ern8tg) 'uorlelsuerl osle aas 'VNutu leuorrcury plerd ot SutssecordseoS:epun(ldrrcsuert l.reurrrd) trnpord VNU Ierrlur (selodrelna aql uy 'aseraudlod yNU lq VNO Jo uorldursuerl Iq pacnpord sr lI '(etntcnrts drerurrd aql ('a'I) utalord e uI spI)P oullue (y51a ratuassatu) y516ur Jo repro aqr sar;rcedsreqr y51g duy 'suralord cr;neds a1d -Illnu Sulleldrogdsoqd dq srsolrurorur IIOr E Jo of,uerluo sraSSrrl leqt'(yq3) tsEurl luapuedep-uqcdr pue uqcdr rrtolru e;o pasod -urol 'uratord JrreurrpoJeleq V (rotoe; Eurlourord-srsolrru) dcIW 'surso^ru pue'sursouDl'suroudp osle ees uolotu tpln)alow pelleJ osle luorloru drelor .ro reeurTraql -ra ateroueSor srsdlorpdq dIV uro{ d8raue asn leql serudzualecr -ruer.lf,oupqf,oru ssEIf, urolord rolotu Jo leneds e yo roquau duy duadord ]euorlf,unJ cr;neds E r{tr^.\peterf,ossesr uel -to pue eJnlf,etlqf,re p leuorsuerurp-Jerqt ;elnrrlred Jo alrteJrpur sr dllensn;rlotu I€rntf,nrtsy 'p1oJlo,tnlcwts paller osle lacuanbesprce ourue d.reurrd a,rrtcurtsrpe dg patereue8 sr uotlo teql ernlf,nrts dre -rtJel pue drepuocas uorlpurquroo 'surato.rduy Jo luJnlrruts Tuoru Rtt-ZZ arn8rg) 'uort€rtuaJuoJ slr Jo uortJunJ e se luaurdola.rap Surrnp sate; uotoqdroru IIel tuoreurp sar;nads teqt elnreloru Surleu8rs y "(OzHf,)

elnurot

erll qrr.41\.re8ns aldrurs duy

'L-€ : uareq]l.^ eprrpqrJesouoru

'seprJpqJJesouoru puE'seprtoepnu'sprle outue epnpur seldruexg 'raudlod e uroJ ot ad,{t aures aql to srar{to qtr^\ dllecrruaqc pa>lul eq uer lerlt elnreloru 1lerus .{uy roruouoru

(71-7an?r.gl 'a8uer a]dltlnruro asolc ruaql uaeratoq suollJeJelul luale,roluou lB o.t\t Jo seruo uorterurot,lAollpteqr JoaJaqtsuorlrodro sJInJJIoru 'sa8reqr'sadeqseqt -dord yecrsdqd reqlo ro/pue lbplqor{do.rp.,{q uaa.^teg lrJ puDI .,ta1-pue-4ro1 ,{lrreluauoldtuof,rplnJalotu Jo 'ouoredpqJees euoredeqJrElnJeloru 'lueluoloy51q elqesodsuErlees lueuala vNq olrqou (9g-91orn8tg)'sntplpddp)tioitLuPell€rosle t1lerSurpr,rrp aqrJo saprsolrsoddoot ruegl s11nd pue seqsnduoql aqt sarnlderteql (srsolruSurrnpsl1accrlodre4 puE seruosoruoJr{J -naur luaserd'ernlcnrts y olpurdsrrlolru drerodrualpazqercads [dw ees rolJEJ Eurlouord-srsolru (yE-g1 ern8rg)'srsoreru puE srsaurloldrosle eas 'selllosoluofl{Jto requnu proldrp aqr r{trl{ relJnuJatqSn€pluale,rrnbadllerrleueBorrrt Sutrnpord 'sopr,r -rp snalJnuagl,{garaq,r.ssacorder{l 'sl]ol rrlodJelneuI slsolllu 'uorlere;qo.rd setotuordteqt 11ac tolce; qr.,rror8e se qcns 'alnraloru relnllacerlxa duy uoEolru 's11ao rrrodrelnaur dIV erJlJo tsotu 0-U pue 9-6 sarn8rg) Suronporddqaroqr'uorleldroqdsoqda,trtuprxotno sarrre) pue 'y51q sureluol'seuelqruoru orrtrdq pepunor radelrqprdrloqdsoqd -rns sr leqt elleue8roa8rel (errpuoqcotttu'1d)uoupuoqrolnu OZ-B pue eg7-gsarn8rg) 'vNuls oslP ees 'uorlelsuerl slr llqrqur or vNur.u o18urse or azrprrq^(qtsntu sVNUrru Iere^es dpce;redtutseztptrqdqVNUIIU aqr qrrr1.,ta or VNUIU te8:ct e Jo uorl€lsuerlstrqlqurteql (3519) xaldurorSurcuaps pelnpur-VNg ue ruJoJot suratord]ere^esr{tr1( y 'sYNu rosrncerd selPrrossE vNuFu srnlerueql to Puerrsa18urs 3uo1ul seJntlnJlsdrepuocesurdrreq;o suot8arpepupJrs-elqnop ruorJ passeJo.rd are leql '3uo1saprloallnu0E-02 'sypg reln1 '11eurs -1ersnoueSopua snoreurnu;oduy (y51g orcnu)VNUIIIT 'stuerrlnu ilod G-et pue 7-41 sorn8rg) Jo 's11ec -sueJtrot €Jreef,e;Jns agl Surseercur lerlaglrdaleurlsolurJo snoJerunN'sluoru ef,etJns arrrldrosqe aql uo tuesoJdoJErTIr^oJf,ftu -EIrJurlJe to eJoJe SururetuocIIer leurrueuE Jo erEJJns aql uo uortca[ordpere^of,-auerqureu'11eu5(rqr,ronlur'1d)snly.torcru 'f,OIWees raluerturzruetro-alnqnlonrtu (St-gt pue yy-g1 sarn8rg)&Ugrlr rror{lsalelnterpueselngnlorrrru ot spurglpql uretord.(uy (4yyrg)urorordpelerrosse-olnqDorrrru rel (t-8I pup 7-91sarn8r4)'sarntf,nrts 'eqrc;o slueuodruoJ -nlleJreqlo pue 'alpurdsf,rtotrruaqr 'e11a8eg drrrelod leuorlJun;pu€ l€JnlJnrls lueuodurr eJp se]nqnloJcry,q dq pourro; slrqrqxepue sreruououuqnqnr-$'pro uorlezrraurdlod sr teqt (;alauelp ur ruu 97-) :eqry 1era1a1sor,(3elnqnlorf,rru 'vNuru ees vNu orrrru (g-41arn8rg)'sernlrnrtspue suortJunt elJsnlu re]nlleJJeglopuE'luarua,roru 11ar'srseurloldJ'uorlcertuof,

sr-9

AUVSSO'19

(69-y arn8rg)'puertsVNq snonurtuorE rurot 01ase8qy11q dq peurotlprde.r ere pue uorlecrldarVNC ul puerlstufEel eqr to srseqtuds Surrnppeurro;ere ler{t slueru8p{ '(saseq vNq pepuels-a18urs 96611) rroq5 sluerutu{ DIeZDIO

oa.rteSau pue a,rrlrsodro uorlnqlrtslp ctrtetutudseJo oBJpqJJIJltola leu due s{J€l leqt ernlf,nrls ro elnra]oru e ot SuurataU Jeloduou (Zt-Z pue 9-7 sarnSrg) 'suorlJole ;o Surreqs eleurlur ue oAIoAuI tou seop lpql uoll -JpJelur uorlJeJelur luolelotuou IEJrrueqJ1ea.Lldlalrreler duy 'sleJnueqJJrxot Jo 'llplrrcale

'snelf,nueqt ur Jnf,rodlqtuasse oruosoqrJ pue vNU;o Surssero.rd '1eeq 'erunerl leJru€qJarudq posner sanssrrlpoq ol IJnluI qtlll\ :seuosotuorr.lJ pue srsaqtuds otur pazrue8roy51q surpluof,ter{l paterJosse ured ol spuodsar lerlt Josuesoupr{Jary JoldaJrtou s11ar orlodrelneur allaue8ropepunog-auerqruaur a8rel snelrnu (€-z etqPr pve 9I-Z arn8rg) dle,rrlredsar'esoqrrpue esoqrr,koepSururel 'ruole uoqrer -uof, soprloa]rnu sraurdl0dore y5a pup 10 YNCI ,S eqt or dlleraua81(larourre8nsaql ol puoq retseue er^ palurl sdnor8 areqdsoqdaroru ro euo qlr-t{ aprsoeltnu V eprloolf,nu (67-9 ern8rg)'pedder.usl VN( Jo tuaruSesdq-*t e qf,rr{.1\4. punorE surato.rdeuolsrqJo eror padegs -{sp e ;o SunsrsuocurleruoJr.p trun }o lernlf,nJls eruosoopnu (E-ZEIqEI) '(asoqrrdxoep ro esogrr reqtro) esolued e ol pe{url eseq eurp -rurrrddro aurrnde;o pesodurorelnraloru IIErusV oprsoelrnu 'lrodxe pue lrodurrreelc -nu ut seledrcrued(suuodoalonu-3g)sselr euo .xoydruocerod reeltnu eqt dn eletu teqt suralord;o dnor8 a8rel surrodoolcnu (egg-9arn8r4)'palguasseer€ strun -qns auosoqrr pue sJnf,f,o Surssarord pue srsoqluds vNUr erer{^\ s11ao rrtodre4no,o snelf,nuerlt ur ernlJnrts a8rel snloelJnu 'pnp f,relf,nu posolrue agr snld prsder 1err,ry prsdecoalcnu 'sllef,ur sprJef,reltnulreurrd rql ere 'spuoq VNU pue VNC ralsarpoqdsoqddq pe4ur1sepnoelrnuJo raru,(1ody pne rralrnu (69-t orn8rg)'f4truopadns lolQant ptoralspeller osle luortdrrsu€rl eletrlJe leqr saxeldurorrotdecar-pue8rl8ur -uro; '(souoturorlprorots''3'a) salnrelou alqnlos-prdr1 purq rpql srotdara.rJelnllef,eJtur Jo sselJp Jo Jaqruaw roldacarJealf,nu (79-91a;n8rg) 'suralordalqnlos preeqrqrr-^sJdN gSnorqrpat.rodsuert dleart Jo -ralesere seprlred urero.rdoalcnuoqrr pue suratordo8rel ts34g q8norqr esntlrpdlaar; selncelour pue suol 'adolaluereolc lletus -nu aqt ssoJJEspuelxaleqt 'surrodoalf,nu .(la8reypasodruor Jo 'ernlcnrts urolordrllnu 'e3:e1 (34111) xaldruoc arod reopnu 9t-OZ arnSrg) 'stuJurelrJ elerpeuJelur urrupl ,o pasodruooedolanuareolf,nu 0rll Jo eJetJnsJeuuraqt uo {ro.4a10u snoJqrc eurluel Jeelf,nu

'eprreqrceso8rlo Po{ul-o oslE eos'uralordof,dlS E dnor8 ourrue ureqf,-eplsaqr ol perlJel ur enprsaraur8eredseue ;o -le ureqr ePuEr{f,rEsoSrloPeqluPrq v ePuEqrresoErlo Po{urI-N '+dqVN

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'sueSoqted Surpezrur reap dlaq pue uorleuuelJul ol elnqlrtuor '(aur.{zosd1''3'e) seru,{zue3ur,(orlsop leqt slcnpord rer{lo pue -prJelJeq'seur4oldc'saurlouraqc snorJEAelaJJes slrqdorlnau 'pa1e,rr1ceaJuO 'anssrl eql otur eter8ru pue a8eruep enssrt Jo salrs ol pelrprlle ore leqt saldf,o{nel rrrdroSErld spqdortnau 'sernllnrls 'so,{rqure elerqeue^ ur G7-ZZpue gE-77 sern8rg) Ier 'elEld -neu olur sdole^ePteqt rurePolf,aoql Jo uorlrod eql IErneu eqr ;o 3utp1o;ut dq aqnl Iernau oql Jo uolleluroC uoBelruneu '(gNCg) tottel crqdortornru Pe^IreP-uIPrq pue (CCN) roloe; qu,r,or8 e^reu epnlf,ur lsuornau Jo le^I^rns roJ perrnbar are pue s{{ pel]El sroldacar ot purg leqt sJolJBJctqdorr Palelar dlleuorloun; pue lllernlcnrrs ;o dlrureg sulqdoJlorneu 'iloc crtdeudsrsodaql uo rotdo)or rcZ-tTpue 51-E7 sern8rg) str ,(q peururelap sr 'drolrqtlur Jo drolelrcxa JOr{lratalllusupJloJ -nau e dq petrorlaosuodsaraql 'ller crtdeudstsodaqt or leu8ts aqr s.(z1arpue esdBuzlsleJrruaqJ e te uoJneu f,Ildeu,(soJd agr dq paseal -eJ sr ler{t elnf,elou Surleu8rs relnlleleJrxg JelllusueJloJnou '(uoxe) ft-tZ pue 1-97 sern8rg) ssecord 3uo1 auo pue !(solrrpuep) sessecord paqcuerg 'l.rogs eldrrlnur :dpog 1ec p sureluof, uornou lecrd,b V 'tuelsls snolrou (11eoerrrau) uornau eql Jo sllar Surtcnpuoc-eslndtur eqr ;o .(uy (q7-g p ern8rg) 'suoxe u.ry\opslerluelod uoltf,e Jo uglssrusuen ]o elBJPu? ernlf,nJts]euoxe ol alnqlJluoJ lEtll'suoJnou ut dluo puno; 'suralord luoruelq elplpalurelul Jo dnor8 V (sg1q)sruerueyrJoJnau 'srsoldodp qlr^\ ts?JluoJ 'slualuof, rleqr Jo esEeleJ r{lr^a sllaf, to Sultsrnq pue Suqlerrrslq pa>1retudllensn :dSologred reqto ro aSeurep enssrt ruo{ Surllnser qlEeP^l]ef, srsoJJou (s-tz arntsrc)'sllor rournl

pue sllef, petf,atur-snrr^ IPI PUE rratap dll€lrtlcedsuou lPql Iuol ( 1-6 arn8rg)'saxelduroc -s,{s aunururr elpuur egl Jo stuauodruo3 qlac (y51) ralll{ Iernteu arodreepnu .(g pereroyrad eJ€seueJqrueu ol{l or{lpue unlnJrleJ (71 - 1 1 e.rn8rg)'dutnd +X/ *oy1 Paller dluoruuror rruseldopueaqt qlr.4r\ snonurtuoosr eueJqrueuretno Jqt :snelf,nu !s11ec lerurue q (qBH) *) pue (.uo1)*eg;o suollerluaruor relnllef, aqt SurpunorrnseJnlf,nJts euerqrueu-elqno( edole^uaJpelJnu -erlur lpruJou agt SururelureruJoJ algrsuodsar.(1a3;e1sI :suol +) 1o lrodurr pue suor *eN Jo t.rodxa ol olnralou dJV euo 1o srsdlorp -dq seldnoc teqr dund pere.nod-41y sselr-d V es"dlv *)/*pN

'snloolJnueqr sr ad{r lueuruo.rdtsoruerll 'saxaldruor(4119)urarordoeltnuoqrr;o,{lquesseaqt ur uortf,unt dueu !sy51g pue surelord rr;rcodsSururetuoc'snJIJnueqt ur uor8arpezqerceds dlleuorlrun;'lerr.reqds dgSnoa [poq reapnu (17-garn8rg)'tunrolg uror{lnososleaag'aqord VNq peleqel e ot uorlezlplrqdq ^(q srseroqdortteledq peler -edessy51g rr;oads Surlralap.royanbrugcal 8ulrrolqurequoN

'elgnlosurrote^\ueqo erepue salnlelou .re1od ueqr 'se8reqc ratel\ ur olqn]osssalere llleraua8 selnrelou re1oduo51

'srsaqlu.{soroqdSurrnp pue sdervrqredcrlaqtudsotq uI roIrrEJ uortrele ue se ,{lo,rrsuetxepesn sI reql +qVN Jo turo; pareldroqd -soq.I (ereqdsoqd opltoolrnulp auluape aprueurtooru) +dCVN (egE-7arn8rg) 'uorlnlos erlt uro{ *H auo PUPalnJolou JouoP E lrrog suorlJele o,ut Surldecce dq rarrrec uoJlf,ale uE s€ suollf,unJ rerll o]nJalou (apuoalonulp auluaPp ePlueunorlu) *qVN crue8ro IIErus y

(7-6 arn8tg) 'srsoldoopuapalerpau-roldarar ruo{ t)urtsrp :uol -alalsordr uIlf,e eqt;o Surlapourarelrsuelxe seAIolur tegl ssarord e uI s]lel rttod.re4na urplrer dq pezqeu.ralurere (s11orlerrelreq ''3'a) saycrued a8rey dlaarlelar qrrq.ry\dq ssaco.r4 srsordroteqd 'sllar lrtrrpuep pue'se8eqdoneru'slrqdorl 'suo8rlue -neu eJE sardro8eqd dreurrrd rer{lo aq1 elplnJrued pue suaSoqled dorrsap pue lseSur uef, ler{t 11ecluy ardrotpqd 'euIIE{lp ere aloqe esoqt Jrprf,e ere srqr .{\olJq senle,r!4 ;o pue '[*H] 3ol- = 11d e ot luale,rrnbasr ftqe.r1ne11 11d :rarr1rad salou ur uollerlue)uor uor ueSorp.(q eqr;o urqtrreSol enrle8eu aqt se paur;Opuorlnlos e ;o ,brur1e11ero dlrprce eql Jo ernseeurV gd 'asEIetef, dq ua8lxo pue JelE.,!Aol peue,ruof, sr r{Jrr{.a\'aprxored ua8 -orpdq elerauaS suorlJeer lq spne ourrue pue sprce z(ttey8ur ter{l -per8ep ro; sorudzuasureluor teqr ollaue8ro 11eur5 euosrxorad 'EurruEI IEsEqeqr;o ruauod -uoc ro(Btu e lsrolce; qrmor8 pue 'selnralou ace;rns-11er'sluau -oduror prq3g dueur or spurq leqr (Wlg) xrrlEru relnllerertxe aqt Jo luauodwoc uec,(16oe1ordureruoprrlnru a8rel y ueoapad (1-91 ern8rg) 'uralord auerqruarulurt -olu osle aa5 :a.{elrq prdrloqdsoqd erll to erof, erqoqdorplg agr Jelue tou seop rng oueJqruarue Jo $e! crruseldoxoro cqosoldr aqt qlra soterJossetegl urotord duy ulelord euerqtuetu preqdrrad ( g 1-7 arnSrg)'(uorrerpdqap) elnra]ou telet'l' e Jo espelerleu aqt qtr^ raqtoue yo dnor8 ldxog -rer Jtlt pup prJe ourrue euo;o dnor8 ourue eql uoa.r\laqpeuroJ splre oultup uao..vitaqa8e4url aprrue luele^or aqJ puoq eprrdod 'apndeddlod oslp eas dlqea8ueqrralur pesn ue5o erc apudado8tlo pue ap4flad surrat ar{J 'spuoq aprldad .(q parrau -uor sprf,eounue Jo pasoduroororudlod reeurl lletus V opDdod ( 9 1 - 7a n -3tc) dle,turadsar'y51q pue vNu ur ruasard are asoqrrdxoeppue esoqrJ sesoluad aq1 'aprreqJJesouoru uoqJEJ-e^rJV asolued (E7-9 arnSrg) 'spupJts dreluaulaldruor aqt eleredes ot tuarulpert leaq yorrq dq pe.lrolloJ sraurrrd aprloalcnuoSrlo tror{s ruor; srsaqtudsVNCI Jo sa1c.(raldrrlnu dq a.rnlxrru xeyduror e ur tueurSasy11q rr;rcads e 8ur,!r1due ro; anbruqtal (uorlrear ureqf, eserarudlod) g34 'Kpoq Surssato,r.d -yy1y tnasoldol(c payet osle !sy51gru pet€rf,osseyo uorreper8ap pu? uollp]sueJl Jo uorssaJdJJur suouJunJ rpqr csJolJEJuorlplsupJl ro seuosoqrr ou Sururetuor'ureruop rruseldotl.t esuaq Ipoq a '8ur.Lrdgrallnq e uo u.re11ed rolor rrll ro pupq p to seuoq agr elrl 'sural]ed lerteds parepro-l]e^aolur odrqua SurdolerrepE Jo senssrl pue 'sue8ro 's11aceqt Surzrue8roJo ssaJoJd uorleruJoJ uralled

AUVSSO'19

9r-9

qrrl Suole'uortrunl pup srotf,eJuottdtrcsuerl apoJua1y'odrque dlrea eqr ur srxe rorratsodotretue aqt 3uo1e sadrrts Surteuralle ur passerdxa saua8 ;o dnor8 e 'ollqdosot(I uI soue8 elnr-rred $7-97en*gl 'sJaf,uef, ur eJe aua8 uprunq,(uew punoJ Egd aql ur suorlelnur 8ur -tE^rlreul 'y51q paSeueP qrr.ry\sl]ar Jo lsorre or{l ur alor lef,rlrrJ e sdeld reqr euat rossarddns-rorunl e 1o rcnpord aq1 ulerord 6gd

'srseqlu^s dJV re.r\oo ol asn slr pue lrodsuert uortJele Surrnp arro; earloru luanbesqns 'erJpuor{Jolrur pue erJoiJeq -uolord e Jo uorleJeue8 sa.t1o,ru1 ul (zO) uo8-{xo ol suorlf,ela to ratsuert ar{r dq ua^rrp dJV ruroJ ol dq1v ;o uortel.(roqdsoqd eq1 uor1e1.{r6qd5egde^Ileprxo 'uorlJear esre^er aql roJ (uorlrnpar) lPrluelod uon -f,nper er{l se u8rs elrsoddo tnq apnlruSeu aureseqt seq Iertuolod (uorlJPeJuorlPPlxo ue.lr3 e Joc 'uoJFele uE esool uorlpprxo oql ot alnJelou e;o Lruapuar aqt Jo ernseJure luortrala ue sesol olnf, -oloru ro ruole up ueq.vreSueqr e8€tlo^ aql prluolod uortppxo 'uontnper ;o alrsoddo :pappe sr uo8dxo ro elnJelou e ruory pelorueJ sr ruole uaSorpdq p ueq.^a sJnJf,o se elnJalou Jo ruole ue uoJt suorlf,Oleto sso'I uorlEPrxo (9-11 ornSrg) 'uorterlueruor elnlos leleet? Jo euo ot ressel p Jo uorlnlos ruort (arnlos ot tou inq role^\ ol alqearurod) ouerq -ureur elqeeruredrtues e ssoJf,EJale.lrl. lueruelour loN srsoruso Jo (gE-E7 arnSrg)'ouoqdorcrur s,dpoq aqr !saslndurr lef,rrtJele olur lueruelotu Ief,ruEr.lf,erupelereueS-punos ef,npsuBrlrEql sl]el rreg;o posodwor Pue rer reuur eql to eaF{roJ eql urqlrl,\ posnog eJntf,nJls drosues Jrlsnof,v r1ro3 ;o ueEro 's11ao ouodre4na fi-G pue q7-1 sarn8rg) ur punot arnlJnrts r€lnllergns pelrur1-euprqueu ,(uy alloueEro (eg1-y arn8rg) 'suratord eldrrlntu ro; secuanbesSurpoc SururBluoc VNUru uE ol osrJso,rr8leql ralouord auo ruo{ peqrJJs 'VNC -uert sauo8 snon8rluoc uoredo lerretreg q to ralsnlf, e (7-7 enfigl'oua3 luace[pe ue ;o uorldrrcsuerl sloJluoJ pue urelord rosserdor e spurq teqt atuoueS a8eqdorretoeqro leuetreq e ur aluanbas VNq uoqs rolerado 'utalord e Sutpocua;o lrrlqeqord q8rq e seq suopoJ 00I rseel le rot spuetxa pue uopor lrels p qtr.{\ sur8eqreqr CUO uV'seruert Surpea.rraldrrr aqr Jo auo ur suopoJ dors dq peldnrralur 1ou sr l€ql VNq pecuanbas;o uor8ag (gag) euerl Eurpear uedo 'ursrue8ro ue ur eceydJo erurl Suorzrraqt ur ro ssaJxeur pernpord sr leql uralord Ieurou e lo 'uralord IeruJou e Jo urroJ peteln8erun luelnu e aq leur fuorlera;qord IIOf,IErurouqe sesner leqt auaSocuo ue dq peporua uralord y urelordocuo

( 17 - 1 1 a r n 3 r 1 ) ' o u e . r q u o r - u (1 1-97 ornSrg)'uorsrirrpro qtrrror8 IIar to Iorluor er{t ur pe^lo^ur urato:d z ro; (oua6oruo-orord) euoS Ieurou E to urro; tuelnur e sr IIar er{r;o qrred llerus p or peqdde sr drl asoq.u euadrdonnu e Jo 'slerurue ur racuel Surlnpur ur ro erntlnJ ur sllef, Sururroy esn dq arltue ue eupJgruetueqt ssorf,Ero uor o13urs dllerauag 11ar Jo lauuErlJ e q8norqr ,!\olJ uor Surururatap ro; enbrugcal -su€rt ur rar{tre pollolur sr trnpord esoqrtr aua8 y 8urduelc qrred auatocuo 'uorsnJJrpdq re8;er eqt seqr?ar pue (s)y1ae dgreau e dg parnpord sr reql (ranrursuerl -orneu lotre; ql,uro.r3''3'a) elncalou Surleu8rse ot spuodser 11er ta8tet e qf,rq^\ ur rusruer{treruSurleu8rs ot SurJJeJeU euucerBd (ttZ-ZZ arn8rg) 'serg ur sluaru8as dpoq eqr Sururturatap ur'seuo6 dlrrelod-luautas pue sauat deE

'saprreqoresotlo pa{url-N osle ees 'uratordor,{y8p ur anprsar euruoanll ro ourras e ur dnor8 ldxorpdq urer.{J-eprs aqr ot parlJel -te sr tpql ureqf, eprrer{coeso8qg eprreqmeso8qo pa{uq-O dlqea8uegcratul pesn uetto rre ap4Qado8tlo pue ap4iai surret aql 'spuog eprrded dg parceuuof, sprf,€ ourrup Jo pasodruocreu.{1od r€eurl pezrs-unlpeu ot llprus V aprtdodoEqo

Lt-9

AUVSSO'19

'sPuoqeldrrl (7y-7y en8;.g)'srnrcouodsuerl Jo elqnoPere spuoq uoqJPf,-uoqrEleqr Jo eroru Jo o^\t qln{.41\ uortcalaoloqd ereq.r\reluef, uouf,per e pue qldqdorolqc Sururel ur (prre drre; ''3'a) punoduor e ot Surrrayag poternlesundlod -uoc saxalduocSurlsarrreg-lg8r1 Jrtaql yo lsrsuorleql 'srusrueBJo -udsoroqd ur uretordrllnyg suolslsoloqd tuesard'saxalduroc 11e 'zg;o uotrnlo,ra Puegz11;o (zOJ uort solerpdqoqrec uondrunsuoceqt qtr.^a.,{11ensn ateraue8 ol pesnsrd8rauarq8l qrqm ur slseldorolqrrupld ur pue errolJeq eruosur SurrrnccosuortleelJo serJes xalduo3 srseqludsotoqd

'stf,osur ue.raldrp GV-9 pue 7y-9 sa:n8rg) rer{to pue olltldosotq Jo sanssrlrer{lo eruospue spue18drerr -IIESeqf uI punoJ luorteredes lnoqtr,1\uorlecrldar lgruosoruorr{J .(ueu VNq Jo salodcaldrrlnur.,{gpaurro;JlestrJo sardoo1e11ered ;o pasoduoo auosoruorqt paS;eyug euosoruorqJ ouerdlod

'saprt oqst aso8rioprl .(cuenrgoaql Surcnpersnql (gy-71arn8rg)'srseqlu.{sologd;o lBr 'zOO Suueraua8pue ueuo are senPrsalsI ueql Ja.r\e,qtr^. esor{J.sanprsersI upql 41y Surunsuocdq (apIc urap3) uorlExrJ zol qrlaosetadurocrpqt de^.\qreduortf,eeJV uorlerrdsoroloqd ororu Sururetuoodllensn pue spuoq crprsordlSdq pa4ur1seprr -er,ltrpsouou reurdlodpeqruerq ro reour.1 oprreqoces,(1od 1o 'srseqtudsotoqd ern8rg) ur slue,ra (EE-71 sa,rrrp tuanbasqns (97-7 arn8rg)'awosClojpoller osletypA ra8uesseur proleldqt oqt ssorJeuorteredas o8reqrp seterauoS el8urse 3urre1 lEr{teuerqruaul -suel Sururetuor xaldruory ouosoqrrdlod leql trodsuert uorlrale uarrrrp-rq8r1 uodsuBrl uorlcoyootoqd IIp'souosoqlJ leJa.,las 'urolordosleees'senprsoJ (q,e9 aJoruro 67 Sururetuor dllensn.spuoq epudoddq penauuor sprJeourrueJo rarudlodreeurl oprrded.{lod -61 arn8rg)'seuerqueuorqIIe rot uorlepunotarll lrotuar aqt ur .reloduoueqr PuB'oprsroqlrouo erpelusno 'Uf,d ees uorlf,Eerureqoeseraudyod er€ sur€rlJ1,lte 1orc1 -enbeoql ot pasodxaare sprdqoqdsoqd yo sdnor8peeqrelod eqr y radepq prdqoqdsoqd qlrq.tr ur ernlrnrts e{rTteaqs'rade1-o.rrt (91-7ern8rg)'spuogJuele^orlg pe1ur1(sreuouoru)slrunrelnurs ro ]PrllueProldrrlnu yo pesodurocrlnrelolu e8relluy raudlod 'eS-gI 6 Z - Z p u eq 'serrteurrudse sern8rg)'sprdqo8urqds pue soprracdltoqdsoqd Surpnpur'sauerq dq pa4reu eJnltrnrls IerntJnllspue ]euorlf,uny -rueuorq ur tuasard sprdrl ;o sselc :oleu eq1 prdrloqdsoqd r?lnllergnsro ylar due or Surrra;e.r i{8o1orq u1 pazuelod 11ec 'tueuodruorJelnlloJJo (0€-St pue 67-91sorn8rg)'€dI p,te 5yq 'sre8uasseur lf,urtsrpur seJueJeJJrp IIeJp Jo suorSor I"rnt -rnrts rol?ue puof,eso,r.t oteraue8or ereqdsogdslq-S'tlolrsourldprlegdsoqd eqr {3010rq11ec u1 drFBIod Ieuorllunt;o acuasard prdq euerqruetueql saAeJJJ teql (oDCro boC raqrradq pal€^rtre 'rele.a.r .soSreqc 'asedqoqdsogd ur elqnlosdllensner€ selnralou.re1o4 patercosse-ouerqruou v (C'Ia) C osedqoqdsoqd e,rrle8aupue e,rrtrsodJo uortnqrrlsrp crrteurur,{sero a8reqc (71-6ysarn8rg)'sprd[oqdsoqd;opue crpqdorp,ttlaq] ur spuoq f,rrlJoleleu e qtr.{\ eJnlJnJlsJo alnf,alou e o1 Surrreteu re10d snorrel e,teelf,teqt sarudzualerelas Jo euo asedrloqdsoqd 'aruB{ turpeor aqt ur rJrr{sE esnef,IIr.^a Gz-gt pue 6z eprloeltnua18urs e Jo uorlalepro -91 uorlJnpsuert-leu8rs ur sarn8rg)'sderrrqted srotuassaru uorrrppv'uopof,dols e ro prre ourue tuereJtrpe 8ur.(;rceds lera,res uopor 8ur p uorteruroJur llnsoJuer furoto.rdro1Surporuor8are ur d11en puoJosse uorlf,unJaruoslsa.LrleiuJep lolrsourpareldroqdsoqd Jo -uretuofsprdrlpunoq-ouergrxou;odnor8y saprtrsouroqdsoqd -edsa'y51q ur oprloelf,nue18urse a8ueq3 uonelnru lurod 1o (eg-g1pue g7-7 sorn8rg)'souerqruaru ( 8€ - 6r ern8rg)'sller Ieurrupur suorpunl ds6 or snoSoleuedlleuortcun; -orqur sprdrltuepunqelsorueqt lereqdsoqderltor peqletrednor8 ur sdnor8 ldxorpdq eql ol per;rrelse peeq relod e pue 1oracd13 er€ pue sl1arrueld luef,Elpe;osruseldordo eql tteuuorrelurt?ql o.nrJo lsrsuof,dllereua8reqr areqd sureqcldce.&re1crqoqdorp.,{g suorlcun[ ]latr e{rTaqnl (eursapouseld.3urs)eleusepouseld -sogd-glorerdp Jo se^rle^rrep crgredrqdruy saprrerdy8oqdsoqd '6uruop VNq ur rolre^ (7-y arn8rg) e se pasndluounuor !11ec e ur uorterrldarsnoruouotne;oelqed 'epls -eJ alntrelourvN( ,t eqt uo rrr{touepue eteqdsorldeqr;o epls ,S eql uo euo relnorrc,11erugpruseyd Ieuosoruorr{Jerlxe 'spuoqretseoqdsoqd oirg Jo slsrsuoc:yNa pue vNC[ ul sePrloel) ft-Ot pue 1-91 sarn8rg) -nu tuacelpeuee^teq a8elurl ]EJIruetlJ Puoq rolsolPoqdsoqd 'surolordpue sprdrleuerqruaupeterf,ossp pue rodellq prdqoqd n pue S aqt pue (19-7arn8rg)aJV ul saleqdsoqd -soqd e to stsrsuoJllueuruorrirua leuJelxestr ruoJJIIef,er{l seteJ 'sdnorSoreqdsoqdoal uoa.4ataq sareqdsoqd -edasteqt lyace Surpunorrnseuprgrueru fl pue /" eql sp qrns eq1 ouerqrueueuseld addr y puoq apupdqueoqdsoqd peulrot puoq ,(treua-qtlq Jo (gy-y arn8r4).do1a,rap (senbeld)s11ec pesdl teqt (Eg-Earn8rg) Jo sperereelr er{tSurlunocueql pue sllartsoq elgrtdersns ;o .ra.(e1 'suratord dueur JEInI]ef, ;o ,ftr,rrtleoqt Iorluof,ot saseur{uratord e uo aldues polnlrpe 3urrn11nr,(q alduese ur solcrtredlerrl snorl 'srsdlorpdqdq eterlsqnse r{tr^.\tf,e sesereqdsoqd urelordoqdsoq4 -f,aJur reqrunuoqt Surururralap ro; enbruqoel Iesse anbeld Jo uor; dnorSareqdsoqdB seloruerteqt eudzuauy osereqdsoqd 'seJnlJnJls Jeqlo 01 sluolu -plrJ elerpaurelur r{relte dleq reqt surelo.rd;o .(prue; y sur4eld 'lurod f,rrlreleosr ees

1d

(Ey-31 arn8rg) 'tueql uee.4taq IIEI\^ IIat ^.\au aqt olur dola.rap sluoluor esor{1rlpue slyacrarq8nep aqr ,aseqdolal Jo seueJgruarueurseld oqt euotraq soueJqrueu osoq.u Surrnp peurro; 'arnlcnrls drerodurel e ,stueld u1 lseldouteJqd

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AUVSSO'r9

8r-9

(67-9 arn8r4) 'selnf,eloururlrnbrqn eldltlnur Jo tuowl{reDe dg uorlrnrtsep roJ prlreru surelo.rdrplnlloJerlur saper8ep]eql Iosol -do aqr ur xaldruor eseatord leuollJunJll]nu a3re1 auoseoloJd

ouo SururetuocspunodurocsnouaSorlru Jo ssp]JV sourprurrdd Q1-7 amtug)rred oseqosleeas '(C) 'VNU pue VNC ul punoJsrprloelrnuto stueuodtuoresEqare euruenSpue (V) aururpe'saur:ndo.nr1's8urrcqrdrorateqpesn; o.vq Surureluor spunodruocsnouaSorlruJo ssEIJv sourrnd 'durnd paraarod-4ayea5 durnd

'etels elltf,e dlecrSolorq (e IlEu str ur (uoBeuroluoc) adeqs Jeuorsuaurp-eerqtrrtsrretrerer{l e orur peploJ pue sulpqf,epude&(1od eJoru Jo euo ;o pesodtuor alnJolouroJJplu V uralord JEOuTT

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'suretord laSret esealord

ur spuoq aprrdaderoru Jo euo sa^€olf,leql eudzue duv

(99-91arn8rg) 'rurot ol sur8aq olpurds f,Ilotlru agt pue 'sa1odelpurds eqt eluoJ -eq ol at€JedasseruosoJtuacparectldnp eql (osuepuo) souos -oruoJr.{f,eqr grrr{A\ Surrnp slsolllu ur a8ets lseIIrEE aseqdord $y 1 anfu g)'slueua]e leurtxo.rd-ralourord eldrrlnur lq pellorruor sr soua8 .{ueur 1o uoltdlrf,sue{ 'atls tr€ls uortdr.rcsuerlaql;o srred espq002: urqll.Aapelprol sI terlt VNq JIto -dre>1nour acuenbasdroteln8ar duy luauele purxord-ralourord (11-7 arntrg) 'aserarudlodVNU e rot uoIlEIlIuI JaloluoJd uorldrrcsuerlJo etrs aql soulure]ep regr aruonbes y51q (yg-91 ern8rg)'saroqcolounl pellet serntrnrts POZITeIrods te srred euosoruorr{J ,.arntdel,, salod olpurds eql urorJ palgures -sE selnqnlorf,rru pue 'urLop learg eulruel Jeelfnu pue odolJluo reelJnu eqt qlrq.{\ Surrnp srsolrurur a8els puoJes espqdeleuroJd (g-1 ern8rg) 'selo^rp{ne oslp eas 'sellaue8roraqlo puE snalrnu pelulll-aueJqluetuent] E Ile[ ]eqr'eaeqtre PUE(Eua] -ceqna) Erralreq erJrSurpnpul'stusrueSlo to ssel3 selolrp{ord 'srsotdode oa5 qrpep IIer petuuerEord 'uotreztPtrq'(q '{q (peleqel dlpl secuanbesprf,e rrelrnu cryrcedslratep ot pasn sr reqr -rueqr ro dla,rrlreorper 'tuaru8e.rl VNC ro yNU peuIJeC aqord 'PuPrls ateldual oqr .{doc ol saPlloellnu Jo uorlrppe ro1 lurod Surl:ets oqt s€ suollf,unJ pue puerls otelduol dretuerualdurof, p t{Il.ry\srrud aseq surro1 teql dno;3 ldxorpdg

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(acuanbas)lueure8uerreJeJurl Jr{l'suralord u1

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auat e dq peporuesuratordsnoSolouroq;olag dyrue; uralord

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6t-D

AUVSSOl9

(67-97 erntrg) 'a.{eeqr ur deru eqr alrl sr urerg er{t ur detu oql 'urerq 01 o/a ruo{ uorleuro;ur 3ur.(: -r€r sllor uorlSue8 leurler dq peleerr (unrcal eqt) urprq eqt Jo ued Iznsr^ eql ur euo pup tq8rl Suruocur dq pareoro Eurler aqt ur auo 'uorleurrolur lensrl Jo sdeu Surpuodsorro3 sderu IElJelourlJJ 'tseed ur dlleuorr JUVJS o1 lua1e.,rrnba -cun; fsrotre; qr,r,ror8 af,uesqe er{t ur uele epdr eqr Surtalduror Jo pue aseqd 5 aqt Surrelue ol peltnuuoJ euro)oq sllaf, uerleurrueru qlrqrrt rE alodc 11acaqr 1o tg atel ur rurod aq1 lurod uorlrrlsar 'sd'IJU ees srusrqdroudlod qrtual luourtery uorlrrJtsar '8uruolc y51q pue selnf,eloruVNC rueurquof,er to uortrnpord aqt ur pesn aJp stuaru8erl asaql 'orudzue uorlJrJlsal relncrued e qtrztr a8e.tealc ruo:y Surtlnser tuau8erJ VNC peurJep V luetute{ uorlJrrlsoJ (I-S elqpl:1p-g ern8rg) 'aspapnuopuauotilt4sat pel1erosle !o.rrr.,r ul VNC lueurqruorar ernpord o1dla,trsualxepasn lselnrelou vNC papuerts-olqnop ut 'aps uotpt4sar aqt (af,uenbastroqs cr;rceds e saleop pue sazruSocar leqt aur,{zua duy atulzua uorlJrrlsaJ 'sl]eJ Iprurue ur yerlualod auerqruotu Suttser a.r.rle8au-aprsur aqt Surtereua8 ro; elqrsuods -er .,{l.reurrrdere 'ese4ay +)/+EN eqr ,{g pocnpo.rd uorle.rtuac '1eq1aueJqrueru -uoJ *) crTosoldr qSrg aqr qlr.u uorlrun[uor ur euseld aql ur slauueqf, uor +) pere8uoN sleuueqr *) tullsar 'srsaqtuds dIV ro1 Id pue dqv;o ,{yddnseqt uo ZHCVC pue HCVN Jo uorleprxo Ierrpuor.{Jolru1o eruepuedaq IoJluoJ,(rolerrdsar 'ureqo lrodsuerl uoJlJalo eos ureqc drolerrdsar tamod Surnlosat pellel osle fsnte.reddeIerrtdo ue dq paqsrn3urrsrpaq uEf, tEI{l slf,efqo ol.\l uea-^a]eqaJuetsrp runrururu ar{J uorlnloseJ 's.ros.rnrardJrroruouoruagl yo oBelurl tueleAof,req€ urEruaJ reqr ;audyod € ur strun Surleadar aqr JoJ turat IeJOueC enprseJ .UOI]OIJJ

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'PaTull sI lr r{f,rq.a ol retoruo.rd er{l Jo uorip^rlJe elef,rpur ot stuerurredxa;o saddt snor ,.3.e) -JeAur pesn are sauaSratrodaa'(asera;rcn1'eseprsolreleS-d po.(essedlrsea sr legt uralo.rd e Surpocue ouo8 V auet ralrodar 'auo lsnI ure]uoJ dllensn sprtuseldpue seruosoruoJqJ sEaJar.{.Aa'sur8rro aldrrlnruureluoJ seruosoru lprJelr€q -o;qr rtto,{re4nE 'sur8aq uorleoqder VNCI rlJlqna 1e auroue8 s,rusr -ue8ro ue ur luaserd sluaur8as y51q anbrun urtrro uorlecqdar (gg-g ern8rg) '4,to] Sutmo8 pallzr osle fsrseqruds y51q Surrnp patecrldar pue pateredas ere spuprts o^u eql qf,rr{.4a le VNC paPuErls-elqnop ur uor8e.r pedeqs-1 >FoJ uouecrlder

'po^oru 'uorlPPlxo Jo etrsoooo oql -er sr uo8.(xo ro elnreloru e or poppe sr urote uaSorpdq e uel{^\ srnf,f,o sE e]nJalolu ro ruote ue .(g suortcale Jo urEc uorpnPaJ 'Jor{loue ol auo Iuort luElf,eeJ PaJJa}sueJlaJ€ suoJlJele eJoru Jo uonJEeJ xoPeJ auo qf,rq^.\ ur uorlJeeJ uorlf,nPeJ-uorlEPrxo uv (91-g a.rn8rg)'saurdzuapue VNq perltrnd qll.4aortrl ur lno .rreda.r-yyg Ielalos Suunp .rnlo paureJ Jq uEJ pue srusruer4Joru osle (od,b crSoloqdrour ruereJJIPJo seuosouorqr uao-tuoq ''a'r) uoneurquof,er snoSolouoquou pue uorleurquof,ar snoSolourog 'srs 'sauosoruoJr{t snoSo]ouJorl Jo JeAo Surssorl ol osrr 3ur,lr3 -oreru Surrnp sJnJJo uorleurqruoJer sno3o1ouro11'suollpulgruof, .Llaue,rr3ol pauro[e.rere slueruSerl eql PUEPaAealJJre selnJolou uollpulqluoJeJ VNC Jo saruosoruol{f, rlJrrfl\l. uI ssarord duy 'saJrnos tuereJlrp uror; sluaur8ery ypq 3u1 -uro[ dq or]rl ur peruro; elnreloru VNC IUV VN(I lu?ulqruorer (7-9 arn8rg) 'uorlf,unJ s.aua8aql Jo ssol E ur tlnser dlyeroua8 se]allea^rsseJorecnpord leqt suortetntrJ'se]e]le elrssoler o,r.-t8ur -trtrct (ato8trzouroq)lenpriupur ue;o addrouaqd eql ol sraJeroslp llueserd sr elellp lupuruop eqt ueq,t\ ed,$ouaqd eql ut pessardxe tou sr teql eua8 e;o elel]Eter{t ol Surrra;ar'srtleue8 u1 ealssaJeJ 'sde-tnqred 8ut Ut-gt pue 9y-91 sorn8rg) -leu8rsrelnllererlur Surletlrurdgeragr'utetuop rrlosol.(r s,roldacer eql ur dlr,rrlce eseut4 uratord cgtteds-autsordt sete,rtlceSutputg pue8rl 'srolreJ gtmor8 lueu pue uIInsuI roJ esoqt Surpnlour 'ureruop eueJqruerusuert e18urse qlr.rrrdllensn 'sroldecar ef,eJJns to sselc a8rel € Jo regruew (yag) eseurl aursordl rolderer -IIar

(67-y1 antu1) '(aurosopuodlrea) e]Jrse^ popunoq-eueJqrueu IIErus € ruro; ol eupJqrueu eruseld uorleur8e.rurdg sroldarer ereJrns-llef,cryrcadsor punog sle arlt Jo -rJalpru relnllaJeJtxe srsol,bopua pelerperu-roldacar ;o elerdn 'roldoral Jeelrnu pue roldacor uolsaqpe fi-9l pxe 1-91 sern8rg) osl? aes 'asuodser rplnllel e Surlerlrur dqeraqt totdacar aql ur e8ueqc leuortpruroJuor e sernpul ueuo qlrq^.'(puetq) elnf,elou rplnllef,€Jlxe cr;rcedse sPurq leql snelJnu ro '1osoilc 'auerqureur euseld eqt ur pateJol utelord E setouep .(1uouruo3 'ssecordre1n1 -letrraqto ro 'srsoilcopua 'uorseqpe 'Sutleu8tsllel-llal elerPeruol roldoror elntelouI raqtouE spurq dllerrtrrads reqr uralord duy (91-y arn8rg) 'selue{ Surpeartuare;Jlp oml ut Sutpear dq soptr -dad,{1odtuaroJJrpotul pelplsuerl aq uef, sVNUtu euos 'uopol dots e ol VNUru up uI uopof, tJets uolte]suerl cryrcadsE luorJ sunr teqt (suopor) staldrrt apltoelrnu yo ecuenbeseq1 aurery8urpear -Jeer

'uortf,eor IeJruraqJ e lo efer aql ot sluel luelsuof, a}PJ to suorteJlueJuor eql selE]eJlel{l luelsuof, v

'sroldocer ef,BJrns-llef, ralllo GZ-gt pue 67-91 sarn8rg) euos pue saseuDleursordl roldeoar or Surpurq pue8rl lq Patelrlre lsde.vrqtedSuqeu8rs relnllelerlul uI suoltf,unJ pue .roqrue prdrl

(qOf -E ern8rg) 'suralord (trunqnsrtlnru) crreurrtlnru uI suleqr eptrdaddlod aqr alnlJnJls dreurolenb ;o suorlrsod e^rleler pup Jeqlunu aql

'uortf,€ar (uorreprxo) asJeler er1l .roJIerluelod uorlepxo eqt se u8rs etrsoddo lng epntruSerueues eqt spq g'uorlcear uoltf,npeJ ue^,rr8 e roC 'uorlra]e ue ure8 ot elnl -elou ?;o dcuapual egt Jo aJnseeu e luorttelo ue sure8 elnJa]oru ro ruole uE uer{^\ a8ueqr e8€tlo^ eql (g) prruotod uorpnper

e dq auerqureu etuseld aqr ot pereqral sI tEI{t sutalord qrll-ry\sJo dlrtueyradns as?dl5 el{t Jo requreru f,IJeluouou V uralord seg (t-g elqet) 'selnJelou lerr8olotq uI slaq€l se .,(11er -uarurJedxepesn dluouruor a.re sadoloslolpeJ IeJe^aS'sderep lr se uon€rpEJ slrure leql luolP ue ,o tuJoJ elqElsun adolosrorper

(17-p antugl'(srseqrudsuretord) uorlplsuerl Surteururratdqaraqr'ureqc eprtdeddlod peraldruoc eql Jo aseeleratouord pue VNUrU ur suopor dols azruSora.rleql suralord Ieurosoqrruou ;o seddr o-AuJo auo (gg) rorey oseeler

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(gg-g sern8rg)'eruanbaslueror1rpE r{lr.ry\ sVNUur drel ,o teql tou lnq ouaSaqr .{q pepocuavNuru papuerrs-elturs -uarualduroreqt Jo uortpper8epro uonelsuertto uortrqrqurreql -re sernpurleqt VNU pepuerls-elqnop Surpuodserroc e dq euet rr;rceds? Jo uortelrtreur leuortJunC (IVNU) oJuoreJrolurVNU

(y1-7 ern8rg) 'f,not8 g pellel osle lpne ourue qree Jo saruedord relnrrr;ed aql saurrurelapdla8rel leql ruole uoqrer (o) eqdle oql ot paqtet -te dnor8 luenlrlsqns a]qerJel eql (sprJe ourue uI ur"qJ oprs

aprsoelrnuoqrr selerlsqnsse 8ur (11-y arn8rg)'sereqdsoqdrrl -sn puprtsy51g dreruaualduroroqt o{pru 01(puerrsaLoldutala'qt) VNC Jo pu€rls euo sardor leqt ourlzuo uy oseraurdlody11g

'sllaJ Jerllo ot Jo lueuuoJr^uo IeuJOlxe slr ol IIer E Jo asuodsoraql Surlprpetuur pe^lo^ur elnrrloru relnlleJ -€JtuI Jo Je]nllef,erxe due ro; ruJel IpJeueO alnJelou tuqeutrs

(41-g arnSrg)'stsoqluoJet -Jrpo.{\r ur uorte8edord;o alqedeorolJo^ prwseld ropaa ellrnqs gt-6I pue 7-61 sarn8r4) 'suralordocdlSr€lnllarerlxe ur ro sllrr tuerelpe Jo ereJrns aqr uo sprdrloodlSpue surerordordlS ur serta -roru aprJerlJJeso8rlocr;nads qtrl\ suorlJeJelur luapuadep-*zeJ eterpeur ler{t selnrelou uorsaqpe-lleJ Jo ,{1rue; y surlreles 'srsorerupue srsotru Suunp s11atJerq8nep ol saruosoruoJqJ to tuerueldruor lenba ue satnqrJlsrpteqt ssacord aq1 uorlBtartas 'odrqrua dlrea eql ur srxe rorralsodorratue aql 3uole suotalalsordc 1o lrrrelod aqr pue sote1IIel aruanl;ur reqr sualsds Surleu8rs;o stueuodruoo 8ur -porua saueS;o dnor8 e 'oyqdosotq u1 sauat dlrrelod-luaru8as 'llel eql ruort Peseel -er eq ot pourtsap selnralou Sururetuor {ro^rleu rt1o5-suo,4 ayl ruort Pa^rJePelJrse^ Punoq-eueJquaru lleurs alcrsarrfuoloDos (1-91 arn8rg) 'llrl aqt tuog peterf,es,{11enrua,ra suratord pue !suratord euEJqruerueuseld !saruososdlpue'r31og 'unlnrrtar rrruseldopua oql ot pazrlelol surelord euerqruorupue alqnlos 3ur -lros pue Surzrsagluds ro; de.Lr,qredrelnlla3 de,ra.qredfuolortos 'suJnl 'leeqs 'xleq o aql Surpnlcur sernlrnrts reln8a.rotur urer.lJ d pue d aPrtdaddlod e ;o 8urp1o; 1erol 'suralord u1 ornltuls drepuores (5-91 ern8rg) 'uorlf,npsuert leutrs ur suortrunJ teql pue leu8rs r€lnlleJeJlxe ue ;o Surpurq ol esuodsa.rur (sasearcap 'dWCr ro) saseanur uorl€Jluef,uoJ esoq.u (e41 pue 'Cy('*ze) '4yqyc ''3'e) elnrelou relnlla)Ertur llerus V retuasseu puoros 'spuoq a13ursere spuoq uoqreluoqreJ er1l ''3'a) punoduror e ol Surrra;ea pelernlps II€ rlJrr{.{\ur (prce ,bre1 'y51q aruanbos-oldurrs aa5

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(79-71 en?r.g) 'uorlterluor sraSSrrluorlelnurls elf,snu .,iqparnp -ul *zEJ perols Jo eseeler:suor +zp3 sratsanbesreqr IIeJ alJsnru e ;o useldordc ur seueJgrueuJo )Jo.rtraN runlnJrloJrnuseldocres 'uorlrertuot 67-71 sen?r.g)

(0€-l.I pue Surrnp suetror{s lauo tuarelpe ue

or Isrp Z euo rnor! Surpualxe pue sluaurelrJ (ursodru) Ilrqr pue slueru€lrJ (ur1re) urqr Surddelre,ro 'pazrue8ro ;o pasoduroc e1c -snu (1era1a4s) paterrrs Jo trun Iprnlf,nrls Surleadeg ororuorrps 'apdo ler ea5 aseqd (slsaqru,(s)5 'aseldxoqree areqdsoqdsrq-S'I esolnqu ees

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(77-y a:nF;.gl'solo.{.re1nereqSrq ur VNUr SS pup 'Sg'S 'S8I 'S8Z :luelrrJJaor uorletuarurpes rreqr dq pareu8rsap uar;g 'saruosoqlJ Jo stuauoduor IEuorlf,unt pue IerntJnJts eJeteqt selnJ -elour VNU e3.re1 (ygg ptuosoqlr) VNUr lerarres;o auo duy (6-8 Pue g-g sern8rg)'auosoacqds oslz aas 'sy51Aru-ardur suoxe yo Sururol turcrlds yNg Pue suolur Jo lE^oturr ur sllnsar reqr sseoord y

'VNUur drelueuraldtuof,-Jpau ro drelueurelduroce ro uorleper8apselerperu reqr (VNUrru ;o uorssordar IeuorlelsuBrt ro y51grs) vNU pepuerrs-a13urs lrorls E rllrl( pelerf,ossexolduroc uralordrllnu e8rel (CSIU) xalduroc turcuays pernpur-VN1 'pererlesr ygau-ord e;o aruenbas eqr qrrq.{ ur SursserordVNU Jo ed,{rlensnun 8urrrpoy51g (11-p an8r.7\'uorldrrcsuerl pup erntrnrtsurtpruorqrSurllortuoc ur pue 'sy11aurJo uonelsuerrpue drrlqersaqtSurllortuorur selor deld sy51a l]erus Jo ,(torre,te fsrsaqlu.{suralord ur selor tueroJ -1rpdeld pup 'VNUr 'VNU* 'seprtoelrnuesogrr;o pasod VNUr -uoc'raudlod papuerts-a13urs l€our.1 (prre rrelf,nuoqlr)VNU 'xoldurocEuroueysparnpul-VN1 ees f,SIU (Ey-71ern8rg) 'o)stqnf,pellel osle lereracllSoqdsoqd-g selnrelou o1r\.1 urroJ Jo or (ereqdsoqdsrq-g'1 asolnqrr)re8nsuoqrpf,-o^rt e ot zOJ Jo uorl 'a1odr -rppe aqt eql ur uouf,eer eql saz(letet ur,rp3 slseld tsrrJ reql -orolqr ur petef,olaur.,{zug aseldxoqrecoreqdsoqdsrq-S(I esoFqu 'srseqlu,{s uralordpue SunrldsVNU ul uorlf,unJ soulzoqra 'dtr,rrlcecrtlletec qlr^^ alnf,elou yNU uy orudzoqrr 'vNur ees vNu leuosoqrr '(srs G.e-, pue 77-y sorn8rg) -aqludsurato.ld)uouelsuerl;o eur8uaagr ltrunqns]leluspue lrun 'suratord upql oJorupue selnf,alou -qnsa8rele olur pozrueSro 0S VNUr tuare;Jrplera^osSursrrdtuocxelduror e8rel y aruosoqrr 'sdNU ruro; rqr ur Jo llol or{tur tueserd are selnreloruVNg rsoyq 'vNU pue surolord;o pesodruocxeld -tuoc due roJ rurol Ierauog xoldruoc (4111a)urerordoapnuoqrr 'vNu ees (vNu) PIrErralrnuoqrr (96-9 ern8r4)'serpnlsate -{uq uErunqur r€lnJololuPOs€q-vNCse e^resuer teql sro{r€ru slenpr^rpuruea.r\tegsef,uereJJrp acuanbes;osed.{rIera OsJo auo ery 'saudzuouorlJrJlseJrelncrlred lq pozruSocersalrsdorlsop Jo elEeJJrpql VNC rnuoueS;oeruenbaseqt ur slenprlrpurSuoue serueretJrq (srusrqdrour,(1od qrtual lueutery uortorrtsar)sd'I{U (y1-9 arn8rg)'ereldurarVNU pepuerls-e18urs e uor; pazrsaqruds sl VNq PePuerts-elgnoP e rlllq./!\ uI uolrJear xalduoc e sazlle -teJ lErlt sesnrrloJtalur punoJ eu:dzug aselduf,sueJlasJeAeJ $g-g a:nfig\'suralordlerr^ roJ sVNUU eqt se se rnuoua8Jar1tJnJ ol esrrse,rr8pue 'snrr,rorde Sunu.roy VNU Ile.,rn .VNC Ipruosoruorrlrrelnller olur peuasur sl VNC IErr^ srql 'vNU eql ;o ddor VNe e Sur4eurrsrr; dq sller ur salerrlderreqr auroue8VNU ue Sururetuoc snrr,rorlodrelne;o edl.1 snJraoJleJ (qg-9arn8rg).uos 'dots -odsueJl osle JaS uondrrcsupJt esJeeJ E se^lolur pu€ elerp -eurrelurVNU u€ .{q pererparu sr aurou:3rqt ur lueruelou esoql\ crlodre4na;oadll uosodsuerlorlar lueuele yll.Iq alqesodsuerl

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dq pa,rou ere leql rro3 Jo uetro aql ur sller rrprl Jo suorsnrl -ord pa11ry-lueu€lrJ alrtouuoN (run{rroorals'8urs)erpooarols (7-17an*gl slertualodletueudolaaaptuoreJJrpqrr^aslleJ;arq8nepalereue8 o1 Kllwrualuu.tCsp to IIer urots le]urrpd eql ol Iprrluepr sr lprl -ualod esoq.ry\ sllel rarq8nep o,r\t ot esr.r a,rr8o1 leluarudole,rap Kllotulauu(s epr^rpuef, legl 11erSur.trauar-JlJs V IIOrtuols $7-7enBr.g) 'luelsuoJ '1enbeaJEeoupls sur€rual uorleJtuJ)uoJ str os leql -qns € Jo uorldrunsuoc eler pue uortpruJo! ewJ eqt uerln\ Jo Jo uortrpuor aqt 's.,te.u,qted rrlogetau relnllel uI elels dpeets (71-91 en?;.g) 'sroldoceraur4ol(l ot Surpurgpue8rl3umo1 -1o; eqt ur pele^rtJeeJeteqt srotJeJuorldrrcsuertJo sse]J losordc 'uorldrrsuerl Jo uorle^rtrv pue uorlf,npsuera1eu8r5 IVIS (97-71ern8rg)'s11er rueld ur arerpdqoq;era8erolsdreurrd eqrsTteqt 'slrunesocnlS yo dla,rrs -npxa pasoduoc'eprreqcces.{1od peqruerq'3uo1drerry rlf,rpls ( g g - 9 1e r n -8tg) 'sptdtlrer1to srseqluds se IIa^{se trodrurpue srsaqluds Jo IorerseloqJur pe^lo^ur surelordSurporuasoua8;o uorsserdxa atelntultsueql pup sla^el re]nl]ef,,violol asuodsarur lorotseloqr 'sJolreJuortducs pate^rtf,ear€leqt 'aue.rqrueur UA eWur pazrleJol -ue.rl luepuedep-lorerseloq3(saggUS) suralord turpurq-ggg (11-garn8rg)'EurcrldsVNU rno serrr€rpue y51gur-arde uo salquesseleqt xaldruocuratordoalcnuoqrJ aSJe'I otuosoarqds (q9-61orn8rg)'(seprsoq8ue8'saprsorqoroc) dno.r8peaq ererp,{qogrec ;o (uqo.(uro8urqds) dnor8 peaq parell.roqdsoqde rar{trepue surpqf,uoqJEJorpdq 8uo1o,!u ureluoJl?rll'ourso8urqds ruo4 pa^Irap 'sptdrl auerqwaur;o dnor8 .lo[eyrq prdqoturqds 'surelo.rd cr;neds8urre1.(.roqdsoqd trq apdc I]ar eqr;o aseqdS eql otur 11orcrrodrelneE to eruprtues.re33rrl pue uqr,b t9 e pesod teqt '(yq3) aseuDl luepuedap-uqcdc ;o -uroc'uretordJrrerurporetaq V (rotteyEurtourord-aseqd 5) g45 'ftt-ZZ pue 'Sururelted ZI-ZT.sarn8r4) IErlue^-lesrop ur suorlJunJteql sodrgua uerqrqdue Pue rorratsod-JorJelue .(1reayo eprslpsrop rrlt uo ratueJSuqeu8rg nzruetto uueruads

(61-y1 arn8rg) 'uortrsodde osolr otul souerqlualu 'saxelduroc elgEls dra^ sluroJ auerg teSret pup olf,rse^eql Sur8urrg -ruaru la8rel e uo SEUVNS-I eteuSoc qll^{ olJIseAP uo sgUVNS-^ 'seuEjqtuetu leSJel qll^\ soJJIsJA uOISnt alou JO to uorlJBJelul -ord reqr surolord ou€rqruaru ler8atur Pue lllosoilJ SSUVNS fiZ-07, pue gE-9 ern8rg) 's11ecrelq8nep ol sauosouortlt I€Iratreg Jo uolleSer8es radord 'aseqdeue uI uollere eql ur uorlf,un; sutelord JWS IeIJerJEg 'sutsaqo) pue 'srsolrtu -des rraqt lrlun sprtpruoJtlJJatsIs{uq glII{^\ Surrnp soruosoluoJl{f,esuJPuoJ dlag qrq,,n 'sutsuaPuo) ePnlf,ur dpue; srql Jo sregruory 'slsotllu Surrnp uorte8arSos radord rreqt pup saruosotuorr{l Jo eJnlf,nrls lecrSoloqdrou eqt Sululelureur JoJ Ief,rlrJf, oJe leql sutelord ult€uroJqf, euolslquou ;o dpue; IIEIUSe suralord 31415 isuralord atuosoruoJtlf, Jo eJueuelul€Iu lernlf,nJls (y-91 arn8rg)'sroldacar of,€Jrns-llef, rrer1t ol salnreloru Surleu8rs ;o dpue; (dgCf) $ roree; qr.trort tururro;suerl eql to srequreu ;o Surpurq 3urmo11o; uorleldroqd -soqd dq pelp^rlJe ere lBql srolJe, uotldtrcsueD Jo ssEll sPelus (qg7-g arn8r4) 'VNUttu osl€ aes 'seue8 rr;rcads ;o uotsserdxe lIgIquI dlletuaurrredxe ol peuSrsap aq uer sVNUIs 'yNU Klottqtqut llpus Pue yyly Suuapaw ilputs ro Ltoqs pa11erdlsnorrerr fd1roe;redsrted eseq VNUIS aql qrlg.4a ot SVNU 1a8re1saleelc lPql (f,SIU) xolduroc Sulcuelts Paf,nPuI -VNU up rurot ot sutatord Iereles qll.4t salelJosse VNUIS eql Jo puErts alSurs V 'pua rlJeo te seplloalJnu pepuerrs-a18ulso^\,1qll.{\ ';1eursy 3uo1 saprloepnu tZ-lZ 'y11a pepuerrs-rlqnoP VNUIs 'SANIS IIE rsuElunq uI 'VNCI lnoq€ roJ luno)Je slurlurle n/y to sPrrrll-o.ry\r (8uol dq uerunq lelot Jo ruerrod €I: elnlrlsuol lPql ool-oot 'suosodsuerlorler Jo ssPIJ (stueurelaPesredsrelulrroqs) s9515 .YNC anpps peller oslE :Peqursuerl lou erE Pu€ suollProl letuosolu -oJqJ reqlo le sP lle/r\ sP soJeluolal Pue saJaruorlueJ lE Punot aJe 'rroq5 y5q eruanbas-aldurrs leqt seruanbospaleadar ,(luapuer

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Jplnllaf, Jr1rladseJour ro auo ut SurleululnJ PuP roldecar P ol IEU -3rs relnllacerua ue eql Surpurg dq palerrrur ssacord lelluanbos to 'ratpoue olul ruro1 ot sreter .{luouruor l.3o1otq IIaf, uI IEJIIUaqT ro yecrs.(qdeuo luo{ leu8rs e Jo uolsJe^uo3 uorlcnpsuerl pu8rs

(I-€I etqer) 'atuanbas osl€:llel pu8rs pa11et pue apaQad 3ur7.agw1-a4u1in

(II-8 pue eqt urqlrll uorlpJol rr;rcads e ol utelord eql slf,aJIP tetll uratord 'yNlXu-ard Surrrldsur uorlf,unJpue otuosoocqds e ulqtr^\ ecuanbospne oultue rJoqs .{le^IreloJ V acuanbes putrs 6-9 sern8rg) to er{t Jo stuouoduor a.resvNuus e^rc 'snalf,nueql or Pezrl?rol SVNUalgets'lleurslere^esJo auo (ypg realonuleus) yg1us 'snloelJnu eql uI uorleJrtrpouaseqpue Surssecord VNUt ur suouJunJter1l '1yeus;o addr y (y515 reloapnu VNU elqers lleurs)VNUous

(9-91 arn8rg) 'paralduror er€ UE alp olul uorlerolsueJt pue utelord Jql Jo slseqluls areqrrrou€Jqtuetu uE aqt ol xoldruoJ Jruosoglr/uleqf, tuaJseu aql sre^IIePpue urelord drola.rras tuef,seu e ut ecuenbaspuErs gg eql or sPurq legl aprtred (4g5) ollrrred uorlrutocarleuErs urolordoolcnuoqrr cqosotdr y

'lduJsueJl ,fueuud el8urs e otul peqrrJsuert sr teqt 'elrs uorlBunuJet pue elrs (;rers) uorlerlrur ue dq pepunoq 'VNq ur uor8er y lrun uorldrrcsuerl 'secuanbaslroleln8er rroqt ol Surpurq .(q seueBrelnrrtredyo uortdrrcsuerl (srossorder)lrqrqur ro (srole,rrpe) elelnwrts srotrej cltcadg'atrs lrels eqt reau xalduoc uopetlrurard-uorlducsuerl eql ro uortEruJo,ur etedrcrued ,saua81e;o uorldrJ]suerl ro; parrnber 'srotf,EJ rrrodrelne ur uorl lpnuaS's1Iar -drrcsuert atelnSar to elertrur ot parrnba.r'aseraur.{1od VNU ueqr raqlo 'uralord due .ro; rurat IEreueC (ga) rorcey uorldrnsuerl 'aua8 Jplnf,rued e;o uorldrrcsuerl aleln8ar teql seouanbas.(roleln -8er y51q eqt uortar lorluoo-uondrrrsuel ]lE rot urrat e^rrrallo3 'aseraudlod y51g fifV pue gp-y sarn8rg) /q VNU dreluaualdruoJ e Jo srsaqtudsro; eleldurel e se pasn sI alnJe]ou VNe e Jo puerts euo r.{Jrr{1r^. ur ssef,ord uorldrrlsuerl (E1-91 arnSrg)'auerqueruunlnJrlar cnuseldopua aqt otur suretord euerqrueusuer Jo sass€lJsnorJEA Jo uoueluJlro pue uouJesuraqt tJJJrp luauaSue:re pue lequnu 'ecuenbasasoq.Lruralord e urqtrA.stuauSa5 secuanbasrruatodol (gg-y7 arn8rg) 'addr ylar eqr uo Surpuadapsasuodsarsnorrel sef,npurreqr de.rgted 3ur1eu3rsp selertrur Surpurq pue8rl .strn -pord lerqo.rrrur;o dlerre,r B azruSoce:tEgl sJolda)er relnllafpJtur pup aJe;rns-llel Jo sself,e Jo raqruery (911) rordarer e{q1lol (9p-61 ern8rg) .euerq -uaru eurseld eqt;o suor8er lereleloseq pue leorde oqt uee^\teq stuauodruoc eueJquau Jo uorsnJJrp pue sllel uaarr,rtaqseceds eql ur suor pue selnJalou lletus ,{ueur pue solnlaloruoJf,eru Jo uorsnJJrpstue,rardlpqt sller leqeqrrda luerefpe Jo seuprqurau eruseld aql uee^4,taquortcunf l]er-llel ;o eddr y uorlounl lqtrl (67-71 an?r.g) 'suelsdsoloqd pue sluau8rd rrraqlu,{sotoqd aql uretuoc puE sTtpls ur pa8uerre aq uer teqt rseydorolqcE ur sres snouprgruorupeueuelC spro1e1,(ql (e61-g ern8rg) .sureqcaprs uee.trteqsuortreretur luolelof,uou a1dt1lnrulq pazrpqerssr r{r1{1r\'ureqc aprldaddlod e .suralo.rdu1 alnlJn4s ,fuerlrol Jo ruJot leuorsueurp-eerr{tIIpJaAo (9-g a;n8rg) 'eJIIroJ Ierruessesoua8Jo uorlef,rtrtuapr ur InJesndllercedsasr uonelntu;o adlr srql '(arnleradural o,rrssnu -:aduou eql) aJnleradtuetror{toue rE addtouaqd luetnru e 1nq (arnt -zradruel arrrssrturad eql) ernt?Jeduet auo t€ ad.&ouaqd adlr-ppn e saonpord teqt uortetnru v uorlelnu (sl) orrprsues-arnleredruel (79-91 ernSrg)'paralduoc sr (srseu ,asuapuocepsaruosoruorqJ -qordc) tuseldotdc eqt Jo uorsr^rp pue egl (sauosoruorqr pale.ledos Jo sles o,trt aql punore sruJoJ-er adoyaauereelf,nu eqt qlrq,/y\Surrnp aSelsJrlotru leurC eseqdolal $y-9 anfu g)'uorlectldar ylrlq Surrnp sauosoruorrlf, ;o Suruot.roqs slua,rard leqt sserord lercads e 19 paleclda.r ere pue uonBtar8es euosoruoJr{J redord JoJ parrnber ere seJeruolel .acuanb -es ('IgJ) rrreruolet E uoqs ;o sreader uapuel eldrtlnur Sururel -uoJ eruosoruorqc crtodre>1nee ro pue rlf,eo le uor8ag eJeruolel

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-3rg) 'xalduror tqJ Jrreurtlnu Surcnpsuert,leuSrsaql qlr.,rapoterr -osse pue suorSor pu€ olqerJel tuelsuoJ 3urureluoc uralord aue.rq -ruelusueJt Surpurq-ua8rtup JrJaurpoJ3far{ V roldecar IIet-I OE-VZ pue yg-97 sa;n8rg) '(sllal g ,seurloldc ecnpord ,pelcrrlser to uorlelrtf,e ro; pa;rnber

A U V S S O Il

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'(sllar rorunl pue IHW II sselote4reur tqf,) sllor TnQaq pue 'palcrrlser 're>1reur pelre;ur-snrr^11r>1 ace;rnsgq3) IHW I sselc sllerL)txotoTd: :sasselo ro[eruo-n]arpereql'solnJeloruf,HW or pexaldurorsaprrdad rrua8rtuepurqreql srotdecercr;rcads-ua8rlue sossardxa pue snurdqreqt ur serntetuteqt et/Joqdu,{l V IIor I (71-7 an?r.g)'selgruesse xelduoc uort€rlrur-uorldrnsuerl eql eJeqA\seua8Surpoc-urelord f,tloIJe{ -na dueru;o ralouord aqt ur aruonbospo^Jesuof, V xoq VIVJ

(c'q6-Sern8rg) 'aua8petelarp Jo erueseql ur uorlelnur Jaqtoue roegecrddr ;o -ouoqd oqt saseeJf,ur trreqrur(s teqt uortelntu V uorlelnru 1eqra1 'seIJJqS luaJOJJIPeJotu Jo o./r\l uI OIuOS -oruoJrlJ e uo repro orues aql ur seueS ro eJueJJnJf,O i(ualuds 'SurleuBrs 'sleu8rs Ilel-lloJ ur Surledrcrtred .,{geraqr Ieurolxe purq deru pue 'uolela>lsoldr aqt r{ll^{ lf,eJetur 'uorsaqpe xrrleru-]lef, ur uortf,unt teqt supl^ltoalord are;rns-llar Jo sself, v suecapuds 'eueJqrueruetuseld a13urs e dg pasopue ruseldotdr to sseru paleo]rnurllnu V unr1lbuz(s (y-E7 arn8rg) 's11ec crldeudstsod pue -e.rdeql 3urlcouuoo suorlrunI det er.t sJnJf,ouorssrrusuerl aslndrur 'osdeuds )t4)ap ue le frollrursup4ornau e dg parcnpuoc sr eslndurr egl 'osdeudsp)tuaq) E tV 'peltrursuert ere saslndrul glH,1a ssone (y1er elf,snru ''3'a) 11ecalgplrf,xe ror1lo Jo uoJneu tuecelpe u€ pue uoJ -neu e Jo lpurruJatuoxe ue uee^\teq uor8er pazrleroad5 osdpuds

( [s e]'g- r r

ernSrg) 'godrlue osle ees 'uortrorrp auos eqt ur auerqruaru IIar P ssoJJe suor ro selnJslor.utuaJeJtrpornl slrodsuen (tauodutcsl urelord eu?Jgrueru e r{Jnl1\^ur ilodsuerloe ;o addr y trodruds

(e6-g ernBrg) 'suretord Surlreretur Surpocuasaua8dyrluaprol pesn dlruanbar; ere suorlelnu rossarddng 'uort€lnru puooes E to rle;ta crd,bouaqd eqt sesJelal ter{l uorlelnu V uorlelnru rossarddns 'dnot7 ptqt e pa]]Er osle lurole rn11ns E ot pepuog dlluele,ror uote uaSorp,iq e;o Surlsrsuof,selnf,elou reqlo pue ourelsdr prJe ourue eqt ur lueserd dnor8 tuentrlsqns V (gg-) dnor8 l,trpdqyps 'uo8dxo Jelnf,elou ro o)Jo, ar'rtoru-uotord e uo puedep lou op teqt suorlf,eer ur sour,(zuacrlosotlc dq pezdprec t4 pue dCV uro{ dJV to uorl?ruroC uor1e1{seqdssqd leirelarerrsqns 'atulzue ue dq pez,(p -leJ uortJeal e ur e8regc e soo8repun reqr olnJOIoW oleJlsqns (ca-91 arn8rg) 'sdno.r8ldxorplq eroru ro auo Sururel -uof, sproJots erc s1rc.,.als'sproJels ere (euorelsoSord pue ueSorlse ''3'a) 'spunodruoc seuourroq .(ue14 lueuodrur patelor pue Iorel -salor{r Surpnlcur suoq.recorpdqSurr-rno; yo dnor8 y sprorals 'puoq elqnop e Sururetuor srlnf,elour surro1suotl lo pue sn eqr apnlJur staLuost )r4awoa7 'uorqspJ e8erur-.rorrrur e ur paBue.rre ale ruole uoqJer lrrlaunu,(se uB ot papuoq sruolp eql (-I PUE C peleu8rsop 'statuost \oil1flo u1 'sluaura8ueue len -eds ur JepJo erues aql ur aJe ruaragrp polurl sruole esoq^^sel lnq -nuroJ Je]nf,eloru sJaruosroeJels l€Jrtuopr qrr,n spunodruoJ olnl .11errreq r{rea r1lr.r\peteroosse ftt-tZpue gE-97 sern8rg) suoxe eql ur uorlezrJelodep3ur.ra88rrt'suorlerqr,r pef,npur-punos

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AUVSSO't9

-nrurur rprlord ol rep.ro ur asuodsar aunrurur ue ol pau8rsap IJIJIO pue uaSoqredp ruo{ pelrrepuortBrederd snonf,ouur uv eurJJea

$y1an8r.g) 'salodre4naraq8rq ur tueruele leurxo.rd-ralourordro raJu€r{ua u€ ol lual€,rrnbeluorssardxa aua8leurrxeruro; d.ressereu sr lpql seto,fue4na aldrursraqlo pue lseadJo VNC aql ur aruenbas.{rote1 -n8a.rSurpurq-urelord duy (gyn) eruenbesturle.,rrlce rueorlsdn

-r{reru selodrelna pue satodrelord uI selotu dq euoua8 aql e tegt 'uosodsuerl ul tuaserd lueuele y51q olqesodsuerl v vNq (g-9 arn8tg) 'tueruelo yo addr egt uo Surpuadep uslueqJalu elsed-Pue-ddor Jo rusrueqJeru etsed-pue-lnc e ,(q srnoco ieuouaS eql ulqll^\ uoDlsodsuErl elqesodsuerl e Jo luauo^o,^{ luauela vN( (f -g atqet) 'padal Pashdsnlut pue lualuaP VNQ afqou PaIV) osye luorlrsodsuerl ,(q uotttsod A\ou e ot elolu ueJ pue satceds

'rueorlsu^{op ''3'a) sdars E JO slenPr^rpur IIe uI uolleJol leuosouroJrlf, elues Oql uI lue osle aes'(de.uqredSurleu8rs ;o epeJsetr E uI JaIIJBa Jnf,f,o leqt sfueaE(7) ')te'Z-'1- pateu8rsap -sard tou sr l?gl ef,uonbasy11q duy luetuelo ypq alqesodsuert are (aprtoalonu peqrrf,suprt rsrg aql) uorlrsodI + eqt ruorJrueorls 'euErgrueru ]eSreteql qll.&\ uolsnj dq stuat -dn saprloapng 'uorldrrcsuerlSurtnp saloru esererudlody51g ur lpqt ol elrsoddouorlf,errpaql'eua8e rog (1) ueerlsdn qrrq.ry\ 'puoq eldrrrro elqnope sr spuoguogr€J-uoqreJer{t euo Jo qlrr{^r.ur (poe drre;''3'e) punodurotE ot Surrra;aA petprnlesun ([VE]'E-f f arn8rg)'srat.rodrun;o sald -rupxeparpnls-lle.la. erp (surelordIn.1O) sratrodsue.rt esornlSeq1 'uodsupJl pelelrlrf,pJer,r uortertueJuoostr u,r\op eupJg tuarperS -rueruE ssoJJpe]nJelour llerus€ Jo tuetua^oruseterporu(.tayod -run)utalotd euerqruauE qJrq-a ur trodsuerl;o ad.{ty ilodrun 'srseqtu^s dIV Sunrqrqurdqaroqr'slseldorolgrto euerquou proleldqr ro eu€rq -ueru Jeuuraql ssoJJeoJJoJa,rrlour-uolordeql lerJpuoqf,olnu setedtssrp ro (urue8 teqt (louaqdo:lturp-l'Z''3'a) lua8elef,rruerlf, -oruraql uralord eqr ''3'o) ef,uelsqns lernleu ,{uy raldnooun (67-9arnSrg)'uretordre8reregrJo uorlrun, eql ur uorteJatlero 'auososdlaql ol Suruos'eruoseolord eqr dq uorr -eper8ap;ot suralordasaqr3ur33erdqareqt'surelordrelnllef,erlur rar{loot pa1ur1dpualelof,aq uer leql uralordlleursV ulrmbrqn

'raruef, sadft reqlo pue fit-SZ pue 5-97 sarn8rg) ;o letrero -lor Surdolo^opro; lsrr eqt sesearf,ur .{yrear8(f ylUS pue 'JdV 'gU ''3'e) seue8;ossa.rddns-rorunt dueu yo alallptuelnru a13urs e 'rruaSocuosr uonelnruuortf,un;-Jo-ssol e qf,rr{.la ur to aJu€trrer{u1

-uor rraql espelerpu€ elleue8ro rouoP eql ruo{ Jro Sutppng .{q urrot salf,rse1'de,r,lqreddrolenas aql uI uoItJeJIP esJeAeJJo PJEru\ -Jo, aqr ur suralord ,,o8re),, aueJgluotu Pue elqnlos serJreJleql tueruuedruoJ pepunoq-auerqureu IIEurs V elJlse^ lJodsu€Jl 'urelord uodsu€Jl eueJqruou aes uralord ilodsuerl 'urolord eueJqlueru pJtelur oes

uralord eueJqlueusueJl

(4-91 ern8rg) 'pazrsaqtudsSuraq st lI sP uotunl Ug eql sroluo urato.rd drotaroas tueJs?u e gJltl.^aq8norqr runlnJllar crtuseldopue q8no.raqr;o euerqrueu eqt ur xelduor uIOlordIllnIAJ uorolsu?rl (41-y ern8rg) 'VNdur ue uI ef,uonb -as eprtoalf,nu eql trq Peltllads st eruenbes PIre oullue esoq.t\ dlqruassepetelPeru-euosoqrr aqJ uollelsuerl aprrdaddlod e ;o 'alotpaunlut alo$ uorl.ts -upu eql po]ler osle !1aire1d8reua rsaq8q stl le sI rualsds eql ueq^4' uorlJeoJ IeJrrueqJ e Surrnp sluelf,?eJ oql Jo erBls alPls uolllsueJl (tt-W pue 1-y1 sarnttg) 'soruososdlol Jo eJetrns IIaJ eql ol sulaloJd elqnlos Pu€ ousrqlualu drrel luatu -lredruoc r31o3 prsrp-rsou slql uor; Surppnq selrlsa1 'z(er'rqred drolarces aql ur lurod rllueJq roleru e se selJes leql selJlse^pue (NOf) {ro^ lau tE1o5-suo'4 seuerqlueu to lro^.\lou xalduo3 'ouatsuerl e Sutdrrer lerulue Jo lueld due ol SuuJOloU f,ruotsueJl 'suonBreuaB

elrssaf,f,nsot uo pessed sI pu€ IeuIuE ro lueld e olur pelerod slrqrqurtrltcarrpur ro dlloar pue e1c.(c IIar eqt q8norqruorsserSord -rp urelord papoJuaasoq.la, aua8duy aua6 JosseJddns-Jorunl -rocur dlqers pup paJnporrut sr legt euo8 Peuolr V ouatsuerl 'ref,u€Jlseerq SulPnlrut 'taruef, ueur 'lueu8t1eturo uEruoq b-9I pva g-91 sern8rg) aq derulqluor8 ]leJJo sror?ln8arleruroueqrto ssolot enp sesrre -nq ur peteJrldtur ere slueuoduof, uorlJnPsuerl-1eu8ts Sggl ur suortelntrAJ'tI elelnurlls ueql l{r^{or8 llglqul uauo arolu e luorJpa^rrepdllereua8's11ec sseruV rorunl teqr 'llef,e18urs 1o dlue1 Sggl el{r Jo sraquatr J 'sletulue I]e ro lsou uI senssll (g-91arn8rg)'selnqnlorf,rru eqt ruroJot azr Jo IIe^\ lecr.rpurldr lsoru Jo ruerudole,rap eql uI Pesn er€ leql sutalord 3ur1eu3rs -raur.{1od relnqop;o dpue; y uqnqnl tegl surerord1era1a>lsordc ,tyue1 y (dEcr) eleg rolrEJ qrmor8 Eunuro;suerl petarf,es ;o 'luaBP SuIsnef,-Jef,uEJ

'srsoldodBdg o8rapunueqo sl]of,'sleu8rs gcns ..aprcrns,, egt ur fsursrue8ro .re1n11acrr1nru ur sller Jo le^r^rnseql Jo of,uesge ro; parrnbersurato.rd Suqeu8rs snorerunu;o.,{uy rolcel rrqdorl

'VNUrJIreourruE ue Suru.roy 6Z-V pue 61-y sa;n8rg) 'prle ounue relncrtred e ot palurl dlluale,rocseruoJeq VNUr gf,EE'srsaqtudsurelord Surrnp srouop prf,eourue se uorlrunJ lpgl selnf,elou VNU lletus ;o dnor8 y (y5111ro;suerl) VNUI

Jer{lo Jo snJIA ? tllrlr\ luaruleorf dq pocnpur dllensn sellJodord elrl-ref,uEf,r{1r^\IIal e olul I]eJ uellPlurueru .(lPlurou,, e to uols -reluo3 (7) 'uoucaJsuoQalqp$ Paller osle lauoua8 Ilrr-lsoq eq] otul VNC u8rero; e;o uotte:od;orul PuP a{Pldn aql ruort Surrlns -eJ IIef, E ur uollEJelle elqerlJal{'lueu?uIJad (I) uollEruJo}sueJl

'vNur aas vNu reJsuErl

P ot perJ 'VNC fieV'd)'elnrelotu1o;ard13 ern8rg) pornPorl (79-g -rJelsesurpqJ11oe1o]r-1 earqtJo stsrsuoJlsleurue ur palrodsuerl -ur 'arnllnc uI sllar ,{11ensn eql ur seuaS;o uotsserdxedq pe.vro11ol pue perots ere sprf,e.{ue1qrrq.r,lur ruroJ ro[eIAJ eprror,(18rrt otur VN( uglarot to uoIlJnPoJluI JeluJlulredxE uorlteJsueJl 'eprrao,(1trrlaa5 yorer,(ltldrerrr (0I-9 pue 5-9 sarn8rg)'uosodsuerl -orlor osleoo5'uorlrsodsuerl ursrue Pupsrsoqtulsylqq 8ur,tlo,rur

'srsodcoxa pue srsolzb 6t-VZpue g7-g1 sarn8rg) -opue pelerpou-roldacar seulqruof,reql leeqs IErloqllde ue ssorJe se)uelsqns uleuoJ Sutlrodsuert Jo' IuslueqJery stsolfcsuBrl

'JIEIUPE AIEUTO' PUE EIBUI

e uorsnJwo{ Surtlnser proldrp t33apezrlrra; y etot(z to ilel 'srotrq uoudrrcsuer Jnodre1na snorerunu GT,-Lpueq6-Esarn8rg) ur luoserdluor Jurze punorepeplo,sernrJnrts drppuores;opesod -tuor slrlotu PrnDnrls Surpurq-yNq petslar lEre^e5 raturl curz (7y-E an?r.g) 'sllnsarteql slodsolarrsrp Jo ure$ed uorrrp{Jrp aqt Surzdleuepue salnralou par;rrndaqr Jo lelsdrc e q8norqr sder-xSursseddq (sprcef,ralrnupue surelord ,(lrelnrrued)solnf,elouroJf,eru'o eJnpnrtsleuorsueurp-eorqt aql Bur -unuratapro; anbruqcalpasndluouuro3 r(qdertogelsi(rJdur-x (79-91 arnSrg)'stuaru8as eu?rqruarusuert ueAOs qtrznsurolordsselJ-palzzrJ{ eresrotderagraf,ueouo1oc,{llercadsa 'Jef,uef,uerunq ur pelecrldrurare stuauoduroJuorlf,npsuerl-leu8rs lulN ur suortelnry 'slerurueIIe ro lsou ur senssrllsour to luarudo -la^epaqr ur pesn surelord Suqeu8rs palarf,as;o dpure; y rul6, 'tusrueBro ro '1101'upto.td 'auo8e Jo urot luetnuuou ,1euro1q edft ppq (gg-gern8rg)'8ur.1go1qounuut palIl.coslelserpoqrlue peleqel;o asndq petrerepar" ueqt suratordcr;nedspue .euerq -rueruraqto Jo asolnllaf,oJlru e ol peqtepe ere srseroqdorDele .,(qpeteredassuralord qrrq.{t ur anbluqcel turpolq urelsa I

AUVS50'19

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sarn8rg) 'ouerqruau p ssorf,e selnJolou Jo uodsuert polerpeur -uratord se qcns ssacord Joqlo Jo uorlf,poJ paztrlele>austlzua ue ;o dtrcolal lerurxeru eql seqrrJsep leql JatarueJed xeu1

@y-yentugl 'qrrpeser dSolorqIIer ur pesnl.lepmr:llel tsoq olqrldacsns e ur

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CD28 in T-cellactivation,1,094-1095 in T-cellkilling, 1095 cD40, 1096, 1.101. cD80,1094,1099 cD86, 1.094,1.099 cD133,1111 ddc mutations,1,70-1,7l,'1.70f, 85 1,-852, 8 52f, 860-862 in cell-cycleregulation,859-853 Cdc2.853t,859-863,86l f Cdc6,879 Cdc138 , 5 3 t ,8 5 1 Cdc1.4,871 Cdcl4 phospharase, 858, 879,886t Cdc20,869-870, 888-889, 889f Cdc25,861-852,8621 Cdc25phosphatase, 861, 862, 883, 886t Cdc25Aphosphatase, 883, 886t Cdc25Cphosphatase, 886t Cdc28,853t,873 Cdc42 in actin filamentassembly, 725,726f in cell migrarion,746-748, 748f-7 5Lf cdhl, 858, 859f, 870, 871,,876 Cdh14phosphatase, 858, 859f CDK-activatingkinase,862, 862.862f,862f CDK inhibitoryproteins,851, 883-884,886t CDK mutant,ATP analog-dependent, 866-867,867f C D K 1 ,8 5 3 t ,8 6 1 ,8 8 1 ,8 8 3 CDK2, 853t, 862-863,863f,881 cDK4, 881, 884, 1134-1135 c D K 5 , 8 8 1 ,8 8 4 ,1 1 3 4 - 1 1 3 5 cDNA (complementary DNA) amplificationof, 189 definitionof, 181 cDNA libraries,1.79-1.82, 1,80f,1,82f screeningof, 1.81.-182, 1,82f CED-3,in apoptosis,939-941,939f CED-4,in apoptosis,939-940,940f CED-9,in apoptosis,939-940,940f Cell(s),1-30 birth of, 906-921..Seealso Cell division blood,2f red.SeeErythrocyte(s) stem,8 synthesisof, cytokinesin, 672-673 white. SeeLeukocyte(s) cancer,1107-1119,1,1,08L \ee also Cancer as chemicalfactories,15-20 cloningof, 9, 9f, 372, 394 cohesionof, 16 competent,951 daughter.Seealso Cell division;Stemcell(s) germ-line,913-91,4 in meiosis,167, 1,68f in mitosis,L67, 1.68f,872, 8721 retroviral,158 from symmetricvs, asymmetriccell division,906-908 deathof, 19-20,20f,88. Seealso Apoptosis development of, 8, 8f diploid1 , .9,1.66,849 diversityoI, l,2f endothelial, in Ieukocyre exrravasation, 837-838 epithelial.SaeEpithelialcell(s) eukaryotic,3, 3f evolutionof, 4, 4f, 6-7,23-25 excitable,1003-1004 muscle,1004-1005 neural,1003-1004 extracellularmatrix of. SeeExtracellular matrlx l-8

.

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fluorescence-activatedsorting oI, 394-39 5,

395f founder, 908-909 functionsof,15-20 germ,905 divisionof, 1,67,1,681 in oogenesis, 9 53-955, 9 53f primordial,953 germ-line,13-1.4,91.3-91.4, 950 fate of,907-908 segregation of, 953 stem,91-3-9L4 haploid,1.9,1.66,849 horizontal,7027f, 1029, 1030-1031 hybrid,401 immortal,398,398f integrationinto tissue,801-843 lumen of, 410 microclimateoI, L4-15 microscopicappearance of, 2l}Jll moleculesof, 9-1,4 movementof. SeeCell movement/migration necrosisof, 937 nucleusof. SeeNucleus parietal,472,472f pH in, 52 plant,2f elongationof, 378 growth of, 840 properties of, 839-842, 839f plasma,3f in immuneresponse,1,061,1061f, 1075 polarityof, 8, 47L,71,4 cytoskeletonand,7 14-715, 7 l4f postmitotic, 264,849 postsynaptic,1005 precursor,905 presynaptic,1004f, 1005 primary,definitionof, 394 progenitor,905 prokaryotic,structureof,2-3, 3I protein contentof, 11,23 quiescent,781 reproductionof,7-8,7f, 8f, 1.8-1.9,1.81. Seealso Cell cycle;Cell division; Reproduction satellite,cultureof, 396,3971 secretory, in rough endoplasmic reticulum, 376,376f senescent,1115 s h a p eas n d s i z e so f , 7 , 2 f , 1 6 somatic.SeeSomaticcell(s) steady-state reactionsin, 50 stem.SeeStemcell(s) structureof,2-3, 3, 3f,1,6.Seealso Cytoskeleton transformed,397-398, 399f transientamplifying,905 typesof, 2f Cell-adhesion molecules(CAMs), 16, 395, 803-805.Seealso Cell-celladhesion; Cell-matrixadhesion adaptorproteinsfor, 803 in adhesivestructures,833, 834f cadherins,803, 804f. Seealso Cadherins diversityof, 808 domainsof, 803, 804f, 808 evolutionof, 807-808, 807f familiesof, 803, 804f fibronectins,830-833, 831,f,8321 functionsof, 803-804, 804f heterophilicbindingby, 803, 804f homophilicbinding by, 803, 804f Ig superfamily,803,804f, 1.067 immunoglobulinfold in, 1057 immunoglobulin,836-837

integrins,803, 804f,816-81.7,817t. See a/so Integrins intercellular,836-837 isoformsof, 808 laminins,805t,821, 821f,822f in mechanotransduction, 843 neural,836-837 selectins, 803, 804f. Seealso Selectins in signaling,803, 807, 807f, 833-835, 843 in synapticcommunication,1019 synthesisof, 803-804 vascular,835 Cell biology,20-21 Cell-celladhesion,802f, 808-819 adaptorproteinsin, 803 cadherin-mediated, 810-814 calciumionsin, 811, 811f cell-adhesion moleculesin. SeeCelladhesionmolecules(CAMs) cis,803-804,804f disruptionof,806-807 formationof, 803-804, 804f heterotypic,803, 805 homotypic,803 IgCAMs in,835-837 integrinsin,8l6-817 intercellular,804, 804f intracellular,803-804, 804f lateral,803-804,804f in leukocyteextravasation, 837-838, 838f motile, 833 nonmotiie,833 oligosaccharides in, 552 overviewof, 803-808 in plants, 847-842, 842f propertiesof, 804-805 signalingin, 833-835 tightnessof, 804-805 trans,804, 804f Cell colonies,396 Cell cortex,7L6,71.6f Cell cultures,372 adherentcellsin, 396-397 animal-cell,395-396 for artificial tissue,404 moleculesin, 395 cell-adhesion in cell differentiationstudies,396,3971 cell linesfor, 398-400 definitionof, 398 differentiationin, 398-400 immortalized,398, 3981 cell strainsfor,397 clonesin, 372 disadvantages of,400 embryonic stem cell, 91.'1.-91.2, 91.1.f epithelialcellsin, 399400,401f in expressionsystems,1.94-196,195f fibroblastsin, 396, 397f future researchareasfor,404 hybrid, 400-402,402f life spanoI, 396-397, 398 MDCK cellsin, 399400,4011 mediafor, 395-396, 40L, 402f in monoclonalantibodyproduction, 400-402,401.f myoblastsin, 396 nanotechnology for, 404 nonadherentcellsin, 396 primary cellsin, 394,396-397 in protein factories,L94-L96,'1.95f satellite cellsin, 396,397f stagesof, 398f transformedcellsin, 397-398, 399f viral, 155, 156f

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Clathrin,structureof, 598, 598f Clathrin/AP-coated pits, in receptor-mediated endocytosis, 606-607, 607f, 609-61,0, 1,023 Clathrin/AP-coated vesicles,393, 586-589, 587r, 598-600, 598f-600f in organellepurification,393-394, 393f pinchingoff of, 599-600,599f,600f,1.023 dynamin in, 1,023 in receptor-mediated endocytosis, 606-607, 607f uncoatingof, 600 Claudin,814f,815-815 Cleavage proteolytic, 91-92 in secretoryparhway,603-604, 603f, 604f of zygote,9 50, 9 60-9 61, 9 50f Cleavageand polyadenylation, in pre-mRNA processing, 329, 3291,335-336, 336f Cleavage factors,335, 336f Cleavagehrrow,790 Cleavagelpolyadenylation complex, 335-336, J.J6T

Cleavagestimulatoryfactor,335, 336f CLN genes,874-876, 874f1 Clonal selectiontheory,1066-1067,1066f Clones/cloning, 7, 9, 176 cell,9,9f, 372, 394 DNA, 176-190 in cDNA library consrruction,179-\82, L80f, 782f definition of, 176, 1,79 gel electrophoresis in, 184, 185f Okazakifragmentsin, 178 restrictionenzymesin, 1.76-1.77 restricrron tragmenrs in, 177-178,178f sequencing of, 1.85f-187 f, 1,87 subcloningin, 184 transformation in, 1,78-1,79 vectorsfor, 176-1.79,176f-1,791. See a/soVector(s) in Northern blotting, 192, 1,921 nucleartransfer,908 of receptors,631,632 in Southernblotting, 191-192, l91I transformationin in planrs,242 in yeast,1,78-1,79 viral, 155 Clotting integrinsin, 834 platelet-derived growth factorsin, 745 von Willebrandfactor in, 834 Clusteranalysis,193, 1,94f Co-activators, 293, 305-31.0 genes,clusteranalysisof, 1,93, Co-regulated 194f Co-repressors, 294, 304-305, 305f Co-Smads, 670-572 Coagulation integrinsin, 834 platelet-derived growth factorsin, 745 von Willebrandfactor in, 834 Coat assembly, in vesicular rransport, 587-588,588f Cocaine,1023 Cochlea,1032, 1.033f Cochlearhair cells,339-340,340f, 1032-1033,1033f Codingregions,definitionof,217 Codons,127-1,29,128t, 129f basepairingwith anticodons, 130-131,131f definitionof, 127 in geneticcode,127-1,29,1,28f,1,29t,240, 24lt l-12

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in plants,240,241.t in readingframe,128, 1,29f start,127-128, 128t stop,127-128,128t in wobble position,730-737, 1.31.f CoenzymeQ (CoQ), in electrontransport, 4 9 5 t , 4 9 6 ,4 9 6 f CoenzymeQH2-cytochromec reductase, in electrontransport,49 5t, 497-498 Coenzymes, 84 Cofllin, 721-722, 722f, 745, 747f Cognitivefunction,1005-1005.Seealso Neuron(s) Cohesins,781.,869-870, 870f, 894-898, 896f Coiled-coilmotif, 67, 69,70[, 292 Colchicine axon extensionand, 1040-1041 mechanismoI action of,766 Cold perception, 1031,-1,032 Collagen,15, 805, 805t anchoring,823t in basallamina, 821-823, 827f, 822f, 823t in basementmembrane,821 in bonesand teeth,826 in cartilage,825-827 classificationof, 822, 823t crossJinking in, 822, 823t disordersof,826,827 in extracellularmatrix, 820 fibril-associated, 822, 823t, 825, 826-827, 827f fibrillar, 822, 823t, 825-827.Seealso Collagenfibrils fibular,805t host defense,822, 823t, 825-826 interactionof, 826-827, 827f procollagenand, 825f, 826 propertiesof,823t sheet-forming and anchoring,805t, 821-822,823t in soft tissue,826-827 structure of, 821-822, 821f, 823t synthesisof, 825-826, 825f in tendons,826 transmembrane, 822, 823t, 825 triple helix of , 821.-822,822t, 825-826, SZsf defectsin, 827 typesof, 822,823t Collagenct chains, 821.-822,822f, 823t Collagenfibers,825, 825f Collagenfibrils, 825, 825f definitionof, 825 synthesis of, 825, 825f,826 in typeI collagen,823t,826 in typeII collagen, 823t,826-827 in type III collagen,823t in type IV collagen,823t in type V collagen,825 in type XI collagen,826 Collagenpro-ctchains,825 Colon cancer,148 development of, 1,1,16, 111,7f DNA-repair defectsin, 1.1.42,'1.1.42t inherited,1124 metastasis in, 1,1,1,6, L1,1,7f mutationstn, \116, 11,24,1125 Colonies,cell,396 '1,027f, Color vision, 1028 Combinatorialdiversity,815 Combinatorialjoining, 1071 Commissureless axons(Comms),1.046,10471 Competentcells,951 Competition assays,for binding affinity, 629, 630f

Complement, 1059-1060 Complementarrty, 32f, 3940, 39f protein binding and,78 Complementarity-determining regions (CDRs), 79,1,067 Complementary base pairs, 114-1.15. See also Base pairs/pairing Complementary DNA. See cDNA (complementary DNA) Complementation tests, 171, 172f Computer algorithms, for microscopy, 387 Concentration gradient, 54-55 diffusion nte and,439 electrochemical gradient and, 439, 464,

46sf ion channelsand. 438f. 458 in membranetransport,438f,440, 447 Condensins, 866 Conditionalmutations,1.70-1.7 l, 1.70f Cones,retinal, 1.027f,1,028 retinotectalmapsand, 1.042,1.042f Confocalmicroscopy,386, 386f Conformation,of proteins,22, 63, 67-70 X-ray crystallographyof, 103-1.04,104f Congenicmice,MHC, 1077-1,078,1.077f Congestive heart failure,468 Congression, 783, 783f Connectivetissue,801, 825-833 basallamina and, 820-825 collagenin, 82L-823, 821f, 822f, 823t extracellularmatrix of, 825-833 glycosaminoglycans in, 827-830 hyaluronanin, 829-830 proteoglycansin, 827-830 turgor pressurein, 830 Connexin,383, 383f, 802f, 819, 1026 Consensus sequences, 329-330, 329f Conservedsynteny,259 Constantregion,of light chains,1.0661,1,067, 1068 Constitutivegeneexpression,290 Constitutivesecretoryvesicles, 602 Constitutivetransportelement(CTE), 346-347 Contractile bundles,7 41,-742 in cell migration,T4Tf Contractilering, 71.6,71.5f, 742, 742f, 789 Contractilevacuoies,444 Coomasieblue, 98 Cooperativity,in protein regulation,89, 89f Coordinate rcgulation,27 | COPI vesicles, 586-589,587,587t,589t,595 COPII vesicles, 586-589, 5871,587t, 5881, 589t, 592-593, 592f-594t 495t Copperions, in ATP synthesis, Cortical neurons,structure of, 1.002f Cortical reaction,957 Cos2protein,702 Cotranslationaltranslocation,537-538,537f, 539.Seea/soProteintranslocation of integralmembraneproteins,544-546, 544f. 545f of secretoryproteins,537-538, 537f, 539 Cotransport,440, 440t, 465470 antiportersin, 440, 466, 468470. Seealso Antiporters symportersin, 440, 466470 Coupledreactions,57, 58 Covalentbonds,32-35. Seealso Bonds definitionof, 32 formation of, 33, 33f geometryof, 33, 33f, 34, 34r high-energy,58 40, 41f in macromolecules, nonpolar,34 Dolar.34

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denaturationof, 1.1.6-1.1.7, 11.7f,1.41., 1.41.f, 142f directionalityof, 1.14,1.14f DNase digestion of, 251.,251.1 duplex,139 5' end of, 1.14,1.741 functions of, 111.-1.12,1.13f hyperchromicityin, 1.17 intermediate-repe^t, 226 interspersed-repeat, 224, 226, 265-266. See a/so Mobile DNA elements junk, 215 lengthof, 21.6,247 234-235 linker,248-249 monogenetic, 200f,203 meltingtemperature of, 117,1.L7f multiple endocrineneoplasiatype2, 1127 methylationof, in genomicimprinting,958 musculardystrophS1.99r,200, 200f, microsatellite, 201, 224, 224f, 225f 234-235, 7 31, 795, 827, 835-835 in DNA fingerprinting, 225,225f mutatlonscausing,14 in geneticdiseases, 224,340 myotonicdystrophy,224, 340 minisatellite,225, 225f osteogenesis imperfecta,827 mitochondrial.SeeMitochondrialDNA pemphigus vulgaris,813-814 mobile.SeeMobile DNA elements polycystickidney disease, 780 moderately repeated,226 polydactyly, 992,993f natural,115,115f polygenetic,203-204 noncoding, 215115, 221.t,223-226,224f., rerinitispigmentosa,204 225f RNA editingin, 341 amountof, 223 scurvy,825 evolutionof, 223-224, 225-226 sicklecell anemia,157 microsatellite, 224, 224f sicklecell disease, 1,67,199,l99t satellite, 224,225f signalingdefectsin, 671,-572,680-682, in nucleosomes, 248-249, 248f 706-707,710 nucleotidebasesof, 11. Seealso Base(s); situsinversus,9 54-955 Nucleotide(s) spherocyticanemia,730 organelle,236-242 spinalmuscleatrophy,334 chloroplast, 236,242 spinocerebellar ataxia,224 mitochondrial,236-242. Seealso stemcell therapybt 912 mtDNA (mitochondrialDNA) Tay-Sachs disease, 374 packagingo1,216, 21.7,247, 378. Seealso thalassemia, 345 Chromatin tuberoussclerosis, 354, 355 palindromic,1.76,1.76f Ushersyndrome,1033-1034 parental,139 vaccines for, 1.1.01-1\02 phosphodiester bondsin, 40, 41.f,'1.14, 'Wilms' tumor, 290, 290f 11.4f xerodermapigmentosum,748-1,49,1142, plasmid,1.78-179 71,42t recombinant,23, 176. Seealso Zellweggersyndrome,568 RecombinantDNA technology Disheveled p athway,667f definitionof, 175 Disintegrin,705 experimentalorganismsin, 25 Dislocation, 555 expression vectorsin, 194-197 Disruptionconstruc, 205, 205f relativeamountsof, 223 Drssociation constanr(Kd),50-51 renaturationof, 117 for acids,52, 53f repetitious.SeeDNA, noncoding for binding affinity, 628 replicationof. SeeDNA replication Dissociation(Ds) elements,228-229 selfish,226 Dissociationreactions,52-54, 53f (satellite), simple-sequence 224, 225f Disulfidebonds,43, 552 in DNA fingerprinting,225,225f in proteins,68 stability of, 1.16,1.1.8 DNA, 11, 111-112 strandseparationin, 116-117, 117f A-form,115, 115f structureof, 1.0f,1.1.,llf B-form,115,115f doublehelix in, 11, 11f. Seealso bacterial,l2 Double helix basesin, 11,,44,44f.Seea/soBase(s) supercoiled,117-1.1.8,11.8f bendingof, 3f, 12, 117-1,18,ll8f, 122, 1,231 synthesisof. SeeDNA replication centromeric,785 template,120, 1.21.f chloroplast, 1,3,236, 242 3' end of, 1.14,L1.4f in chromatin,247-256. Seealso Chromatin unclassifiedspace6220t, 225J26 circular,3f, 1.2,13, 11,7-118,1,1,8t. See unwindingof, 1.1.6-11.7, 11.7f,1.41., 1.41.f, a/so MitochondrialDNA 1.42f classificationof, 220t vector,176 cloned,L79. Seealso Clones/cloning Z-form, 115, 115f codingregionsof. SeeGene(s) DNA affinity chromatography,288, 288f complementary strandsof, 11, 11f DNA amplification copyingof. SeeDNA replicarion by cloning,176-1.90.Seealso daughter,1,39,849 Clones/cloning synthesisof, 739,1.41.-1.44. Seealso by polymerase chainreaction,188-189, DNA replication 188f,189f Hutchinson-Gilford progeria syndrome, 795,866 I-cell disease,602 inheritance of, 199-200, 200f, 203-204 Kartagener's syndrome, 9 54-9 5 5, 968 kidney disease,780, 822-823 Leber's hereditary optic neuropathy, 241 leukocyte-adhesion deficiency, 838 lysosomal storage diseases,502 microsatellite repears in, 340 m i s f o l d e dp r o t e i n s i n , 7 7 , - 8 f mitochondrial mutations in, 241,-242 m o b r l e D N A e l e m e n t si n , 2 3 2 - 2 3 3 ,

in polytenization,261,,26'l,f 1120 in proto-oncogenes, DNA-basedmolecularmarkers,200 DNA-binding domains of activators, 288-290, 289f linkage of, 289-290, 289f linker scanningmutationanalysisof, 290 of nuclear receptors,312-313, 31.2f,31.3f of repressors, 290 structuralmotifs in, 290-293,292f, 293f structureof, 289-290, 289f types of, 290-293 DNA-binding motifs,69-70, 70f, 290-293, 291.f-293f basic-zipper,292 coiled-coilmotif, 67, 69, 70f, 292 helix-loop-helixmotif, 69, 90, 90f, 290-293 helix-turn-helix moti[, 69, 70f, 290 leucinezipper,69, 70f, 291,-292,293f zinc finger, 69-70, 70f, 291.,291.f DNA chips,192 DNA clones,1,76-1.90. Seealso Clones/cloning DNA damage apoptosisand, 891 in cancer,1.1.36-1.1.37, 1.1.37f p53and,891 repairof. SeeDNA reparr DNA-damagecheckpoint,888t, 891 DNA end-joining,149-150, 150f DNA fingerprinting, 225, 225f DNA flow cytometry,395 DNA hybridization,11, 1\7 definitionof, 181 in DNA library screening,181.-1.82,1,82f DNA ladder,185f DNA libraries,1.79-182,180f, l82f screeningof, l8l-182, 782f DNA ligase,141 in cloning,L76, 1.77-1.78, 1.78f DNA microarrayanalysis,23,24f, 1.92-1.94, 194f, 795f in cancer,1.1.1.6-1.'1.1.9, 1.1.'1.8f, 1.127 in diseasegeneidentification, 202 in geneamplification,1121 DNA Pol B, in cancer,1143 DNA polymerases, 12-13 in DNA repair,1.45,L143 in cancer,L143 in DNA replication,12. 140-141, 141f,

r42f,r43 proofreadingby,'1.45,l46f DNA polymorphisms definitionoi 200 DNA fingerprintingand, 225,225f in linkagemapping,200J01, 200-202, 201.f restriction fragment length, 201, 207f singlenucleotide,20l DNA recombination.SeeRecombination DNA repair,745-154 baseexcision,147, l47f defective,in cancer,145, 1108, 11,36-1137, |137f , 1.14L-Ll43,1.1.42t of double-stranded breaks in cancer,L143 by homologousrecombination, 1.52-1.53, t52f by nonhomologousend-joining, 149-150,150f of r{diation-inducedmutations,1,49-1,50,

1s0f recombinationin. 149-153 homologous,150-153, 157f, l52f npnhomologous, 1.49-150,150f of replicationfork collapse,150-152, 15lf TFIIH in,298 transcription-coupled, 149 INDEX '

I-15

DNA replication,172, L1.3f,1.39-1,45 autonomouslyreplicatingsequences in, 261 backwardslippagein, microsatellite repeats and,224,225f,340 basepairing in,139-140. Seealso Base pairs/pairing conservative, 139 cyclin-dependent kinasesin, 144 directionof, 140-141, 1.411,1,43-744, 143f,1.44f D N A p o l y m e r a s iens. 1 4 0 - 1 4 1 ,l 4 l , l 4 l f , t42I errorsin. SeaMutations in eukaryotes,144 helicases in, 1.41.,143, 1,44,l44f inhibition of, 895 initiation of, 1.40-741,877-879, 878f at replication origins,141,261,262f, 877-878,878f laggingstrand in,'1,41,141f, 1,42f shorteningoI, 263-264, 264f leadingstrand in, 1.41,141f, 1,42f MCM proteinsin, 144 mitochondrial,236 Okazakifragments in, 141, 141f,143 originsin, 141 in polytenization, 261, 261f prereplicationcomplexestn, 878-879, 878f primersrn, 141, 143 replicationforks in, 141, 141f, 1,42f, 1.43-144,t44f in S phase,876-879,875t-878f semiconservativ e, 139-140, 140f telomerasein, 263-254, 265f telomereshorteningin, 263-264, 264f templatesIo4 1,39-140,140t, 1.41.,141.f, L42f unwindingin, 1.1.6-1,L7,117I, 141, 141f, L42f in viruses,1.42-144,1.42f,1,43f in lytic cycle,156-158,1.55f,l57f in yeast,877-879,878f DNA replicationinitiation factors,877-879, 878f DNA responseelements,31.3,31,3f DNA sequencing,243-247. Seealso Genomics dideoxychain-termination methodof, 1,8sf,186f,187 polymerasechain reactionin, 188-190, 1 88 f whole genomeshotgun,187 DNA transposition,226J35. Seealso Transposition DNA transposons, 227-229, 227f-229f, 265-266,350.Seealso Retrotransposons bacterial,227-228, 227f definition of,227 eukaryotic,228-229, 229f in exon shuffling,235 multiplicationof, 229, 2291 DNA viruses,154. Seea/soVirus(es) oncogenic,1,1,22-1,1,23 DNase,chromatindigestionby,25l, 251.f DNaseI footprintng,286,287f Dolicholphosphate, 550, 551f Domains.SeeProteindomains Dominant-active pr oteins,747 Dominant alleles,155-157, 1.67f Dominantmutations,1.66-1.70, 167I, 860, 1r1,3 genefunction and, 166-167 segregation of, L67-169, 1,68f,169f Dominant-negative mutations,L67, 209, 674, 747,1.L36 l-16

.

rNDEX

Dominant-negative pr oteins,747 Dopamine,structureof, 1,020f. Dopaminereceptor,cocaineand, 1023 Dorsaltranscriptionfactor,971,,972f Dorsal-ventralpatterning in Drosophilamelanogaster, 970-971, 97'tf,972f. in Xenopuslaeuis,963-965 Dosagecompensation, 253, 9 58-959, 959f Double helix, 1.1.,1.1,f,114-116, 115f, 1.1.6f basepairs in,1,1,4-116,115f.Seealso Base nairc/neirino

bendingof, 1.1.5-176, 1.16f left-handedorientationof, 115, 115f major/minorgrooveof, 115, 115f right-handedorientationof, 115 Double-mutantanalysis,1.71.-172,1.73f double-sex,alternativesplicingin, 339, 339f Doublet microtubules,760, 7 601 Down syndrome,887 Downstreampromoterelements,298 Downstreamtranscription,1.20,12Lf, 277 Drosha, 348 Drosophila melanogaster asymmetriccell divisionin, 931-935, 932f-934f body segmenta tion in, 974-9 83 developmentin, 970-979 pamernlngrn, anterlor-posterlor 971.-974, 973f,974f patterningin, 970-971, dorsal-ventral 9711,972f of eye,685-687,587f lethal mutationsin, 999-1000 as experimentalorganism,26 geneticscreensfor, L71 germ-linestemcellsin, 914 heat-shockproteinsin, transcriptionof, 315 Hedgehogsignaling in, 701.-702,7 01.f Hox genesin, 979-981.,980f mutationsin, lethal,171 neurogenesisin, 929, 931,-935, 932f-9 34f oogenesis in, 953-955, 953f P elementsin, insertionmutationsand, 1,89-1,90 polytenechromosomes in, 261.,261.f Ras/MAPkinasepathwayin, 685 retinalneuronsin, 340 sexualdifferentiationin, 338-339, 3381, 339f stem-cellnichesin, 913-91.4,91.3f protein in, 1097-1098 Toll 'Wnt signalingin, 699-700 Drugs agonist, 629 antagonist, 629 membranetransportof, 455 stereoisomers of, 33-34 Dryer,V., 1105 Ds elements,228-229 Dscam isoforms,retinal neurons and,340 DSL complex,in neuroblastdivision,933-935, 933f Duchennemusculardystrophy,199t, 200, 2001,234-235,835-836 Duplex DNA, 139 Duplicatedgenes in genefamilies,217, 218f, 220 segmental duplicationand,221in tandem anays,220t, 221.-222 Duty ratio,of myosin,735,737 Dynactin,774-775, 775f in mitosis,787, 787f, 788f, 789 Dynamin, 1023 in vesiclebudding, 599-600,599f,600f, 609f

Dynamitin,775 Dynein(s),71.5,774-776, 7741,775{ in axonaltransport,775-776 axonemal.

/ / /. / /61

cytoplasmic, TT4 958 in heart development, rnner-arm. ///. //61 in mitosis,787, 787f, 788, 788f, 789 outer-afm.///. //6I power stroke of, 774, 775f structureof, 774-775, 774f, 775f Dystroglycan,835-836, 836f Dystrophicglycoproteincomplex,835-836 Dystrophin,7291,731, 835-835, 835f in musculardystrophy,835-836 F. box,926-927 E-cadherin,803, 804f. Seealso Cadherins transitions and, epithelial-mesenchymal 81.2-813,81.3f E-selectin,838 E site,of ribosome,1.33,134f, 1.36,1.37 E2A, in myogenesis, 927 E2F factors,882-883, 882f E5, human papillomavirusand, 1-L37 E6, human papillomavirusand, 1137 E7, human papillomavirusafld, \137 Ear. Seealso Hearing structure and function of. 1.032-1034. 10331,r034f genes,690, 881 Early-response cyclin-CDKcomplexes,853t, Early S-phase 877, 877f Ectoderm,907, 907f developmentof, 907, 907f, 9 51, 962, 962( in limb development,99I-992, 991.1 in neurulation,985, 985f, 990 Edmandegradation,103 EF handproteins,69,70f,90,901 in signaling,534 Effector proteins, in cell migration,747 Effector specificity,in receptorJigandbinding, 628 EGF domain,71.-72,71.f Egg, 1.9.Seealso Fertilization;Oocyte(s) EGL proteins,in apoptosis,940 eIF2 kinases,355-356 eIF3,133, 1.34,1.35f eIF4 cap-bindingcomplex,133, 134, 135f eIF4E,351-352,351f eIF4E-bindingprotein,353 34, 35f Electricdipole, .Waals interactions,38, 38f in van der Electric energS54 Electricpotential, 55.Seea/soMembrane potentral 1,025-1026.Seealso Electricalsynapses, Synapses gradient,439, 464 Electrochemical Electrogenicpumps, 453. Seealso Pumps Electron carriers.Seealso Electron transport in glucosemetabolism,487, 489491' reductionpotentialof,499, 500f in respiratorychain,495499, 495t, 497f Electrondensitymap, 103-104 Electrongainlloss,59-60, 591,60f of secretory Electronmicroscopeautoradiography, pathway,582-583, 62L-622, 622f Electronmicroscopy,2'1,,388-390, 388f, 404. Seealso Microscopy electron Electron shuttles,49049L e, 49049 1.,490f malate-aspartat Electrontransferflavoprotein(ETFI,497 Electrontransferflavoprotein:ubiquinone (ETF:QO),497 oxidoreductase

Electrontransport,493-503.Seealso Protonmotive force ATP synthasein, 504-505, 504f in ATP synthesis, 504,504f cell damagefrom in chloroplasts,521-522 in mitochondria,502-503 chemiosmosis in, 503-504, 504f coenzyme Q in, 495t, 496,496f CoQH2-cytochrome c reductasein, 495t, 497498 cytochromec oxidasein,495t, 498499, 501f cytochromesrn, 495495, 49 5t, 498499 directionof, 495 in glucosemetabolism, 487,48949L iron-sulfurclustersin, 495496, 495f in mitochondria,493494, 494f multiproteincomplexesin, 495-502,495t, 497f NADH-CoQ reductase in, 495, 495t,496, 497f oxidativephosphorylationrn, 494 in photosynthesis, 512, 513f-515f, 514-515,517-520,51,8f,520f, 521f in bacteria,517-520, 51.7f,5l8f cyclicvs. linear flow in, 519-520, 520f, 521f, 522-523,523f, 524f prosthetic groupsin, 495,495t proton pumps in,493494, 494f stoichiometryof, 499-500, 501f Q cyclein, 500-502,501f stepwiseflow in, 493, 499, 500f succinate-CoQ reductasein, 49 5t, 496497 supercomplexes in, 498f, 499 toxic by-productsof, 502-503, 521-522 uncouplers in,510 Electron transport chain, 480, 493 Electronegativity,34 Electrophorectic mobility shifr assay(EMSA), 286,287f Elecrrophoresis. SeeGel elecrrophoresis Electroporation,195 Electrosprayionizationion-trapmass spectrometry,101,-1,03,102f Elongationfactors,135-135, 136f EMBL Sequence Data Base,243 Embryo animal pole of, 963 polarity of, 950-951 vegetalpole of, 963 Embryogenesis , 908f, 91.1,949, 9 50-969.See a/soDevelopment asymmetriccell divisionin, 8, 905-908, 930-936.Seealso Cell division, asymmetrlc in Caenorhabditis elegans, 908f. cleavagein, 950, 960-961,960f definitionof, 930 dosage compensarion in, )53. 958-959,9591r in D rosophila melanogaster, 970-97 7 eventsin, 962f future researchdirectionsin, 995 gastrulationin, 961.-963 genomein, 951 key developments in, 994 neurulationin, 98 5-987, 98 5f, 986f polarizationin, 950-951 signalingin,963-969 somaticcellsin, 951 Embryonicstemcells,8,960. Seea/soStem cell(s) experimentalusesof, 912 in geneknockout studies,207-208, 2071, 208f

from innercellmass,960,962f mouse,25 therapeuticusesof, 912 Emersoneffect,519 Emerson,R., 519 Emery-Dreifuss musculardystrophS795 Emphysema, misfoldedproteinsin, 555-556 EMSA (electrophorectic mobility shift assay), 286,287f ENaC channels,1034 Enactin, in basallamina, 821.,821f End-productinhibition, in protein regulation,89 Endergonicreactions,55, 55f coupledto exergonicreactions,57 Endocrinesignaling,77f, 1.8,31.2-31.3, 31.2f, 625, 625f, 626. Seea/so Hormone(s); Signaling Endocyticpathway,579-580, 581f,606-607, 61.L-612.Seealso Receptor-mediated endocytosis iron transportvia, 61,1.-61.2, 61.1.f Endocytosis, 373 actin polymerizationrn, 726 rn antlgenpresentatron, 1085-1086 definitionof, 506 multivesicular endosomes in, 61.2-514,51.31 1019, 1021f, 1022 of neurotransmitters, pinocytosisand,606 receptor-mediated, 373, 606-612, 683-684.Seealso Receptor-mediated endocytosis in synapticvesicleformation,1019 in transferrin cycle,611,-61,2,61,7f vesiclebuddingin, 41.4f,581,581f,610, 61.2-614,613f Endoderm, 907,907f developmentof, 907, 907f, 951.,962, 962f., 963 Endogenous retroviruses(ERVs),in transposition,230 Endoglycosidase D assay,583-584, 583f Endonucleases, in DNA cloning,1.76-1.77, 1.76f,1.77t Endonucleolytic pathway Endoplasmic reticulum.15. J7Jf, 375-376, 375t,376f actin filaments and, 731. fatty acid synthesisin, 375, 37 5f, 430 functionsof, 375 interconnected membranesof, 375-376 lipid synthesisin, 432, 433 membraneof, 41.8,418t protein folding in, 534 protein modificationin, 534-535 proteln targetrngto, 535-555. Seealso Protein targeting protein translocationacross,535-555 roogh,373f, 375-376, 37 5f, 376f, 535 functionsof,375-376 membraneorientationin, 543, 545-546,546f proteinfoldingin, 541, 542f,552-555 proternrnsertloninto, 542-549, 543f-s49f protein modification in, 376 proternsecretlonfrom, 37 5-376, 3761 protein synthesisin, 376 structureof, 535, 536f unfolded-protein response in, 555, 555f smooth,373f, 375, 37 5f, 376f Endosomes, 372-373, 374f late,580 in endocyticpathway,609f, 61.0-61.1. in secretorypathway, 601.-602,501.f multivesicular, 612-614, 6731,61.4f Endosymbionthypothesis, 415

Endosymbionts, 13, 236, 237f Endothelialcells,in leukocyteextravasation, 837-838 55f, 56 Endothermic reactions,55, Energy,54-60. Seealso Cellularenergetics activation,57, 57f, 79, 80f in concentrationgradient,54-55 electric,54 potential,55 tree,))-)tl in energycoupling,58-59 in membranetransport,464465, 465f reactionrateand, 56-57, 57f kinetic,54 mechanical,54 potential,54-55 chemical,54 electric,55 radiant,54 sourcesof, 58-59 thermal,54 transformationof, 55 units of measurefor, 55 Energycoupling,58-59 Engulfment,in apoptosis,937 Enhancers,I 8, 274-275. 274(. 284-285,285f, 2861,676, 676f exonicsplicing,333, 334f multicomplexeson, 29 5-296, 296f Enhancesomes, 295-296, 296f Entactin,805t in basallamina,821, 821f Enthalpy(H), 55, 56 Entropy /S),55, 56 hydrophobiceffectand, 39 Enveiopedviruses,154-155, 155f. Seealso Virus(es) buddingof, 614,615f lytic replicationof, 158 retroviruses as, 158, 159f Enzyme(s), 10 activesite of, 80, 80f, 81-84 catalytic action of,79 commonpathwaysfor, 84-85, 85f compaftmentation of, 92 definition of, 79 lysosomal,374 deficiencies of, 602 targeting of, 600-602, 601f membranebinding of, 427 modification,176-177 in multienzymecomplexes,85, 85f pH and, 84, 84f propertiesof, 79 reactionrate and, 80, 80f restriction,1.76-1.77, 1.76f,1.77t in signaling, 639,640t specificityof, 80 turnovernumberfor, 81 Enzymeassays,98 Enzymecofactors,84 Enzymeinhibitors,84 Enzyme-substrate binding,39, 39f, 80-84. See a/so Protein binding mechanisms of, 467468, 4571 Enzyme-substrate complex,80, 81f Eosin,385. Seealso Staining Ephrins,1043-1044,1044{ Ephs,1043-1044,1044f Epiblast,962,962f Epidermalgrowth factor (EGF),525, 580 in cancer,706 in heart disease, 706 in Ras/MAPsignaling,693-694 receptortyrosinekinasesand, 680-682, 681f,682f INDEX '

I-17

Epidermalgrowth factor domain,7L-72,71f Epidermalgrowth factor receprors,680-681, 58l.f Epidermalstemcells,914-91,5,91,5f Epidermolysis bullosa,795-796, 795f, 810 Epidermolysis congenita,795-796, 795f, 81,0 Epifluorescence microscopS380f Epigeneticinheritance,303 Epigeneticprocesses, 254 Epimerases, 45 Epinephrine,10 in glycogenolysis, 648-549 receptorsfor" 636-637, 636f desensitization of, 651 structure o\ 10201 Epithelialcell(s) apicalmembranein, 471,,471f apicalsurfaceof,399,471, 808, 809, 809f,810f basallamina of, 808 basalsurfaceof,399,808, 809, 809f,810f basolateral-apical sortingin, 505, 605f basolateralmembranein. 471,.471,1 basolateralsurfaceof, 471, 808, 809f cell-celladhesion/cell-matrix adhesionin, 808-819. Seealso Cell-celladhesion; Cell-matrixadhesion culture of, 399400, 401,f developmentof. 960 functionsof, 713, 808 intestinal,470471, 471.f lateralsurfaceoI, 399, 808, 809, 809f, 810f membranetranspoft in, 470472, 47l,f, 472f polarized, 471,,808 structureof, 808, 809f Epithelialcell junctions,372, 399400, 471., 471f, 802f,803,809-819,810f Epithelial growth factor, in breast canceg631, Epithelial-mesenchymal transitions,812-813, 81.3f,960 Epithelialtissue,801, 802 Epithelium,399,802 basallamina of, 399, 401.f definitionof, 713 developmentof, 951 extracellularmatrix of, 81,6-817,820-825 paracellular transportin, 815-816,816f simplecolumnar,808, 809f simplesquamous,808, 809f stratifiedsquamous,808, 809f transcellulartransportin, 471,,471,f, 81,4, 81,6f transitional,808, 809f typesof, 808, 809f Epitopetagging,98, 198, 385 Epitopes, 78, 198,401,1.068 Epo receptor.SeeErythropoietin (Epo) receptor e heavychains,1055. Seealso Immunoglobulin(s), heavy-chain Equilibrium chemical,49-50 Equilibriumconstant(K.o),32f, 49-50, 52 free energychangeanii, 56 Equilibriumdensity-gradient centrifugation, 94, 1.06,1,07 f, 392, 393f, 407408, 408f ERG1,290,290f Ergosterol,structureof, 412f, 416 ERVs(endogenous retroviruses), in transposition,230 Erythrocyte(s) cytoskeletonof, 729-730, 730f definition of,728-729 erythropoietinand,572-673, 673f functionsof, 729 glucosetransportin, 442 l-18

.

rNDEX

productionof, 672-674, 673f, 674f, 917-920,91,9f Erythrocyte membranq 423 actin filamentsin, 729-730, 730f Erythropoietin,672-673,6731,6741,91.8,91.9f supplemental, 679 Erythropoietin(Epo)receptor,673-676 in cancer,1,1,28-1,129, 1,1,28f JAK kinasesand,674-676, 674f ligandbindingto, 673, 673( mutationsin, 679 in signaling, 674-616,674f structureof,673,673f Escherichiacoli cell structurein,2, 3f expressionsystemsof, 1,94-196,195f genecontrol in, 1,7,271-275, 272f, 273t, 274f,275f lac operon in, 271.-273, 272f membranetransportin, 454, 454f plasmidvectorsof, 1.78-179 T4 phagein, lytic cyclefo! 156-1.57,l56f trp operon in, 1.24f vitamin B12permeasein, 454, 454f ESCRTproteins,573-674,614f, 61.5f Essentialfatty acids,4748 Esterification, 48 Estrogenreceptor,293,293f,31.2f.Seealso Hormonereceptors Ethylmethane sulfonate,1139 Eubacteria,2f evolutionof,4,4f Euchromatin,249, 252, 252f, 253, 258f definition of,299 Euglenagracilis, mitochondrial DNA in, 237, z5/l

Eukaryotes,1-2 cell structurein, 3, 3f definition of, 3 genecontrol in, 27 5-281, genesin, 216f,217-222 kingdoms of, 1-2 unicellular,4-5,5f eue,in body segmentation, 976-977 gene,in body segmentation, euen-skipped 97 5f, 976-977, 1,000 Evolution of apoptosis,938-939, 940f of asymmetriccell division,934 of cells,4, 4f, 6-7, 23-25 of chloroplasts, 236, 237f,242, 505, 505f of chromatin,249 259, 260f of chromosomes, conservedsynteny and, 259 development in, 952 endosymbionthypothesisfor, 505, 505f exonshufflingin, 235, 235f,335 geneconservationin, 28-29, 29f of genefamilies,221 geneticvariation and, 7, 28-29, 29f genomeperspective on, 28-30 of genomicimprinting,958 of Hox genes,980-981.,981f nf

inteorinc

R-lA

of kinesins, 774,774f of mitochondria,235, 237f, 240, 505, 505f mobileDNA elementsin, 1,4,226.Seealso Mobile DNA elements mutationsrn, 7, 1.4,28 of myosin,774,774f of noncodingDN l\, 223-224, 225-226 of organelles, 505, 505f of plants,242 of prokaryotes,4, 4f of proteins,72-73, 73f, 244

of ribosomes,133 sequence drift in, 220-221 sequencehomology and, 244, 245f of snRNA, 334-335 of vision, 1028-1029 Evolutionarycancer,1115 Excision repair,1.47-1.49 base,1.47,1471 1.48f mismatch,1.47-1.48, nucleotide,148-149, 1.48f,149f Excitablecells,1003-1004 muscle,1004-1005 neural,1003-1004 Excitatoryreceptors,in axon potential generation,1025 Exergonicreactions,55, 55f coupledto endergonicreactions.57 Exocytosis,41.4f,580, 591 1020-1021.,1.020f, of neurotransmitters, 1021.f,1.022 vesiclefusion in, 41.4f E x o n ( s )1, . 2 3 , 2 L 6 duplicationoI, 217, 21.8f. joining of. SaeSplicing lengthof, 333 stzeot. zt / skipping of,333-334 Exon-intron junctions, 21,7,329-330, 329f Exon-junctioncomplexes,332-333,357 Exon shuffling,235,235f,335,336 333, 334f Exonicsplicingenhancers, Exonucleases, 336-337 Exoplasmicface,of phospholipid bilayer, 41.44',t5, 414f, 532, 543f 336-337 Exosomes,in pre-mRNAprocessing, Exothermicreactions,56 Experimentalorganisms,25-27, 26f Exportins,573-574, 574f Expressedsequencetags,245 Expressionassays,in receptorpurification, 631-632,632f Seealso Gene Expressionvectors,1.94-1.97. expressionstudies;Vector(s) 6, 1.95f bacterial,1.94-1.9 eukaryotic,1.96-198, 1.96f-198f 198f in gene/protein tagging,1.97-1.98, plasmid,79 5-196, 1,95f retroviral,1,97,1,97f Extensin,840 External iace,of membrane,414, 41.41 matrix,16, 16f,372,373L \ee Extracellular also under Matrix adhesionreceptorsand, 820 adhesiveinteractionsof, 805, 806f 820-825 in epithelium, 81.6-8'1.7, basallaminaof,820-825. Seealso Basal lamina in cancer,1110 cell movementthrough, 805 cell vs. matrix volumein, 805 componentsof, 805-807, 805t of connectivetissue,825-833, 825f definitionof, 803 dynamicnatureof, 805 fibronectinin, 830-833, 831.f,832f functionsof, 805-807, 805t, 820 glycosaminoglycans in, 827-829, 828f hyaluronanin, 829-830 805-807, 806f in morphogenesis, of nonepithelialtissue,825-833 of plants,839-840, 839f.Seealso Plant cell wall proteoglycansin, 827-830, 828f, 829f in signaling,805 structureof,802f Extracellularmatrix proteins,805-807, 8051

Exrravasation,1097 leukocyte/lymphocyte, 837-838, 838f, 1097 Eye.Seea/soVsion developmentof, in Drosophila melanogasten 68 5-687, 687f function o1,7027-1031 lack of iris in,29,29f structureof, 1027f, 1.028 eyeless,29f F-actin,717, 7L7f, 718f. Seealso Actin filament(s) proton ptmps,447[,448,453.Seealso F-class Pumps F factor,179 FeFl complex,505-509, 505f, 508f. Seealso ATP synthase Fab fragments.1063f,1065 Faciliratedtransport,440, 440t Facultativeaerobes,485 FAD (flavin adeninedinucleotide) in ATP synthesrs, 482, 495r as electroncarrier,59-50, 50f FADD, in apoptosis,943-944 FADH2 electrontransportfrom, 493497, 499-500.Seealso Electrontransport productionof,60,60f in citricacidcycle.489490,489f, 490t Familialhypercholesterolemia, 608-610 Fanconianemia,l\42t Fasreceptor,in apoptosis,943-944 Fat cells,in glucosemetabolism,649, 550t Fattyacid(s),4749,47t. SeealsoLipid(s) definitionof, 47 essential, 47-48 esterification of, 48 incorporationinto membrane,431 isomersof, 48 as membraneprotein anchors,422, 424426, 425f,430 metabolismof, 430, 430f peroxisomaldegradationof, 374-375 polyunsarurate d, 4748, 47t saturated 4 ,7 , 4 7 t , 4 3 0 structureof, 47, 47f, 481 synthesisof, 375, 37 5f, 430 trans,48 transportof,430, 430f unsaturated, 47, 47t, 430 Fatty acid oxidation, 487,488f, 491492 mitochondrial,491, 492f peroxisomal, 491492, 492f Fatty acid synthase,430 Fatty acid-bindingprotein (FABP),430,430f Fatty acyl-CoAdehydrogenase, in electron transport, 497 Fatty acyl group, 48 Fatty acyl-CoA dehydrogenase, in electron transport, 497 Fc fragment,1063f, 1.065 Fc receptors, f055, 1058 Feed-forwardactivation,in glycolysis,483, 483f Feedbackinhibition,in protein regulation,89 Fermentation, 484f, 485 FerritinmRNA, 356,357f Ferrotransferrin, 5 11-6 12, 6l1f Fertilization,8, 8f, 19, 19f, 9 50, 9 50f, 955-959 acrosomalreactionin, 956, 957 definitionof, 955 gametefusion rn, 95 5-957, 9 56f in vitro, 8 Fetalantibodies,1055-1066

F(ab)fragments,1063f, 1065 FG-nucleoporins, 342, 342f, 572 F G F 1 0 i.n l i m b d e v e l o p m e n9t9, l . 9 9 l f, 9 9 2 . 992f Fibril-associated collagen,822, 823t, 825. See aiso Collagen Fibrillar adhesions,833 Fibrillar collagen,822, 823t, 825-827.See a/soCollagen Fibroblast(s), 825 cultureof, 396,397f integrinsin, 833-834, 834f movementof, 745 Fibroblast-derived fibronectin,'1.26, 126f, 338 Fibroblastgrowth factor (FGF),680, 680f i n l i m b d e v e l o p m e n9r9. 1 .9 9 l f , 9 9 2 , 9 9 2 f , 993f,994 in patterning,966-967 receptortyrosinekinases,580, 680f Fibronectin, 805t,830-833 alternativesplicingin, 126, 1.26f,Zl8, 338 antibodiesto, 806f classes of, 831 functionsof, 830-831 hepatocyte-derived, 1.26,1.26f,338 integrinsand, 831-832,83lf intronsin, 217 isoformsof, 1.26,1.26f,338 RGD sequences in, 815, 831, 831f s t r u c t u roef , 8 3 1 , 8 3 1 f synthesisof, 832-833 Fibrousproteins,68 Fight-or-flightresponse,9-10 Filaments.SeeCytoskeleton, filamentsof Filamin,728, 729f, 747I Filopodia,71.6,71.6f, 745, 7 50f Filopodium,1040, 1.040f Filters,in ion channels,461.463, 462f Fimbrin,728,729f Fingerprinting,DNA, 225, 225f Fingers,extra,992, 993f FISH (fluorescence in situ hybridization),258 Fishvectors,310, 311f Fisher,Emil, 80 5' cap, 1,24,125f in pre-mRNAprocessing, 324f, 325-336, 3 2 5 f , 3 2 7 13, 3 7 shortening of, 352-J53,352f in transcription,280-28L, 282 in translation,134 5'end of DNA strand,114, 114f of Okazakifragment,741, 141f, 1,42f 5' untranslatedregions(UTRs),124 55 rRNA, processing of,359-363 Flagella,415 basalbodiesin, 761 beatingof, 778-779, 779f deftnition of,777 in intratlagellar transporr.r02, 779-780, 781.f microtubulesin, 777-7 80, 778f-7 9lf of sperm,954,954f, 955, 968 structureof, 777, 778f Flavinadeninedinucleotide(FAD) in ATP synthesis, 482 as electroncarcter,59-60,60f Flavins,in ATP synthesis,482, 495t Flippases, 420, 43'1.432,456, 4 56f Floral meristem,983. Seea/soMeristems Floral organ-identitygenes,983 Flow cytometry,394-395, 395f Flowerdevelopment,983-984, 984f Fluid mosaicmodel,410f Fluorescence-activated cell sorter(FACS), 394-395,39sf

Fluorescence in situ hybridization(FISH),258 Fluorescence microscopS 382-386 of actin polymerrzation,7'1.9 in Ca"' measurement, 383-384, 384f confocal,386,386f deconvolution.386. 387f in H2* measuiement, 384 immunofluorescence, 385, 385f SPED,385 total internalreflection,404 vs. electronmicroscop5388 of VSV G proteins,582f, 583 Fluorescence recoveryafter photobleaching (FRAP),4L74L8,477f in microtubulehalf-lifemeasurement, 785 Fluorescence resonanceenergytransfer ( F R E T )6, 3 9 , 6 3 9 f Fluorescence spectroscopy,of actin polymerization,719 Fluorescent antibodies,21 Fluorescent staining,382 Fluorochromes, 383, 385, 385f Focal adhesions/contacts, 7 16, 7161,833, 834f in cell migration,745, 747f Focalcomplexes,833 Folding,protein.SeeProteinfolding Follicles,953 Footprinting,133 iormlns. /25-/z+- /z+l fos oncogene,1131, 11.321 Foundercells.908-909 Fovea,L027f, 1028 FOXO 3A, 596 Fractionation,21 Frameshiftmutations.1,28.1,67 Franklin,Rosalind,114 FRAP (fluorescencerecoveryafter photobleachingl,41.741.8, 41.7f in microtubulehalf-lifemeasurement, 785 Freeenergy(G), 55-56 in energycoupling,58-59 in membranetransport,464465, 465f reactionrateand, 56-57,57f Fringeproteins,705 Frizzled,in Wnt pathway,599, 699f Frog. SaeXenopus laeuis Frog oocyte expressionassay,for ion channels, 464,464f Fructose2,5-bisphosphate, in glycolysis,482f, 483,483f,485 Fruit fly. SeeDrosophila rnelanogaster ftz (fushi tarazu), in body segmentation,975f, 977, L000 Fumarate,succinateoxidation to, 59, 59f Functionalcomplementation studies,of JAK/STATpathway, 677, 677f Functionaldomains,70 Functionalexpressionassays,in receptor purification,631.-632,632f Functionalgroups,34, 35t Fungi. Seealso Yeast disease-causing, 5 functions of, 5 Fura-2fluorescence microscopn383-384, 384f Furanoses, 45,45f. Furin, 604 fushi tarazu (ftz), in body segmentation,975f, 977, 1.000 Fusionproteins,1.98,1.98f G-actin.717-718.717f. Seea/soActin polymerizationof, 71.8,71.9-721,719f G bands,258,2581,261 INDEX

t-19

G protein(s), 90,9lf activation/inacrivation of, 637-639,639f. Seealso GTPaseswitch proteins (GTPasesuperfamily) classificationof, 639, 640t cyclingmechanisms of, 637-639 diversityof, 639 functionsof, 90 G" subunitof, 637-639,638f,640t,644,644f Gp"ysubunitof,537-638, 6381,64'1,,641,f, 644, 644f monomeric(small),354-355 in signaling,634 monomericsmall,354-355 muscarinicacetylcholine receptorand,641, 64'l.f in nucleartransport,571 in signaling,633-657, 633f. Seealso Gprotein-
.

INDEX

G1 phase,18, 18f, 848, 848f, 849,850f.See a/so Cell cycle arrestof, by DNA damage,11.36-l'1.37, 1137f changesin, in cancer,1134,1136-1.137, t1.37f regulationof,872-879 G2 phase,18, 18f, 781,,848f,849.Seealso Cell cycle changesin, in cancer,1134 in mammals,881, 882f in yeast,877,877f G",90,91f GABA (gamma-aminobutyric acid),structure of, 1.020f Gag protein,51.4,61.5f G"'GDP complex,637-638, 538f, 644 G"'GTP complex,637-638, 638f, 641, 643-644,643f in GPCR./adenyly cyclasepathway,646-652 Gain-of-functionmutations,167 in cancer,L1.19-1.L2l GALL prornoter,206 GAL4 transcription factor, 288-289, 289f Galactose, 45,45f,46f membranetransportof, 443 GalT protein, in fertllization, 957 Gametes, 19,'1.67,950 parentaltype, 175 recombinanttype, 1.75 Gametogenesis, 953-955 genomicimprintingin, 958 Gamma-aminobutyric acid (GABA),structure of, 1020f Gammacarboxylation,of amino acids,43 Gamma-secreta se,705-706, 705f Gamma-tubulin,761.,762 Gamma-tubulinring complex,761, 762 1 light chains,1.065.Seealso l m m u n o g l o b u l i n {l si g) ,h t - c h a i n Ganglionmother cells(GMCs), 932-935 Gap genes,972 discoveryof,999-L000 in Drosophiladevelopment, 974-977, 975f Gap junctions,16, 383,383f,809, 810f,811t, 81.7-819,81.8f 1.026f 1,025-'1,026, in electricalsynapses, vs. plasmodesmata,84L proteins),90, 637, GAPs(GTPase-activating 644,585 in asymmetriccell division,935 Gastricacidification,472, 472I Gastrulation,951 Gatedchannels.SeeIon channels GCN2, in translation,356 GCN4 transcriptionfactor,289, 305-306 GCNs, 305-306 GCPRkinases,644 neurotrophicfactors), GDNF (glia-derived LL27 GDP (guanosine diphosphate) GTPaseswitchproteinsand,637-638, 6381 764-765 in microtubuleassembly, in nucleartransport, 571.-572,572f, 573, 574t in signaling,633-634, 633f, 637-638, 638f.,691. in vesiculartransport,587-588, 588f Geigercounter,100 Gel electrophoresis,22f, 9 4-9 6 in cloning,184, 185f in Northern blotting, 1,92,1.92f procedurefor, 185f mide,9 4-95, 294f SDS-polyacryla 1.91f in Southernblotting, 1,91,-1,92, two-dimensional, 95-96, 9 5f, 106

Gel filtration chromatography,221,96, 97f Gel-shift assay,286, 2871 Gelsolin,723 Geminin,883 GenBank,243 Gene(s).Seealso Genomeand specificgenes abundanceof, biologicalcomplexityand, 246,246f allelesof. SeeAlleles autosomal-dominant,199-200, 200f 200, 200f autosomal-recessive, co-regulated,clusteranalysisof, 193,1.941 codingregionsof,217 of,217 components conservationof, 29 definitionsof, 1'1.2,120, 2L7, 2L9 densityof, 221f, 223-224 951 in development, Seealso Cancer;Diseases disease-causing. and conditions examplesof, 1.98-204 identification of, 198-204. Seealso Geneidentification inheritanceof, 199-200, 200f, 203-204 mapping of , 200-203, 201f-203f. See a/so Mapping 747 dominant-negative, duplicated in genefamilies, 217, 218f, 220 segmentalduplication and, 221 in tandem arrays,220t, 221J22 690, 884 early-response, eukaryotic,2161,217-222 floral organ-identity, 983 gap,972, 999-1000 974-977. in Drosophiladevelopment. 97sf globin a,220J21.,221.1 p,220J21.,2271 evolutionof, 235 167 haploinsufficient, heat-shock,76-77, 76f, 31.6 homeotic,979,983 282 housekeeping, i--'i.terl

9SR

jumping.SeeMobile DNA elements 884 late-response, linked, 175 mutationsin. SeeMutations organizationoI, 223J26, 224f, 225f in eukaryotes,'1.23-1.24 in prokaryotes,L22-123 pair-rule 97 5f, 976-977, in body segmentation, 976f discoveryo1,999-1000 pattern-formation, 999-1000 polycomb, 982-983 protein-coding,120, 2L9-221, 220t in eukaryotes,123-724 organization of, 122-124 in prokaryotes,1,22-1'23 solitary,219220 pseudogenesand, 220-221. relativenumber of, 245-246, 246f reporter,277, 283 in functional complementationstudies, 677 segment-polarity,977, 999-1000 s\zeof,217 solitary,21,9-220 structure of in eukaryotes,21.6f,217 in prokaryotes,217

tandemlyrepeated,227-222, 221t in DNA fingerprinting,225 tumor-suppressor, 882, 1107, 1122t inheritedmutationsin, 11.23-1124, 1.1.23f loss-of-function mutationsin, 148, L1,23 lossof heterozygosity in, 1124,1125f unlinked,175 Genecontrol,1.3,17-1,8,112 cytoplasmic,323 genomicimprintingin, 958 at individualsynapses, 325 post-transcriptional, 323-357. Seealso Post-transcriptional genecontrol specificiryin, 922-923, 923f transcriptional.SeeTranscriptionalgene contfol Geneconversion, 153 Genedensity,221f Geneexpression, lT-18 constitutive,290 coordinate, l23 definitioo nf,17,269 differential, 24,24f in eukaryotes,122-1,23 rn prokaryores, 122-l23 regulationof. SeeGenecontrol Geneexpressionstudies clusteranalysisin, 193-194, 195f DNA microarraysin, 192-194, 194f, 195I expressionsystemsfor, 194-1,97, 1,95f-197f gene-inactivation, 204-21,1.Seealso Gene lnacuvailon gene/protein taggingin, 197-198, l98f Northern blotting in, 1,92,1,92f in situ hybridizationin, 192, l93f Southernblotting in, 1,91-192,Iglf vectorsin, 194-198 bacterial,194-196,195f ^1-96-198, eukaryotic, 196f-1.98f plasmid,L95-1.96,195f r e t r o v i r a l1. 9 7 ,1 9 - { Genefamilies,220 Geneidentification BLAST algorithmin,243 clusteranalysisin, 1,93-1,94, 195f databases for,243 DNA microarraysrn, 1.92-1,94, 194f, 1,95f expressed sequence tagsin,245 expressionsystemsfor, 1.94-197, 1 95 f - l 9 7 f future researchareasfor, 265-266 gene/protein taggingin, 197-198, 19Bf for geneticdiseases, 198-204 mappingin, 200-204, 20lf-203f. Seealso Mapping Northern blotting rn, 1,92,l92f ORF analysisrn, 244-245 sequence homologyin, 244, 244f, 24Sf in situ hybridizationin, 1,92,193f Southernblotting in, 1,91-192,1.91f Geneinactivation,204-211 disruptionconstructin, 205, 205f geneknockout in, 207-208,207f , 208f homologousrecombinationin, 204, 204f promotersin, 205 RNA interference in, 270, 211f in sex determination,253-254 somaticcell recombinationin, 208-209, 209f Geneknock-in,241, 241,f Geneknockout, 207-208,207f, 208f Geneloci, 175 oeflnlnon ot, I /) of linked vs. unlinked

genes, 175

Gene mapping, 174-175,200-203. See also Mapping Gene regulation. See Gene control Gene tagging, 197-1.98 e p i t o p e ,9 8 , 1 9 8 , 3 8 5 green fluorescent protein rn, 197-198, 198f,382-383,383f by insertion mutations, 189-190, 790f G e n e r a li m p o r t p o r e s , 5 5 9 , 5 5 9 f , 5 6 1 General transcription factors, 253, 296-297, 297f, Z98f Genetic analysis, 1.65-21.2 breeding experiments in, 767-1,70, l69f complementation tests in, 17l, 772f c o n d i t i o n a fm u r a t i o n si n , 1 7 0 - l t l , 1 7 0 f diploid organisms in, 166, 167f, 771 DNA cloning in, 176-1,90. See also Clones/cloning d o u b l e - m u t a n t , 1 7 l - 1 7 2 , 7 73 f expression vectors in, l9l-198 t u n c t i o n - b a s e d1, 7 4 ,2 l 2 future research areas for, 211,)12 gene inactivation in,204-21.1. See also Gene inactivation gene knock-in in, 241, 2411 gene knockout in, 207-208, 207f, 208f in genetic diseases,798-204 genetic screens in,170-171,, 170f haploid organisms in, 765, 167f, 1,69-1,71,, 170f inbreeding experiments in, 171 mapping in, 174-17 5, 174f, 200-203. See also Mapping position-based, 774-17 5 suppressor mutations in, 173-174, 173f synthetic lethal mutations in, 173f, L74 t e m p e r a t u r e - s e n s i t i vm eutarions in, 170-171, 1.70f wild type organisms in, 166, 1,571 G e n e t i cc o d e , 1 1 , 1 . 2 7 , l 2 8 t codons in, 1.27-1.29,1281, l29t oelrnrtron oI, Iz,/ oegenerate, l/ / deviations from, 128-129, 129t mitochondrial, 240, 24lt reading frame fot 128, l29f universal nature of, 28,128-129 Genetic complementation, 17 l, 1,72f Genetic complementation tests, 219 Genetic diseases.See Diseasesand conditions Genetic diversity, from recombination, 892, 955 Genetic engineering. See Recombinant DNA technology Genetic heterogeneiry,204 Genetic linkage mappin g, 200-202, 201,f-203f. See also Mapping Genetic map unit, 175 Genetic markers, 175 in linkage mapping, 200-201, G e n e t i cm u t a t i o n s .S e eM u t a t i o n s Genetic recombination. See Recombination Genetic screens,25, 170-171,170f for pattern development, 959 for recessivelethal mutations, 171 Genetic variation, 7. See also Mutations in evolution, 28-29, 29f Genetics,22-23 developmental, 28-29 Genome complexity of, gene number and,246,245f definition of,12,64 DNA amounts in, 223 gene/protein function and, 246, 246f individual variation in, 247 interspeciessimilarities in, 29, 291

in nucleus,378 proteinsin, 64 s e q u e n c i no gf , 6 4 , 1 2 2 viral,154 progeny,158 Genomicimprinting,958 Genomiclibraries,180-181,1.82-184,l83f screening of, 181-182 Genomics, 23,243-247 BLAST algorithmfor, 243 databases Ior,243 oerrnlllon o\

25, zt /

expressed sequence tags in,245

future researchareasfor, 265-256 mousemodelsin, 245 protein/gene identificationin, 243 sequence homologyin, 244, 244f, 2451 singienucleotidepolymorphismsin, 246-247 Genotype,166 definition o1,22, 166 vs. phenotype,166 wild type, 166 Germ cells,905 division of, 1.67,1.68f in oogenesis, 9 53-955, 9 53f primordial,953 Germ layers development of, 951, 962-963,962f fatesof, 907-908,907f Germ line, 907 definitionof, 913 Gerrn-linecells,13-14, 91.3-914,950 fate of,907-908 segregation of, 953 stem,91-3-974 Germarium,stemcell nichesin, 913,973f GFAP(glial fibrillary acidicprotein),793t, 794 GFP tagging,L97-198, 1.98f,382-383, 383f GGA protein, 598-599 Giant, 974-977, 975f Gibbs,J.W.,55 Gleevec, 1130 Glia-derivedneurotrophicfactors(GDNF), r1,27 Glial cells,917, 1003 astrocytes,1,01, 6, 1,01,7 f microglia,1014 myelinating, 1014,1015f oligodendrocytes, 1014, 1015f Schwanncells,1014-1016,1,01,5f structureof, 1003, 1003f typesof, 1.014-1017,1015f Glial fibrillary acidicprotein (GFAP),793t, 794 Glioblastoma, 1109 Gliomedin,1015 glnA promoter,274, 274f B-Globin,RNA processingin, 125f Globin genes a,220-221,221.f 9,220-22't, 221.f evolutionof, 235 Globin proteins evolutionof,73,73f structureof,73,73f Globular actin. SeeActin, G form of Globularproteins,68 Globulin actin (G-actin),717-71,8,7 1.7f Glucagon,658-660, 6591 in cAMP synthesis, 663-654 Glucocorticoidreceptor,312f, 31.3,31,4f.See a/so Hormone receptors Glucofuranose, 4546, 451 Glucopyranose, 4546, 45f INDEX

t21

Glucose fermentationof, 484f, 485 membranetransportof, 44'1.443,441f, 442f. Seealso under GLUT rransepithelial. 47 1, 47 1f oxidation of, 59 structureof, 45, 45f, 45f metabolism,1.7,1.7f, Glucose/glycogen 481-485, 482f484f acetylCoA oxidation in, 487489, 4881 acetylCoA synthesis in,487,488f aerobic,483-485,484f,485, 488f allostericregulationof, 483-485 483,484f,485 anaerobic, in ATP synthesis, 481-485, 482f484f calciumin, 558, 658f cellularrespirationin, 59, 487-489,489f citric acid cyclein, 487489,489f electrontransportin, 488f, 489491, 490f, 493-503 feed-forwardactivationin, 483, 483f fermentationin, 484f, 485 glucagonin, 658-660, 659f glycolysisin, 481-485, 482f484f, 488f insulin in, 558-660,659f, 696-697. See a/soInsulin in mitochondria,378, 485487, 486[ multiple secondmessengers in, 657-660, 658f oxygendeprivationand,484f,485 protein kinaseA in, 698 pyruvate oxidation in, 487, 488f pyruvatesynthesisin, 481-485, 4821484f rate of, 483-485 regulationof, 483-485,483f cAMP in, 648-550,648f, 649f stagesof, 481.482, 487, 4881 in Glucosemetabolism,glycogenolysis multiple secondmessengers in, 657-660, 658f regulationof, 648-650, 6481,649f Glucosetransporters,441443, 441f, 442f, 471, 471.f.Seealso under GLUT hydropathyprofile for, 548,549f as multipassintegralmembraneproteins, 547 in protein folding, 553 Glucosyltransferases, 1, in plant cell adhesion, Glucuronyltransferase 842 GLUT(s),441.443, 441.f,442f, 471.,471.f as multipassintegralmembraneproteins, 547 GLUT1, 441,443, 44Lf, 442f hydropathyprofile for, 548,549t GLUT2, 441f, 443, 471.,471.1 GLUT3,442,443 GLUT4, 442, 443, 659, 696-697 insulin and, 443 Glutamate,42, 42f. Seealso Amino acid(s) structureof, 1020f Glutamatetransporters(VGLUTs),1020 Glutamic acid,42,42f Glutamine,42L 43. Seealso Amino acid(s) 10f Glutaminesynthetase, 3-phosphate, in Calvin cycle, Glyceraldehyde 525,526f Glycine,42f,43. Seealso Amino acid(s) in collagentriple helix, 822,822f structureof,1,020f Glycobiology,843 Glycogen,17, 46 Glycogenphosphorylase, 648 kinase,649, 649f Glycogenphosphorylase Glycogensynthase,697 Glycogensynthasekinase3 (GSK3),697, 699 l-22

.

INDEX

Glycogenolysis. Seealso Glucose/glycogen metabolism in, 657-660, multiple secondmessengers 658f regulationof, 648-650,648f, 549f Glycolipids,416 tight junctionsand, 815 transmembrane, orientationof, 426 Glycolysis,481-485, 482f484f, 488f definitionof, 481 productsof, 481, 482f, 490t Glycolyticpathway,481, 482f, 488f GlycophorinA, o helix of,422f,423 GlycophorinC, 730, 730f Glycoproteins, 550 definitionof, 824 folding and stabilizationof, 552 perlecanas, 824 proteoglycan,824 transmembrane,orientation of , 426 (GAGs),46, 843 Glycosaminoglycans chain elongationin, 827 definitionof, 827 in extracellular matrix, 827-829, 828f functions of, 827-829, 828f in perlecan,824 in proteoglycans, 827-830, 828f, 829f structure of, 827-829, 828f in Wnt signaling,700 Glycosidicbonds,40, 41f, 45f, 46, 46f 43I Glycosphingolipids, Glycosylation,376, 426 of amino acids,43 (GPI)-anchored Glycosylphosphatidylinositol 425426, 425f' 543f, 545, membranes, 547,548f (GPI)-anchored Glycosylphosphatidylinositol proteins proteins.SeeGPI-anchored Glyoxisomes,375 Glypicans,829 Goat anti-rabbitantibody,385 golden,469,469f Golgi, Camillio, 376 Golgi complex, 1.5,373f, 376-377, 376f cis regiono(,3-6-37-, 3-7f vesiculartransportin, 592-596, s92f-596f cisternalmaturationin, 580, 596,597, 597f functionsof,595-596 medial regionof, 376-377, 377f, 59 5-596, 595f,s96f structureof, 595-596, 596f transregionof,376-377,377f,580, 581f, 59r, 595-596,595f,596f, 597-604 protein aggregation in, 602-503 protein targetingfrom, 588-591, 6oo-505,605f vesiculartransportin, 597-604, 598f-601.f,6031,604f Golgi membrane,418, 41.8t Gonads,developmentof, 91'3-914 Goodpasture's syndrome,823 GPl-anchoredproteins,425426, 425f, 543f, 545,547,548f targetingof, 605 basolateral-apical on lipid rafts,420, 605 425-426,425f,543{,545,547, GPI anchors, 548f signaling,954 Gradient-mode Graft rejection,MHC moleculesin, 1.078f 1.077-1.078, Grana,379 factor, Granulocytecolony-stimulating L94-195 geneticallyengineered, Granzymes,in T-cellapoptosis,1060f, 1095

GRB2 adapterprotein,in Ras/MAPpathway, 685, 6861,687-688 prorein,2 l. 98, 382-383,383f Greenfluorescent in gene/proteintagging, 197-198, 798f GroEL, in protein folding, 77, 771 Ground tissue,in plants,839, 839f GroupI introns,363,364f Group II introns, 334-335,335f, 363, 363f Growing forks, 141, 1.41.f,1.42f,1.43-L44, 1.44f Growth cone,1040-1049.Seealso Axon(s), growth-coneextensionof Growth factors.Seealso TGFp superfamily in cancer,631,,680-682,681f,706, 1 1 2 7 - 11 2 9 ,l 1 3 4 , i l 3 4 f , t 1 4 2 - 1 1 4 3 in cell division,913 in cellmigration,746-748,748f-757f epidermal.SeeEpidermalgrowth factor (EGF) eoithelial.63l fibroblast.SeeFibroblastsrowth factor (FGF) 706 in heart disease, 918, 9191 in hematopoiesis, hepatocyte,928 nerve,593, 938, 938f,942 olatelet-derived.746 in Ras/MAPkinasesignaling,693 626,680 in signaling, in cell division,914 Growth hormone,receptorbindingof, 626f,627f Growth hormone receptors,679 triphosphate) GTP (guanosine 724,724f, in actin filamentassemblv. 725-726,726f discoveryof,663-664 GTPaseswitchproteinsand, 637-638, 6381.Seea/so GTPaseswitch proteins (GTPasesuperfamily) hydrolysisof, in microtubuleassembly, 763-755,765f in nucleartransport,57 l-572. 572(,573, 574f 633-634,633f,637-638, in signaling, 638f,663-664 in translation in elongation,135-1'36,136f in initiation, 1,33,134, 135f in termination,137-1'38,1'37f in vesiclepinching, 599-600,599f,600f in vesiculartransport,587-588, 588f, s99-600, s99f, 600f GTP-bindingproteins in cell migration,746-748, 7 481 tubullnas. /Jy. /bur in vesiclecoating,587-588 GTP exchangefactor, 90, 9Lf proteins(GAPs)'90' 637' GTPase-activating 644,685 in asymmetriccell division,935 GTPaseswitch proteins(GTPasesuperfamily)' 90,91,f,r38, 587,637-638 in cell migration.246-748,747f in mitotic exit network. 890, 890f Rasprotein rn, 684. Seealso Ras protein in signaling,633-634, 633f, 637-638' 6381 in vesiculartransport,587-590' 590f GTPases,inactivation of, 209, 210f GTP-B-tubulincap, in microtubuleassembly, 764-765,765f Saea/soBase(s) Guanine,44, 45t, 1'1'3-11'4. in doublehelix,114-115,115f structureof,44,44f proteins,90, 91'f Guaninenucleotide-binding factor (GEF) Guaninenucleotide-exchange in mitotic exit network, 890

in signaling, 633-634,633f,637, 685 in cell-cycleregulation,890 rn cell migrarion,74Guaninenucleotide-binding proteins,90, 9i,f Guanosinediphosphate. SeeGDP (guanosine diphosphate) Guanosinetriphosphate.SaeGTP (guanosine triphosphate) Cuidanceproreins.in axon extension, 7043-t046, 1.044f-1.045f Guillain-Barre syndrome,1015 Gustaducin,1035 Gustation,1034-1036,1035f H* measurement of. fluorescenr microscopy in. 384 p H a n d ,5 1 - 5 2 , 5 2 f H-2 complex,1,078,1078f H* ATPase, 447f, 448, 453454, 453f,460 H - c h a n n e4 l ,4 7 f , 4 4 8 ,4 5 3 4 5 4 , 4 5 3,f4 5 0 H- pump,447f,448, 453454, 453f H1 kinaseassay,for mitosis-promoting factor, 8 5 7 ,8 5 7 f H3, centromeres and, 263 Hair, sremcellsfor, 914-91,5,915f Hair cells,cochlear,339-340,340f, 1032-1033,1033f Hairpins,118, 1191,210,224 in somaticrecombination, 1,071,1,07|f hairy, in body segmenta rion, 977 Half-life, of radioisotopes, 99 Hand, developmenr of, 991-992,997f. See also Limb development Hanson-Huxleyexperiment,755-756 Hanson,Jean,755 H a p l o i dc e l l s 1, 9 , 1 6 6 , 8 4 9 Haploid chromosomes, 849 Haploid organisms,165 geneticanalysisin,769-171, 170f Haploinsufficie ncy,157 Haplotypes,202,202f Harmonin,1034 Hartwell,L.H., 170 HAT medium,401,402f HB-EGR 706 Hearing,1032-1034 cochleain, 339-340,340f, 1032-1033, 10 3 3 f hair cellsin, 339-340,340f, 1032-1033, 1033f K--channelproteinsin, 339-340, 340f impaired,1033-1034 stereocilia in, 1032-1034,1033f,1034f Heart. Saea/so Cardiacmuscre developmenrof, 957-969, 967f, 958f kinase-associated proteinsin, 652, 552f muscarinicacetylcholine receprorsin, 64j,, 641f Heart disease,740 Heart failure,458 Heat, generationof, brown-farmitochondria in, 510 Heat perception, 1031-1032 Heat-shockproteins as chaperones, 76-77, 76f transcriptionof, 316 Heavy-chainimmunoglobulins. See Immunoglobulin(s), heavy-chain Hedgehog(Hh) parhway,667f, 697-699, 700-702, 700f, 701f in axon guidance,1046 in bodysegmentation, 977,1,000 in cancer,1.124-1125

in cell division, 91.3-91.4 in neural development, 987 Hedgehog protein, 92, 416 Hedgehog receptors, 666f HeLa cell line, 398 H e l i c a l v i r u s e s ,1 5 4 , 1 5 5 f Helicases in replication, 141, 1.43, 144, 1.44f in transcription intitiation, 298 Helix alpha. See cr helix DNA, 11, 71.f, 114-1.1.5,1.1.5f,l16f . See a/so Double helix r e c o g n i t i o n ,2 9 0 , 2 9 1 f sequence-reading,290, 291,f triple collagen, 821-822, 822f, 825-826,

82sf Helix-loop-helixmotif, 69, 90, 90f, 292-293, 293f Helix-turn-helixmotif, 69, 70f, 290 HelperT cells,1076-7077,1096 CD4 and, 1080 cytokineproductionby, 1.097 MHC moleculesand, 1080 Hemagglutinin folding and assemblyof, 554-555, 554f structureof, 70-71, 71f, 72 Hemarocrit,579 Hematopoiesis, cytokinestn, 672-673 Hematopoieticstemcells,917-920, 9l9f Hematoxylin,385. Seea/soStaining Heme,89, 89f cytochromesand, 395f, 495-496 Heme-regulated inhibitor,in translation,356 Hemicellulose, 839f,840 Hemidesmosomes, 796, 802f,809-810,810f, 811t,816 Hemoglobin B-globingenesin, 220-221,227f evolutionof, 73f oxygenbinding by, 89, 89f structureof, 70f, 73, 73f HemoglobinS, 199, 799t HemophiliaA, 1.99t Henderson-Hasselbalch equation,52, 54 Heparansulfate,827, 828f,829 Heparin,827, 828-829, 828f, 829f Heparocyte(s) basolateral-apical sortingin, 505, 605f in glucosemetabolism,649, 550t Hepatocyte-derived fibronectin,126, 126f, 338 Heptad-repeat motif, 69, 70f HER receptors, 580-683,681f,682f HER2, in breastcancer,631, 680-682,681f Herbicides,52l Hereditaryhypomagnesemia, 816 Hereditarypolyposiscolorectalcancer,1,1,42t Hereditaryretinoblasroma, 882, 1L23-1124, 1 r 2 3 f ,1 . 1 3 5 hes7,978 Heterochromatic murants,in cell lineage studies,909-911,910f Heterochromatin,252-253, 2 52f, 37I centromericr224 definrtionof,299 formation of, 252, 253f, 350-351 X-chromosomeinactivationand, 253-254 Heterochromatinprotein 1 (HPtl, 252, 302-303 heterocyclic amines,in cancer,1l4l,1.l4lf Heteroduplex,153 Heterogeneity, genetic,204 Heterogeneous RNA (hnRNA), 327 Heterogeneous RNPs (hn RNPs),325, 327, 328f nucleartranspot of,345,345f, 573

Heteroplasmy, 240 Heterozygosity, 166 Hexokinase,in glycolysis,483, 483f Hexoses,4445,45f HIF-1, in cancer,1L12-1113 High-densitylipoproteins,regulationof, 707-709,708f High-mobilitygroup (HMG) proteins,257 High throughputliquid chromatography-tandem mass spectrometry, L06-L07, l06f, 1.07f Histamine, structure of, 1020f Histidine,4243, 42f. Seealso Amino acid(s) Histone(s),247, 248, 299 acetylationof, 250f, 251, 251,f,307, 307 in transcriptionrepression, 303-305, 304f,305f amino acid sequences in, conservation of, 249 centromeres andr263 deacetylation of, 300, 307 in transcriptionrepression, 300, 303-304,305 definition of,247 evolutionof, 249 hypoacetylation of, 301 methylationof, 250, 250f, 251.-252,306, 307 in transcriptionactivation,305 in transcriptionrepression, 304-305, 305f,306 in nucleosome, 248-249, 2481 phosphorylationof, 250, 2501,251 post-translational modificationof, 250-252,250f, 299-307, 307 typesof, 248 ubiquitinationof, 250, 250f, 251,-252 variant,250 Histoneacerylases, 251.,305,307 Histonecode,250, 252-253 Histonedeacetylase, 304, 304f, 307 in myoblastdifferentiation,928 in transcriptionalrepression, 300-301, 301f Histonelysinedemethylases, 307 Histonemethyl transferase, 252 HistonenRNA, in oogenesis, 957 Histone talls,299. Seealso Histone(s) post-translational modificationof, 250-252, 250L 299-, 299-307, 307 HIV. SeeHuman immunodeficiency virus H L A c o m p l e x1. 0 7 8 ,1 0 7 8 f HMG-CoA reductase, in cholesterolsynrhesis, 432,432f HMG proteins,257 HMGI protein,in enhanceosome formation, 295-296,296f HMI locus,in transcriptionrepression, 299, 300f HMR locus,rn transcriptionrepression, 299, 300f hnRNA (heterogeneous RNA), 327 hnRNPs(heterogeneous RNPs),325, 327,3281 n u c f e atrr a n s p o rot f . 3 4 5 .3 4 5 f , 5 7 3 HO, transcriptionof, 309, 309f Hollidaystructure,151, 151f,153, 153f resolutionof, 153, 153f Homeodomainproteins,291 Hox genesand,978 Homeosis, 979,983 Homeostaticchemokines,1096 Homogeneity,biochemical,407 Homologouschromosomes, 167, 168f Homologousrecombination in DNA repair,150-153, 151L 1.52t in geneinactivatton,204, 204f I ND E X

t-23

Homology,72-73,73f HomozygositS166 Homunculus,1.032,1032f Horizontalcells,1027f, 1.029,1.030-1.031. Hormone(s) cAMP regulationand,,649, 650t proteolyticprocessing of, 91 receptor binding of, 312-31.3,31.2f,31.3f secretionof, 37 5-376, 376f in signaling,l7f, 18, 312-313, 31.2f,625, 625f, 626. Seealso Signalingmolecules steroid,416 in transcription activation, 312-313, 31.2f Hormonereceptors heterodimeric vs. homodimeric,313, 31-3f, 31.4f in nuclear-receptorsuperfamily,312-31.3, 3L2f, 373f. Seealso Nuclearreceptor(s) response elements and, 313, 313f genes,282 Housekeeping Hox genes,29f, 919-920, 978-983 definitionof, 978 in D r osophila melanogaster,979-9 8 l, 9 80f evolution of, 980-981,,981,f 990, 99Lf,992-994, in limb development, 994f mutationsin, 979 organizationof, 979-980, 980f Polycombproteins and, 302, 306 regulationof, 302-303, 982-983 Trithoraxproteinsand,302,303, 305 in vertebrates, 980f-982f, 981-983 Hozumi,N., 1105-1105 HP1.,252, 253f, 302-303 HPV infection,159 1.1.29 cancerand, 11.22-1.723, p53 and, 11.37 Hsc70chaperones, 559f, 560,561 Hsc70 proteins,206 /b, /oI rlsp/U cnaperones, H s p 7 0p r o t e i n s , 7 6 , 7 6 f HSP7OB,in photosynthesis, 522,5221 HSV tk gene,283,284f Hubel,David,1031 Human growth hormonereceptor,hydropathy profile for, 548, 549f Human immunodeficiency virus b u d d i n go f , 6 1 . 4 , 6 1 . 5 f membraneinvasionby, 409, 409f replicationof, 3'1,5-31.6, 3'1,5f, 326 Human immunodeficiency virus infection,159 AIDS and, 159 mRNA transport in, 346-347, 346f Human papillomavirus,159 in cancer,1,1,22-1,723, 1,1,29 p 5 3 a n d ,1 1 3 7 Human T-celllymphotropicvirus (HTLV), 159 hunchback,972, 973f-975f, 974, 999-1.000 Huntington'sdisease,199t, 200, 200f Hutchinson-Gilfordprogeriasyndrome,795, 866 Huxley,Hugh,755-756 hY RNA,222t Hyaluronan, 827, 828f, 829-830 Hybrid cell cultures,400402,402f Hybridization definitionof, 181 in DNA library screening,181-1.82,1.82f 1.951 DNA microarraysand,1.92-1.94,794f, in situ,192, 193f in diseasegeneidentification,202 Hybridomas,400-402, 402f, 1.068 Hydrocarbons,insolubilityof, 38 Hydrochloricacid,gastric,472, 472f Hydrogenbonds,34, 34t, 37, 37f, 38f in proteins,66, 66f, 67, 67f l-24

.

r ND E X

Hydrogenion(s) measurement of, fluorescentmicroscopyin, 384 pH and, 51-52,52f 447f, 448, 4 53-454, Hydrogenion ATPases,

4s3{ Hydrogenion channel,447f, 448,453454, 453f,460 Hydrogen-potassium ATPase,in parietalcells, 472,472f Hydrogenpump,447f, 448, 453-454, 453f Hydropathyprofiles,for integralmembrane proteins,548-549, 549f Hydrophilicaminoacids,4243,42f, 68, 68f,69 '1.4, Hydrophilicends,of phospholipids, 1.4f' 41.f,48, 41j.414, 4l3f Hydrophilicmolecules,31, 37, 37f Hydrophobicamino acids,42, 42f, 68, 68f, 69 Hydrophobic effect, 38-39, 39f in proteins,68 Hydrophobicends,of phospholipids,14,'l'4f, 411,48,41.141.4,413f Hydrophobicmolecules,31,,38-39, 39f Hydrophobicity, diffusion and, 439 Hydroxyl /O-) linked polysaccharides, structureof,828,828f Hydroxylation,of amino acids,43 Hydroxyproline,in collagentriple helix, 822, 822f I 99t Hypercholesterolemia. familial, 608-61.0 Hyperpolarization membrane,541 in actionporentialgeneration, 1007-1008,1025,1.026f of photoreceptorcells,1028 Hypertonicity,372, 444 740 Hypertrophiccardiomyopathy, Hypervariableregion of Ig light chains,1056f,1.067,1068 of light chains,1066t,1.067 Hypoblast,962,962f hereditary,816 Hypomagnesemia, Hypotonicity 372, 392, 444 Hypoxia-induciblefactor (HIF-I), in cancer, 1L1.2-L1.13 Hypoxic tumors, 11.09,1,112-1.113 I-celldisease, 602 I-domain,816 I-nB kinase,667I, 703-704, 7041 I-Smads,670-672 IAPs,941 ICAMs, 837 Icosahedral viruses,154, 155f Id protein,928 molecules,803, Ig superfamilycell-adhesion 804f, 1,067.Seealso Cell-adhesion molecules immunoglobulinfold in, 1067 IgA, 106s,1065f IgCAMs, 836-837 IgD, 1065 IgE, 1065 IgG, 1065-1056,1065f IgM, 1065 Imaginal discs,970, 97lf Imatibin,1130 Ime2,895 Imidazole,43 Immortalcells,398, 398f Immortalizedcell lines,398 Seealso Immunity lmmune response. adaptive,1058, 1058f, 1.062-1063 affinity maturationand, 1073

interactionsin. See antigen-antibody Antibodies;Antigen(s) antigenpresentationin, L082-L087. See a/so Antigen processingand presentation evasionof, 1062 future researchdirections for, 1'1'02 inflammation in, 1061-1062, 706Lf opsonizationin, 1060 in, 374, 374f, 606, 1.059, phagocytosis 1060,1084-1085 specificity oI, 1062-1063, 1'066,10561 vaccinesand, 1.1.0L-1'1,02 Immunesystem,21, 1055-1102 antibodiesin. SeeAntibodies cellsin, 1059.Seealso antigen-presenting and presentation Antigenprocessing activationof,1.099 professional,1080 antigensin. SeeAntigen(s) in, 1059 chemicaldefenses chemokinesin, 1061' circulatorysystemand, 1'057, 1057f' 1058-1059 complementin, 1059-1060 c y t o k i n eisn , 1 0 6 0 - 1 0 6 1 dendriticepidermalT cellsin, 915 Fc receptorsin, 1068 interferonsin, 1060-1061 in, 1057-1058,1058f leukocytes in, 1057-1058,1057f,1058f lymphocytes in, 1059, 1097 mechanicaldefenses 'l'060-106'l'' 1060f natural killer cellsin, overviewof, 1055-1057 pathogenentry and, 1057 phagocytes in. SeeImmuneresponse' phagocytosis in primary lymphoidorgansin, 1057-1058 routesof infectionand, L057 secondarylymphoidorgansin, 1058 Toll-likereceptorsin, 1'097-1099, 1098f Immunity adaptive,1058, 1058f, 1061'1,1'062-1'063, L097-1,1,01 definitionof, 1055 in, 1055-1063 host defenses innate,1059-1052, 1'0611 Immunization, L'1.01.-1,1'02 95, 97f Immunoaffinitychromatography, Immunoblotting,98, 99f microscopy,388 Immunoelectron microscopy,385, 385f Immunofluorescence 1063-1068.Seealso Immunoglobulin(s), Antibodies of, 1064f, 1065-1'066'1065f classes clonal selectiontheory and, 1066-1067, 1.066f discovery of, L062-1063 heavy-chain,1063-1064, 1063f in, classswitchrecombination 1075-1076,1075f D segmentsin, 1'069,1069f, 1071-1073,1072f regionsof, L067, 1068 hypervariable isotypesof, 1065 J segmentsin, 1069, 1'069f,1'071'-'l'073, r072f V segmentsin, 1069,1'069f, 1077-1073,1.072f variableregionsof, 1'065f,1'067 isotypesof, L064f, 1065-1065' 1'0651 light-chain,1,063-1064'1063f constantregionof, 1'0661,1067, 1068 hypervariableregionof, 1'066f,1067 isotypesof, 1065 J genesegmentsand,1069-107L, 1069f,1070f

somatic recombination and, t069-1.07 1., 1.069f, 1070f V gene segments and, 7069-1071,, 1069f, 1070f variable regions of, 1,066-1067, 1066f maternal-fetal rransfer of, 1055-7066 as multimeric proteins, 554 s t r u c r u r eo f , 1 0 f , 5 5 4 , 1 0 6 3 - 1 0 5 8 , 1063f-1057, 1064f transcytosis of , L06 5-1.066, 1055f Immunoglobulin A (IgA), 1065, 1065f Immunoglobulin cell-adhesion molecules (IgCAMs), 836-837 Immunoglobulin D (IgD), 1065 Immunoglobulin E (IgE), 1055 Immunoglobulin fold, 7065f, 1,057 Immunoglobulin G (IgG), 1.055-1.066, 1.065f Immunoglobulin M (lgM), 1065 Immunoprecipitation, 100 chromatin, 255 Immunoproteasomes, 1083-1084 Immunoreceptor tyrosine-based activarion motifs (ITAMs). Sea ITAMs Impila, 509 Import receptors m r r o c h o n d r i a l5, 5 9 - 5 6 l , 5 5 9 f peroxisomal, 567-568, 567f stromal, 555 Importins, 57 1-572, 572f Imprinting, genomic, 958 In situ hybridization, l92, I93f in diseasegene identification, 202 In vitro fertilizarion, 8 Inbreeding experiments, 171 Indirect-acting carcinogens, 1 139 I n d u c t i o n , 9 5 1 , 9 6 3 - 9 6 4 , 9 6 3 1 . S e ea l s o Signaling Infections bacterial. Sea Bacteria rmmune response in. See under Immunel I m m u n e r e s p o n s e iI m m u n i t y i n t e g r i n si n , 8 3 8 l e u k o c y r ee x r r a v a s a r i o inn . 8 3 7 - 8 3 8 . 8 3 8 f , 1097 protozoal,5 routes of, 1057 s i g n a l i n gi n , 8 3 8 viral. See Virus(es) yeast, 5 Inflammation, 1.061-1062, 1061f I n f l a m m a t o r y c h e m o k i n e s ,10 9 6 Inflammatory T cells,1096 Inheritance autosomal dominant, 199-200, 200f autosomal recessive,200, 200f epigeneric, 303 of genetic diseases,1,99-200, 200f,

203-204 X-linked recessive, 200, 200f Inhibirorof apoptosis proteins(IAPs),941 Inhibitory receptors,in axon potential generarion,1025 Initiationcomplex,134, 135f Initiationfactors,133-135,134f Initiators,282 INK4s,884, 885t Innateimmunitn 1059-1052.SeealsoImmune response Inner cell mass,960, 962f Inner ear, 1032-1034,1033f,f034f. Seealso Hearing Inorganicphosphate(P1) from ATP hydrolysis, 57,57f in ATP synthesis, 59 Inosine,basepairing with, 131

(IP3),in signaling, Inositol 1,4,5-triphosphate lamins,378, 793t, 794-795, 864f-8671, 634, 635t, 640t,654-657,654f,655f 865-867 Insertionmutations overviewof, 791-792, 791f genetaggingby, 189-190, 190f plakinsand, 796 mobileelementsand,226, 228-229, propertiesof, 7 58f, 791-792 232-233,234-235 protofibrils and, 792, 7921 P elementsand,189-190 protofilamentsand,792, 7921 qrrlrcrrrre Insertionsequences, of. 759f 79) 7921 227-228, 228f Insig-1(2)/SCAP/SREBP pathway,707-709,708f Intermediatefilament-associated proteins Insulators,254 (IFAPs),796 Insulin,17, 658-660,659f Intermediatemesoderm,991 activationof,696-697 Intermediate-repeat DNA, 226. Seealso deficiencyof, in diabetesmellitus,660 Mobile DNa elements disulfidebondsin, 552 Intermembrane space,in mitochondria,486 GLUT4 and,443 Internalface,of membrane,41,4,41,4f regulationof, 17, 17f, 1004-1005 Internalribosomeentry site (IRES),134 secretion of, 17, 17f,1004-1005 Interneurons,1005.Seea/soNeuron(s) structureof, 10f v is:;.al,7027f, 1029-1 03 1 Insulin receptor,activationof, 696-697 Interphase, 782f,783, 848, 848f, 849, 850f. int, 699 Seealso Cell cycle Integralmembraneproteins.SeeMembrane in meiosisvs. mitosis,894, 894f in nlentc 7gO 79Of proteins,integral(transmembrane) Integrase,in transposition,230, 233 Interphasecells,centrosomes in, 760-761,, Integrins,745-746, 807, 807f, 81.6-81.7, 81.7t. 76lf Seealso Cell-adhesion molecules(CAMs) Interphasechromosomes, 255 activationof, 834, 835f DNA amplificationof, 260J61, 261,f acrive/inactive forms of, 834-835, 835f nucleardomainsof, 255,255f adapterproteins|or, 876,817t polytene,260-261, 261,f in adhesive structures, 833-834,834f structureoI, 254-256, 255J56, 25 5f, 2561 in axon guidance,7048-1049,10481 Interspersed repeats,224, 226, 265-266. See bindingcapacityof, 810 a/soMobile DNA elements in blood clotting, 834 Intestinalepithelialcells,470477, 47lf in cell-matrixadhesion, 816, 831-835, Intestinalvllli, 471.,91.6,91.61 832f,834f, 83sf Intra-Sphasecheckpoint,888, 888t conformarionsof, 834 Intraflagellartransport,779-780, 781,f diversityof, 815 Intraflagellartransport(ITF) proteins,702 evolutionof, 816 Introns,123,216,21,7 expressionof, 835 in bacteria,123 in fibroblasts, 833-834,834f exosomedegradationof, 336-337 fibronectinand, 831-832,83lf g r o u pI , 3 3 4 , 3 6 3 , 3 6 4 f functionsof, 81.6-817,832 g r o u pI I , 3 3 4 - 3 3 5 3 , 35f,363,3631 in hemaropoietic cells,835 lengtn oI, zl/,5J5 I-domainin, 8.[6 in pre-rRNA,363,363f in leukocyteextravasation, 837-838 in pre-tRNA,364,365f ligandbindingbn 816, 817t,834-835,835f self-splicing, 334-335, 335f, 363, 364f. See plateletsand, 834 a/so Splicing regulationof, 834-835,835f in viruses,123 in signaling, 807, 807f,817, 833-835 I n v a d i p o d i a1,1 1 0 ,1 1 1 0 f type IV collagenand, 821.-823,821.f,823t Inversionalloining, 1070f,'1,071 virusesand, 838 Invertedrepeats,in IS elements,227-228,228f Intercellularcell-adhesion molecules,836-837 Iodine-125,100 Interferons,672, 1050-7061,.Seealso Ion channels,440,440t Cytokine(s) action potentialand, 1004. Seealso Action Interleukin(s),672. Seea/so Cytokine(s) potential T-cellproductionof, 1095-1096 calcium Interleukin1 (IL-1,1,703 IP3-gated,554 Interleukin-2(IL-2) in musclecontraction,1.024,1.024f functionsof,7096 in neurotransmitter release,1022 in T-celldifferentiation,1091 store-operated, 655, 655f T-cellproduction of, 1096 structureof, 1010-1011 lnterleukin-4(IL-4), 1.09 6 in vision,642 Interleukin-7(IL-7), 1.096 conformationalchangesin, 440 (IL-8),1096 Interleukin-S directionof flow through,464465, 4651 (IL-15),1096 lnterleukin-15 future researchareasin, 473 lntermediarefilamenr(s),16, 1,6f,71,5,775f, G protein-coupledreceptorsand,,641-645 7s7, 7 58f, 791.-796 hydrogen,447f, 448, 453-454,453f, 460 a s s e m b loyf, 7 9 2 , 7 9 2 f ligand-gated,540, 1018.SeealsoNicotinic classes of, 792-795, 793t acetylcholine receptor definitionof, 791 in mitochondria, 486-487 desmins, 793t,794 as neurotransmitter receptors,640, 1018 disassembly of, 795 in nicotinicacetylcholine receptor, di:ease-causing defectsin, 79 5-796,795f 1024-1025,1025f dynamicnatve of,795 nongated,438f, 440, 458465 in epithelialcells,809 nonselective, 541 functionsof,758f novel,464 keratins,793-796, 793t, 794f, 795f oocyteexpressionassayfo\ 464,464f IN DEX

t-25

Ion channels (continuedl in patch-clamp experiments, 463464, 463f, 464f potassrum ball-and-chain model domain of, 1013,

10r3f delayed,1007 diversityof, 1009 in heart muscle,641 inactivation of, 1013,1013f membranepotentialand, 438f, 460,460f in hair cells,339-340,340f in musclecontraction, L023-1.024, 1.0241 in nicotinicacetylcholine receptor, 1024-1025,1025f opening/closing of , 1.007-1.009, 1008f propertiesof, 1008 resting,450, 461f,462f selectivityof, 461463, 462f shakerm:utations and, 1009-1013 structureot, 461463, 46'1,f,462f, 1010-1011, 1.01.1.f, 1.01.2f subunitsof, 461.,461.f,462f typesof, 1009 voltagesensingct helix of, 1007, 1011-1013,1.0rLf,1.0r2f restingpotentialand, 1004 selectivityof, 461463, 462f in signaling,639, 640-645,640t, 641f-643f sodium actionpotentialand, 1005-1007 inactivationof, 1007, 1009, 1013 membranepotentialand, 458460, 459f in nicotinicacetylcholine recepto! 1.024-1.025, 1.025f opening/closing of, L007, 1007f, 1008f, 1009 salt perceptionand, 1034-7036 selectivityof, 461463, 462f structureof, 461.463, 462f, 1010-1011,1011f in vision,542 voltagesensinga helix of, 1007, 1011-1013,1.01.1.f, 1.01.2f store-operated, 655, 655f structureof, 461.463,461f,462f, 463f 462f subunitsof, 461, 461.1, voltage-gated, 440 action potentialand, 1006-1014 a helix of, 1007,1011-1013,1011f, 1.01.2f. coordinatedaction of, 1.024,1.024f inactivationof, 1.007,1009,1013, 1013f openingand closingof, 1007-1009, 1008f,1011-1013 structureof, 1009-1011, 101.1.f, 1.012f voltage-sensitive domainsin, 1 0 11 - 1 0 13 Ion concentration,in cytosolvs. blood, 448449,448t Ion concentrationgradient.SeeConcentration gradient Ion-exchange chromatography, 22f, 96, 97f Ion pumps.SeePumps Ionic detergents, 428 Ionic interactions,36-37, 36f Ionizingradiation leukemiadue to, 1139-1140 mutationsd]ueto, 146-L47 Ionophores,502 IP3/DAGsignalingpathway,653-657, 654f, 655f,667f, 694 IPLG complex,in neuroblastdivision, 933-935,933( l-26

.

INDEX

IRAKs,1098,1098f Iris, congenitalabsenceof, 29, 29f Iron, intracellular,regulationof, 356-357 protein (IREIron-response element-binding BP),356 Iron-sulfurclusters,495f, 496 Iron transport,via endocyticpathway, 61.1-6L2 6 ,t L I IS elements,227-228, 228f Isoelectricpoint (pI), 96 Isoforms,219,338 fibronectin,1.26,126f, 338 proteln alternativesplicingand, 1,26,126f, 338 definitionof, 808 productionof, 125-126, 126f, 338 Isoleucine,42, 42f. Seealso Amino acid(s) Isomers amino acid,34f,4L optical,33 Isoproterenol, mechanismoI actionof,629 Isotonicsolutions,444 6, 1'075f Isotypeswitching,B-cell,7075-1,07 ITAMs 1073 in B-celldevelopment, in B-celllymphocyteactivation,1091 in B-cellproliferationand differentiation, 109L, 1092f in T-cellproliferationand differentiation, 1.091.,1092f IZUmO proteln, y J /, >J / r

J chain,1065 J segments in light chains,1069-1071,1069f, 1070f 1088,1089-1091, in T-cellreceptors, r090f JAK/STATpathway,667f, 674-679, 674f-678f, 1.096 enhancersin, 656f, 676f studiesof, functionalcomplementation 677, 6771 negativefeedbackin, 678-679, 678f SH2 domainsin, 675, 682, 683f ST.{Tactivationin,674-676, 675f 728 Jasplakinolide, Jumpinggenes.SeeMobile DNA elements Jun N-terminalkinase(JNK-1),692-693, 698 iun oncogene,1.1.30 (JAMs),814f, 815 Junctionadhesionmolecules Junctionalimprecision,in antibodydiversity, 1,076 Junctions cell. SeeCell junctions exon-intron,217, 329-330,329f l0l9 neuromuscular, action potentialgenerationat, L025 ion channelactivationat, 1024, 1'024f postsynapticdensityand, 1019 Junk DNA, 21,5-216,223-226, 224f, 225f. Seealso NoncodingDNA K* channels.SeeIon channels,potassium K-zasmutations,in cancer,1116 r light chains,1.065,1.069.Seealso light-chain Immunoglobulin(s). Kartagener's syndrome,968 Karyomeres,871 Karyopherins,573 Karyotype,257 Karyotyping,spectral,258, 259f Katanin,790 768, 768f in microtubuledisassembln K6 (dissociation constant),50-51, 628 IOr acros, )l,, )Jr

for binding affinity, 628

KDEL receptor,589t, 594-595, 5941 KDEL sortingsignal,589, 589t,594-595, 594f Kearns-Sayre syndrome,241 Kendrew,John, 103 K"o (equilibriumconstant),32f, 49-50 freeenergychangeand, 56 Keratan stlfate, 827, 828 Keratinocytes,793, 91,4-91,5 Keratins,793-794, 793t. Seealso Intermediate filament(s) bullosasimplex,795-796, in epidermolysis 7951 KEX2 mutarions,604 KH motif,327 Kidney disease,822-823 polycystic,T80 proteins,in signaling,552, Kinase-associated 65Zr Kinasecascade,684, 688-690, 689f switch,in protein Kinase/phosphatase regulation,91,,91.f I 41-842 Kinases,wall-associated, 4f Kinesin(s),7 1,5, 769-774, 771.1-77 in axonal transport,770-771' classes of, 771,-772,772f dyneinsand,775-776 evolutronol, / /+, / /+r functionsof,771'-774 movementof, 772-773, hand-over-hand //Jl

headdomainof, 771, 77lf linker domain of, 771, 771'f transport,796-797 in melanosome in mitosis,787, 787f, 788, 788f, 789 myosinand,774,774f 772, 772f +/- end-directed, processivityof, 772-773. 772f, 7'73f structureoI, 770f-772f, 771, 77lf, 772 tail domain of, 771.,777f Kinesin-13proteins,in microtubule d i s a s s e m b7l y6.8 . 7 6 8 ( Kinetic energy,54 Kinetochore,263, 786-788, 786f-7 88f' 869' 887 attachmentto microtubules,887 in meiosisvs. mitosis,892, 894f orientationof, 898 in sisterchromatidseparation,869-870 structureol, /6b, /dbr Kinetochoremicrotubules,784, 7 84f, 787-788,787f 1.062-1.063 Kitasato,Shibasaburo, KKXX sortingsignal,589, 589t,594f,595 Kleisins,255, 869-870, 896-897 80-81, 8lf, 628 constant), K- (Michaelis Knee-jerkreflex, 1005, 1005f Knirps, 974-977, 975f, 999-1000 134 Kozak sequence, Krebscycle,487489, 489f Krs protein,in MAP kinasepathway,593 Kriippel, 974-977, 975f, 999-'1.000 L cells,810-811 Labeling,radioactive,99-1'00,99f Iac operator,27L lac operon,2Tt-27 3, 272f, 307f lac prornoter,271 system,195,l95f in E. coli expression 271, 307f /ac repressor, 485 Lacticacid,in musclecontraction. 46,46f Lactose, Laggingstrand,141, 1'41'f,l42f shorteningof, 263-264, 264f Lamellipodium , 71'6,71'6f,745, 745f, 746f, 1040, 1040f

Lamin(s), 378, 793t, 794-795. See also Basal lamina; Intermediate filament(s); Nuclear lamina c l a s s e so f , 8 6 5 defects in, 865 depolymerizarion of, in mitosis, 864-867, 864f-867f Laminopathies, 7 9 5-7 9 6 L a n g e r h a n sc e l l s , 1 0 9 7 Large T-antigen, 143 L a r i a t s t r u c t u r e ,i n s p l i c i n g , 3 3 0 , 3 3 0 f L a t e e n d o s o m e s ,5 8 0 receptor-ligand dissociation in, 6091, 610-61,'l in secretory pathway, 601-502, 601.1 Late G1 cyclin-CDK compleres,853t, 874-878, 874f-877f Late mitotic cyclins, 877 L a t e - r e s p o n s eg e n e s ,8 8 1 Late S-phase/earlyM-phase cyclin-CDK c o m p l e x e s8, 7 7 , 8 7 7 f Latent TGFB-binding protein, 568 Lateral inhibition, 705 i n a s y m m e t r i cc e l l d i v i s i o n , 9 3 2 in neural development, 988-989, 989f L a t e r a l p l a t e m e s o d e r m ,9 9 1 L a r r u n c u i l n !/ 2 6 - / L / Lbx1, 928 L D L r e c e p t o r ,5 0 8 - 6 1 0 , 6 0 9 f , 6 1 0 f i n f a m i l i a l h y p e r c h o l e s t e r o l e m i a6,0 8 - 6 1 0 L e a d i n ge d g e , 7 7 5 , 7 7 6 f L e a d i n gs t r a n d ,1 4 1 , 1 , 4 1 f , 7 4 2 f L e a f l e t s ,o f p h o s p h o l i p i d b i l a y e r ,4 1 1 , 4 1 3 f L e a r n r n g t. r a n \ l a t i o ni n . 1 i 2 , 3 5 Leber's hereditary opric neuropathy, 241 Lectins,838 definition of, 426 in protein folding, 553, 554f L e n s , m i c r o s c o p e ,r e s o l u r i o n o f , 2 1 , 3 8 1 Lentivirus expression systems, 797, 197f Lethal mutations in development, 999-1000 identification of, 171 recessive,171 s y n t h e r i c 1, , 7 3 t , 1 7 4 L e u c i n e ,4 2 , 4 2 f S e ea / s o A m i n o a c i d ( s ) Leucine-rich repeats, in Toll-like receptors, 1097 Leucine ztpper, 69, 70f, 291-292, 293f L e u k e m i a , 2 5 9 , 1 1 . 0 9 ,n 3 A c h r o m o s o m et r a n s l o c a t i o n si n , 1 1 3 0 , 1 1 3 2 f chronic lymphocyric, 1 13 8 H o x g e n e r e g u l a r o r si n , 9 8 3 imatinib for, 1130 P h i l a d e l p h i ac h r o m o s o m e i n , 2 5 9 , 1 1 3 0 r a d i a t i o n - i n d u c e d ,1 1 3 9 - 1 1 4 0 sremcellsin, 920 t r a n s l o c a t i o n si n , 2 5 9 , 2 5 9 f L e u k o c y t e ( s ) ,1 0 5 7 - 1 0 5 8 , 1 0 5 8 f e x t r a v a s a t i o no f , 8 3 7 - 8 3 8 , 8 3 8 f, 1 , 0 9 7 movement of, cell-cell adhesion in, 837-838,838f Leukocyte-adhesion deficiency, 838 LG domains, 821 L G L p r o t e i n , i n a s y m m e t r i cc e l l d i v i s i o n , 9 3 4 Libraries cDNA, 179-182, 780f, 182f g e n o m i c ,1 8 0 - 1 8 1 , 1 8 2 - 1 8 3 , 1 8 3 f s c r e e n i n go f , 1 8 1 - 1 8 2 , 1 8 2 f L i g a n d ( s ) ,5 0 cellular sensitivity to, 631 concentration of, 629-530, 630f definition of,78, 624 lysosomal degradarion of, 512 receptor binding of, 50-51, 51f. See also R e c e p r o r - l i g a n db i n d i n g in signaling, 624

Ligand-gated ion channels, 640, L0L8. See also Nicotinic acetylcholine receptor in muscle contraction, 1023-L024 Light visual adaptation to, 644-645 w a v e l e n g t ho f , 5 1 4

Locus, 175 definition of, 175 of linked vs. unlinked genes, 175 MHC, 1078, 1078f Long interspersedelements (LINEs), 230-234, 232f,2331

in photosynthesis, 514, 514f Light-chainimmunoglobulins. Sae Immunoglobulin(s), light-chain Light-harvesting complexes,513f, 5f 4, 5 1 5 - 5 1 65 , 1 6 f ,s L 9 distributionof, 523, 524f Light microscopn380-385.SeealsoMicroscopy Light perception,1027-1031.Seealso Photoreceptors; Vision rhodopsinin, 641-645,642f-645f Light reactions, in photosynthesis, 512-513,513f Lignin,840 Limb development. Seea/so Development f i b r o b f a sgrr o w t hf a c r o ri n . 9 9 l , 9 9 1 1 , 9 9 2 , 992f Hox genesin, 990, 9911,992-994, 994f limb bud axesin, 991 miRNA in,349,349f at proper srte,990-991, 991f signalingin,99l-992 LINEs, 230-234, 232f, 233f in exon shuffling,235 Linkage,175 Linkagedisequilibrium studies, 202, 202f Linkagemapping,200-202, 201.f-203f . See a/soMapping Linkedgenes,175 Linker DNA, 248-249 Linker scanningmutation analysis of repressorbindingsites,290 of transcription-control elements, 283, 284f Lipid(s).Seealso Lipoproteins;Phospholipid(s) cellularuptakeof, 606-617,507f-609f. Seealso Receptor-mediated endocytosis fatty acidsin, 4648, 46t Seealso Fatty acid(s) membrane.SeeMembranelipids reguJarion of, SRE-binding proreinsin, 707-709,7081I triglycerides,48 Lipid-anchoredmembraneproteins,422, 424426,425f Lipid anchors,422, 424426, 425f, 430 for Hedgehogproteins,599 for Vnt proteins,699 Lipid-binding motifs, in rr'otein targeting,427, 427f Lipid rafts,420,605 Lipid solubility,hydrophobicityand, 439 Lipid transport flippasesrn,431432 betweenorganelles,433, 433f tight junctionsand, 815 transferproteinsin, 433 Lipoproteins.Seealso Lipid(s) amphipathic shellof, 607,608f definitionof, 606 low-density endocytosis of, 606-610, 607f-609f structureof, 606-607, 508f structureof, 606-607, 608f Liposomes,477, 41,3f vesicularfusion in, 591 Liquid chromatography, 96-97, 97f Listeria,movementof, 726, 727f Liver.Seea/soHepatocyte(s) cancerof, 1141 in glucosemetabolism, 649, 650t Loco,935

Long-latencyretroviruses,1122 Long terminalrepeats,in retrotransposons, 229-230, 229f, 230f Loss-of-function mutations,157 in cancer,148, 1123, 1135-11,37 Lossof heterozygosity, in cancer,1,124,1,1,25f Low-densitylipoprotein(s)(LDLs) endocytosisoI, 607-61.0,607f-609f. See a/so Lipid(s);Lipoprotein(s) regulation proteinsin. of. SRE-binding 707-709,708f structureof, 606-607, 608f Low-densitylipoproteinreceptor,608-610, 609f,61.0f ligand dissociationfrom, 61,0-611,610f loxP-Crerecombinationsystem,208, 208f LRP receptor,599 LTR retrotransposons, 229-230, 229f, 230f Luciferaseassay,98 Lumen,373, 410 Lunatic fringe,706 Lung cancer,smokingand, 1140, 1140f Lymph, 1058 circulationof, 1.057,1057f,1058-1059 Lymphnodes,1058-1059,1058f Lymphaticsystem,1057-1059,1057f Lymphaticvessels,1057f, 1058 Lymphocytes, 20, 1057-1058,1057f,1058f, 1,062-1063. SeealsoB cell(s);T cell(s) chemokinesand, 1.096-1097 circulationof, 1057,1057f,1058-1059 in clonalselection,1.066-1067,1,066f definitionof, 1056 future researchdirectionsfor, 1,102 interleukinsand,1096 migration of, 1096-1097 in monoclonalantibodyproduction,401 productionof, 1058 Lymphoidorgans primary,1058-1059 secondarS1059 Lymphoidstemcells,9L8,91.9f Lymphoma,1109 apoptosisin, 939 Burkitt's,L1.31., 1.132f,11.35 microarrayanalysisof, 1.1.1.8-11.19, 111,8f Lysine,42, 42f. Seea/soAmino acid(s) Lysinee-aminogroups,in chromatln condensation, 250f, 251-252 Lysis,in viral replication,155, 155 Lysogeny,158 Lysosomalenzymes,374 deficienciesof, 602 targeting of, 600-602, 501f Lysosomalstoragediseases, 602 Lysosomes, L5, 86-87, 373-374, 373f-375I, 374f acidificationof, H* MPasesin, 453-454 definitionof, 373, 580 discoveryof, 407408, 408f functionsoI, 374, 374f, 612 phagosomes and,612 plasmamembraneof, 41,0 primar%374,374f protein targetingto, 612-676 in autophagic pathway,6L4-616,616f in endocyticpathway, 610-61,1,, 61.2-61.4, 689f in secretory pathway,600-602,600f,501.f secondary, 374,374f Lysozymes, 1059 INDEX

t-27

Mating-typetranscriptionfactors,in cell-type specification, 922 Matrix chloroplast,55T extracellular. SeeExtracellularmatrix mitochondrial,378, 3781,486, 557 nuclear,378,378f Matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry,701.-1.02,L0Lf Matrix-attachmentregions(MARs), 254 Matrix metalloproteinases, 703, 7 05-706, 70sf Matrix-targetingsequences, 558, 550-551, 560f Maturases,335 Maturation-promotingfactor,895 in Xenopuslaeuis,855 Maximal velocity(V-"*), 80, 80f McClintock,Barbara,226-227, 228 MCM helicases, 144, 879 MCMI, in yeast,922-923,924 MDCK cells,399-400,40lf Mdm2 protein, 1137 MDRI protein,455 flippases and.456, 456f Mechanicalenergy,54 Mechanosensitive nonselective cation channels, 843 Mechanosensors, 1031-1032 843 Mechanotransduction, Media, culture,395-396, 401.,402f medial-Golgicisternae,580, 581f. Seealso 60rf Golgi complex Mannose-lectin bindingpathwaS 1059, 1060f Mediator complex,in transcriptionregulation, MAP kinase(s),684. Seea/so Ras/MAPkinase 299, 307-308, 308f pathway and Medical conditions.SeeDiseases functionsof, 690-691,,69lf conditions structureof, 690,6901 Meiosis,19, 1,9f,150, 767, 1.68f,892-898. in transcription,690-591, 691f Seealso Cell cycle MAP kinasecascade,684, 688-690, 689f chromosomecohesionin, 892, 895-896, Mapping, 1.74-1.7 5, 1.74f,200-203 895t,8961,897f chromosome,'1,74-17 5, 175f crossing overin, 150, 153, 168f genetic,200-202,20lfJ03I definitionof, 892 physical, 202,202f eventsin, 167, 168f, 892-895, 893f 953-955,953f,9 54( 203f cytogenetic, in gametogenesis. geneticmarkersin, 200 Ime2 in, 895 homunculus,1,032,10321 key featuresof, 892-895 linkage, 200402, 2011-203f monopolincomplexin, 898 physical,202,203f oocytematurationin, 854-856, 855f recombinational,175 Rec8in, 896-898,896f retinotectal,l04Lf-1043 recombinationin, 150, 153, 892-895, of sensoryareas,1.032,1032f 894f,895f sequence, 203f spindleattachmentin, 898 MARIUPaT-1,767 stepsin, 1,67,1,681,892-895, 893f MASPproteins.in complement activation. vs. mitosis,167, 1,68f,892-895, 894f 1059,1050f in yeast,895 Mass spectrometry, 101-103, L0lf, L02f Meiosisl,892,893f in proteomicanalysis,1.06-1.07, 1.06f Meiosis11,892,893f Mast cells,1051 Meiotic maturation,of oocytes,854-855, 854f MAT locus,931,9311 MEK proteins,688-689,689t, 691, 692f in cell-typespecification, 922, 922f MEKK proteins,591.-692,691.f in yeastmating-typeswitch,299, 300f Melanin,469 Maternal mRNA, 971 Melanocytes,796-797 Mating factor, 923 Melanoma,1.48-149 469 Mating type(s),ala, 1.69,1.70f Melanosomes, in asymmetriccell division,930-931,931,f, transportof,796-797 of DNA, l1'7,1'1'7f 932f Meltingtemperature, 46-47 haploid/diploid,specificationof, 922-923, Membrane(s), 922f,923f basement,821 pheromonesand,923-924, 924f in cancer,1110 transcriptionin, 922-923, 9221,923f functionsof, 1110 Mating-type switching buddingof. SeeVesicles,buddingof in asymmetriccell division,930-931,931f, 415,5121 chloroplast, 932f cltoskeletalsupportfor. SeeCytoskeleton in transcriptionregulation,299, 300f, 309 cytosolicfaceof, 41,4,414f, 415

M phase. See also Cell cycle; Mitosis of cell cycle,18, 18f Macroautophagy, 355 Macromolecules, 10. See a/so Molecules b i n d i n g s i t e so n . 5 1 . 5 1 1 construction of, 32f, 4041, a s m o l e c u l a r m a c h i n e s ,7 2 , 7 2 f proteins in,72,72f Macroorganisms, relative size of, 20f Macrophages, 7 1.3-714, 7 14f, 1059 Mad cow disease,77 Mad2 protein, 886t, 888-889, 889f Madin-Darby canine kidney (MDCK) cells, 3 9 9 4 0 0 , 4 0 1 f , 8 1 . 1 ,8 ' t 2 f MADS transcription factors in plants, 984 in yeast, 922-923 Magnesium ions, in cytosol vs. blood, 448, 448t Major groove, 115, 115f Major histocompatibility complex. See under MHC Malaria, 5, 5f, L67 Malate-aspartate shuttle, 490491,, 490f M a l i g n a n t t u m o r s , 1 1 0 9 - 1 1 1 0 . S e ea l s o Cancer Malnutrition, mTOR in, 355 Manganese, in photosynthesis, 521 Manic fringe, 706 M a n n o s e ,4 5 , 4 5 f membrane transport of, 443 Mannose 6-phosphate (M6P), in vesicular transport, 589, 589t, 600-602, 600f,

l-28

.

|NDEX

dynamicnatureof, 409,4'101,41.5 in endoplasmicreticulum,375-376, 41'8. Seealso Endoplasmicreticulum erythrocyte,4Z3 actin filamentsin, 729-730, 7 30f exoplasmicfaceof, 41.441.5,414f, 415, 532, 543f external face oI, 414, 41.4f fluid mosaicmodel of, 410f functions of, 409 Golgi,418, 418t GPl-anchored,425-426, 425f, 543f, 545, 547,548f internal face of, 41.4,4l4I leafletsof, 41L,413I mitochondrial,378, 378f, 415, 418t, 485487,486f nuclear,342-343, 342f, 343(,414f, 415 organelle,409, 4'1.0,410f, 415 permeabilityof, 437, 438f, 444 aquaporinsand,423-424, 44444 5 phospholipidbilayerof, 411.415,415f. Seealso Phospholipidbilayer physicalproperties of, 418-41,9 plasma,437 actin filamentattachmentto, 728-73I, 730f activezone of,1.019 apicalsurfaceof, 47L, 47lf protein targetingto, 604-605, 605f surfaceof, 471.,47|f basolateral protein targeting to, 604-605, 6051 cell .junctionsin, 372 definitionof, 409 in eukaryotes,409 functionsof, 372, 373f, 409 lipid contentof,418t. Seealso Membranelipids in, 7 | 5, 7 lif microfilaments permeabilityof, 444445 of plants,839f in prokaryotes,2, 31,409 protein anchoringto, 422, 424426, 425f protein flip-floppingin, 420, 431432, 456,456f for, 39l-392 rupture techniqlues structureof, 372 synapticvesiclefusion with, 1022, 1023 protein attachment ro, 422. 424-426, 425f, 430 sizeand shapeof, 41,5,415f structure of, 47, 409434 thylakoid,379,3791,511,5'l'21 photosynthesisin, 51.1,512f slLf structureof, 51'1., typesof, 409,4L8t vacuolar,3-7-378, 377f vesicledockingin, 589-590,590f fusion of vesiclefusion with. SeeVesicles, Membraneattack complex,1'059,1'060f 641 Membranedepolarization, in action potentialgeneration,1004,1004f, 1007-1009,1007f,1008f,1011-1013, 1025,r026f alphahelixesand, 1011-1013 Membrane-hybridization technique,1.8l-182 on, 641Membranehyperpolarizati in action potentialgeneration,1007-1008, 1025,1026f Membranelipids,14, 1'4[,4648,46t, 41.L420 amphipathicnatureof, 4L1.,472f'41,6 cholesterol,416 classificationof, 411.,41.2f diffusion of, 41.6417, 4171,420

distributionof, 479420 future researchareasfor, 434 hydrolysisof, 420, 420f in lipid rafts, 420 membranepropertiesand, 418419 movementof,433,433f. SeealsoLipid transport;Membranetransport; Membranetransportproteins betweenorganelles, 433 i di l a y e r4,1 6 - 4 1 - 74.1 6 f , in phospholipb 477f, 420,431432 in phasetransition,417 phosphoglycerides, 4849, 49t, 471,,41,2f , 415416 in phospholipidbrlayer,41641.7,416f, 417t,420, 431432. Seealso Phospholipidbilayer ln protelntransport,433 relativeamountsof, 418479,419r, 433 sphingolipids, 416 synthesisof, 429431, 430f, 431, 431f, 432433,432f sitesof, 420 typesof, 415416, 478t Membranepotential,439, 458455, 1004-1005,1004f in animalcells,450 definitionof, 439, 1003-1004 electrochemical gradienrand, 439, 464, 465f generationo\ 458460, 459f magnitudeof, 460 measurement of, 450, 460f in membrane transport,439,458455 Nernst equarionfot 459450 potassiumion channelsand,460, 460f resting,458-455 sodiumion channelsand, 458450, 459f voltageand, 463, 4531,464 Membraneproteins,409,410f,421429 aquaporins,423424, 424f, 44444 5, 444f446f classificationof, 427422 detergent-solubllized, 427428, 428f diversiryof, 421 integral(rransmembrane), 68, 421, 422f, 542-549 o helix of, 422423,422f,423f, 543, s43f asymmetricorientationof, 41447 5, 41.4f,426 classification of, 543-547, 543f definitionof, 421 degradationof, 556 glycosylation of, 550-551,551f GPl-anchored, 425426, 425f, 543f, 545,547,548f hydropathyprofilesfor, 548-549, 549f insertioninto endoplasmicreticulum, 542-549, 543f-546f modificationof, 549-555 multipass,423, 423f, 543-544,543f, 546-547, 546f, 547 orientationof, 543, 545-546,546f porins,424, 425f, 485487, 571 predictionof,549 sequence homologyof, 549 signal-anchor sequence in, 545-545, 545f, 546f in signaling, 625f,626 single-pass, 422f, 423, 543f-545f, 545-546 stop-transferanchorsequence in, 544, 544f-546f structureof, 422-423, 422f synthesisof, 543-544, 543f

topologyofltopogenicsequences of, 414415, 474f,426, 543-549, 543f-546f lipid-anchored. 422, 424-426,425f. 430 misfolded,77, 78f, 86-87,555, 555f in emphysema, 555-556 quality control for, 553, 556 hrrlfrfiarr.

\\4-\\\

overviewof, 421 peripheral,422 purificationof, 428 purificationof, 427428 single-pass, 422f, 423, 543f-545f, 545-546 solubilityof, 428 targetingof,427. Seealso Protein tarBeting;Proteintranslocation tight junctionsand, 815, 815f transport.Seealso Membranetransport protelns unassembled, degradationof, 556 Membranereceptors,372,409. Seealso Membrane(s);Receptor(s) Membranerecycling,in cell migration,746, 746f Membranerepolarization,in actionpotential generation,1.004,1.004f , L007-1.009, 1008f,1.025,1.026f Membranerilfles,746 Membranetransport,63, 372 active,440,440t antiport,440,466,468470. Seealso Antiporters aquaporinsin, 423424, 444445, 444f445f ATP in, 58-59,438, 440,440t,450451, 45tI bacterialpermeases in, 454, 4 54f concentrationgradientsin, 438\ 447 conformationalchangesin, 440 cotransport,440,440t cotransporters in, 440, 440t by diffusion,438439. Seealso Diffusion directionof, 464465, 465f of drugs,455 gradientin, 439 electrochemical pathwayin, 579,606-512.See endocytic a/soEndocytosis energyfor, 58-59, 503-510. Seealso Proton-motiveforce in epithelialcells,470472, 471f, 472f exportinsin, 573-574,574f facilitated,440,440t flippases in,456,456f free energyin, 464465, 465f of glucose,44L443,441f, 4421 transepithelial, 471.,471.1 in glucosemetabolism,441.443,441f, 442f, 471.,471f, 488f, 489491., 490f, 545f,547 importins tn, 571,-572,572f ion channelsin, 439, 439f, 440, 458465. Seealso Ion channels mechanismsof, 440t membranepotentialin, 439 betweenorganelles,433, 433f overview of, 439-441, 440t phospholipidbilayerpermeabrlityand,437, 438f i n p l a n r s4. 6 9 4 7 0 , 4 7 0 f proteinsin, 438, 439440,439f. Seealso Membranerransporrproteins proton-motiveforcein, 480,480f, 503-510. Seealso Proton-motiveforce pumpsin, 438, 439440, 4391,447-458. Seealso Pumps rate of, 440441

secondaryacrive,440, 440t secretorypathwayin, 533-535, 534f, 579-606. Seealso Secretorypathway; Vesiculartransport symport,440 in transcellulartransport,471.,471.f transepithelial, 470472, 47Lf, 472f transferproteinsin, 433 transporters(carriers)in, 439, 439f, 440, 447t, 448,45 5456, 45 5t. Seealso Transporters uniport,440,447443, 447446, 44lf , 4421,444f446f vesicular,579-606. SeealsoVesicular rransporr of water,444445, 444f4461 Membranetransportproteins,63, 372, 409, 437438 ABC,447f, 448, 4 554 56, 455t MP-powered, 438,4391,440. Seealso Pumps cargo in nucleartransport, 571.-574,572f,574f translocationoI, 572, 572f in vesiculartransport,580, 588-589,

s93 channel,440,440t,458465. Seealsolon channels definitionof, 409 enriched,443 experimentalsystemsfo5 443 exportins,573-574, 574f in facilitated diffusion, 440, 440t GLUT,441.443, 441.f,442f. Seealso under GLUT importins,57L-572, 572f overview of, 439441., 439f in plants,469470,470f purification of, 443 ptt^tive, 469 transporter,439, 439f, 440. Seealso Transporters water-channel, 423424, 444445, 444f4461 Memory,translationtn, 352,357 Memory T cells,1096 MEN2 syndrome,L1.27 Menten,Maud Leonora,80 Meristems, 840, 842, 920, 921.f floral, 983 Meromyosin,732-733, 7321 Meselson,M., 140 Meselson-Stahl experiment,140f Mesenchymalcells,development of, 960 Mesenchyme, development of, 951 Mesoderm,907,907f developmentof, 907, 907f, 951, 963-964, 964f intermediate, 991 lateral plate,991. in limb developmenq991, Mesophyllcells,in carbonfixation, 529-530, 529f Messenger RNA. SeemRNA (messenger RNA) Metaboliccooperation,818 Metaboliccoupling,818 Metabolicintermediates, 481 Metabolism aerobic,483,484f anaerobic, 483,484f Metal shadowin1,390, 3911 Metamorphosis, 952 Metaphase,849, 850f. Seealso Cell cycle chromosomestructurein, 24715 5, 254f, 256,256f.SeealsoChromatin in meiosisvs. mitosis,892-894, 894f in plants,790,7901 INDEX

t-29

M e t a s t a s i s1, 1 0 8 , 1 1 0 9 - 1 1 1 0 , 1 1 0 9 f , 1 1 1 0 f . See also Cancer i n c o l o n c a n c e r .I I 1 6 , I l l T f Methionine, 42, 42f . See also Amino acid(s) start codons for, 127, 1.28f,1,33 Methionyl-tRNAlMet, 133-13 4, 13 5-136 Methotrexate, in protein targeting, 560-561 5-Methycytidine (mC), 305 Methylation of amino acids, 43 of histones, 250, 250f, 251-252, 306,

307 in transcriptionactivation,306 in transcriptionrepression, 304-305, 305f, 305 Mevalonate,432, 432f, 433 Mg*, in cytosolvs. blood, 448, 448t MHC (major histocompatibilitycomplex), 1,076-1087 classrestrictionand, 1080 cytotoxicT cellsand, 1076,1078-1079, 1,079f,1,080 in graft rejection,1.077-107 8, 1,078{ H-2 complex and, 1078, 10781 helperT cellsand, 1080 HLA complexand, 1,078,L078f loci of, 1078,1078f mice congenicfor, 1.077-1078,1,078f organizationof, 1,078, 1078f in pregnancy1078 in T-cell differentiation, 1091-1,094 T-cellreceptorand, 1081-1082, 1 0 8 8 - 1 0 9 11, 0 8 9 f ,1 0 9 0 f MHC molecules,1055, 1076-1087 classI, 1079-1082,1080f in antigenpresentation,1082-1084, 1083f classII, 1079-L080,1080f,1082 in antigenpresentation,1,084-L087, 1085f,1086f MHC restriction,L079 Mice chimeric,207,207f as experimentalorganisms,25, 25 in genomicanalysis, 245 knock-in,241,241f knockout,207-208,207f, Z08f MHC-congenic,1077-1078,1.077f transgenic,209,209f Micelles,411,4L3f, 428 Michaelisconstant(K^), 80-81, 81f,628 Michaelis,Leonor,80 Michaelis-Menten equation,80 Micro RNA. SeemiRNA (micro RNA) Microarrays,DNA, 23, 24f, 192-194, 1.94f, 1 95 f in cancer,111,6-1119,l1l8f, L1.21. in diseasegeneidentification,202 in geneamplificarion,1121 Microfilaments, 16, 1.6f,715-745,757,7 58f actin,71.6-731.. Seealso Actin; Actin filament(s ) definitionof, 715 in melanosome transport,795-797 myosin,731-745. Seealso Myosin overviewof, 7 | 5-- 16,7 l6f propertiesof,758f in short-rangetransport,796-797 structuresmadeof organizationof, 728-731 typesof, 716,71.6f Microglia,1014 Microorganisms,relative size of, 20f Microsatelliterepeats,201,224, 224f, 340 in DNA fingerprinting,225,225f in geneticdiseases, 224,340 l-30

.

INDEX

Microscopes, 20-21 cryoelectron,104 electron,21, 388-390, 388f-390f light,380-385,380f optical,380f resolution of, 21.,381. Microscopicreversibility, 49 Microscopy bright-field,381 computeralgorithmsfor, 387 confocal,386, 386f deconvolution,386, 387f differentialinterference contrast,38L, 382, 908f digital imagingrn, 387, 387f electron,21.,388-390,388f-390f, 404 cryoelectron,389, 390f immunoelectron, 388 metal shadowingin, 390, 391.f 607, of receptor-mediated endocytosis, 6071 scanning, 388f,390 specimenpreparationfor, 388-389, 3891,390,391 transmission, 388-389,389f vs. fluorescence microscopg388 epifluorescence, 380f fluorescence, 382-386 of act-inpolymerization,71.9 in Ca'* measurement, 383-384,384f confocal,386,386f deconvolution,386, 387f in H2* measurement, 384 immunoflurorescence, 385, 38-5f SPED,385 total internalreflection,404 vs. electronmicroscopS388 of VSV G proteins,582f, 583 future researchareasfor, 403-404 immunoelectron, 388 immunoflurorescence, 385, 385f light,380-387,403 Nomarski,908f phase-contrast, 380f, 381-382 refractiveindex in, 382 resolutionin, 381 specimenpreparationfor, 384-385, 384f SPED,385 ctainino

fnr

1R{

timeJapse, 382 Microsomes, rough,535-536,535f,537 Microtubular protein, 763 Microtubule(s), 1.6,l6f, 715, 71.6f,757-791 assembly/disassembly of, 760-751, 76lf, 753 catastrophestagein, 764, 764f drug effectson,766 dynamicinstabilityand,763-766, 764f, /b-)t

GTP-p-tubulincap in, 764-765, 7 65f -6---68. -67f,768f r e g u l a t i oonf . rescuestagein, 764, 764f astral,784,784f in axon growth cone, 1040-7042,1.0401, L041.f -69-77 l, 76c1,7-0f in axonaltransport. -77 axonemal,777 9, 778f, 779f in ctlia,777-7 80, 778f, 7 80f, 7 8lf definitionof, 758 distributionof, 766 doublet,760,760f dynamicnatureof, 762-766 in flagella,777-780, 778f-781f functionsof,758f kinetochore,263, 784, 784f, 787-788, 787f, 887

lifespanof,762-763 in long-rangetransport,796-797 in melanosome transport,796-797 in mitosis,T6'1.,761f chromosomeattachmentto, 786-788, 787f dynamicsof,784-786 in plants,790-791.,790( shorreninsof. 788f. 789 treadmillingin, 785-786, 786f, 788 in mitoticspindle,782,782f,784,784f organizationof, 760-7 61, 761.f overviewof,757,758f +/- ends of, 763, 763f, 764, 764f polarity of, 7 59, 784, 784f properties of,758f protein polymerization in, 763 protofilamentsin, 7 58-760, 759f, 760f, 764 functionsof, 766 search-and-capture of, in mitosis,788f,789 shortening singlet,759-760, 760f stabilizationof, 767-7 68, 767f structureof, 7 58-760, 759f, 7 60f treadmillingtn, 753, 763f in mitosis,785-786, 786f, 788 in vesiculartransport, 593, 769-770, 7701 kinase Microtubule-affinity-regulating (MARKlPar-L),767 proteins(MAPs),758, Microtubule-associated 763, 767-768,757f MPF phosphorylationof, 866 motor proteins,769-776. Microtubule-based Seealso Motor proteins dyneins.774-776, 774f, 77 5f. Seealso Dynein(s) . Seealso kinesins,769-774, 771.f-774f Kinesin(s) centers(MTOCs), Microtubule-organizing 760-761.,761.f,766, 766f in mitotic spindle,783,784f in plants,790-791, 790f Microvilli, 373f, 71.6,71.6f actin filamentsin, 731 oellnltlonor, /lJ intestinal, 47'L Mid-G1 cyclin-CDKcomplexes in mammals,853t, 881-882,882f in yeast,8 53t, 874f-877f, 87 5-876 Middle prophasechromatid,255 (MM) proteins, Minichromosomemaintenance 744 MinisatelliteDNA, 225, 225f Minor groove,115, 115f miRNA (micro RNA), L20, 2Z2, 222t, 347, 367, 910-971. basepairingby.347. 348( in cell differentiation, 9 10-9 l1 in cell division,910-91.L diversityof,348-349 functionsof, 347-349, 910-911' in geneinactivation,210 in limb development,349,349f in neurulation,986-987 processingof, 348, post-transcriptional 349f synthesisof. Seealso Transcription RNA polymeraseII in, 279,279t 347-349, 348f in translationrepression, Misfoldedproteins,77,78f,86-87, 555, 555f 555-556 in emphysema, qualitycontrolin,553, 555 1.48f Mismatch repair,1,47-1,48, Missensemutatrons,157 Mitchell, Peter,503-504

Mitochondria,3731,378 aerobicoxidation in, 479 in ATP synthesis, 15, 15f, 378,378f,379, 48s,487-492 brown-fat,510 cellularrespirationin, 485, 487489, 489f definitionof, 378 dynamicpropertiesof, 485 evolutionof, 236, 237f,240, 557 fatty acidoxidarionin,491,492f fusionand fissionin, 485,486f geneticcode of, 240, 241f in glucosemetabolism, 378,379, 485, 487492 electrontransportin, 493494, 494t matrix of, 557 oxidativedamagein, 502-503 oxidarivephosphorylationin, 504f, 505 as power plants,378 proteinsynrhesis in. 557 protein targeringto, 557-565, 557t. See a/soProteintargeting,to mitochondria proton-motiveforcein, 502, 510, 561 in pyruvateoxidation,485,487489 respiratory controlin, 510 sizeof, 485 structureof, 378, 378f, 485487, 485f transcription in, 317-318 MitochondrialDNA (mtDNA), 1.3,236-242. Seealso mrDNA (mitochondrialDNA) Mitochondrialmembranes,378, 378f, 418t, 485487,486f Mitochondrialproteins,238,239f, 486 Mitochondrialribosomes, 240 Mitogens,880-881,1134 M i t o s i s 1, 8 , 1 8 f , 1 9 , 1 5 7 , 7 5 8 f , 7 8 1 - 7 9 1S.e e a/so Cell cycle anaphase in, 783, 783f, 789, 849, 850f, 857-870, 869f APC/Ccomplexin, 850, 850f,851, 858, 859f,869-870,876-877 centrosomeduplicationrn, 783, 784f chromosomecohesionin, 892-895, 895f chromosomecondensation in, 21 chromosomedecondensation in, 870-87l, 87lf chromosomemigrationin, 21.,2lf chromosomesegregation tn, 167, 168f, 787-7 88, 867-870, 857-87 1., 868f-871f chromosomeseparationin, 21, 2lf condensin in, 855 congression in, 783, 783f cytokinesisin, 783, 7 83f, 789-790 contractilering in, 7 16, 716f, 742, 742f,789 daughtercellsin, 872,872f destruction box in, 858, 867,868f durationof, 849 dynactinin, 787, 787f, 788f, 789 dyneinsin, 787, 787f, 788, 788f, 789 entry into. Seealso Cell cycle,regulationof cyclinA in, 883 cyclinB in, 855-858,855f, 857f cyclin-dependent kinasesin, 859-863 in mammals,883 mitosis-promoting factor in, 855-858, Sssf-ss7f,86r-863, 862f, 863f.See a/soMitosis-promotingfactor (MPF) molecularmechanisms in, 864-871 premarure,861,f,875, 886 preventionof, 888 in Xenopuslaeuis,855-858,856f,857f in yeasr,860-863 eventsin, 1,67,L68f, 782-783, 782f, 892-895,894f

evolutionof, 860-861 exit from, 889-891 APC/C complexin, 850, 850f, 851, 858, 859f, 867-870,869f, 876-877, 878f, 879. Seealso APC/C complex destruction box in, 858, 867,868f regulationof, 858 interphasein, 7 82, 7 821,783 karyomeresrn, 871.,872f kinesinsin, 787, 787f,788, 788f, 789 kinetochorein, 785-788, 786f-7 88f Iamin depolymerization in, 864-867, 864f-867f metaphase in, 849. 850f microtubulesin, 767, 761f chromosomeattachmentto, 786-788, 787f dynamicsof,784-786 in plants,790-791, 7901 shorteningof, 788f, 789 treadmillingin, 78 5-785, 7 86f, 788 molecularmachinein, 791 nuclearenvelopein, 864 disassembly oI, 865-867, 8661 reassemblyof, 870-87l, 8721 nucleoporinsin, 866, 866f, 871. in plants,790-791, 790f prometaphase in, 782f, 783, 786-788 prophasein, 782, 782f, 783 regulationof, in Xenopuslaeuis,856-858, 856f, 857f, 867-869, 868f sisterchromatidseparationin, 869-870, 870f,871.f SMC proteinsin, 866 spindlepolesin, 761, 761f stepsin, 1.67,L68f, 782-783, 782f, 892-895,894f telophasein, 783, 783f, 871 vs. meiosis,167, 168f,892-895,894f Mitosis-promotingfactor (MPF), 855-858, 855f-857f in chromosomecondensation, 866 in condensinphosphorylation,865 cyclinB in, 855-858,856f,857f inactivationof, 87l, 872L 888, 890 in lamin depolymerization, 864-867, 864f-867f in mammals,883 in meiosis,895 in mitotic spindleformation,866 in nuclearenvelopedisassembl5864-857, 864f-867f nuclearprotein phosphorylationby, 864-867, 864f-867f in nucleoporinphosphorylation,866 in oocytematuration,854-858, 854f-857f regulationof, 861-863, 862f, 863f pompe, 861,,883 in Schizosaccharomyces in vesiculartransport,866 rn Xenopuslaeuis,855f,856-858 Mitotic apparatus,849 Mitotic asters,782, 782f, 783, 784f Mitotic cyclin-CDKcomplexes,850f, 851-853,852f Mitotic cyclins,853t, 877, 877f cyclinB as, 856-858,856f,857f definitionof, 856 late, 877 in mammals,883 mitosis-promoting factor and, 857-858, 857f polyubiquitinationof, 869-870 pompe,862, 862f in Schizosaccharorlxyces in Xenopus,856-858,856f, 857f in yeast,862, 862f, 877, 877f Mitotic exit network, 890

Mitotic spindle, 782, 7821,849. Seealso under Spindle assemblyoI, 761.,7 611,789, 789f in absence of centrosomes,789, 789f mitosis-promoting factor in, 856, 866 in, 789, 789f self-assembly asymmetryo1,933, 934f microtubule-organizing centersin, 7 83, 7 84f orientationof, 933, 934,934f Mobile DNA elements,14, 226-235,265-266 definitionof, 216 orscoveryo\ Lz6-/z/ DNA transposo ns, 227-229, 227f-229f in evolution,14, 216, 226, 234-235 geneticdiseases and,232-233, 234-235 in genomicDNA, 234 mutationsand,226, 228229, 232-233, 234-235 retrotransposons, 227-234 in genomicDNA,234 I:IR, 229-230, 229f, 2301 non-LIR (nonviral),230-234, 231.f-233f Model organisms,25-27, 26f ModeratelyrepeatedDNA, 225. Seealso Mobile DNA elements Modification enzymes,176-177 Molds, 5. Seealso Fungi Molecularchaperones. SeeChaperones Molecularcomplementarity, 321, 3940, 39f protein binding and,78 Moleculargenetictechniques,1.65-21,2 classicalgenetic,165, l66f cloning,1.76-1.90. Seealso Clones/cloning in disease-causing geneidentification, L98-204. Seealso Mapping geneinactivation,204-21J.. Seealso Gene lnactlvailon geneticanalysis,166-'1.76. Seealso Genetic analysis recombinantDNA technology,L76-1.90. Seealso RecombinantDNA technology reversegenetic,1,65,1,56f Molecularmachines,64, 72, 72f macromolecules as,72, 72f in mitosis,791 proteasomes, 87-88, 87f ribosomesas, 132-1.33 structuralanalysisof, 108 Molecularmarkers,DNA-based,200 Molecularmotors,63,85,769-775.Seealso Motor proteins Molecularresolution,92 Molecules.Seealso Macromolecules; Protein(s) amphipathic,31.,49, 69 binding of, 39-40 bind.ingsiteson, 51, 51f bondsof. SeeBonds functionalgroupsof, 34, 35t hydrophilic,31, 37, 37f hydrophobic, 31.,38-39, 39f iarge, construction of, 32f, 4041 polarity of, solubility and,39 solubility of, 37, 38f, 39 stereoisomers of, 33, 34f MonocistronicmRNA, 2l 7 Monoclonalantibodies,1068 in affinity chromatography,22f, 96-97, 401, production of, 400402, 401,f usesof, 401 Monocytes,extravasationof, 837-838, 838f Monogenetictraits, 203 Monomericactin,71.7-71.8,7L7f . Seealso Actin Monomeric(small)G proteins,354-355 in signaling,534 INDEX

t-31

Monomers, 10, 40, 41f, 113 Monopolin complex, 898 Monosaccharid es, 44-4 5, 4 5 f structure of,40,41,f Morgan, T.H., 175 Morphogen(s), 700. See a/so Hedgehog pathway definition of,964

in neuraldevelopment,987,987f Morphogenesis definitionof, 969 extracellularmatrix in, 805-807, 806f Morula, 960,960f Mother cells,ganglion,932-933 Motifs, 69-70,701,290-293 definitionof, 243 DNA-binding,69-70,70f,290-293, 291f193f basic-zippeg292 coiled-coilmotlf, 67, 69,70f,292 helix-loop-helix motif,69,90,901, 290-293 helix-turn-helixmotif, 69,70f,290 fprreine 'innc' Aq 7qf,291.-292, 293f zinc finger,69-70,70f,291, 291f RNA-binding,327, 3281 sequence analysisfor,243 vs. domains,69 Motor homunculus, 1032,1032f Motor neurons,1005,1005f structureof, 1002f,1003,1003f survivalof,937-938,937f targetfield and,937-938, 937f trophic factorsfor, 938 Motor proteins,63, 85, 715,769. Seealso Cell movement/migration and specific proteins in axonal transport,769-777,769f,770f, 775-776 in crlia,777-781 cooperativeactivity of, 745-7 5L, 775-776, 776f,796-797 dyneins,774-776,774f,775f,776f.See also Dynein(s) in flagella,777-781, kinesins,770-774,771f-774f,776f..See also Kinesin(s) microtubule-based,769-776 in musclecontraction,732,738-743. See a/soMusclecontraction myosin,71.5,731.-745 in nonmusclecells,74l-742,742f MPSBcomplex,in neuroblastdivision, 933-935,933f MRF proteins,926-929,927f,928 mRNA (messenger RNA), 11, 11,2,1,1,2f,11,3f. Seea/so RNA basepairing with snRNA, 330, 331,332, 332f circular,1,38,l39f conformationsof, 118 cytoplasmicdegradationof, 352-353 cytoplasmicpolyadenylationof, 351-352, 351f 5' cap of, L24, 1.25f in pre-mRNA processing, 324f, 325-336,325f,3271,T7 shorteningof, 352-353, 3521 in transcription,2S0-28L,282 in translation,134 functionsof, 1.12,113f,727,1,27f halflives of, 352 histone,in oogenesis,957 identificationof, by in situ hybridization, 1,92,1,93f l-32

.

INDEX

improperly processed,elimination of, 357 marernal,971 monocistronic, 217 nonsense-mediateddecay of, 357 nuclear transport of, 341-347, 367. See a/so Nuclear transport n u c l e a r p o r e c o m p l e x e si n . 3 4 2 - 3 4 3 ,

342f,343f polycistronic,217 production of,723-L24,125f. Seealso RNA processing from simplevs. complextranscription units,21.7-21.8,21.9f splicingof. SaeSplicing siructureof, 118 synapticlocalizationof, 352,357-358, 358f synthesis of. SeeTranscription in translation.SeeTranslation unrranslated regionsof, 124 viral,transportof, 346-347,346f mRNA surveillance,357 mRNP,325 Balbianiring, nucleartransportof, 344-345 nuclear,341-3Q, 3Q--342 rransporro1,342-347,343f-346( mRNP exporter,342-343,343f,573-575,574f mRNP remodeling, 574, 574f mtDNA (mitochondrial DNA),236-242,957 codingcapacityof, 239 cytoplasmic inheritance of,237-238,238f in Euglenagracilis,237,237f mutationsin,240-242 rn planrs,239, 240,24k proteinsencodedby,238,239f replicationof, 236 sizeof,238,239 structureof,239-240, 239f rn yeast,237,238{ centers), MTOCs (microtubule-organizing 760-761.,76lf, 766, 766f in mitotic spindle,783,784f in plants,790-79'1,,790f mTOR, in translation,353-355 Muller, H., 171 Multiadhesivematrix proteins,805, 805t, 820, 821,822t fibronectin,830-833.Seealso Fibronectin laminins,805t, 821, 821.f,822f MulticoloredFISH. 258. 259f l26f Multidomain proteins,1,25-1.26, Multidrug-resistance rransportprotein (MDR1), 455 flippasesand,456,456f Multimericproteins,72 Multipassmembraneproteins,423, 423f, 543-544,543f, 546-547, 546f Multiple endocrineneoplasiatype 2, 1127 Multipotent stemcells,907 Multivesicularendosomes, 612-614,613f, 6l4f p. heavychains,1055.Seealso Immunoglobulin(s), heavy-chain Muscarinrcacetylcholine receptors,in cardiac muscle.641. 641f Muscle cardiac in, 706 ADAM proteases proteinsin, 652, 652f kinase-associated in, receptors muscarinrc acetylcholine 641,64lf sodium-linked Ca2* antiporterin,468 collagenin, 826 desminin, 793t,794 developmentof,925,9251 dystrophitrin, 835-836, 836f

proteinsin, 740, 740f skeletal,accessory smooth,contractionrn, 742-743, 743f vascular,protein kinaseG and,656-657, 6571 Musclecalciumpamp, 44945L, 450f Musclecells calciumions in, 449452, 450f,451.f,658 cultureof, 396,397f as excitablecells,1004-1005 specificationof,924-929 synapses in, 1019 Musclecontraction 1024f acetylcholinein, 1.023-1.024, ATP hydrolysisin,735,736f,755-756 calciumions in, 449452, 450L 451,t, 740-743,743f, 1024,7024f in cardiacmuscle,740 glucosemetabolismand, 484f, 485 Hanson-Huxleyexperimentin,755-756 ligand-gatedion channelsin,1023-1024 myosinin, 732,738-741 receptorin, 1,023-1,025 nicotinicacetylcholine in skeletalmuscle,740-741 slidingfilamentmodel of, 738-740,739f in smooth mtscle,742-743,743f regulationin,740,741.,741.f thin-filament Muscleregulatoryfactors(MRFs),926-929, 927f Musculardystrophy1.99t,200,200f,731., 827,835-836 Duchenne, 1.99t,200,200f,234235, 835-836 795 Emery-Dreifuss, Musculartissue,801 23, 166, 1.67.Seea/so Mutations Mutagenesis, and,1139 carcinogenesis 1,67-169 Mutants,true-breeding, Mutations,1.3-14,22-23,1'45 autosomaldominant,1.99-200,200f 200,200f autosomalrecessive, in cancer.SeeCancer,mutationsrn cdc,170-171,170f, 851-852,852f, 860-862 chemicalcausesof, 1.45-146 testsfor,219 complementation conditional,1'70-1'7I,170f correctionof. SeeDNA repair deamination,146, 1'46f definition oI,'1.3,L56 depurination,147 l'4,29-30. Seealso disease-causing, and conditions Diseases identificationof,200J03,201'f-203f dominant,166-170, L67I, 860' 1,1'13 genefunction and, 166-1'67 of,1'67-1'69,168f,1'69f segregation 1.67,209,674' 747, dominant-negative, 1136 in evolution,7, L4, 28 frameshift,128' 167 gain-of-function,167 in cancer,1L19-112'1, geneticanalysisof,166-176. Seealso Geneticanalysis 167 haploinsufficient, in cell lineagestudies, heterochromatic, 909-91'L,9L0f rnsertlon genetaggrngby,189-190,190f mobile elementsand,226,228-229' 232-233,234-235 P elementsand,189-L90 lethal in development'999-1'000 of,171 rdentification 171 recessive' synthetic,1'73f'174

linkage mapping of , 200-202, 201.f-203f linkageof, 175 linker scanning,283, 2841 Ioss-of-function, 167 in cancer,148, 71,23,11,35-1,737 in lossof heterozygositS 1124 mapping of , 200-202, 201.f-203f missense, 167 in mitochondrialDNA, 240-242 mobile DNA elementsand,226, 228-229, )t)_)11 )14-)1S in noncodingDNA, 14 nonsense, 138,167 p o i n t ,1 4 6 ,1 4 6 f , 7 5 7 identification of , 202-203 in proto-oncogenes, 1120 preventionof, 145 radiation-induc ed, | 46-1.47 repairof, 149-150,150f recessive, 166-170, L67f , 850, 1122t, 1123 genefunction and, 1.56-167 identificationof, 171 lethal, 1.71. segregation of, 167-169,1681,l69f sourcesof, 145-1.47 suppressor, l- 3-174, 173f temperature-sensitive, 23, 170-171, 170f in yeastcell cyclestudies,851 transcription-u nit, 21,8-219 Xlinked recessive, 200, 200f myc oncogene,1115,1L22, 1.1.31., 1132f Myelin sheath,1003,1003f in action potentialpropagarion, 1013-1016,1014f defectsin, 1015 formationof, 7014, 1.016,1015f by oligodendrocyres, 1014,1015f by Schwanncells,1014,1015f structureof, 1014,1016,l0l6f Myeloid sremcells,918,91,9f Myelomacells,401, 402f MyfS,926,927 Myoblasts definitionof, 925 cultureof, 396 differentiationof, 925-929, 925f-929f migration of, 928-929, 929f Myocyte(s) calciumionsin, 449-452,450f,451f,658, 740-743,743f c u l t u r eo f , 3 9 6 , 3 9 7 f as excitable cells,1004-1005 specification of , 924-929 synapses in, 1019 Myocyte-enhancing factors(MEFs),927, 927f myoD, 926-929,926f Myofibrils,739,741.f earfysrudiesot, 755-756 Myogenesis cell-typespecificationin, 924-929 eventsin, 925,925f MyogenicbHLH proteins,926-929, 926f-930f Myogenicdeterminationgene,926-929, 926f Myogenin,926, 928, 929f Myoglobin, 73 Myosin,715, 731,-745 in actin filamentmovement,733 in contractilebundles,741-742, 742f cntfcalconcentratron ot, 7 33, 7 33f in cytokinesis, 7 42, 7 42( in cytoplasmicstreaming,744, 744f definitionof, 731 outy ratlo olr /JJ, /3/ evolution of,774,774f torce generated by, 7 35-- 31

functionsoI,734f headdomainsoI, 732f, 733 conformationalchangesin, 735, 736[ kinesinand, 774,774f in melanosome transport,796-797 as motor protein,733-735 movementpoweredby actinbindingin. 735. 736f in asymmetriccell division,931,,931,f -ATPhydrolysisin, 7 35, 7 361,774, 774f hand-over-hand vs. inchworm model of, 737-738,738f in musclecontraction,732, 738-743. Seealso Musclecontraction processive, 73-,737f stepsizein,735-t37, -37f. neck domain of, 732f, 733 s t e ps i z ea n d , 7 3 5 . 7 3 7 f in nonmusclecells,74l-7 42, 74L-743, 742f overviewof,731.-732 power strokeand S-1motor domainsof,733-735,733f assayfor, 733, 733f sliding-filament in stereocilia,1034 structureof, 732-7 33, 7 32f, 734-735, 7 34f tail domainsof, 732f, 733, 734-735 rhick filamentsin, 739-740, 7 391,742 types/classes oI, 73 l, 733-735, 7 34f M y o s i nI , 7 3 4 - 7 3 5 , 7 3 4 f Myosin II, 7 31.-733, 732f, 734-735, 7 34f MyosinLC kinase,742-743,743f Myosin motor protein,93L Myosin regulatory lighr chain,742 Myosin superfamily,7 33-735, 7 34f Myosin V, 734, 734f, 735, 737-738 cargotransportby, 743-7 44, 743f Myotonic dystrophy,224, 340 Myotubes,396, 925, 925f N-cadherins,81.0.Seea/so Cadherins N-linked oligosaccharides, 550, 550f, 827, 828f. Seea/so Oligosaccharides N-region,of heavychain,1.073 N-terminus,of protein,65, 65f Na*. SeeSodiumions NADin citric acid cycle,487489,489f, 490t in electrontransport,59-60, 60f, 493, 495 in glycolysis,482, 482f in pyruvatesynthesis,482,482{' NADH in citric acid cycle,489,490t electrontransport ftom, 493-502.Seealso Electrontransport oxidation of, in respiratorycontrol, 510 productionof, 60, 60f,487489, 489f, 490t NADH-CoQ reductase, in electrontransport, 495,495r,496, 497f NADP-, in photosynthesis, 512-513,515, 515f,519-520,522-523,523f NADPH, in photosynthesis, 512-513,515, 5 1.5f , 51.9-520,522-523, 523f Nanosprotein,974, 974f Nanotechnologgfor cell cultures,404 Nasalcancer,1139 Nascentproteins,translocationof, 534, 536-537,537f Nascenttranscript,cappingof, 324f, 325-326, 3271 Native-stateconformations,74 NaturalDNA, 115, 115f Natural killer cells,1060-1.061,1060f,1095 Nebulin,740,740f Necrosis,in apoptosis,937

Nernstequation,4 59-460 Nerve growth factor (NGF) in neuronsurvival,938,938f,942 in Ras/MAPsignaling,693 Nervous system cellsof. SeeNeuron(s) development of, 9L6-917,917f, 985-990. Seealso Neural development in, 1005-1006,1005f interconnections plasticityof. 1005-1006 tissueof, 801. Netrins, 1.044-1.04 5, 1.044f Neu protein,71 molecuies,836-837 Neural cell-adhesion Neural development, 916-917, 917f, 985-990, 9 8 6 f , 9 8 7 f ,9 8 9 f axon guidancein, 1,046-1047 moleculesin, 835 cell-adhesion in D rosophila melanogaster,93 1.-935, 932f-934f lateralinhibition of, 988-989, 989f neuraltube formation in, 986-987, 986f neuronmigrationin, 988 neurulationin, 985-987, 985f, 986f signalingin, 965, 987-988 Neural plate,986 Neuraltube,91,6,917f formationof, 986-987,986f Neuraminidase, 546 Neuregulins,68l Neuroblast(s), 9 16-9L7, 9 17f asymmetricdivisionof apicalcomplexesin, 933-935 basalcomplexin, 933-935 daughtercell sizein,934-935 in Drosophila melanogaster,931-935, 932f-934f eventsin, 935 ganglionmothercellsin, 932-935,933f lateralinhibition rn, 932 mitotic spindleorientationin, 934-935, 934f differentiationof, 929, 930f Neuroblastoma,1144 NeuroD,929,930f Neurofascin155,1015 Neurofascinl86,1015 Neurofilaments,793t, 794. Seealso Intermediatefilaments Neurogenesis, 929,930L Seea/soNeural development in Drosophila melanogaster,931.-935, 9321-934f neurulationin, 98 5-987, 98 5f, 986f signalingin, 965, 987-988 Neurogenin, 929,930f Neuromusculardiseases. SeeDiseases and conditions Neuromuscularjunction,1019 action potentialgenerationat, 1025 ion channelactivationat, 1.024,1.024f postsynapticdensityand, 1019 Neuron(s),1001-1050 abundanceof, 1001 action potentialof, 1003-1005,1004, 1.004f,1006-1.01.4. Seealso Acrion potential in circuits,1005, 1005f coordinatedaction of, 1005, 1005f definitionof, 1001 as excitablecells,1003-1004 functionsof, 1003 informationflow through,1003-1005 interneurons,1005 vistal, 1027f , 1029-1.031. light-sensitive, 1027 INDEX

r-33

Neuron(s)(continued) migrationof, 988 morphologyof, 1003f motor,1005,1005f structureof, 1,0021, 1003, 1003f survivalof, 937-938, 9371 targetfield and,,937-938,937f trophic factors 6or,938 nociceptive,938 olfactoryreceptor,1037 presynaptic, 1004f,1005,1018 projection,1037 properties of, 1001,1002-1003 restingpotentialof, 1004 rctinal,1027-7031.,1027f , 1.029 f sensory,1005, 1027-1,039 in hearing,1,032-L034 in smell, 1036-1.039 in taste,L034-1036 in touch, 1031-1.032 in vision,1027-1031 in signaling, 1001-1006 structureof, 1002f, 1003, 1003f survivalof, 937-938, 937f. synapticconnectionsof, 1004f, 1005 targetfield and, 937-938,937( in thinking, 1001 typesof, 1002f voltagein, 1004 Neurotransmitter(s), 10. Seealso Signaling definitionof, 1005 degradation/reuptake of, 1023 drug effectson, 1023 Iow-molecular-weight, 1019-1020, 1,0201 mechanismof action of, 1005 packaging of, 1019-1.022,1020f, 7021f releaseof, 1020-7022, 1021f structureof,1020f in synapticvesicles,1019-1,022,70201, 1021.1 synthesis of, 1020 transportof, 1019-1.020 Neurotransmitterreceptors densityof, 1019 G-protein-coupled, 423, 624, 63 5-657, 1018.Seea/so G-protein-coupled receptors ion channels as,640, 1018 ligand bindingby sloq 1018 Neurotrophins in cellsurvival,937-939,937f in development, 938 Neurulation,985-987, 9851,986f Neutrophils,1.059,1.061. extravasation of, 837-838,838f Nexin, 777, 7781 NF-rcBsignalingpathway,703-705, 704f activationof, 656f,667,703 in antigen-presenting cell activation,1102, Lr02f Nicastrin,705-705 Niches,stem-cell.SeeStem-cellniches Nicotinamideadeninedinucleotide. SeeNAD* Nicotinamideadeninedinucleotidephosphate (NADP-), in photosynthesis, 512-513, 515,515f Nicotinamidedinucleotidephosphate (NADP*), in photosynthesis, 512-513, 515, 515f, 519-520, 522-523, 5231 Nicotinic acetylcholine receptor,1018, 1023-1025 as ligand-gatedion channel,1018, 1023 in musclecontraction, 1018,1023-1025 structureof , 1,024-1025,1025f l-34

.

INDEX

Nidogen,805t in basallamina, 821, 82lf Nieuwkoop center,963, 964f Nipkow confocalmicroscopy,386 Nitric oxide/cGMPpathway,656-657, 657f Nitrogen mustard,1139 Nitroglycerin,656 Nkx proteins,in heart development, 967-968, 967f Nociceptiveneurons,survivalof, 938 Nociceptors,1031-1032 Nodal cilia, 968-969 Nodal proteins,963, 966-967,967f Nodal vesicleparticles(NVPs),968-969 Node,in developmental 955,968, signaling, 968f Node of Ranvier,in action potential propagation, 1013-1014,1014f,1015 Noggin, 986 9081 Nomarskimicroscopy. NoncodingDNA, 215-216, 221.t,223-226, 224f,225f amountof, 223 evolutionof, 223-224, 225-226 microsatellite, 224, 224f satellite, 224,225f Noncovalentinteractions,32-33, 3540 hydrogenbonds,37,37f i n p r o t e i n s6,6 , 6 6 f , 6 7 , 6 7 f hydrophobiceffect,38-39, 39f ionic, 36-37, 36f in macromolecules, 40-41, 4lf molecularcomplementarity and, 3940, 39f strengthof, 35, 37, 38 van der Vaals,37-38, 38f in phospholipidbilayer,411, 41.8-419 Nondisjunction,chromosomal,887 lossof heterozygosity and, 1124 Nonhomologousend-joining,149-150, 1s0f Nonpolar bonds,34 Nonsense-mediated decay,357 Nonsensemutations,138, 1,67 Nonviral retrotransposon s, 230-234 Norepinephrine, structureof, 1.020f Northern blotting, 792, 7921 in diseasegeneidentification,202 Notch cystolicdomain,activationof, 666f, 667f Notch/Deltapathway,703, 705-707, 705f in cell division,91.4,932 in neuraldevelopment,988-990, 989f Notch protein,71, 705-706 c l e a v a goef , 7 0 5 , 7 0 5 f synthesisof, 705 Notch receptors,666f Notochord,986f NPXY sortingsignal,589, 589t,608-609,609f NTCAM, 1016 NRIF protein,943 NSF prorein,in vesicular transporr,591 NtrB protein in transcriptioninitiation,274, 274t NtrC prorein,in transcriprion initiation, 274-275,274f Nuclearbasket,570, 570f Nuclearbodies,364-356, 365f Nuclearcap-bindingcomplex,337 Nucleardomains,364-366, 355f Nuclearenvelope,342, 373f, 569-570 definitionof, 864 disassembly oI, 865-867, 866f microscopicappearance of, 390f reassemblyof, 870-87l, 872f Nuclearexport factor 1 (NXFl), 342-343, 343f,573

Nuclear-exportreceptor,573 Nuclearexport transporter1 (Nxtl), 342-343, 3431,573,574f Nuclear-importreceptor,571 Nuclearlamina,570, 5701,865,866f definitionof, 865 disassembly of, 864f, 865 reassembly of, 871 Nuclear-localization signal,57L, 57Lf, 670 Nuclearmembrane,342-343, 3421,343f, 4141,4L5 NuclearmRNPs,325, 341,-342 transportof, 342-347, 343f-346f Nuclearpore complexes,342-343, 3421, 343f, 363, 390f,464, 570, 570f assemblyof,871.,872f microscopicappearance of, 390f Nuclearpores,342, 378,570,570f Nuclearreceptor(s), 291.,31.2-31.3, 31.2f, 31.3f,975 activationdomainsof, 293, 2931 heterodimeric, 313 homodimeric,313, 31.4f repressiondomainsof , 294 Nuclearspeckles, 355 Nucleartransfercloning,908 Nucleartransport,342-347, 367 of Balbianiring mRNPs,344-345 cargoproteinsin, 571-574,572f, 574f constitutivetransportelementin, 346-347 directionof, 343-344,572 exportinsin, 573-574, 574f of hnRNPs,345,345f,573 importinsin, 5a l-572, 5a2f of mRNA, 342-347, 343f-346f mRNP exportercin, 342-343,343f, 573-575,574f mRNP remodelingin, 343 nuclear-export signalsin, 573 nuclear-localization signalsin, 571--572, 572f nuclearpore complexin, 342-343,342f, 343f,363, 3901,464, 570,57of assemblyolr6/lr6/21 microscopicappearanceof, 390f nucleoporinsin, 342-343,343f, 570, 572, 572f RNA export factor in, 342-343,343f SR proteinsin,343-344, 344f of viral mRNA,346-347, 346f in yeast,343-344,344f 719, Nucleation,in actin filamentassembly, 71.9f Nucleicacid hybridization,LL, 1,1'7 Seealso Nucleicacids,71-1,2,40, 1.1.L-11.2. DNA; RNA basesin, 11,,44, 44f, 45t. Seea/so Base(s); Basepairs/pairing definitionof,1.1,40 directionalityol, 114, 11.4f functionsof, 1'l.l-1.L2,L73f overviewof,111-L12 phosphodiester bondsin, 40, 41.f,114, 1.1.4f as polymers, 40, 113-1,1,4 purine,44, 44f, 45t, 113 pyrimidine,44, 44f, 45t,'1.L4 structureoI, 40, 41f, 44, 441,45t, . Seealso Helix, 1.1.3-11.9, 1.1.4f-11.9f DNA synthesisof, 44 154-155, 155f Nucleocapsids, Nucleolar organizer,pre-rRNA as, 359 Nucleolus, 373f,378 pre-rRNA processing in, 359 Nucleoplasm,378

Nucleoporins,342-343, 3421,343f, 378, 570, 570f,572, 572f FG,342, 342f,572 MPF phosphorylationof, 866 phosphorylationof, in mitosis,866, 866f, 871, Nucleosides, 44, 45t Nucleosome,248-249, Z48f definitionof,248,299 structureof, 248-249, 248f Nucleotide(s), 11, 44,44f, ll3. Seea/soBase(s) as monomers,40,47f phosphodiesterbonds of, 40, 41.f,11.4,71.4f phosphorylated, 44 purine,44,44f pyrimidine,44,44f structureo1,44, 44f. terminologyof, 45t Nucleotideexcisionrepau,748-149, 148f, 149f Nucleotidesalvagepathways,402, 403t Nucleus,373f,378 protein targetingto, 557t structureof,569-570 NuMA protein,933 Nurse cells,953 Niisslein-Volhard, Christiane,171, 999-1000 NXF1, 342-343,343f O bloodgroupantigen,426,426f,427t O-linked oligosaccharides, 550, 827, 828f.See a/soOligosaccharides Objectivelens,381 Obligateaerobes,485 Occludin,815,841f Oct4,960 Octylglucoside, 428, 428f odd-paired,1.000 odd-skipped,1.000 Odorant receptors,1,036-1,039, 1039f Oil drop model,68, 68f Okazakifragments, 1.41,1.41L143,778 Oleate,48f Olfaction, 1036-7039 primarycilia in, 780 Olfactoryreceptorneurons,1037-1039, 1037f-1039f Oligodendrocyres, 1014,1015f Oligonucleotide probes,in DNA library screening,181-182 Oligopeptides, 56 Oligosaccharides, 550, 827, 828f in cell-celladhesion,552 N-linked,550, 550f, 827,828f structureof, 550, 550f synthesisand processing of, 550-552, 550f,551f O-linked, 550, 827, 828f structureof, 550 in protein folding and stabilization,552 sidechainsof, 552 structureof, 550, 550f synthesis of, 550, 551f Oligosaccharyl transferase, 550 Oncogenes, 158, 11.07,11L4, 1127-1131 abl, 11.29-1.130, 1.130f bu, 1.1.30 cell survival and,939 definition of,1107 fos, 11,31,L1.32l iun, 1,1,30 myc, 1l15, 1122,113L,1.132f proto-oncogene conversionto, 1107-1108, 7173, 11.19-1121 ras, 1113-L1.14,1114f, 1115f, 11.29

in signaling,11.29-7130,1.130f sis,1,1,27 src, 1.L29,1130f trk, 11.28,L1.28f vir al, 1L21--L123, 11,28-1,1,29 1108 Oncogenesis, Oncogenicmutations. SeeCancer,mutations in Oncogenicreceptors,1,727-11,28 Oncogenicretroviruses,1128-1,1,29 1.11.4f Oncogenictransformation,1.11.3-1114, mechanismsof, 1L19-L12l 1.L32f transcriptionfactorsin, 11.30-1.131, Oncoproteins, 671, 67Lf in oncogenictransformation,L120-1.L21 vftal, 1.1.2'1.-1.1.23, 71.22f,1128-1.129,11.29f Oocyte(s), 950 differentiationoI, 9 1.3-91.4 fertilization of, 8, 8f, 1,9,19f,950,950f, 955-959 acrosomalreactionin, 956, 957 definitionof, 955 gametefusionin, 955-957,956f in vitro, 8 maturationoI, i?tXenopuslaeuis,854-858, 854f-857f mitochondrialDNA from, 957 primary,953 OocyteexpressionassaSfor ion channels, 454, 464f Oogenesis, 9 53-954, 953f, 954( Op18/stathmin,in microtubuledisassembly, 768,768f Open readingframes(ORFs),232-234, Z33f definition of,244 in geneidentification, 244-245 in transposition,232-234, 233f Operons bacterial,transcriptionunits in, 217 definitionof, 122 lac,271-273,272f transcriptionof, 1.22-123,l24f trp, 124f,21.7 Ophthalmoplegia, chronicprogressive, 241 Opsin, 535-635, 541.,643f, 644-645, 645f, 1028 Opsonization,1060 Optic tectum,1042 Optical isomers,33 Optical microscopes, 380f Opticaltraps,735, 737f Oral rehydrationtherapy,471.472 Orexins,550 ORF analysis,244-245,245f. Seealso Open readingframes(ORFs) Organ of Corti, 1032, 1033f,1,034f Organelle(s), 3, 3f, 372-379,373f, 4L0 acidificationof, H* AIPasesin, 453-454 cell fractionationin, 407408 definitionof, 371 evolutionof, 505, 505f,557 membranesof, 409, 470, 410f proteinsof,410 purificationoI, 391-394 antibodiesin,393-394 centrifugationin, 392, 392f, 393f clathrin-coatedvesiclesin, 393-394, 3931 homogenizationin, 391.-392 membranerupture in, 39'L-392 sonication in, 391 sequence homologyin, 557, 557t transportoI, /6y-/ /6 dyneinsin,774-776 Krneslns ln, /6>-/

/1

OrganelleDNA,236-242 chloroplast, 236,242 circularshapeof, 3f, 1.2,1.3,117-1.1.8, 1.1.8f

mitochondrial,236-242. Seealso mtDNA (mitochondrialDNA) Organisms diploid,1.66,171,950 25J7, 26f experimental, haploid, 166, 169-17L, 1.70f geneticanalysisin, 1.69-771., 1.70f relative sizeof,20f Organogenesis, 95I-9 52 Organs definitionof, 801 developmentof, 9 51.-952 in, 801-803,802f. See trssueorganrzatron a/so Cell-celladhesion;Cell-matrix adhesion Origin, replication,141 complex(ORC), 144, 878 Origin-recognition receptors,660 Orphan G protein-coupled 244 Orthologoussequences, Osmosis,444,444f Osmoticpressure,444, 444f 1033f Ossicles,'1.032, Osteogenesisrmperfecta,827 Ouabain,468 OvarS developmentof, 91.3-91.4 Ovum, 19. Seealso Fertilization;Oocyte(s) Oxal protein,in mitochondrialprotein taryeting, 562 Oxidases,375 Oxidation aerobic,479480,480f in, 480, 481f chemiosmosis proton-motiveforce in, 480,481f peroxisomal,375 Oxidation potential,50 Oxidation-reductionreactions,59-60, 59f, 60f 494. Seealso Oxidativephosphorylation, Phosphorylation ATP-ADPexchangein, 509, 509f in ATP synthesis,494-510 in bacteria,504f, 505 in brown-fattissue,510 chemiosmotichypothesisand, 503-505, 504f in chloroplasts,504f, 505 definition of, 494 in electrontransport,494-503 in mitochondria,504f,505, 510 proton-motiveforceand, 503-510 rate of, 510 in thermogenesis, 510 uncouplingof, 510 Oxidativestress in chloroplasts,52L-522 in mitochondria,502-503 Oxygen cytochromec oxidasereductionof, in electrontransport,495t, 498499, 5011 hemoglobinbinding of, 89, 89f product,480, 511, as photosynthesis 519-521..Seealso Photosynthesis triplet, 521 Oxygendeprivation,glucosemetabolismand, 484f,485 Oxyntic cells,472, 4721 P-450enzymes, 1139 P bodies,348,353 P-cadherins, 81.0.Seea/so Cadherins P-classion pumps, 447-448, 447f, 460. See a/soPumps Ca*, 450452,450f,45Lf catalyticsubunitsof, 450451., 45'l.f

INDEX .

I-35

P-classion,pumps,(continuedl Na-'K-, 449,452453 P elements insertionmutationsand, 189-1,90 in transposition,229 P-nucleotides, in antibodydiversity,1071 P segment,101.0,1.01.1f,1012f P-selectin, 838 P site,of ribosome,133,134,1,34f,135-136 p-values,243 P1 (inorganicphosphate) from ATP hydrolysis, 57,57f in ATP synthesis, 59 p16, in cancer,1 134-1135 p 2 1 t ' 0 ,8 8 3 - 8 8 4 8. 8 6 r .8 9 I i 2 7 * t o t , 8 8 3 - 8 8 48, 8 6 i i27*t",88J-884,886t p38 kinases,693 p53 in apoptosis,891 in cancer,1,11,5, 11,36-L1,37, 11.38 of lung, 1.140-1141., 1140f in cell-cycleregulation,891 HPV infection and, 11,37 p75**, in apoptosis,942-943 PABPI,343, 343f, 351, 351f PABPII,335, 336f, 343, 343f Pain perception,1,031-1032 Pain receptors,1031-1,032 Pain-sensing neurons,938 Pair-rulegenes in body segmentation, 975f, 976-9-77,976f discovery of, 999-1,000 paired,in body segmentation, 977 Palade,George,582, 621-622 Palindromicsequences,'1,7 6, 176f Palmitate,48f Pancreas, insulin secretionfrom, 17,77f, 1004-1005 Pap smear,159 Papillae,tastebudsin, 1034, 1035f Par complex,in neuroblast division,933,933f pathway,in transepithelial Paracellular transpoft,815-815,816f Paracrinesignaling,625f, 626 Parietalcells,472, 472f Parkinson'sdisease,77 Passivediffusion,438439 Patch-clampexperiments, ion channelsin, 463464, 463f,464f patched,702 Pathologoussequences, 244 Patternformation,951. Seealso Development anterlor-postenor in Drosophila melanogaster,971,-974, 973f,974f in Xenopuslaeuis,965-9 66, 966f in axon extension,1046-1,047 body segmentation in,969, 974-983 dorsal-ventral in Drosophila melanogaster,970-97 1,, 977f,972f in Xenopuslaeuis,963-965, 964f in embryonicperiod in D r osophila melanogaster,97 0-977 in Xenopuslaeuis,963-969 Hox genesin,979-983 left-right axis in, 966-969, 967f in limb development,990-994, 991,f-994f somitesin, 977-978, 978f, 9791 translationinhibitors in, 973-974 Pattern-formationgenes,999 -1,000 Pax3,928 Pax6,29f, 269, 277, 277f, 284 PCZPC3 endoproteases, 504 PCNA (proliferatingcell nuclearantigen), l-36

.

INDEX

142f, L43 PDGF receptor,in cance11.1.27 PDKI/PDK2, 595-696 Pectin,839f, 840, 842, 842f PEK, in translation,356 Pemphigus vulgaris,813-814 Pentoses, 44-45 Peptidebonds,37, 38f, 40, 41.1,65, 65f, 66 protein folding and, 74, 74f Peptidemassfingerprint,103 Peptides,65. Seealso Protein(s) Peptidyl-prolylisomerases, 553-554 PeptidylIRNA, 137-138, 73719 Peptidyltransferase reaction,L36, 136f Perforins,in T-cellapoptosis,1.0601,1095 Pericentriolar material,7 61, 7 67f Peripheralmembraneproteins,422. Seealso Membraneproteins purificationof, 428 Perlecan,805t, 805 in basallamina,821, 821.f,824 Permeases, bacterial,in membranetransport, 454,454f Peroxisomal-targeting sequences, 567-568,567f Peroxisomes, 15, 373f,374-375, 37 5f, 557 discoveryof, 407408, 408f fatty acid oxidation in, 491492,492f protein targetingro, 534f, 557r, 567-569, 567t-569f synthesis o1,569,559f in Zellweggersyndrome,568 Pertussis, 540 Perutz,Max, 103 P e t a l s9, 8 3 , 9 8 4 f petite yeastmutants, cytoplasmicinheritance of, 237, 238f Pexproteins,569,569f pH,5l-54,52f buffersand, 52-54,53f cytosolic,448 regulationof, 468 definitionof, 51 dissociationconstantand, 52, 53f enzymaticactivity and, 84, 84f regulationof, 52-53, 53f pH gradient.SeeProton-motiveforce Phage(s), 154 temperate,lysogenyin, 158 PhageT4, 155f lytic replicationcyclein, 156-1.57,156f Phagocytes, 1059 in opsonization,1060 Phagocytosis, 374, 374f, 606 in antigenpresentation,1084-1085 Phagosomes, lysosomesand,61.2 Phalloidin,728 Phase-contrast microscopy,380f, 381-382 Phenotype definition of,22,166 vs. genotype,156 Phenylalanine, 42,42f. Seealso Amino acid(s) (PKU),199t Phenylketonuria Pheromones, 623, 923-924, 924f Philadelphiachromosome,259, 1130 PhoR/PhoBtwo-componentregulatorysystem, )7\

)7\F

Phosphatases, 91,91f in signaling,634 Phosphate, inorganic from ATP hydrolysis,58 in AIP synthesis, 59 Phosphatetransporter,in ATP-ADPexchange, 509,509f Phosphatidylch oline,47f , 48 membranecontentof, 418t Phosphatidylethanolamine, membranecontent

of, 418t Phosphatidylinositol, 412f, 420 in GPI anchors425f.426 in signaling,654-657, 654f, 655f Phosphatidylinositol 3-phosphates in signaling,695-696, 695f-697f synthesisof, 695, 695f. 420 Phosphatidylserine, membranecontentof, 418t Phosphoanhydride bonds,57-58, 58f 1.1.4f Phosphodiester bonds,40, 41f, 1.1.4, in carbon Phosphoenolpyruvate carboxylase, fixation.528f.529 Phosphofructokinase-1, in glycolysis,483, 483f,485 in glycolysis,483, Phosphofructokinase-2, 483f Phosphoglyceri des,47f, 4849, 49t, 41.1,41.2f, 415416. Seealso Membranelipids in signaling, 694-597, Phosphoinositides, 595f-697f in cell migration, 7 50-7 5'1. Phospholipase C, in signaling,639,640t, 653-657, 567f, 694 Phospholipases , 420, 420f, 427, 427f Phospholipid(s) , 1.4,14f, 4lI, 4649, 48. See a/soLipid(s);Membranelipids annular,424,424f enzymebinding to, 427, 427f fatty acidsin, 4749, 47t hydrolysisof, 420, 4201 hydrophilic ends of, 14, 1.4f, 41.f,48, 41.1., 4121 hydrophobicendsof, 1.4,74f, 41f, 48, 41.1, 412f movementoI, 416418, 431432. Seealso Lipid transport srrucrureof,14, l4f, 41.f,47,471 of,431,43Lf synthesis reticulum,375,375f, 376 in endoplasmic 413f Phospholipidbilayer,14, 41.1.41.5, chemistryof, 414 curvature oI, 419, 41.9f cytosolic iace of, 41,4,4141,41,5 exoplasmiclace of,41441.5,41.4f, 543, 543f fluidity of, 4161,41.7,41.841.9,4L9f formation oI. 41141.4. 41.3f gel form o1,41.5f,41.7 hydrophilicface of, 1,4,1.41,41f, 48, 411.41.4,41.3f hydrophobiccore of, 14, 1.4f,411,48, 4 r 1 4 1 4 - 4 1 2 f .4 l 3 f leafletsof, 411.,4l3f lipid/protein movementin, 41,6-418,41,6f, 417f,420,431.432 permeabilityof, 437, 438f protein anchoringto, 422, 424426, 425f protein flip-floppingin, 420, 431,-432, 456,456f sizeof, 411. structure of, 10f, 1.4,74f, 41.f,41.L4'1.4, 413f,41.4f in viral envelope,154-155 viscosityof, 417 phosphatase, 649, 649f Phosphoprotein inorganic Phosphorus. Seealso Phosphate, in ATP synthesis,494 Phosphorylation, 91.,91.f of amino acids,43 dephosphorylationand, 91.,91.f of histones,250, 250f, 251 in mitosisentry,861-863 of myosin, in muscle contraction,T43 of opsin,644-645.645f oxidative.SeeOxidativephosphorylation protein kinasesin, 91.,91.f

PSII,512, 51,5f,519-523, 520f-522f 657-652,651'f in receptordesensitization, in purplebacteria,517-519,517f,518f in signaling,634, 644-645, 657 reactioncenterin, 513f,514-515,515f' 481.-482,482f substrate-level, 5761,51,9,520-521,520f, 52Lf 355-355 in translationrepression, 791 Phragmoplast, phosphatases, in cytokine Phosphotyrosine Phylogenetictree, 245f 678,678f signaling, Physicalmapping,202,202t. Seealso transport,513f-515f,514-515, Photoelectron Mapping 51.7-520,5r8t, s20f, 52rf PI-3 kinasepathway,667t, 694-697, 69 51, 517f,518f in bacteria,51,7-520, 6961 cyclicvs. linear flow in, 519-520, 520f, in cell migration,7 50-751 52rf, 522-523,523f, 524f 518 absorptionspectroscopy, Picosecond Photoinhibition,52L-522,522f Pigmentgranules,transportof, 796-797 Photons,513-514 469, 469f Pigmentation, 1027f Photoreceptors, retinal,rhodopsinand, 641,-645,642f-645f Pinocytosis,505 Pinsprotein,935 retinotectalmapsand, 1042,10421 Piwi proteins,914 527-529,527f, 528f Photorespiration, Plakins,796 479, 480, 480f, 511-529 Photosynthesis, Plant(s) a n t e n n ai ns , 5 1 4 ,5 1 5 - 5 1 65, l 6 f adhesiveproteins in, 841'-842, 842f ATP synthesisin, 9, 15, 59, 511,,51,2, -ATPsynthesisin, 511-529. Seealso 5 r 3f, 520-521,523f Photosynthesis in bacteria, 511, 516f, 517-579,51'7f,51'8f cell-celladhesionin, 841-842, 842f Calvin cyclein, 525-527, 525f-527f celljunctionsin, 811t carbonfixationin, 59, 512-513,513f, cell structurein, 2f s24-529,525f-528f cell wall of, 839-842, 839f carotenoidsin, 514, 514f chloroplastDNA in, 242 in. 5 l4-5 15.5 t5f, chargesepararion dermaltissuein, 839, 839f 5r7-5r8, 577f evolutionof, 242 chemicalreactionsin, 511 flowersof, 839 See chlorophyllin, 514-516, 514f-51.61. of, 983-984, 984f development a/so Chlorophyll groundtissuein, 839, 839f in chloroplasts,379 growth of, auxinsin, 840 in, 512-513,5r3f dark reactions leaf structurein, 51.2f Emersoneffectin, 519 leavesof, 839 in, 514,519 energyconsumption membranetransportin, 469470, 470f future researchareasfo4 529 meristemsof, 840, 842, 920, 921'f,983 herbicideeffectson, 521 mitochondrialDNA in, 239, 240, 241,t light absorptionin, 511-512,513f mitosisin, 790-791, 790f complexesin, 513f, 514, light-harvesting in, 51'7-529.Seealso photosynthesis 515-516, 51.6f,523, 524f Photosynthesis light reactions in, 512-513,513f plasmodesmata in, 840-841,841f overviewof, 511 proteintranslocationin, 557-558, 557t, oxygen-evolvingcomplex in, 520-521.,521f 5 6 5 ,5 6 6 f 5I 1 oxygen-generating, roots of, 839 photoelectrontransportin, 51,2,513f-515f, signalingin, 840-841,841f 514-515, 51.7-520,5181,520f, 521.f tissuein, 839,839f sporogenous in bacteria,517-520, 51.7f,51.8f stemcellsin, 920,921'f cyclicvs. linear flow in,51.9-520,520f, stemsof, 839 521.f,522-523, 523f, 524f in. tissuestrucfureand organization photosystems in, 51.9-523.Seealso 839-842 Photosystems transgenic,470 primaryelectronacceptor(Q) in, 512, 513f turgorin, 377-378,444 productsof, 511, 511f,525,526f vacuolesin, 377-378,377( proteintargetingin, 565,566f vasculartissuein, 839,839f proton-motive forcein, 480, 481f, 511, Plantcell(s) 512, 5t3f elongationof, 378 in bacteria,51,7-520,517f, 518f growth of, 840 Q cyclein, 518-519,520,521f,522-523, propertiesof, 839-842, 839f 523f Plant cell wall rute of, 514f, 519 cell growth and, 840 reactioncentersin, 513f,514-515,515f, componentsof, 839f, 840 51.6t,s20-s21.,52rf functionsof, 840 resonance energytransferin, 515-516, 516f plasmodesmata in, 840-841',841'f stagesof, 511-513,513f strengthof, 840 in thylakoid membrane,51L, 51,2f structureof, 839f, 840 Photosystems, 51,4,51,9-523 turgor pressureand, 444, 444f a n t e n n a sI n , ) l + , J I J - ) I C r ,J I O r Plant tissue i n b a c t e r i a ,5 1 6 f dermal,839, 839f coordinated activity of, 523,524f organizationoI, 839-842 cyclic electron flow in, 522-523,523f structureof, 839, 839f definition of, 514 Plant viruses,154 discovery of, 519 Plaqueassay,155, 155f light-harvesting complexes in, 513f, 514, Plasmacells,3f s76f,s23, 524t 515-516, 1.075 in immuneresponse,1,061,1.061.f, PSI,512,515f,519-520, 520f,522-523, Plasmamembrane.SeeMembrane(s),plasma 523f

4lzf, 416, 433 Plasmalogens, Plasmidvectors,L78-179,178f, l79f expression,1'94-196,195-196, 195f in protein production,1'95-1'96,1'95f 16, 350, 811t' 840-841, Plasmodesmata, 84Lf Plasmodium life cycleof, 5, 5f mitochondrialDNA in, 238,238f 318 Plasticpolymerase, translocationof, 565 Plastocyanin, Plastoqinones Platelet-activatingfactor, 837 growth factors,in cell Platelet-derived m\gration,746 Platelets,thymosin-Bain, 722, 722f Pleatedsheet,67,671 Plectin, 796, 796f Plexins,1045 Pluripotentstemcells,907,960 +TlPs, 767, 768f PML nuclearbodies,356 833 Podosomes, Point mutations,1,46,1.461,1.67 identificationof, disease-causing, 202-203 1120 in proto-oncogenes, Pol E, in DNA replication,1'42f,743,744f Pol I. SeeRNA polymeraseI II Pol II. SeeRNA polymerase Po[ II preinitiationcomplex,296J99,297f, 298f of, 307-308,308f assembly III Pol III. SeeRNA polymerase Polar bonds,34 solubility and,37 Polar microtubules,7 84, 7 84f Polarity of actin filaments,71,8,718f 977,999-1000. See in body segmentation, aiso Body segmentation in cell migration,746-747 of cells,8, 471,,714 cytoskeletonand,7\4-7 1'5,714f of embryo,950-951 of molecules,solubilityand, 39 759, 760f of protofilamenrs. Pollen,adhesionof, 842, 842( Poly(A)-bindingprotein I (PABPI),343' 351 336,336f' Poly(A)bindingproteinII (PABPII), 343,343f Poly(A)polymerase,1.24,335, 336f Poly(A)sites,217 Poly(A)rail, 124 lengtheningof, 351-352, 351f processing of, post-transcriptional 335-336,336f shorteningof, in mRNA degradation, 352-353,353f Polyadenylation in antibodyproduction,1074-1075 35l-352. 351f cytoplasmic. 324-325, 329, in pre-mRNAprocessing, 3291,335-336,336f PolycistronicmRNA, 217 Polyclonalantibodies,401 Polycombcomplex,254 in transcriptionrepression,302-303,302f, 306 Polycombgenes,982-983 780 Polycystickidney disease, 992,993f Polydactyly, Polygenetictrairs, 203-204 Polygenictraits,204 Polylinker,178f, 1.79 Polymer(s),10, 40, 41'f, 1'13 INDEX

.

I-37

Polymer(s)(continuedl nucleicacidsas,40, 113-114 Polymerase chainreaction(PCR),188-190, 188f,189f rn genetagging,189-1,90 rn genomicDNA isolation,188-189,189f in probepreparation,189 procedure for, 188, 189f reversetranscrlptase, 189 Polymorphisms. SeeDNA polymorphisms Polypeptides, 65f, 65 Polyps,colonic,1116 Polyribosomes, 138, 139f Polysaccharides, 10, 46 N-linked, 827 O-linked,827 strucrureof, 40, 4lf, 46, 461 Polysialicactd,837 Polysomes, 1,38,1,39f Polyspermy, preventionof, 957 Polytenechromosomes, 250-26I, 267f Polytenization, 251, 261f P o l y u b i q u i r i n a r iS on e .eU b i q u i r i n a r i o n Polyunsaturated famyacids,4748, 47t Porins in bacteria, 424,425f in chloroplasts, 511 in mitochondria,486487 Post-transcriptional genecontrol, 323-367. See a/so RNA processing cytoplasmicmechanisms in, 347-358 cltoplasmicpolyadenylation in, 351-352,35lf future researchareasfor in,366-357 heterogeneous RNPsin, 327,329f at individualsynapses, 325 miRNA in, 325 miRNA-mediatedtranslationrepressionin, 347-349,348f mRNA transportrn, 341,-347 nuclearbodiesin, 364-356 overviewof, 323-325, 3241 pre-mRNAprocessingin, 325-341.See a/soPre-mRNAprocessrng pre-rRNA processing in, 358-363 pre-tRNA processingin, 363-364 primary transcriprin, 323, 324f processes tn,326f RNA-bindingmotifsin, 327,3281 RNA interference in, 349-351 rRNA processing in, 358-364,3S9f-362f siRNA in, 325 IRNA processing in, 353, 3631,364f Post-translational translocation,540-541,S42f Postmitoticcells,264, 849 Postsynaptic cells,1005,1018,1019 Postsynaptic density,1019 Potassiumion channels.SaeIon channels, potassrum Potassiumions, in cytosolvs. blood, 448,448t Potentialenergy,54-55 chemical,54 rormsoI, )u) Potentiometer, 450, 450f PPZA,869,870f PRC1 complex,302, 302f PRC1/PRC2, 912 PRC2complex,302,302f Pre-Bcells,1073 pre-miRNA,348,349f Pre-mRNA,1L2f, 123-124 definitionof, 326 exosomedegradationof, 336-337 5 ' c a po f , 3 2 4 f , 3 2 5 - 3 2 6 , 3 2 7 f in RNA processing,113f,123-124 Pre-mRNAprocessing,324f, 325-337, 325-347 carboxyl-terminal domain in, 280, 32G l-38

o

TNDEX

c h a i n e l o n g a t i o ni n , 3 3 3 , 3 3 3 f cleavage and polyadenylation in, 324-325,

329,3291,335-336, 3361 co-transcriprional, 325 exon-junctioncomplexin, 332-333 exon recognitionin, 333-334, 333f exonucleases in, 3J6-337 5' cappingtn, 324f, 325-336,325f, 327f, 337 hnRNPsin, 327,328f regulationof,337-347 RNA-bindingmorifs in, 327,328t RNA editingin, 340-341,347f RNA export factor in, 332-333 splicingin. 329-3JJ.SeealsoSplicing stepsin, 326f Pre-mRNPs,325 Pre-RNPs,360 Pre-rRNA as nucleolar organizer,359 processing of, 358-364, 359f-352f structureof, 359, 359f. synthesis of,278,359. Seealso Transcription terminationof, 314-315 Pre-rRNAtranscriptionunit, 359 Pre-tRNA,processingof, 363-364, 364f, 3651 Pre-B-cellreceptor,1073-1074,'1,074f Precursorcells,905 PrecursorrRNA. SeePre-rRNA Pregnancy maternal-fetal antibodiesin, 1065-1,066 MHC in, 1078 from in vitro fertilization,8 Preinrtiationcomplexes,134, 135f,297 Pol II,297-299, 297f, 298f assembly of, 307-308,308f,309 Prenylation,of membraneproteins,425,425f Preprophase band, 790-791, 790f Prereplication complexes,878-879, 87Sf Presenilin1.,705-706, 705f, 707 Pressure, perceptionof, 1031-1,032 Presynaptic neurons,1004f,1005,1018,1019 Pre-T-cellreceptor,1091 Primarycells cultureof, 396-397 definitionof, 394 Primary cllfum, 702, 780f Primaryelectronacceptor(Q), in photosynthesis, 51,2,5l3f Primarylysosomes, 374, 374f Primaryoocytes,953 Primaryspermatocytes, 954 Primarystrucrure, of proteins,65-66,65f Primary transcript, 127-722, 1,22f,323 Primase,141, 1.43 Primates,evolutionof, 259, 260f Primers,replication,147, 1,43 Primitivestreak,962 Primordialgerm cells,953 Probes antibodS21 in chromosomepainting,259 definitionof, 181 in DNA library screening, 1,81,-1,82 preparationof, polymerasechain reaction in, 189 Processed pseudogenes. 234 Processomer 36l Procollagen, 825f,826 Proenzymes, 603 Professional antigen-presenting cells,1080 -21, -22f, -45, l)rofilin, 747f Progenitorcells,905 Progenyviral genomes,158 Progenyvirions, .156

b u d d i n go 1 , 1 . 5 6 , 1 5 81,5 8 f ,1 5 9 f Progeriasyndrome,795 Progesteronereceptor,31,2f.Seea/so Hormone receptors Programmedcell death. SeeApoptosis Prohormones, 91 Proinsulin, proteolytic processingof , 60 3-604, 603f, 604f Projectionlens,381 Projectionneurons,1037 Prokaryotes,2-3. Seealso Bacteria; Cyanobacteria;Eschericbia coli abundanceof, 2 cell structurein,2-3, 3f circularDNA in, 3f, 12 cytoplasmicstreamingin, 744, 744f definitionof,2 evolutionr4, 4f geneexpressionin, 122-123 geneorganizationin, 1,22-1,23,2'17 plasmamembranein,2, 3f, 409 protein-codinggenesin, 122-123 reproduction in,7,7f ribosomesin, 1321,1.37f transcriptionin, 122-1,23,'1,23f translationin, 123 Prolactin, 672 Proliferatingcell nuclearantigen(PCNA), 142f,143 Proline,42f, 43. Seealso Amino acid(s) in collagentriple helix, 822,822f Prometaphase, 782f, 783, 786mi-788,787f Promoter(s). SeealsoActivator(s) definition of, 1.94,275 in geneinactivation,206 initiator,282 t4c,/,/ I mitochondrial,318 preinitiationcomplexeson, 297-299, 297f, 298f in protein production,195,'1.95f splicing,339 in transcription,1.20,270, 273t in bacteria,270, 273J7 5, 273t in eukaryotes,275 strong,272 weak,272 in translation,351-352,351f, 355-356 Promoter-fusion, 198 Promoter-proximal elements,282-283,283f, 285,286f in heat-shockgenetranscription,316 Promyelocyric leukemianuclearbodies,366 Proofreading,1.33,134, 1,45,1461,355 Prophase,782, 7821,783 chromosomestructurein, 256, 2561 in meiosisvs. mitosis,892,894f in plants,790,790f Proprioception,103l-1,032 Proproteins,proteolyticprocessing of, 603-504,603f, 604f Prostheticgroups,84 Proteases, 70 complementas, 1053 in inflammatoryresponse,1061,-7062 Proteasome(s), 87-88, 87f in JAK/STAT pathway,678-679 in mitosis,858 Proteasome inhibitors,87-88 Protein(s),63-1.09.Seealso Gene(sland specificproteins adapter.See Adapterprotein(s) allosteric,89 alpha helix of, 66-67, 661 anchoring, 652,652f B sheetin, 67,67f

B turn in,67,671 C-terminusof, 65, 65f catalytic. See Catalysis; Enzyme(s) cellular amounts of, 11 chaperone. Saa Chaperones chimeric, definition of, 371 coiled coils in, 67, 69,70f conformation of,22, 63, 67-70. See also Protein folding n trve-state,74 X - r a y c r y s t a l l o g r a p h yo f , 1 0 3 - 1 0 4 conservation of, 29 definition of, 55 d e g r a d a t i o no f , 8 6 - 8 8 d e n a t u r a t i o no f , 7 5 dominant-active, 747 dominant-negatwe, 747 engineered,21 evolution of , 72-73, 73f , 244 families of, 10 fibrous,5S folding of. See Protein folding functions of, 10, 53-64,78-85 structural correlates of, 64-65, 64f,74 generically engineered, 194-1.95 in genome, 64 global analysis of, 23, 705-107 globular, 58 glycosylation of, 425 h o m o l o g o u s ,72 - 7 3 , 73 f , 2 4 4 h y d r o g e nb o n d s i n , 6 6 , 6 6 f , 6 7 , 6 7 f hydrophobic interactions in, 68 identification of. See Protein purification/identification integral. See Membrane proreins, integral (transmembrane) i n t e r s p e c i e s i m i l a r i t i e si n , 2 9 , 2 9 f isoformsof, 808 alternative splicing and, 726, 125f, 338 production of, 125-1.26, 126f, 338 Iocalization of b y m R N A l o c a l i z a t i o n ,3 5 2 , 3 5 7 - 3 5 8 , 358f by transport mechanisms See Protein targeting; Protein translocation i n m a c r o m o l e c u l a ra s s e m b l i e s7, 2 , 7 2 f mass of, 66 m e a s u r e m e n to f , 1 0 1 - 1 0 3 , 1 0 1 f , 7 0 2 f matrix, 16, 76f m e m b r a n e .S e e M e m b r a n e p r o t e i n s membrane transport. See Membrane transport proterns m i s f o l d e d ,7 7 , 7 8 f , 8 6 - 8 7 , 5 5 5 , 5 5 5 f . S e e a/so Protein folding i n e m p h y : e m a ,5 5 5 - 5 5 6 quality control in, 553, 556 mitochondrial, 238, 239f, 486 models of, 68, 69f molecular weight of, 66 motor. See Motor proteins multidomain, 125-126, 7261 multimeric, 72 N-terminus of, 65, 65f nascent, translocation of, 534, 536-537, )-t /l

numbersof, genomiccomplexityand,246, 245f orthologous,244 overviewof, functionsof, 63-64, 64f paralogous,244 as polymers,40 productionof, 15 proteolyticprocessing of. 9l-92. 603-604, 603f, 604f in proteome,64 purificationof. SeeProtein

purification/identification query,243 regulatory,63 scaffold,63 in chromatin,254-255, 254f in MAP kinasepathwag692-693,6921 in skeletalmuscle,740, 740f of, J-5-3-6. 3'6f secretion constitutive,602 regulated,602-603 535-556. Seealso Secretory secretory, protelns self-splicingof,97-92. Seealso Splicing shapesof,22, 63 77, 171.Seealso Cell signal-transduction, signaling sizeof, 10, 10f, 66 structural,53 structuralmotifs of. SeeMotifs s t r u c t u roef , 1 0 , 1 0 f ,2 2 , 6 3 , 6 4 - 7 4 , 6 4 f -5f. foldingand, 74--5. Seealso Protein folding functionalcorrelatesof, 64-65, 64f,74 irregular,66 levelsof, 64-65,65f primar5 65-66, 65f -2, 7 quaternary. 5. 5( 65f. 7 lf, representations of, 58, 69f 6 5f, 66-70, 66f-70f, 68-70, secondary, 75,75f tertiary,65f, 67-72, 68f, 69f,71.f,75, /JI

X-ray crystallography of, 103-104 switch, 90, 90f,91.f, 138, 587 in mitotic exit network, 890, 890f R a s a s , 6 8 4 , 6 8 5 . S e ea / s o R a s p r o t e i n in signaling, 633-634, 633f i n v e s i c u l a rt r a n : p o r t . 5 8 7 - 5 9 0 . 5 9 0 f synthesis of,74. See a/so Translation regulation of, 86 r i b o s o m e si n , 1 3 3 - 1 3 9 , 1 3 5 f , 1 . 3 6 f , 5 3 4 f transfer,433 transport of. See Transport viral, 6 water-accessiblesurfaces of, 68, 69f Protein aggregations, in budding vesicles,602 Protein binding, 64, 78-84. See also Receptorligand binding affinity in, 39-40,78, 92 allosteric effectors in, 89-90, 89f,90f antigen-antibody, 7 8-7 9 , 79f c o m p l e m e n t a r i t y a n d , 3 9 4 0 , 3 9 f, 7 8 enzyme-substrate,80-84. Seealso Enzyme(s) sites of, 78 specificity of, 40, 7 8-79, 79f Protein cleavage in lipid regulation, 705-707, 705f i n N F - r c Bp a t h w a y , 7 0 5 - 7 0 7 , 7 0 5 f P r o t e i n - c o d i n gg e n e s ,1 2 0 , 2 1 9 - 2 2 1 , 2 2 0 t in eukaryotes, 123-124 organization of , 122-124 in prokaryotes, 122-1,23 solitarS 279-220 Protein disulfide isomerase (PDI), 552, 553f Protein domains, T0-72 alternative splicing and, 125-1,26, 126f functional, 70 modular natve of, 71, 77f repeated, 1,25 -1,26, 1,26f structural, 70-7 1,, 7 lf ropological, 72 vs. motifs, 69 Protein expression profiling, 105 Protein factories, 194-196, 79 5f. Protein families, 220 evolution of ,244

homologyin, 244 sequence Proteinfilaments,16, 1.6f Proteinfolding,64,64f, 68-70,68f,701, 74-78 in, 75-77. 76f. 541,542f, chaperones 552-553. Seealso Chaperones rn, / /, / /l cnapefonrn translocationand, 539f cotranslational in endoplasmicreticulum,534, 539f errorsin, 77, 78f, 86-87, 555, 555f in emphysema, 555-556 quality control for, 553, 556 in hemagglutinin, 554-555, 554f limitationson, 74-75 74 native-state, in, 552 oligosaccharides peptidebonds and, 74, 74f quality control in, 553, 555 refolding and,75 regulationof,74-75 responseand, 555, 555f unfolded-protein unfolding and, 75 Protein-fusion,198, 198f SeeProrein Proteinidentification. icatron purification/identif Proteinkinase(s) activationlip o(, 6-4, 675f cascadeof, 684, 588-690, 689f in cell cycleregulation,848 in phosphorylation,91, 91,f in signaling,634. 652. 652f 841-842 wall-associated, ProteinkinaseA, 698. 698f functionsof, 698 698 in glycogenolysis, in signalng, 667f ProteinkinaseB, 659 in PI-3kinasepathway,695-696,696f ProteinkinaseC activationof, 696, 696f anchoringproteinsfor, 652, 652f in cell survival,696 diacylglycerolactivationof, 656 functionsof, 656, 696-697 in glycogenolysis, 648-650,649f in GPCfucAMPpathway,647-649,647f srructureof, 647,647f ProteinkinaseG, 656-657, 657f ProteinkinaseRNA activated(PKR),in translation,356 in signaling,634 Proteinphosphatases, 92-105 Proreinpurification/identification, antibodyassaysin, 98 centrifugationin, 92-94, 93f cryoelectronmicroscopyin, 104 rn, 94-96, 94f, 95f electrophoresis enzymeassaysin, 98 future researchateasfor, 108-109 in, 101-103,101f massspectrometry NMR spectroscopy in, 104 Northern blotting in, 1.92,1.92f peptidemassfingerprintin, 103 radioisotopesin, 99-L00, 99t in situ hybridizationin, L92, 1.93f 791.f Southernblotting in, 1.91.-1.92, 'Western blotting in, 98,99f in, 103-104 X-ray crystallography Proteinregulation allosteryin, 89-90, 89f,90f switchingin, Ca'*/calmodulin-mediated 90,90f covalentmodificationsin, 89 of degradation,86-88 feedback(end-product)inhibition in, 89 GTPasesuperfamilyin, 90, 91,f 92 higher-order, INDEX '

I-39

Protein regulation (continuedl methods of, 86 noncovalent modifications in, 88-90, g9f-9lt phosphorylation/dephosphorylation in, 91, 9lf proteolytic cleavage in, 91-92 reversible vs. nonreversible processesin, 91 switch proteins in, 90, 90f, 91f. See also P r o t e i n ( s ) ,s w i t c h of synthesis, 85 Protein sequencing, 103. See also Amino acid sequences P r o t e i n s o r r i n g . S e eP r o t e i n t a r g e t i n g Protein tagging, 197-198, 1.98f P r o t e i n t a r g e t i n g , 3 5 7 , 5 3 3 - 5 7 6 . S e ea l s o Protein translocation to apical membrane, 604-605,605f to basolateral membrane, 604-605, 605I t o c h l o r o p l a s t s ,5 3 4 f , 5 5 7 - 5 5 8 , 5 5 7 t , 5 6 5 , 566f in class I MHC pathway, 1082-1084, 10 8 3 f d i s l o c a t i o ni n , 5 5 5 in endocytosis, 608-61.0, 609t to endoplasmic rericulum, 534f, 535-556, 5 57t evolunon oI, J),/ t u t u r e r e s e a r c ha r e a s f o r , 5 7 5 - 5 7 6 rn Golgi complex, 376-377 lipid-binding motifs in, 427,427f to lysosomes, 600-602, 500f, 601f, 6 0 8 - 6 L 0 , 6 0 9f , 6 1 2 - 6 1 . 6 m e mb r a n e - b a s e d5, 3 5 - 5 7 6 s t e p si n , 5 3 4 f , 5 3 5 of misfolded proteins, 556 to mitochondria,534f, 55--565, 557t c e l l - t r e ea s s a y sf o r . 5 5 8 , 5 5 8 f c h a p e r o n e si n , 5 5 8 - 5 5 9 , 5 5 9 f , 5 6 0 , 5 6 1 chimeric protein studies of, 560-551, 560f c o n t a c t s i t e si n , 5 5 0 , 5 6 0 f , 5 6 1 energy sources for, 561 g e n e r a li m p o r t p o r e s i n , 5 5 9 , 5 5 9 f , 5 6 1 rmport receptors in, 559-561, 559f to inner membrane, 561-553,5621, 563f t o i n t e r m e m b r a n es p a c e ,5 6 2 f , 5 6 4 ,

s64f to matrix,558-561, 559f,5601, 562f matrix-targeting sequencesin, 558,

5 6 0 - 5 5 1s,6 0 f to outermembrane, 562f,564-565 overviewof, 559f SAM complexin, 564-565 signalingin, 558 Tim proteinsin, 559, 559f,561,-563, 562, 563f Tom proteinsin, 559, 559f, 550f, 561-563,563f transloconsin, 539-540, 540f uptake-targeting sequences in, 557, 5 57t to nucleus, 534f, 557r,569-5?5.Seealso Nucleartransport overviewof, 533-534 pathwaysfor autophagic, 614-616,676f endocytic,579-580, 5811,606-607 nonsecretory, 534[,556-576 secretory, 533-555, 534f, 579-606, 579f. Seea/so Secretoryparhway to peroxisomes, 534f, 557t,567-569, s67f-s69f post-translational, 5 40-541, 542f pulse-chase experimentsfor, 535-536, 536f retrotranslocation in, 556 l-40

.

INDEX

in secretory pathway, 533-541, 534f of secretory proteins, 535-556 s e q u e n c eh o m o l o g y i n , 5 5 7 , 5 5 7 t s i g n a l i n g i n , 5 3 6 - 5 3 9 . 5 5 7 , 5 5 7 r . S e ea l s o Signaling; Sorting signals to thylakoids,565, 565f transcytosis in, 505 vesicular, 535, 589-591, 600-603. See also Sorting signals; Vesicular transport Protein translocation. See aLso Protein targetlng across endoplasmic reticulum, 535-555 of cargo proteins, 512, 572f cotranslational, 537-538, 537f, 539, 539f o f i n t e g r a l m e m b r a n ep r o t e i n s . 544-546, 544f, 545f o I s e c r e t o r yp r o t e l n s ,) J / - ) J U , ) J / f , 539 energy sources fo4 539-541 future research directions ior, 575-576 i n m i t o c h o n d r i a ,5 5 7 - 5 6 5 , 5 5 7 t , 558f-560f i n n u c l e u s ,5 3 4 f , 5 5 7 r , 5 6 9 - 5 7 5 . S e ea l s o Nuclear transport overview of, 534 i n p e r o x i s o m e s ,5 3 4 f , 5 6 7 - 5 6 9 ,

s67f-s69f i n p l a n t s5, 5 7 - 5 5 8 5, 5 7 t , 5 6 5 , 5 6 6 f post-translation al, 5 40-547, 5421 ribosomal,1,35-137,1361 Sec61complexin, 539-540,540f,541.f srgnalrngln, )Jb-)Jv,

JJ/t-JJyt

transloconin, 539-540, 540f in yeast,540-541,542f Proteintyrosinekinases,activationhp in,674, 6 7s f Proteoglycans, 426, 805, 805t,824 in basallamina, 824, 827-833 in cartilage, 830, 830f collagens as,823t,826 diversityof, 829 in extracellularmatrix, 820 glycosaminoglycans in, 827-830, 828f, 829f. Seea/so Glycosaminoglycans (GAGs) membranebindingof, 425 perlecanas, 805t, 806, 82L, 82Lf, 824 structure of, 824, 827-829, 828f, 829f in \7nt signaling,700 Proteolysis, regulatedintramembrane, 555 Proteolyticcleavage/proces sing,9 1,-92 in secretorypathway,603-604, 603f, 604f Proteomes, 23, 64 Proteomics, 23, 705-107 future researchareasfor, 108 Proto-oncogenes, 699, 1107, 1L14, 11.22t conversionto oncogenes, 1,1,1,9-11,21, definition of,1.1.07 gain-of-functionmutations in, 11.19-1.127 proteins encodedby, 11.19,11.20f. Protocadherins, 843 Protofibrils,792, 7921 Protofilaments, 7 58-760, 759f, 760f, 792, 792f Proton,51 Proton-motiveforce,480, 481f, 503-510 in ATP-ADPexchange,509, 509f in bacteria, 505, 517-520 definitionof, 480 functionsof, 505 in mitochondria,502, 561 in photosynthesis, 480, 481.f,511.,51.2, 513f in bacteria,51.7-520,51.7f,51.81 in respiratorycontrol, 510

in thermogenesis, 510 Protonpumps in electrontransport,493494, 494f F-class,447f, 448, 453 stoichiometryof, 499-500, 501f V-class, 4471,448 in synapticvesicletransport, 1,019-1.020 Proton/sucrose antiporter,46947 0, 470f Protozoar4-5, 5f Proviruses,158 Pseudogenes, 220-221, actin, T'1,7 processed, 234 Pseudoknots, 1L8, L1.9f pfcl mutations, in cancer,1L24-1.125 PTEN phosphatase in apoptosis, 1138 in PI-3 signaling,697 in cell migration,750-75 1 PTS1/PTS2 targetingsequences, 567-568, 567f Puffs, chromosomal, 280, 281,f Balbaniring, 345 Pulse-chase experiments,100, 101f, 913 label-retaining cellsin, 913 for receptor-mediated endocytosis,507, 508f for secretoryproteins,535-536, 536f, 582-583 Pumps,438, 439440, 439f, 440,447458. Seealso Membranetransportproteins ABC superfamily,447f , 448, 4544 56 in classI MHC pathway,1084 ATPases and,447,449 calcium calmodulinand, 451452, 451.f muscle,449-451, 450f catalyticsubunitsof, 450451., 451f classificationof, 447448, 447f conformationalchangesin, 440 electrogenic, 453 functionsof,449 hydrogen,447f, 448, 453454, 4531,460 P-classion, 447448, 447f, 450453, 4501, 451f, 460 proton in electron transport, 493494, 494f F-class,447f, 448, 453 stoichiometryof, 499-500, 50lf V-class,447f, 448, 453-454, 453f in synapticvesicletransport, 1,01,9-1,020 449, 4 52453 sodium-potassium, V-classproton, 447f, 448, 453454, 453f 44,44f Purines, Purkinjecell, 2f Putple bacteria,photosystemoI, 51.7-519, 51.7f, 51.8f Pyranoses, 45,45f Pyrimidines,44,44f. Pyrophosphate, inorganic,from ATP hydrolysis,58 Pyruvate glycolytic synthesisoI, 481.483, 4821484f oxidationto CO2, 487489 structureof,482,482f Pyruvatedehydrogenase, 487 Pyruvatekinase,in glycolysis,483,483f Q (primaryelectronacceptor),in photosynthesis , 5L2, 5'1.3f Q cycle in glucosemetabolism,500-502, 501.f,522 in photosynthesis, 518-519, 520, 521f, s))-s)? \?lf Quaternarystructure,65f, 7lf, 72, 75, 75f 243 Query sequence,

cells,781 Quiescent in photosynrhesis, 512, 513f, 515f Quinones, in bacteria,517-51.9,518f, 519f

as switch protein, 685 Rate constant, 50 Rate-zonal centrifugation, 93-94, 93f Rb protein, 882, 882f, 886t,1.1.23-1124,

1.r23f,1.1.35 R bands,258,259f splicingand,339,339f R-Smads, Rbp1,alternative 670-672,670f 513f, Rab proteins Reactioncenter,in photosynthesis, membranebindingof, 425 514-51s,515f, 516f, 51.9,520-521 SeeChemicalreacrions in vesiculartransport,589-591,590f Reactions. Reactive oxygenspecies, damagefrom of neurotransmitters, 1.021-1022 Rab3A, 1021-1022 in chloroplasts,521-522 Rabiesvirus,replicationof,157f in mitochondria,502-503 Rac protein,in cell migration,746-748, Readingframe,L28, l29f 748f-7 51.f Rec8,896-898,896f R a d 5 1 ,1 5 1 ,1 5 2 ,1 5 3 R e c A ,1 5 1 ,1 5 2 , 1 5 3 Radialspokes,in axoneme,777,778f Receptivefields,in vision, 1028-1030,1.029f RadiantenergS54 Receptor(s). Seealso specificreceptors Radiation adhesion, 803, 807, 807f,820 leukemiadue to, 1139-1140 of basallamina,820 integrinsas, 816 mutationsdueto, 146-747 multiadhesive matrix proteinsand, 805, Radicalfringe,706 Radioactivity,measurement of, 100 805t, 821, 822f perlecanand,824 Radioisotope studies,99-100,99t in signaling, 803, 807, 807f, 833-835, Radiolabeling, 99-1.00,991 Raf kinase,in Ras/MAPkinasepathway, 843 6 8 8 - 6 8 9 6, 8 9 f , 6 9 0 type IV collagenand, 821 RAG recombinases, in somaticrecombination, adrenergic, in signaling,636-637,637f, 640t t070f, 1071,7073,1089 bindingaffinityof, 628-629,629f Ran GAP,787 cellularsensitivityand, 631 Ran-GEF, 871 measurement of, 629-630, 630f in mirosis,871 Ran-GTP, cell-surface, 623-624 Ran GTPase activationof, 626-627, 626f in chromosome transport,787,787f affinity chromatographyfor, 631. microtubule-kinetochore interactionand, affinity labelingof, 631 787, 787f of, 666f classes in nucleartransport, 571-572,572f, 574 cloningof, 631.,632 Ran proteins cytokine,672-679 in chromosometransport,787-788, 7 87f functionalexpressionassaysfor, in nucleartransport,571-572,572f, 574 631-632,632f RAP1,in transcriptional 423, 545f , 547, repression, 300-301, G-protein-coupled, 301f SeealsoG 624, 635-657,101.8. Rapamycin,TOR pathwayand, 353-355, 354f protein-coupledreceptors Ras/MAPkinasepathway,567f, 684-694 genomicstudiesof, 631.-632,6321 adapterproteinsin, 685, 686f,687-688, receptortyrosinekinases,679-694 6 88 f recyclingof,610,652 alternativedownstreampathwaysin, 694 seven-spanning, 697-7 02 diversecellularresponses in, 693 structureof,666f in Drosophilamelanogaster, 685 typesof, 524 G protein-coupledreceptorsin, 697-692, desensitization of, 631, 644-645, 651-652, 692f 651.f growth factorsin, 593 hormone,3 I 1-313, 312f, 3L3f kinasecascadein, 684, 588-590,689f in nuclear-receptor superfamily, MAP kinasesin,690-693. Seea/soMAP 317-313, 31,2f , 31,3f kinase(s) response elements and, 313, 313f activationof, 690, 690f intracellular,623 regulatoryactionsof, 690,690f membrane,372 in transcriptioninitiation,691 neurotransmltter Raf kinaseactivationin, 688-689,689f, densityof, 1019 690 423, 624, 63 5-657, G-protein-coupled, Ras activationin, 667f, 684, 685, 686f, 1018. Seea/soG-protein-
in vision, 1027-1031 Receptor-ligand binding, 50-57. Seealso Ligand(s);Proteinbinding;Receptor(s); Signaling activationdomainsand, 293-294 in agonistvs. antagonistdrugs,629 bindingaffinity in, 628-629, 629f cellularsensitivrtyand, 631 of, 629-630, 630f measurement bindingspecificityin, 39-40 in, 631.,644-645, 651-652, desensitization 651.f dissociationconstantfor, 51,628 606 in endocytosis, and, 1005 neurotransmitters in signaling. 624-627,624f binding affinity in, 628-629,629f effectorspecificityin, 628 receptors,627 in G-protein-coupled in growth hormone, 626f molecularcomplementarityin, 627-628 receptoractivationin, 626-627, 526f specificityof, 78-79, 79f, 627-628 sitesof, 51, 51f specificityof, 78-79, 79f, 627-628 in T cells,1090-1091 in transcriptionfactor regulation,312-31-3, 312f,313f,665-668,667f.Seealso Signalingpathways in endocytosis, Receptor-ligand dissociation, 670-611,6l0f 373, 606-6'1.2, Receptor-mediated endocytosis, 683-684 1085-1086 in antigenpresentation, pits in, 506-607, 6071, clathrin/AP-coated 609-610 electronmicroscopyof, 607, 607f of low-densitylipoproteins,606-61,0, 607f-609f pulse-chase studiesof, 607, 608f receptor-ligand dissociationin, 6L0-6L1, 61.0f receptortyrosinekinasesand, 608-610, 609f, 683-684 in signaling,608-610, 6091,683-684 sortingmotifs in, 684 stepsin, 607, 607f, 6091 complexin, transferrinreceptorJigand 61.L-512 6 ,L r f Receptortyrosinekinases,659, 566f, 679-694 activationof, 679-680, 681f adapterproteinsfor, 685, 6861,687-688, 6 88 f in cancer,680-682,681f, 1.127-1.128 definitionof, 679 epidermalgrowth factor and, 580-682, 681f, 682f fibroblastgrowth factor and, 680, 680f in HER family,680-682, 681f, 682f in IP3/DAGpathway,694 ligand bindingto, 680, 680f in PI-3kinasepathway,694-695,6961 PTB domainsand, 682-683, 683f in Ras/MAPkinasepathway,684-694. See a/so Ras/MAPkinasepathway signalingdown-regulationand, 683-684 structureof, 580, 680f,681.f Recessivealleles,166-1 67, | 67f Recessive mutations,166-170, 1671,860, 1,122t,1123 genefunction and, 1.66-167 identificationof, 171 lethal, l7l segregation of, L67-L69,L68f, 1.69f Reclinomonas americana,240 INDEX

t-4'l

Recognitionhelix, 290, 291f Recombinant DNA,23 definitionof, 176 expenmentalorganismsin, 25 expresslonvectorsin, 194-197 RecombinantDNA technology,176-1,90 cloningin, 176-190.Seealso Clones/cloning, DNA dideoxychain-termination methodin, [86f, 1 . 8 7t,8 7 f DNA librariesin, 179-182, 180f, 182f DNA microarraysrn, 192-1,94,194f, 1951. Seealso DNA microarrayanalysis expressionsysremstn, 1.94-1,97 animalcell, 196-1.97,796f E. coli, 194-196, 1.95f retroviral,197,7971 Northern blotting in, 192, 792f in situ hybridizationin, 192,193f Southernblotting in, I9l-192, 191f transfectionin stable,195-1.97,795f transient,196,196f Recombinases, in somaticrecombination, 1070f, r071 Recombination, 1L2, 145,150, 153, 892-899, 894f, 895f classswitch, 1075-1076, 1075f c r o s s i nogv e ri n , 1 5 0 ,1 5 3 ,1 6 8 f , 1 7 5 , 8 9 2 , 893f,894f g e n em a p p i n ga n d ,l t 4 - l - 5 , 1 7 4 f oennttron oI, I /J

in DNA repair, 1.49-1.53 h o m o l o g o u s , 1 5 0 - 1 5 3 , 1 , 5 1 f ,1 5 2 f nonhomologous,149-150, 150f in exon shuffling, 235 in gametogenesis,954f, 955 in gene inactivation, 204, 204f,208-209, 209f genetic diversity ftom, 892, 955 loss of heterozygosity and, 1124 o f m o b i l e D N A e l e m e n t s , 2 3 5 . S e ea l s o Mobile DNA elemenrs somatic, 208-209, 2091, 1069-1.073. See a/so Somaticrecombination Recombination signal sequences in heavy chains, 1073 in light chains, 1.069-1.071.,1.070f Recombinational mapping, 175 Red blood cells. See Erythrocyte(s) R e d o x r e a c t i o n s ,5 9 - 6 0 , 5 9 f , 6 0 f Reduction potenrial (E), 60 of electron carriers, 499, 500f Reduction reactions, 59-50, 59f, 60f R e f l e x , k n e e - j e r k ,1 0 0 5 , 1 0 0 5 f Reflex arc, interneurons in, 1005 Refractive index, 382 Regulated intramembrane proreolysis, 555, 705 Regulator of G protein signaling proteins, 634 R e g u l a t o r yT c e l l s ,1 0 9 6 Rehydration therapy, 477472 Relay-mode signaling, 964 Releasefactors, 137 Renal disease,polycystic, 780 Renaturation, of DNA, 117 Repear formation, 977-97 8 R e p e t i t i o u sD N A , 2 1 5 - 2 1 6 , 2 2 3 - 2 2 6 , 2 2 4 f , 225f . See a/so Noncoding DNA Replication D N A . S e eD N A r e p l i c a r i o n vnal, 156-757, 156f, 1.057 lytic, 156-158, 156f, 157f nonlytic,158,159f i n p l a q u e a s s a y ,1 5 5 Replication fork, 141, 141f, 1,42f, 143-144, 144f l-42

.

INDEX

collapsed, repairof, 150-152,151f Replication origins,141,261,,877 in yeast,261, 262f, 877-878, 878f Replicationprotein A (RPA),142f, 143 Replisome,12-13 Repolarization, membrane,actionpotential and, 1004, 1004f, 1007-1008,1008f, 1025, 1026f Reportergenes,277, 283 in functionalcomplementation studies,677 Repression domains,290, 294 Repressors, 270, 270f, 27l, 290 in alternativesplicing,339,339f co-repressors and,294, 304-305, 305f lac, 271 in translation,355-356 Reproduction asexual,l9 in eukaryotes,8, 8f in prokaryotes,7, 7f sexual,19 of stemcells,8 Resolution of microscope lens,21, 381 molecular,92 Resonance energytransfer, 515-515,516f Resonance hybrid, 34 Resonance hypothesis,7042, 1043f Respiration,cellular,59, 485, 487-489,489f, 511 Respiratorychain,493. Seealso Electron rransport electroncarriersin, 495-499, 495t Respiratory control,510 Response elements, 313, 313f RestingK* channels, 460, 461f,462f Restingpotential,458465, 1004.Seealso Membranepotenrial Restrictionenzymes,in DNA cloning, 1 7 6 - L 7 7 , 1 . 7, 16.f7 7 t Restrictionfragmentlengthpolymorphisms definitionof, 201 in linkagemapping,201.,201.f Restrictionfragments,777-178, 1.78I Restriction points,880-881,880f, 1134 Retina,1027f,1028 tectumand, 1.042-1.043, 7042f Retinal,641,,1028 Retinalneurons,1027-1031,1027f, 1.029f Dscam isoforrnsand, 340 Retinitispigmentosa,204 Retinoblastoma, 882, 1135 inheritanceof, 1,1,23-1,1,24, 11,23f Retinoicacid receptor,312f. Seea/so Hormone receptors Retinotectal maps, 1.042f-1043 Retrograde transport,580, 581f,582, 594-595, 596, 596f Retrotranslocation, 556 Retrotransposons, 227-234, 265-266. Seealso Mobile DNA elements oelrnrtronoI, Lzl in exon shuffling,235 in genomicDNA, 234 I:IR, 229-230, 229f, 230f non-LIR (nonviral),230-234,23lf-233f Retroviralexpressionsystems,1.97,1.97f Retrovirus(es), 158-159,159f.Seealso Virus(es) b u d d i n go f , 6 L 4 , 6 1 5 f in cell lineage studies,916-91.7,91.81 definitionof, 158 endogenous, in transposition,230 life cycleof, 158, 159f long-latency, 1122 mRNA transpot rn, 346-347,346f

oncogenic,158-159, 1128-1129 reversetranscriptionin, 230J34, 230f-233f SFFV 1128 slow-acting,1122 transducing,1122 Retrovirus-likeelements,230 Rev protein, in mRNA transport, 346-347, 345f Reverseenzymetranscriptase, 181 Reversetranscriptase, 158 in transposition , 226-227, 230, 230f, 231,1 Reversetranscriptase polymerasechain reaction,189 Reversetranscription,230, 231.f telomerase in, 263-264, 2551 in transposition,230-234, 2301J331 RGD sequences, 816, 831, 831f RGG box, 327 RGS proteins,634 Rheb protein,354-355, 354f Rho-GTP,in actin filament assembly,724, 724f Rho kinase,743 Rho protein,in cell migration,746-748, 748f-7 51.f Rhodobacterspheroides, photosystemof, 5 1 7 - 5 1 9 , 5 1 75 fl ,8 f Rhodopsin, 635-636,641-645.642f-645f. 1,028 structureot,644,644f Rhodopsinkinase,644 Ribbon models,of proteins,68, 69f Ribonuclease P (RNaseP), 363 Ribonucleicacid. SeeRNA Ribonucleoproteins. SeeRNPs RibosomalRNA. SeerRNA (ribosomalRNA) Ribosomaltranslocation,135-1.37,1361.See a/soProteintranslocation Ribosomes, 11, 1.13f,1.27,1.27f, 1.32-1.39, 3s8-359 A siteof, 133, 134f,1.35,136, 136f,1.37, 137f assembly of, 1.33,1.35f componenrs of. 127f. 132f,133 conformationalchangesin, 1.32f,1.351, 136f E siteof, 1.33,1.34f,1.36,L37, l37f in eukaryores,132f evolutionof, 133 functionsof, 1,27,1,27f,132-133 mitochondrial,240 as molecularmachines,1.32-L33 135-1.36,137f P site of, 1.33,'1.34f, in prokaryotes,132f, L37f in protein synthesis,1.33-139,1.35f,1.361, 534f,557 recyclingof, 1.38,1.39f in rough endoplasmicreticulum,375-376, 534f, 535,s36f sizeof, L32f, 1.33 structureof, 1.33,1.34f subunitsof, 1.27f,1.32f,733 assemblyand maturationof, 362-363, 362f,378 in translation,1.33-139,1,35f,136f, 5341 translocationof, 135-1.37,1.36f Ribozymes,79, 1,1,9, 363 Ringer,S., 90 RMP motif, 327,328f RNA, 11 hydrolysisof, 1.16,1.L6f base-catalyzed catalytic,1,19,363 cleavage of, 118 1,1,9f conformationsof, 1,1,8, exosomedegradationof, 336-337

functionsof, 1.2,7L1.-1.L2, 1.73f messenger. SeemRNA (messenger RNA) micro. SeemiRNA (micro RNA) from mitochondrialDNA, 240 oocyte,in fertilization, 957-958 phosphodiester bondsin, 40,41.f, 11.4, 714l proteinkinase,356 relativeinstabilityof, 116, 118 ribosomal.SeerRNA (ribosomalRNA) self-splicing of, 119 short interfering.SeesiRNA (short interferingRNA) smallhairpin,210 small nuclear.SaesnRNA (smallnuclear RNA) smallnucleolar, 222, 222r in pre-rRNAprocessing, 361-362,367f splicingof. SeeSplicing s t r u c t u roef , 1 1 8 - 1 1 9 1 , 19f synthesis of,120-126.Seealso Transcription tandemlyrepeatedarraysof, 221-222 telomerase, 222t,264, 265f transfer.SeeIRNA (transferRNA) virus-associated,356 RNA-bindingdomains,327, 328f RNA-binding morifs,327, 328f RNA-bindingproreins.sequence-specific. 356-357,357f RNA domains,118 RNA editing,340-341,,341f RNA erporr factortREF).132-333,342-343. 343f RNA flow cytomerry,395 RNA hairpins,118,119f,21.0,224 in somaticrecombination,1.071,1.071.f RNA helicases, 134 in nucleartransport,574-575, 574f RNA-induced silencing complex(RISC),348 (RNAi), 210, 211.f, RNA interference 349-357,367 RNA polymerase(s), 11, 269 in bacteria,122, 1.23f,271-275, 274f chloroplast,3lS functionsof,259-270 mitochondrial, 318 RNAs synthesized by, 279-280,279t structureof, 1.22,1.23f,279,279f subunitsof, 279-280, 280f in transcription, 120-122,L2Lf, l22f in bacteria,722, 123f, 271.-275, 274f in eukaryotes,278-281.,376-317,3l7f in initiation,296-299,297f, 298f, 316-317,377f in termination,314-316 RNA polymerase I, 278,279f,279t functionsof,278,279t s t r u c t u roef , 2 7 9 , 2 8 0 f in transcriptioninitiation, 316-317, 317f in transcriptiontermination,314 RNA polymeraseI initiation complex, 3L6-317, 317f RNA polymeraseII, 278, 279-281.,279f, 279t carboxyl-terminaldomainof, 280, 287f functionsof,279,279r heat-shockproteinsand, 316 mediatorcomplex and, 299, 307-308, 308f promoter-proximal pausingof, 316 structureof,279,280f in transcription elongation, 315,326 in transcriptioninitiation,280-281 in transcriptiontermination,315 RNA polymeraseII preinitiationcomplex, 296-299,297f,298f

assembly of, 307-308,308f III, 278, 279f,279t RNA polymerase functionsof, 278-279, 279t structureoI,279,280f in transcriptioninitiation, 317, 3L7f in transcriptiontermination,315 RNA polymeraseIII initiation complex,317, 317f RNA processing, 1.1,72f, 11.3f,724, 125f. See genecontrol a/soPost-transcriptional 18 alternative, overviewof, 323-325,324f pre-mRNA, 325-337.Seealso Pre-mRNA processlng stepsin, 326f motif (RRM), 327,328f RNA recognirion RNA-RNA duplexes,137 RNA surveillance, 323 RNA transcripts 3' end of, in translation,135,1.36f 5' end of cappingof, 1.24,125f, 1.34 in translation, 134, 135f RNA viruses,154. Seea/soVirus(es) RNaseMRP, 222t P), 363 RNaseP (ribonuclease RNP complexes,325, 327, 328f RNP remodeling,343 RNPs,325, 327,328f heterogeneous, 327, 328f r oteins,222t Ro ribonucleop Robo protein, L046, 1.047f Rodbell,Martin, 663-664 Rods,retinal,641.-545,542f, 643f, 1027f, 1,028 retinotectalmapsand, 1.042,1042f Roots,meristemsof, 840, 842 Rough endoplasmicreticulum.See Endoplasmicreticulum,rough Roughmicrosomes, 535-536,536f, 537 Roundaboutprotein, 1.045,1047f Roundworms.SeeCaenorhabditis elegans Rous,Peyton,1121 Rousesarcomavirus (RSV),1L21,-1,122 Routesof infection,1057 RPA (replicationprotein Al, 142f, 1,43 RPD3 protein,in histonedeacetylation, 304 rRNA (ribosomalRNA), 71.2,1.1.2f,1.13f, 222t. Seea/so Ribosomes functionsof, 1,1,2, 173f,1,27,l27l large,133 processing of, 358-364, 359f-352f 2-8, 2-9t RNA polymerases rranscribing. small,133 structureof, 118, 133,734t synrhesis of. SeealsoTranscription in nucleolus, 378 tandemlyrepeatedgenesof, 221,-222 IRNA and, 137 Rubisco,525-529 in carbonfixation, 525-527, 525f-527f in photorespiration, 527-529, 527f, 528f runt, in body segmentation,9TT S phase,18, 18f, L44,781,847-848,848f, 849, 849f,851-852,872-879, 873f-879f.Seealso Cell cycle in, in cancer,1134-1135 changes DNA replicationin, 876-879, 8761 entry into, 872-877 G1 cyclin-CDKcomplexesrn, 874-878, 8 75 f SCFin, 850, 850f, 851, 876-877 S-phase cyclin-CDKcomplexes,1.44,850f, 8s7, 872-879 inactivationof, 876-877, 877f

S-phaseinhibitor,876-877, 876f S-phasepromotingfactor (SPF),874-876, 874f,875I cereuisiae.Seealso Yeast Saccharomyces in, 169-770, 1691 allelicsegregation cell-cycle regulationin, 851-852,852f, 853t in S phase,872-879,873f-879f cell-typespecificationin, 92'1.-924, 922f-924f in, 263, 263f centromeres as diploid vs. haploid organism,166, 169-1.70,1.69f as experimentalorganism,26, 1,69-1,70 geneinactivationin, 205, 206f 783t genomiclibrary for, 1,82-1.84, as haploid vs. diploid organism,169 MAP kinasesignalingin, 691,-692, 692f in,7,7f reproduction in, 360-353 rRNA processing homologyrn, 244f sequence transcriptionin, 279, 279t, 281.f repressionof, 299-301.,300f, 301f SAGA complex,305-305 Salt fractionization,22f Saltatoryconducrion,1014 Saltytasteperception,1035f, 1035 SAM complex,564-565 Samplepreparation,for microscopy,384-385, 384f method, Sangerdideoxychain-termination 1.86f,1.87,787f Sansprotein, 1034 Sarl protein,587-588,587t, 593, 593f 740,74LI Sarcolemma, )arcomere,/J2, /J)r early studiesof, 75 5-755 reticulum,740, 741f Sarcop^lasmic Ca'* ions in, 449451., 450t Satellitecells,culture of, 396, 397f SatelliteDNA, 224, 225f in DNA fingerprinting,225,2251 Saturated fatty acids,4748,47r regions(SARs),254 Scaffold-associated Scaffoldproteins,63 in chromatin,254-255, 254f in MAP kinasepathway,692-693, 692f in skeletal mascle,740, 7 40f Scanningelectronmicroscope,388f, 390 RNA), scaRNA(smallCa;al body-associated 365 Scatterfactorlhepatocytegrowth factor (SF/HGF), 928 SCF,in cell-cycleregulation,850, 850f, 851, 876-877,886t pombe.Seealso Yeast Schizosaccharomyces cell-cycleregulationin, 851-852, 853t, 859-863,860f-863f in S phase,873-874 centromeres in, 263, 263f Matthias,371 Schleiden, Schwanncells,1014-1016,1015f Schwann,Theodore,371 Screens DNA librarS 181-182 genetic,1.70-171.,1.70f yeastgenomiclibrary, 183-184, 784f Scrotalcancer,1139 ScurvS826 (SDSgel electrophoresis SDS-polyacrylamide PAGE),94-e5,2e4l Seaurchins,cell-cycleregulationin, 856, 855f Seccoatproteins,587f,588f, 593, 593f secmutants,vesiculartransportin, 584, 584f, 591, Sec61complex,539-540, 540f, 541.1 INDEX '

t-43

SecAprotein,541 Secondmessengers, 634, 635r, 818. Seealso Signaling c A M P a s ,6 3 9 , 6 4 0 t coordinatedaction of, in glycogenolysis, 6s7-659,658f enzymes as,639,640t G-protein-coupled receptorsand,634, 635t,639,540t ion channelsas,639, 640t in srgnalamplification,634-635, 650-651., 6 s 1f synthesisof, 639 SecondaryactivetransporL,440, 440t Secondarylysosomes, 374, 374f Secondarystructure,65f, 66-70, 66f-70f, 75, 75f of proteins,6 5f, 66-70, 66f-70f 705-7 06, 70 5f, 707 1-Secretase, Secretogranin II, 602-603 Secretorycells,in rough endoplasmic reticulum.376, 376[ Secretorypathway,533-535, 533-541,534f, 579-606. Seealso Vesiculartransport definitionof, 580 early stagesof, 592-596, 592f-597f experimentalstudiesof, 580-586 autoradiography in, 582-583, 621,-622, 622f cell-freetransportassaysin, 585, 585f endoglycoside D assayin, 583-584,583f fluorescence microscopyin, 582f, 583 oligosaccharide modificationsin, 5 8 3 - s 8 4 ,s 8 3 f pulse-chase labelingin, 582-583 in vitro assays in, 585, 585f in vivo assays in, 582-584,582f-584f VSV G proteinsin, 582-583, 582f yeastmutantsin, 584, 584f oligosaccharide modificationsin, 583-584, 583f proteolyticprocessingin, 603-604, 603f, 604f yeastmodel of, 584, 584f Secretoryproteins proteolyticprocessingoI, 603-604, 603f, 604f pulse-chase experimenrs for. 535-535,536f synthesis ot, 535, 537, 538-539,538f translocationof, 535-555. SeealsoProtein targeting;Proteintranslocation vesicular transportof,502-605.Seealso Vesiculartransport Secretoryvesicles, 373f, 376-377, 376f, 580. Seealso Vesicles constirutive,602 myosintransport of, 743-744, 7 43f regulated,602-603 Securin,851, 859, 871f, 888-889 Sedimentation assays,for actin polymerization, 71,9 Segment-polarity genes,977, 999-1000 Segmental duplication,221 Segregation of chromosomes, 167 i n m e i o s i s1, 6 7 , 1 6 8 f in mitosis,lo7, 168( of dominanrvs. recessive mutafions, 1 6 7 - 7 6 91 , ,68f,7691 SMC complexin, 255, 256f Selectins, 838 Selectionmedium,401 Selectivity filrers.in ion channels. 461463, 462f Self-splicing. Seealso Splicing of proteins,91,-92 of RNA, 119 l-44

.

INDEX

Self-splicing introns,334-335, 335f, 363, 364f SelfishDNA,226 Semaphorins, 1044-1.045,1.044f,1.048-1.049, 1048f Senescence,1116 Sensoryhomunculus,1032, 1032f Sensoryneurons,1005 in hearing,1.032-1,034 in smell,1036-1039 in taste,1034-1036 in touch, 1031-1032 in vision, 1027-1031 Sensoryorganprecursor,989 Sensoryprocessing, primary cilia in, 780 Sepals,983,984f Separase, 869, 871f, 894 Sequencedrifr, 220-221. Sequence homology,72-73, 73f, 244, 244f, 245f Sequence map,203f. Seealso Mapping Sequenceorthology,244 Sequence paraIogy,244 Sequence-reading helix, 290, 291.f Sequence-speci fic DNA affinity chromatography, 288, 288f Serine,42f, 43. Seealso Amino acid(s) Serineproteases, activesitesof, 81.-84,82f, 83f Serotonin drug effectson,1.023 structureof,1,020f Serrateprotein, 705, 706 Serumresponseelement,690 Serumresponsefacto1 690 Seven-spanning cell surfacereceptors, 697-702. Seealso G-protein-coupled receptor(s)(GPCRs) for Hedgehog,700 br Ylnt, 699 Sexchromosomes, 13, 13f, 19,1,9f,955 dosagecompensarion and. 253, 958-959, 959f inactivationof, 253 in heterochromatin formation,253-254 Sexdetermination, X-chromosome inactivation in, 253 sex lethal,alternativesplicingof, 338-339, 338f Sexualdifferentiation,in Drosophila, 3 3 8 - 3 3 9 3, 3 8 f , 3 3 9 f Sexualreproduction,19. Seealso Reproduction SF/HGRin myoblastdifferentiation,928 SFFVretrovirus,1128 SH2 domains,682,683f in JAI(STAT pathway,675 in Ras/MAPpathway,685,686f SH3 domains,687-688, 688f shaker mutation, K* channeldefectsand, 1010-1013,1013f Shearstress,geneexpressionand, 843 Shepherd's crook cells,1002f shh, 966-967, 987-988, 987f in axon extension, 1045-1047 in limb development,99l-994,992f, 993f in neuraldevelopment,987-988, 987f, 1046-1047 in patterning,966-967, 987-988, 987f shibire, 1023 Shine-Dalgarno 135 sequence, Short interferingRNA (siRNA).SeesiRNA (shortinterferingRNA) Short interspersed elements(SINEs),230-234, 232f,233f SHPI, in JAK/STAI pathway,678,678f shRNA (smallhairpin RNA), 210

Shugoshin,897-898 Shuttlevectors,183, 183f Sialicacid, 837 SialylLewis-xantigen,837 Sicl, 876-877, 876f, 886t Sicklecell disease,1.67,1.99,'1.99t Side-chain-specificity bindingpocket,82-83 o (sigma)factors,271, 273-275, 273t, 274f Signalamplific ation, 634-635 Signal-anchor sequences, 545-546, 545f, 545f 965 Signalantagonists. in development, Signalpeptidase,in protein translocation,540 particles(SRPs) Signal-recognition in protein translocation,537-538, 538f structureof, 538, 538f Signalsequences, 535, 536f Signaltransduction,definition of, 624, 624f. proteins,17 Signal-transduction Signaling,9-1.0,16-17, 623-660 in action potentialpropagation, 1005-1006,1005f adenylylcyclase in, 639,646-652 adhesionreceptorsin, 803, 807,807f, 833-835,843 in apoptosis,936-944 autocrine, 6251,626 in axon guidance,1043-1047, 1.044f-t046f B-..ellreceptorsin, 1.091.,'1092f Ca'*/calmodulincomplexin, 65 5-656 calciumions in, 451-452 in cancer,671,-672,680-682,71,0,1109, 1.1.24-1125, 1.126f,1.1.29-1.130, 1130f CD3 complexin, 1088 moleculesin, 805, 807,807f cell-adhesion in cell-celladhesion,833-835 in cell differentiation in muscle,927-929 in yeast,922-923 in cell division,909, 909f, 913-916, 913f, 915f,932-933 in cell migration,746-748, 747f-7 51.f in cell survival,936-937 in yeast,922-924, in cell-typespecification, 922f-924f rn, 632-633 cellularresponses cellularsensitivityin, 631 748-75 1, 75 lf in chemotaxis, competentcellsin, 951 cross-talkin, 657 cytokinesin,672-679. Seealso Cytokine(s);JAK/STAT pathway c y r o s k e l e t oa n d .7 1 5 .7 l 5 f desensitization in, 631.,644-645, 65'L-652, 65l.f in development, 9 51,963-969 double-mutantanalysisof, 172, 1731 down-regulationin, 651-652, 65lf early studiesof, 663-664 effectorprotein activationin, 637-540, 6381,639f environmentalinfluencesin, 657-660 enzymesin, 633, 639, 640t epidermalgrowth factor in, 626, 680 in erythrocyteproduction,673-674, 674f erythropoietin receptor in, 674-676, 674f 311-313, 312f, 31.3f extracellular, extracellularmatrix in, 805, 807, 807f feedbackrepressionin, 551 future researchdirectionsin, 660, 709-71,0 receptorsin, 423, 624, G-protein-coupled 635-657. Seealso G-protein-coupled receptors G proteinsin, 633-634,533f

gap junctionsin, 818 gradient-mode, 954 growth cone in, 1040-1042, 1040f growth factorsrn, 626,746-747 GTP in, 633-634,633f,637-638,638f, 663-664 GTPaseswitch proteinsin, 637-638 hormonesin, L7f, L8,31.2-313,372f,625, 625f,626. SeealsoHormone(s) induction,95 l, 953-964, 963f in infections, 838 integrinsin, 807, 807f, 817, 833-835 intracellular proteinsin, 633-634,633f,6341 ion channels in, 539, 640-645,540t, 641f-643f in leukocyte extravasation, 837-838,838f ligandconcentrationin, 629-630, 630f in Iimb development,997-992 long-rermresponses ro, 665 mechanisms of, 632-633 mitogensin, 880-881 morphogenic, 700,964 negative feedback loopsin, 671,,671f neural,987-988,1001-1006.Seealso Neuron(s); Neurotransmitter(s) o n c o g e n ei ns, 1 1 2 9 - 1 1 3 01, 1 3 0 f overviewof, 623-527, 624f paracrine, 525f,526 phosphatases in, 634 p h o s p h o l r p aCs ei n , 6 5 1 - 6 5 7 in plants,840-841, 841f in pre-mRNAprocessing, 335 proterncleavage in, 703--09 proteinkinaseA in,667f protein kinasesin, 634 in proteintargeting,557,557t to endoplasmicreticulum,536-539, s37f-539f to mitochondria, 558 PTB domainsin, 682-683, 683f receptor-mediated endocytosis in, 508-510, 609f, 583-684 receptortyrosinekinasesin, 679-694 receptorsin. SeeReceptor(s); ReceptorIigandbinding relay-mode, 964,954f secondmessengers in, 534, 635r,8f8 cAMP as,639, 640t coordinatedacrionof, in glycogenolysis, 657-659,658f e n z y m eass , 6 3 9 , 6 4 0 t G-protein-coupled receprorsand,634, 635r,639, 640t ion channelsas,639,640t in signalamplification,634-635, 650-65L,6S1f synthesisof, 639 SH2 domainsin, 675, 682, 683f SH3 domainsin, 687-688,688f signalamplificationin, 634-635,650-651, 650f signalrangein, 625-626, 625f signalthresholdin, 964 small moleculesin, 633-634, 633f Spemannorganizerin, 965, 965f in stem-cell niches,909, 909f,912-920, 9151-919f.Seealso Stem-cellniches stepsin, 624f, 625-627, 625f, 626f switch proteinsin, 633-634, 633f synaptic.SeeSynapses synchronous,1003 in T-cellactivarion,7094-1095,1094f T-cellreceptorsrn, 1.091,1,092t targetcellsin, 623 Toll-likereceptorsin, 1098-1099 in transcription,312-31,3,31,2f,3131,

665-71,0 transcription factors in, 532-633. See also Transcription factors i n v e s i c u l a rt r a n s p o r t , 5 8 9 . 5 8 9 t , 5 9 4 - 5 9 5 .

600-602 in vision,641.-645,642f-645f Signalingmolecules,523-624, 625-627. See a/soLigand(s) cellularsensitivityto, 631 definitionof, 523 membrane-bound, 625f, 626 receptorbindingof. SeeReceptor(s), cellsurface;Receptor-ligand binding signaling rangeof, 625-525,625f srructureof, 710 synrhesis of, 625 transportof,625-627 Signalingpathways,624. Seealso specific pathxL/ays actrvatfon ol, b6', 66 /l

BMP,965-966 future researchdirectionsin, 650 G-protein-coupled receptor,423, 624, 635-657. Seealso G-protein-coupled receptors GPCR/cAMP,646-652 Hedgehog,667f , 697-69 8, 7 00-702, 7 00f, 701.f highly conservedcomponentsof, 632-640 Insig-1(2)/SCAP/SREBP, 707-7 09, 7 08f IPr/DAG,653-657,6s4f, 655f,694 JAK/STAI,574-679, 674f-67 8I NF-xB,703-705, 704f nitric oxide/cGMP,656-657, 657f Notch/Delta,703 o r d e r i n go f , 1 7 2 , 7 7 3 f PI-3 kinase,694-697, 695f-697f, 696f proteinkinaseC in,647-649,647f p r o r e i nk i n a s eC i n . 6 5 6 - 6 5 7 , 6 5 7 f Ras/MAPkinase,684-694 reversibilityof, 703 terminologyfor, 624 TGFB/Smad,665f, 668-672 \lnt, 667f, 597-698, 699-700, 909, 909f Silencers, transcriptional,1,8,299-301, 300f Simplediffusion,438439 Simple-sequence repeats,207, 224, 225f in DNA fingerprinting,225,225f in geneticdiseases, 224,340 SINEs,230-234,232f, 233f,234 Singlelocationpostulate,407 Singlenucleotidepolymorphisms(SNPs),201, 246-247 Single-pass membraneproteins,422f, 423, 543f-545f, 545-546 Singletmicrotubdes,759-760, 760f SIR proteins,in transcriptionalrepression, 3 0 0 - 3 0 13 , 00f,301f siRNA (shortinterferingRNA), 325, 347. See a/so RNA basepairing by, 347, 348f in RNA interference, 350 siRNA knockdown,350 Sisterchromatids, 257, 258f,782-783,783f,849 alignmentof, 7 83, 7 83f, 7 88-789 cohesionbetween,867-869, 870f, 898 in meiosisvs. mitosis,892-894, 8941 separationof, 869-870, 870f, 871,f. See aiso Anaphase Situsinversus,954-955 Skeletalmuscle.SeeMuscle S k ip r o t e i n6, 7 1 . , 6 7 L f Skin barrierfunctionsof, 1059, 1097 cancerof, 148-149

in xerodermapigmentosum, 1.L42,7142t pigmentationof, 469, 469f stemcellsfor, 9L4-9L5, 9l5f touch and, 1.030-1032 Skou,Jens,477-478 SLBPprotein,in oogenesis, 957-958 SLC24A5,469,469f Slidingfilament assay,7 33, 7 33f Slidingfilamentmodel,7 38-740, 7 39f Slit protein, 1046, 1047f Slo pre-mRNA,processingof, 366-367 Slow-actingretroviruses,1122 SMAC/DIABLO,941 Smads activationof, 665f, 667f, 670-672, 671.f, 672f in cancer,1134 SmallCajal body-associated RNA (sca-RNA), 365 Small(monomeric)G proteins,354-355 in signaling,634 Smallhairpin RNA (shRNA),210 SmallnuclearRNA (snRNA).SeesnRNA (smallnuclearRNA) (snoRNPs), Smallnucleolarribonucleoproteins 361-362 SmallnucleolarRNA (snoRNAl,222, 222t in pre-rRNA processing, 361-362, 361t S M C p r o t e i n s2, 5 5 , 2 5 6 f ,8 6 6 , 8 6 9 Smell,1036-1039,1037 primary cilia in, 780 smo, 701-702, 701.f Smoking,lung cancerand, 1140, 1140f Smoothmuscle,contractionin, 742-743, 743f. Seealso Muscleconrracuon smoothened(smo),7 01-702, 7 011 SNAP proteins,in vesiclefusion,591, 1022 SNAREproteins/complexes, 586, 586f, 591, in synapticvesicles,1022 SnoN,671 snoRNA (smallnucleolarRNA), 222,222t in pre-rRNA processing, 361-362, 361,f SNPs(singlenucleotidepolymorphisms), 201, 246-247 snRNA (smallnuclearRNA), 222,222t. See a/so RNA basepairingwith mRNA, 330,331,,332, 332( evolutionof, 334-335 RNA polymerases transcribing. 279, 279t. Seealso Transcription in splicing,330, 331f SnRNPs,in splicing, 330-332, 331.f,332f Soaps,411 SOCSproteins,in JAK/STM pathway,678, 678f Sodium,in musclecontraction,1023-1024, L024f Sodium-bicarbonate-chloride antiporter,468 Sodiumdeoxycholate,428, 428f Sodiumdodecylsulfate, 428, 428f Sodium-glucose symporters, 466467, 466f, 477 Sodium/hydrogen antiporter,466 Sodiumion channelproteins,438f Sodiumion channels.SeeIon channels,sodium Sodiumions, in cytosolvs. blood, 448,448t Sodium-leucine symporter,467468, 467f Sodium-linkedCa'* antiporter,468 Sodium-linkedsymporters,466468, 465f, 467f, 471. Sodium-lysine transporter,438f Sodium-potassium ATPase.438f, 45 245 3, 452f in cardiacmuscle,458 discoveryof, 477478, 478f (Na*/K*) pump, 449, Sodium-potassium 452-453 INDEX

t-45

Solitary genes,219-220 Solubility, 37,381 of amino acrds,4243 diffusion nte and, 439 hydrogenbondsand, 37,37f hydrophobiceffectand, 38-39,39f Iipid, hydrophobicityand, 439 polarityand,39,42 Solutions aqueous hydrophobiceffectand, 38-39,39f ionic interactions in, 36-37, 36f-38f, 38f pH o{, 57-52, 52f hypertonic, 372,444 hypotonic,372, 392, 444 isotonic,444 rehydration,471.472 Somaticcell(s),14, 905, 950 d i v i s i o no f , 7 6 7 , 7 6 8 f oncogenic mutationsin, 1108,1119 stem,nichesfor, 914 vs. germ-linecells,913 Somaticcell nucleartransfer(SCNT),9 Somaticcell transposition,226 Somatichypermutarion,1073 Somaticrecombination,208-209, 209{, 1069-1073, r069f, 1.070f,10721.See aiso Recombination allelicexclusionand, 1.070f,7071, 1073 antibodydiversityand, 1059-1073 in B-celldevelopment,1069-1073 deletionaljoining in, 7070f, 1071. earlystudiesof, 1105-1106 in geneinacrivation,208-209, 209f hairpin openingin, 1071, 1071f in heavy-chainimmunoglobulins,1069, 1.069f , 1.071-1073, 7072f inversionaljoining in, 1070f, 1071. junctionalimprecisiontn, 1070f, 1.071. in light-chainimmunoglobulins, 1.059-707 I, 1069f, r070f RAG proteinsin, 10701,1077 recombination signalsequence in. 1069-107r, 1070f in T-celldevelopment,1.091,-1,09 3 in T-cellreceptor, 1088-1089,1089f Somaticstemcells,nichesfor, 914 977-978, 978f, 979f Somites, cell-fatedeterminationin, 988 in muscledevelopment,925,925f Sonichedgehog in limb development,987-988,987f in neuraldevelopment,987-988, 987f in patterning,966-967 Sonication,391 Sortingsignals, 589, 589t, 594-595,594f,600. Seealso Proteintargeting;Signaling cytoplasmic, 589, 589t di-acidic 5R9 5R91 591

GPI anchors,505 KDEL, 589, 589t, 594-595,594f KKXX, 589, 5891,594f, 595 Iuminal,589, 589t mannose6-phosphate, 60-f, 589, 589t, 600-602,60tf NPXY, 589, 589t, 608-609, 609f in receptor-mediated endocytosis, 508-610 Tyr-X-X-O,589, 589t, 609-610 Sosproteins,in Ras/MAPkinasepathway, 685, 685f, 687-688, 688f 1032-1034 Soundperception, Southernblotting, 191-192, l9lf Space-filling models,33, 33f 220t, 225-226 SpacerDNA, unclassified, l-46

.

INDEX

Specific activity, of radioisotopes, 99 Specific pathways hypothesis, 1042, 1043f Specific transcription factors, 285 Specificity, molecular complementarity and, 40, g0 Specimen preparation for electron microscopS 388-389, 389f,

390,391 for light microscopy,384-385, 384f Spectralkaryotyping,258, 259f Spectrin, 728,729f picosecondabsorption,518 Spectroscopy, SPEDmicroscopy,385 Spemannorganizer,965, 965f Sperm,950 acrosomal cap of. 954. 955, 955f acrosomalreactionrn, 956, 957 in fertilization,95 5-959 flagello a f , 9 5 4 , 9 5 4 f , 9 5 5 , 9 6 8S. e ea l s o Flagella productionof, 9 53, 9 54-955, 954f, 955f propertiesof, 954, 955f Spermatocytes differentiationof, 914 primary,954 Spermatogenesis, 953, 954-955, 954f Spermatogonia, 954 Spermiogenesis , 9 54, 955f Sperry,Roger,1042 Spherocytes, 730 Spherocyticanemia,730 Sphingolipids,41.1.,4121,476 distributionof,420 Sphingomyelin, 431 membrane contentof, 418, 418t structureot, 479, 41.9f Sphingosine,43l Spinalcord, developmentof, 985-986, 9851, 986f. Seea/soNeural development axon guidancein, 7047, 1047f Spinalmuscleatrophy,334 Spindle-assembly checkpoint, 888-889,888t, 889f Spindlepole(s),757, 7 671,782, 7 83, 7 83f, 7841.Seea/soMitotic spindle in meiosisvs. mitosis,892, 894f separation of, 783, 7831,789 Spindlepole bodn 890, 890f Spindle-position checkpoint, 888, 888t,890, 890f Spinocerebellar ataxra,224 Spleenfocus-formingvirus (SFFV),1128 Splicesites,21.7,329-330, 329f Spliceosomes, 330-333 pre-mRNAin, retentionin nucleus, 345-346 Splicing,1.1.9, 1.24,217, 329-333 abnormal,357 in geneticdiseases, 333-334,334 activatorsin, 339 1.25-1.26, l26f ,2L8,219, 323, alternative, 337-338, 366-367 Dscam isoformsand, 340 in fibronectin,126, 125f, 21.8,338 in hearing,339-340, 340f in isoforms,1.26,1.26f,338 in vision, 340 in bacteria, 338-339,338f, 339f basepairingin, 330, 331f,332,332f branchpoints in, 330 definitionof, 1.24,125f,329 exon skippingin, 333-334 by groupI introns,363,364f by groupII introns,334-335,335f,363, 3641 intron removalin, 329, 3291

of nonstandardintrons,333 in, 339 repressors self-splicing, 334-335.335f oI proteins, 91.-92 of RNA, 119 in sexualdifferentiation,338-339, 3381 339f sitesof, 21,7,329-330, 3291 snRNA in, 330, 331f snRNPsin, 330-332,331f, 332f in, 330-333 spliceosomes SR proteinsrn, 333-334, 334f in, 333 trans-splicing in, 330, 330f, 332, transesterification 332f moleculesin, 805, cell-adhesion Sponges, 806f,807 tissue,in plants,839, 839f Sporogenous Squamouscell carcinoma,1,48-1,49 Squidgiant axons,microtubule-based transportin, 770-771.,770f SR proteins,333, 334f, 343-344, 344f s/c oncogene,1.1.29,'1.L30f Src tyrosinekinases,in lymphocyteactivation, 1091, SRE-bindingproteins,707-709 Stabletransfection,196-197, 1.96l Stahl,V.F., 140 Staining.Seealso Microscopy 258, 258f of chromosomes, fluorescent,382 of tissue,21, 385 in transmissionelectronmicroscopy, 388-389,389f Stamen<

gRl

9R4f

Starch,46 product,511 as photosynthesis structureof, 511, 51lf START,in cell-cycleregulation,872,875 Startcodons,127-L28, 128t STATtranscriptionfactors,666f, 667f, 674-677, 676f, 1.096.Seealso JAK/STAT pathway 768, Stathmin,in microtubuledisassembly 758f Statindrugs,432-433 StelZ protein,924 reactions,50, 50f Steady-state Stemcell(s),8-9, 905, 91'2-920 cell divisionin, 906-907, 906f, 912-913 definitionof, 8, 905 91.1.f, 960 embryonic,8, 91,1,-91.2, cultured,9LL-91'2 experimentalusesof, 912 from innercellmass,960,962f mouse,25 91.5f epithelial,91,4-91.6, future researchdirections for, 944 germ-line,9L3-914 hematopoietic,91.7-920,919f identificationof, 912-913 intestinal,91.5-916, 9 1.6f in leukemia,920 lymphoid,91,8,9191 microenvironmen.tof, 9 12-9 13 myeloid,918,919t 917f, 91'8f neural,91-6-91'7, plant,920, 9211 pluripotent,907,960 in, 907 self-renewal skin/hair,914-915, 91.5f somatic,nichesfor, 914 therapeuticusesof, 912 totipotent,907,960 tumor,1111 1'1'1'8f identificationol, 1'1'1'6-1'l'1'9,

unipotent,907 Stem-cellnrches,972-920, 9131,914f L--^.^-^i^+i^

O)A

intestinal, 91.5-9 16, 91.6f neural,976-917, 9l7f signaling pathways for, 909, 909f, 9 1 2 - 9 2 0 , 9 1 4 t , 9 15 f - 9 1 9 f skin/hair, 914-97 5, 915f subventricular, 91,7, 9791 Stem-loop binding protein (SLBP), in o o g e n e s i s9 , 57-9 58 S t e m - l o o p s ,7 1 8 , 1 . 7 9 f Stereocilia, 1032-1034, 1033f, 1034f Stereoisomers,33 S t e r o i d h o r m o n e r e c e p r o r s ,3 7 3 , 3 7 4 f . S e ea l s o Hormone receptors Steroids,415 Sterol-sensingdomain, 432 S t e r o l s ,4 1 5 . S e ea l s o C h o l e s t e r o l ;L i p i d ( s ) ; Lipoproreins Sricky ends, of restriction fragments, 177-178, 177f, 180f, 18r Stigma/stylar cysteine-rich adhesin, 842,842f Stigmasterol, structure of, 472f, 475 Stimulated emission depletion (SPED) microscopy,385 Stop codons, 1.27-128, 1,28t, 137-1,38 Stop-transfer anchor sequences,544, 544f-546f Store-operated ion channels, 555, 655f Stress,oxidative, 502-503, 520-521, S t r e s sf i b e r s , 7 7 6 , 7 7 6 f , 7 4 1 - 7 4 2 , 7 4 7 f , 7 5 0 f Stroma, in chloroplasts, 379 carbon fixation rn, 524-529, 525f-528f Stromal import sequence,565 Structural domains, 70-7 7, 7 Lf S t r u c t u r a l m a i n t e n a n c e( S M C ) p r o t e i n s , 2 5 5 , 2s6f, 866,869 Structural motifs See Morifs Structural proreins, 63 S t u r t e v a n r ,A . , 1 7 5 S u b c l o n i n g ,1 8 4 Substrate(s) definition of, 79 enzymebinding of, 80-84 Substrate binding. See also Receptor-ligand binding m e c h a n i s m so f , 4 6 7 - 4 6 8 , 4 6 7 f Substrate-binding site, 80, 80f, 82-84 Substrate-levelphosphorylation, 481482, 482f S u b u n i t v a c c i n e s ,1 1 0 1 Subventricular stem-cell niche, 91,7, 9l9f Succinateo , xidation to fumarate, 59,59f Succinate-CoQ reductase, in electron r r a n s p o r r ,4 9 5 t , 4 9 6 4 9 7 Succinate dehydrogenase,in citric acid cycle, 489 S u c r o s e4, 6 , 4 6 f as photosynthesis product, 51,1, 525, 526f Sulfhydryl groups, 552 Superoxide, cellular injury from, 502-503 Suppressormutations, 17 3-17 4, 173f -06 S u p p r e s s o ro f H a i r l e s s , SV40 DNA, replication in, 142-144, 1,42f-144f SV40 enhancer, 284, 285f svedbergs,133 Sweet taste perception, 1035f, 1036 S \ 7 V S N F c o m p l e x e s ,3 0 6 - 3 0 7 , 1 1 3 5 - 1 1 3 6 rn cancer, 1,135-11,36 in myoblast differentiation, 927-929 Switch proteins, 90, 90f, 911, 1,38, 587 in mitotic exit network, 890, 890f R a s a s , 6 8 4 , 6 8 5 . S e ea / s o R a s p r o t e i n i n s i g n a l i n g ,6 3 3 - 5 3 4 , 6 3 3 f

in vesiculartransport,587-590, 590f Sxl protein,in Drosophilasex differentiation, 338-339,338f Symporters,440, 466-470, 471 in transepithelial transport,471, 47lf Synapses, 1004f, 1005, 101.8-1.026 directionalityof, 1005, 1025 electrical,1025-7026 formationof, 1018-10f9, 1018f mRNA localizationto, 352, 357-358, 358f in musclecells,1019 postsynaptic cellsand, 1005,1018 presynaptic cellsand, 1004f,1005,1018, r019 Synapsin,102l Synapsis, in meiosis,892,893f Synapticglomeruli,1037 Synapticvesicles,1019-1022,1020f, 1.021.f cyclingof, 1021f dynamin and, 1,023 formation of, 1021.f fusion with plasmamembrane,1022, 1,023 linkageof, 1021 localizationin activezone,1021.-1.022 neurotransmitter releasefrom, 1020-1022, L02tf recyclingof,1023 SNAREsin, 1.022 synaptogamin,L022-1023 Synchronous signals,1003 Syncytialblastoderm,970, 970f Syncytium,925 Syndecans, 829 Synpolydactyly, 992, 993f Syntaxin,591,1022 Synteny,conserved, 259 Syntheticlethal mutations,173f, 1,74 Systematic linker scanningmutation analysis, 290 T-antigen,large, L43 T cell(s),1057-1058,1057f,1058f, 1088-1097. Seealso under Immtnel Immunity activationof , 1094-1095, 1.094f antigenreceptorson, 1056 apoptosisof, 1050f, 1094, 7094f, 1095, 1095f in B-cellactivation,1099-1101,1100f B-cellcollaborationwith, 1097-1,1.01. in antibodyproduction,1099-1.1.01, 1100f in vaccines,7707-1,1,02 cD8, 1095 chemokinesand, 1096-7097 cytokineproductionby, 1,095-1,096 cytoroxic,1076, 1078-1079, 1079f, 1095 CD8 and, 1080 mechanism of actionof, 1095,1095f MHC molecules and, 1080 targetingof in classI MHC pathway, 1082-1084,1083f in classII MHC pathway, 1 0 8 4 - 1 0 8 71, 0 8 5 f ,1 0 8 5 f definitionof, 1056 d e n d r i r iec p i d e r m a9l .I 5 development of, 1088, 1091-1095, 1092f avidity model of, 1.093 positive/negative selecrion in. 1091-1093 somaticrecombination in, 1091-1093 stepsin, 1093f vs. B-celldevelopment,1093f

differentiationoI, L09l extravasationof, 837-838, 838f function of, 1088 future researchdirections for, 1702 helper,1075-1077,1,095-1096, 1096 CD4 and, 1080 cytokineproductionby, L097 MHC moleculesand, 1080 inflammatory,1096 interleukinsand, 1.096 memory,1096 migration of, 1096-1097 natural killeq L060-106L,1060f, 1095 productionof, 1058 regulatory,1096 somatichypermutationand, 1.073 T-cellreceptors,1088-1091 diversityof, 1089-1091,1093 ligand binding by, 1090-1091 loci organization in, 1088-1089,1.090f signalingpathwaysfor, 1091, 1092f somaticrecombination in, 1088-1089, r090f structureof, 1088-1091,1089f,1090f T-loop,863, 863f I-SNAREs,586, 586f,591 T1R/T2R proteins,in taste,1036 Tactilesensatio n, 1034-1036 Tailless,974-977, 975f Tamoxifen,1133 Tandemmassspectrometry, 101, 102f, 1,03 Tandemlyrepeatedarr ays, 221-222, 22lt in DNA fingerprinting,225 TAP (nuclearexport factor 1), 573 TAP,Nxtl mRNP exporter,342-343, 573-575, 574f TAR element, in HIV infection,315-316,315f Taste,1034-1035,1035f TAT protein,in HIV infection,315-316, 315f T,{TA box, 282-283,282f, 286f, 297-298 TAIA box-binding protein (TBP),297-29 8, 297f Tau proteins,77 76--768, 767f in microtubule srabilization, Taxol,766 Tay-Sachs disease,199t, 374 TCA cycle,487489,489f TEL sequences,263-264 Telomerase, 743, 222t, 263-264, 265f in cancer,L143-1,144, 1,L43f Telomerase RNA, 222t,264,265f Telomeres, 27, 261, 261.f,263-264, 1143 lossof, 1143-1144,1143f,L1.44f shorteningof, 263-264, 264f structureof,11.44f Telophase, 871 initiation of, 890 in mitosis,783, 783f,789-790 in plants,790,790f Tem1,890, 890f Temperatephages,lysogenyin, 158 Temperature, melting,of DNA, 117,1.17f Temperature perception,1031-1032 Temperature-sensitive mutations,23, 1.70-171,, 1.70f in yeastcell cyclestudies,851 Ternarycomplexfactor (TCF),690, 691.1,700 Tertiarystructure,65f, 67-72, 68f, 69f,71.f, 75,7sf of proteins,65f, 67-72, 68f, 69f,71f Testis,developmenrof, 91.3-91.4 Tetrads,892 Tetrahymenathermophila histoneacetylase in, 305-305 replication|n, 263-264 self-splicing intronsin, 363 INDEX

t-47

TFIIA, 98, 297, 298 TFrrB,297, 297l, 298, 298f TFIID, 98, 253, 306 TFrrE,297f, 298, 298f TFIIF,297f,298,298f TFIIH,297f,298,298f TFIIIA, 317, 317f TFIIIB, 3r7, 317f TFilrC, 3L7, 3r7f TGF in cancer,1,1,34, 11,34f,1142-1.1.43 in neuraldevelopment, 965, 966f, 985, 987-988,987f TGFp receptors,in Smad activation,565f, 670-672, 670f, 571.f TGFp superfamtly, 668, 669f in cancer, 671,-672,671f, 1L34,1.L34f, ll42-1143 in cell division,91.3,914 in embryonicdevelopment, 963-964, 964f, 965 functionsof, 668 isoformsof, 668, 659f, 671. in neuraldevelopment, 965,9561, 987-988,987f purificationof, 559-670 Smadsin, 666f, 658-672, 670f, 671.f structureof, 658,669f s y n t h e soi sf , 6 6 8 , 6 6 9 f Thalassemia, 346 Thermalenergy,54 Thermogenesis, brown-fat mitochondriain, 510 Thermogenin,510 Thick-filament r egulation,742 Thick filaments,in sarcomere, 739-740, 739f Thin-filamentregulation,7 40, 74l Thin filamenrs. in sarcomere, 739-740,719f Thinking, neuronsin, 1001 Thiol groups,552 Thioredoxin,527 ThreeNa*/one-calciumantiporter,468 3-D adhesions,833-834, 834f 3' end cleavageof, in pre-mRNAprocessing, 124, r25f, 329,329f, 335-336,336f of DNA strand,114, 114f of Okazakifragment,741, 141,f,l42f of IRNA, 135,136f 3' poly(A) tail, post-transcriptional processing of, 335-336,336f 3' untranslatedregions(UTRs),124 3T3 cells,1713-1,1,14,'Ll1,3f 3T3-L1cellline,398,400f Threonine, 42f,43. SeealsoAmino acid(s) Thresholdpotential,1.025,L026f Thrombospondin,in synapseformation,10L9 Thylakoid(s), 379, 3791 definitionof,511 electrontransportin, 512, 513f oxidativedamagein, 521-522 structureof, 511, 5f2f Thylakoidlumen,511 Thylakoidmembrane, 379, 379f,5l2f p h o t o s y n t h e si ni s. 5 l l . 5 l 2 f structure of, 511.,572f Thymidine.Seea/soBase(s) T h y m i n e4. 4 , 4 5 r , I l 3 - t 1 4 i n d o u b l eh e l i x ,1 1 4 - 1 1 5 , 1 1 5 f structureof,44,44f Thymine-thymine dimers,repairof, 148, 1 48 f Thymosin-Ba,in actin polymerization,722, 722f Thymus,1058 Thyroxinereceptor,3L2f. Seea/soHormone l-48

.

INDEX

receptors Tight junctions,802f, 809,810f, 811t, 8 t 4 - 8 1 6 ,8 l 4 f , 8 t 5 f epithelial, 471.,47|f functionsof, 814, 815, 815f l e a k 58 1 5 , 8 1 6 f structureof, 814-815,814f Tim proteins,559, 559f Time-lapsemicroscopy,382 Time-of-flightmassspectrometry,1.01-1.02,1.01.f tinman,967 +T[Ps,767,768{ TIR fluorescence microscopy,404 Tissue artificial,404 cell integrationinto, 801-843 classification of, 801 connective,801 definitionof, 801 developmentof, 9 51.-9 52 epithelial,801 muscular,801 nervous,801 organizationof, 801-803.Seealso Cell-cell adhesion;Cell-matrixadhesion in organs,801-803 stemcellsfor,91,2-920.SeealsoStemcell(s) Tissuecompatibility,MHC moleculesin, 1077-1078,10781 Tissueplasminogenactivator(TPA),71 titan,2l7 Titin,740f. Titration curve,for buffers,53-54, 53f lk genefrom herpessimplexvirus,283,284f TNFa receptors,566f Toes,extra, 992,993f Toll-likereceptors,703, 1059, 1,061, 1.097-1099,1098f in adaptiveimmunity,1,097-1099,1'098f in antigenpresentingcell activation,1099 diversityof, 1098 in signaling,1098-1099 1098f structureof, 1.097-1.098, Toll protein,1097 Tom proteins,559, 559f Tomography,cryoelectron,389, 390f Tongue,tastecellsin, 1034-1036,1035f Topogenicsequences, 543 proteins.541-549, of integralmembrane 543f-546f hydropathyprofilesfor, 548-549, 548f, 5491 identificationof, 548-549 1.18f Topoisomerase l, 1,1,7-118, Topoisomerase II, 118 Topologicaldomains,72 Topology,of integralmembraneproteins. 41441.5, 41.4f,426, 543-549, 543f-546f TOR pathway,353-355, 354f Total internalreflectionfluorescence microscopy,404 Totipotentstemcells,907,960 Touch,L031-1032 Tra protein,alternativesplicingand, 339, 339f Transfatty acids(transfats),48 trans-Golgicisternae,580, 581f trans-Golginetwork, 580.SeealsoGolgi complex Trans-splicing, 333. Seea/so Splicing Transcellularpathway,in transepithelial transport,471,,471f, 814, 816f Transcellular transport,477, 814 Transcript nascent,cappingof, 3241,325-326, 327f primary,1.21.-L22, L22f, 323 Transcription,1,7,1,21,1,12,113f, 120-L26 in a/ctcells/mating types,922-923,922f,

923f regulation, 881-883,882f,884 in cell-cycle in cell-fatedetermination,971.-973,9721, 973f cell-typespecificationand,922-923, 922f, 923f in chloroplasts,317, 31.8 concurrentwith translation,123 differential geneexpressionand, 24, 24f directionof, 1.20,'1.21.f 277, 298 downstream,1.20,1.21,f, elongationin, 31.5,326 of, 120 energetics in eukaryotes,1,23-125,1,23f initiation ot, 72, 72f, 120, L22f activatorsin, 286-290,287f-289f, 289f,305-31.0 in bacteria,271.-276 in, 306, 307f chromatindecondensation complexesin, chromatin-remodeling 306-307,3071 combinatorialregulationof, 294J9 5, 294f,295f CpG islandsin, 282 downstreampromoterelementsin, 298 enhancersin, 1.8,274-275, 274f , 284-285, 285f, 295-296, 296f in eukaryotes,276-281 in, 298 helicases hormonesin, 31,2-313,312f, 31'4f initiatorsin, 282 MAP kinasesin, 690-691,,69lf PhoR/PhoBsystemin, 275, 275f preinitiationcomplexesin, 134, 135f, 295-299,297I, 298f, 307-308,308f promoter-proximalelementsin, 282-283,283f, 285, 376 reportergenesin, 283 in, 1.20,12Lf, RNA polymerases 271.-275, 274f, 278-281,,279t, 280f , 281.1,296-29 9, 297f-29 8f, 3 1 5 - 3 1 73 , l7f typel, 278, 279f, 279t, 316-3L7, 31.71 typeII, 134, 1.35f,279,279t, 295-299,297f, 298f, 307-308, 308f type III, 278-279,279t,317, 3l7f o (sigma)factorsin, 271,273-275, 273t,274f sitesof, 280-281,,282 two-componentregulatorysystemsin, 274-275,274f.275t in, 285, upstreamactivatingsequences 315 in,254 insulators regionsin, 254 matrix-attachment in mitochondria,3L7-3L8 mtDNA in,240 of operon,122-123. 124f,27 l-27 3. 272(, 307f polymerization in, 120, 121.f pre-mRNA in, 1.23-125,123f L22f primary transcriptin, 1.21.-1.22, in prokaryotes, 1'22-723, 1'23f promotersin, 120, 272, 273-275, 273t rate of, 1-21regulationof activatorsin. SeeTranscription, initiation of chromatin-medrated,299 -307 complexesin, chromatin-remodeling 306, 307f combinatorial,294-295, 294f, 295f extracellularsignalingrn, 312-313, 31.2t

futureresearch areasfor,318-319 mediatorcomplexin, 299, 307-308, 308f repressors in. SeeTranscriprion. repressionof in yeasttwo-hybridsysrems, 310, 311f repressionof, 29-305, 290, 299-305 chromarin-remodeling complexesin, 306-307 co-repressors in, 304-305,305f in highereukaryores, 301-303 Polycombcomplexin, 302-303,302f RNA interference in, 350-351 silencersequences in, 18, 299-30L,300I in yeast,299-301,300f,301f reverse, 230,231f telomerasein, 263-264, 265f in transposition, 230-234, 230f-233f RNA processingand, 123-1,24,725f- See a/soRNA processing rn Saccharomyces cereuisiae, 279, 279t, 281f scaffold-associated regionsin, 254 stagesin, 720-1,22,122f strandelongationin, 120-122, 122f TAIA box in, 282-283, 282f, 286, 297-298 templateDNA in, 120, lzlf terminationof, 721-122, 122f, 314-316 upstream,120, 127f, 277, 285, 31,6 in yeast,691-692 Transcription bub6le, 1,20-721,,1,22f Transcriptionconrrol elements(regions),270, 282-28s as bindingsires,286 enhancers, 1.8,274-27 5, 274f, 284-285, 28sf,286f initiators,282,286f locationof in bacteria,274-275 in eukaryotes,276-278, 277f, 278f promoter-proximalelements,282-283, 2 8 3 f .2 8 5 ,2 8 6 f , 3 1 6 T.ATAbox, 282-283, 282f, 286f, 297-298 Transcription-coupled DNA repair,149 Transcription factors,12, 112, 120,270,277, 286-296 acrivationof, 288-290, 289f, 293-294, 664-710,666f.Seea/soActivation domains;Signalingparhways MAP kinasesrn, 690-691, 691f signalingin,65t-660 activator,923 in body segmenration ln lnsects, > /+-> / /

in vertebrates, 977-97 8, 982-983 in cell differentiation, 905-906 in cell-type specification in muscle, 925-929 in yeast, 922-923, 922-925, 922f, 9231 chloroplast,3lS chromatin and,256 co-activators fo4 293 in combinatorial regulation, 294-295, 294f,295f cooperative binding of, 294-29 5, 294f,

29sf definition of,270,286 DNA-binding domainsof, 288-296, 289f. Seealso DNA-binding domains DNA-binding motifs of, 69-70,70f, 290-292, 291.f_293f in embryonicstemcells,917-9L2 gap genesas,974-977 general,253,296-297,297f , 298f in preinitiationcomplex,297--299, 297f,298f heterodimeric, 294-295

homeodomain, 291 identification of DNase I footprinting in, 286 electrophoretic mobility shift assay in, 286,287f sequence-specificDNA affinity chromatography in, 286, 287f in vivo transfection assay,288 interaction of, 294-295 mating-type, in cell-type specification,

922 mitochondrial, 318 in myoblast migration, 928 i n n u c l e a r - r e c e p r osr u p e r f a m i l y ,2 9 l .

312-31.3, 3t2t, 373f in oncogenic transformation, 1130-1131, 1,1,32f r e g u l a t i oonf , 3 1 1 - 3 1 3 repressiondomainsfor, 290 repressor, 290,923 in signaling,632-633. Seealso Signaling specific,286 Transcription-initiation complex,72f Pol I, 31,6-317, 317f Pol II, 134, 135f,296-299,297f, 298f, 307-308,308f Pol III, 317, 3l7f Transcriptionunirs,217-21I bacterial,217 eukaryotic, 217-221, 2l9f complex,218,279f mutationstn, 21.8-219,2L9f simple,217-218, 279f s t r u c t u roef , 2 l 9 f insulatorsfor,254 Transcriptionalgenecontrol. Seealso Transcription,initiation of activatorsin, 270, 270f, 27| in bacteria,27l-276 enhancersrn, 274-275, 274( PhoR/PhoBsystemin, 275, 275f RNA polymerasern, 271.-275, 274f o (sigma)factorsin, 271.,273-275, 273t,274f rwo-componentregutatorysystemsln, 274-275, 274f, 275f combinatorial,294-295, 294f, 295f coordinateregulationin, 271 in developmenr, 28-29, 291 enhancersrn, 18, 274-275, 274f, 284J8 5, 285f,295-2e6,296( in eukaryotes,276-281 functionsof,275 future researchareasfor in, 318-319 in geneticprogram execution,276 overviewof, 269-270, 270f post-transcriptional, 323-367. Seealso Post-transcriptional genecontrol repressors tn, 270, 270f, 271. silencersin, 1,8,299-301,,300f Transcripts,processingoI, 123-124, 125f. See a/so RNA processing Transcytosis, 505 of immunoglobulins, 1065-1066, 1,06 5f Transducin,rhodopsinand, 641, 644f Transducingretroviruses,1122 Transepithelialtransport,470-472, 47|f, 472f Transesterification, in splicing,330, 330f, 332, 332f Transfection,1.95-1,97 in secretorypathwaystudies,582 stable,196-1.97,l96f transient,196,196f in yeast,261,-263,262f Transferproteins,in lipid transport,

+55 TransferRNA. SeeIRNA (transferRNA) Transferrin cycle, 611-612, 617f Transferrin recepror{TfR).356. 357F Transformation in cell cultures,398 of tumor cells,1113-1114,1,1,141 chloroplast,242 oncogenic,1.1.1.3-1114, 1114f PI-3 kinaseand,694-695 in plasmidcloning,1.78-779 transformer,alternativesplicingof, 339, 339f Transforminggrowth factor B. SeeTGFB Transfusions, blood types and,426 Transgenes, 209 Transgenicanimals,209, 209f Transgenicplants,470 Transientamplifyingcells,905, 916-91.7, 91.7f,91.8f Transientreceptorpotentialcation channels, 843 Transienttransfection,1.96,196f Transitionstate,56-57, 57f, 79, 80f Translation,11, 72f,74, 1,1,2,113f, 1,27-131, 21,7 in cell-cycleregulation,881-882 973-974,973f, in ceff-faredetermination. 974f chain elongationin, 1.35-137,l36f in chloroplasts,557 codonsin, 127-129, 1,28r,129f concurrentwith transcription,'123 cytoplasmicpolyadenylationin, 351-352, 351.1 delnition oI, 127 eIF2kinasesin, 355-356 G proteinsin, 354-355 initiationof, 127-128,128t, 130f, 1 3 3 - 1 3 51 , 34f,355-356 sitesof, 217 in learningand memory,352 in mitochondria,557 mRNA in, L27, l27I mRNA degradationin, 352-353, 3521 mRNA surveillance in, 357 nonsense suppression in, 138 P bodiesin, 348, 353 polyribosomes in, 138, 139f poly(A) tail lengtheningin, 351-352, 3s1f preinitiationcomplexin, 134 in prokaryotes,123 proofreading in, 131, 1,45,146f,355 in protein targeting,537-538,537f rateof, 133, 138 readingframesin, 128, 1.291 regulationof, 323-325, 3241.Seealso Post-transcriptional genecontrol;RNA processlng cytoplasmic, 351-353 global,353-355 sequence-specific. 356-357.357f repressionof eIF2 kinase-mediated, 355-356 miRNA-mediated,347-3 49, 3 48f RNA-inducedsilencingcomplexin, 348 Rhebprotein in, 354-355, 354f ribosome in, 132-139 rRNA in, 1.27,L27f, L32-1.39,1.35f,1.36f. Seealso Ribosomes of specificmRNAs, 356-357, 357f stepsin, 130f terminationof, L37-1.38,l37f 3' regulatoryelementsin, 351-352, 351f' TOR pathwayin, 353-355,354f INDEX

t-49

Translation (continued) IRNA in, 127,1,27f Translocation(s) chromosomal a n a l y s i so f , 2 5 8 , 2 5 9 f in cancer, 1,1,30,11321 in proto-oncogenes, 1 120 protein. See Protein translocation ribosomal, 1.35-137,136f Translocons, 539-540, 540f Transmembrane proteins. See Membrane proteins, integral (transmembrane) Transmissible spongiform encephalopathy, 77 Transmission electron microscopy, 388-389, 389f T-^-^^l^-.^.:^r r drLrPr4u r4 Lruil

bonemarrow,920 MHC moleculesand, L077 Transport electron,493-503.Seealso Electron transport membrane.SeeMembranetransport transcellular, 471.,471.f transepithelial, 470472, 471.f,472f vesicular,579-606. Seealso Vesicular transport Transportproteins membrane.SeeMembraneffansport proteins nuclear,342-347. Seealso Nuclear transport Transportvesicles,376-377, 375f, 579-606, 580. Seea/soVesicles;Vesiculartransport Transporters,439, 439f, 440, 441443, 441.f, 442f. Seeaiso Membranetransport proterns ABC.447f. 448, 45445 6. 4554 50,45 5t flippasesand, 456, 456f in geneticdiseases, 455456 antiporters,440, 466, 468470. Seealso Antiporters conformationalchangesin, 440 cotransporters, 440, 440t exocytosisof, 1020-1021,1.020f in glucosemetabolism,441.443, 441.f, 442f, 471.,47 tf, 488f, 489491, 490f, 545f,547. Seealso under GLUT as multipassintegralmembrane proteins,547 G L U T ,4 4 1 4 4 3 , 4 4 l f , 4 4 2 f, 4 7 l , 4 7 l f . Seealso under GLUT for neurotransmitters, 1020-1022,1021.f phosphate,in MP-ADP exchange,509, 509f symporters,440, 456470, 471 in transepithelial transport,471.,471f uniporters,440, 441.-443,441.f,442f Transposable elements.SeeMobile DNA elements Transposases, 228-229 Transposition,226-227 Ac elementstn,228-229 of Alu elements,234 in bacteria,227-229, 227fJ29f cut-and-copymechanismin, 227 cut-and-paste mechanism\n, 227, 228, 228f,229 definitionof,226 of Ds elements,228-229 in eukaryotes,229-234 integrase in,230,233 of IS elements,222-228, 227-228, 228f of LINEs, 230-234, 232f, 233f P elementsin, 229 retrotransponsin, 227, 229-23 4 reversetranscriptionin, 230-234, 230f-233f of SINEs,234 l-50

o

|NDEX

somatic-cell, 226 Transposons, 227-229, 227f-229f , 265-26 6, 350. Seea/soRetrotransposons bacterial,227-228, 227f definitron of,227 eukaryotic,228-229, 229f in exon shuffling,235 multiplicationof, 229, 229f Transthyretin,282-283, 309-310, 310{ Transverse tubules,7 40, 7 41.f Treadmilling in actinfilamentassemblg7Zl-722,721.f , 722f in microtubules, 753, 763f in mitosis,785, 786f, 787f, 788 Triacylglycerides, 48 Triacylglycerol, 491 Tricarboxylicacid cycle,487489, 489f Triglycerides,48,491,.Seealso Lipid(s) TrimericC proreins.355.637-639,638f in signaling,634 in, 644 subunitdissociatron Trimolecularcargocomplex,573 Triplet code.SeeGeneticcode Triplermicrotubules, 7o0, 760f Triskelions, 598,598f TrisomS 887 Trithoraxproteins,302, 303, 306,982-983 Triton X-100, 428,428f trk oncogene,1.128,1L28f Trks, in neuronsurvival,938 I R N A ( t r a n s f eRr N A ) , 1 1 2 , 2 1 7 , 2 2 2 t in amino acid activation,131 amino acid linkageto, 129, 1.30f,L3L in,127,129 anticodons codon recognitionby, 727-1.31' cognate,131 diversityof, 129 folding of, 129,1.30f functionsof, 127, 1.27{,1.29 13 1 isonine-containing, 133 methionine-containing, nonstandard basepairingand, 130, 131f processing o\ 363, post-transcriptional 363f,364f processing of, 363-364, 365f p r o m o t e ri sn . J l 7 , 3 t 7 f rRNA and, 137 structureof, 118, 1.29,1.30f synthesisof. SeealsoTranscription III in, 278,279t RNA polymerase in translation,1.27-131. Trophectoderm, 960 Trophicfactors,in apoptosis,243f,936, 942-943 Trophoblast, 962,962f Tropomodulin,723, 740, 740f Tropomyosin,740-7 41., 7 41f Troponin,740-747, 741.f trp operon,1.24f ,21.7 True-breedingstrains, L67-1-69 Trypsin activationof, 91 activesite of, 80f, 82-83, 82f Tryptophan,42, 42f. Seealso Amino acid(s) TSCl/TSC2,354-355 Tsix, tn genomicimprinting,958-959 TTR, 282-283, 283f of, 309-310,310f transcription Tuberoussclerosis, 354, 355 Tubulin, 7 58, 760f ct form of, 758,760f B formof,758,760f in neurogenesis , 929, 930f of, 763 critical concentratton 1 formof,761,,762f

homologyin, 244, 2451 sequence Tumor(s),1108 benign,1109 hypoxic,1.L09, 1.1.L2-1113 Seealso Cancer malignant,1.1.09-7110. Tumor cells,identificationof, 7716-1J.13,1.11.8f 1111 Tumor microenvironment, Tumor necrosisfactor alpha (TNFo), 703 Tumorstemcells,1111 l1.l8f identificationof, 1.1.16-1119, Tumor-suppressorgenes,882, 1107, 1122t inherited mutations in, 1-123 mutationsin, 148,'l'1'23 loss-of-function in, 11'24,1'1'25f lossof heterozygosity Rb protein,882-883, 882f Tumor-suppressor in cell-cycleregulation,891 Tumor suppressors, Tumorviruses,158-159 1108 Tumorigenesis, Turgor,377-378 Turgor pressure,444, 830 Turnovernumber,8L 20S proteasome,87-88, 87f gel electrophoresis, 95-96, Two-dimensional 95f, 106 symporteg466457, Two Na*/one-glucose 465f, 471. t/one-leucine symporteg467468, 467f Two Na zJz, zSzr Iy elements, Tyrosine,42, 421.Seealso Amino acid(s) U1 snRNA,330, 331f U1 snRNP,332,332f U2 snRNA,330, 331f U2 snRNR332,3321 U4 snRNP,332,332f U5 snRNl 332,332f,333 U6 snRNl 332,332f Ubiquinone,in electrontransport,495t,496, 496f Ubiquitin, 88, 858 610 in endocytosis, pathway,protein Ubiquitin/proteasome degradationin, 555 Ubiquitin-proteinligases.Seealso APC/C complex in cell-cycleregulation,850, 850f, 851' 8 5 8 ,8 5 9 f ,8 7 6 - 8 7 7 , 8 8 38, 8 6 t Ubiquitination in cell-cycleregulation,850f, 851 in anaphaseinitiation, 867-869, 868f of B-typecyclins,858, 858f in mammals,850, 850f,851, 858' 859f,876-877 in Xenopouslaeuis,858,859f in yeast,876-877 867-859, 868f in chromosomesegregation, of histones,250, 250f, 251'-252 Ultravioletradiation leukemiadue to, 1139-1140 mutationsdte to,'1.46-1.47 Umami tasteperception,1035f, 1036 Unc proteins,1045 spacerDNA, 220t, 225-226 Unclassified Uncouplers,5l0 Unfolded-proteinresponse,555, 555f Uniporters,440, 441'443, 441446, 441'f, 442f. Seea/soTransporters transport,471',47If in transepithelial Unipotentstemcells,907 Unsaturatedfatty acids,47-48, 47t Untranslatedregions(UTRs),124 285, 3L6 Upstreamactivatingsequences, Upstreamtranscription,1.20,121.f , 277, 285, 316 IJracll,44, 45t, 113-11'4.Seea/so Base(s) in RNA, 118 structureo1,44,44f Ushersyndrome,1033-1034

V - c l a s sp r o t o n p u m p s , 4 4 7 f , 4 4 8 , 4 5 3 4 5 4 , 453f. See a/so Pumps in synaptic vesicle transport, 1019-1020 V segments in heavy chains, 1069, 1069f,1071,-1,073, L072f in light chains, 1069-7071, 1069f, 1070f i n T - c e l l r e c e p t o r s ,1 0 8 8 , 1 0 8 9 - 1 0 9 1 , , 1 , 0 9 0 f v-SNAREs, 585, 586f, 591., 1022 v-s/c proto-onc ogene, | 121-1 122 Vaccines, 1 1,07-1,1,02 V a c u o l a r m e m b r a n e ,3 7 7 - \ - 8 , 3 - - f Vacuoles,373f acidification of, H* MPases in, 453-454 contractile, 444 plant, 377-37 8, 469-47 0, 469 f Y a l i n e , 4 2 , 4 2 f . S e ea / s o A m i n o a c i d ( s ) Valinomycin, 502 VAMB 591, 1022 V a n d e r W a a l s i n t e r a c t i o n s ,3 7 - 3 8 , 3 8 f i n p h o s p h o l i p i d b i l a y e r ,4 1 1 , 4 1 8 4 1 9 Variable region of heavy chains,1066f, 1067 of lighr chains,1,066-1067,10661 Variation, genetic, 7. See also Mutations in evolution, 28-29 V a s c u l a rC A M - 1 , 8 3 5 Vascular endothelial growrh factor, in cancerr e l a t e d a n g i o g e n e s i s1, 1 1 2 - 1 1 1 3 Vascular muscle, protein kinase G and, 656-557, 657f V a s c u l a rt i s s u e ,i n p l a n t s , 8 3 9 , 8 3 9 f VASP,726 Vault RNA,222t Vector(s) BAC,179 bait,310,311f definition of, 176 DNA insertion rnto, 777-178 D N A p r o c e s s i n gf o r , 1 7 6 - 1 7 7 , 1 , 7 6 f expression, 1,94-197. See also Gene expression studies bacterial, 194-1.95, 794-196, 195f e u k a r y o t i c , 1 , 9 6 - L 9 8 ,l 9 6 f - 7 9 8 f in gene/protein tagging, 197-198, 1,98f plasmid, 195-196, 795f retroviral, 197, l97l fish,310,311f plasmid, 178-179, 178f, 179f e x p r e s s i o n ,1 9 4 - 1 9 6 , 1 95 f in prorein production, 195-196, 795f shuttle, 183, 183f viral, 6 Vector DNA, 176-178. See also Clones/cloning, DNA Vegetal pole, 963 VEGF proteins, in cancer-relaredangiogenesis, 1112-1713 VEGF receptor antagonists, for cancer, 1113 VelocitS maximal (V",",), 80, 80f Ventricles, cerebral, 916 Vesicles aggregatedproteins in, 602 a u t o p h a g i c ,5 1 , 4 - 6 1 6 , 6 l 5 f budding of, 580, 581f, 586-587,586f, 587f c o a t a s s e m b l ya n d , 5 8 6 - 5 8 8 , 5 9 2 f donor/destination organelles in, 585 dynamin in, 599-600, 599f,600f i n e n d o c y t o s i s ,4 1 4 f , 5 8 1 , 6 1 0 ,

612-614, 6r3f, 1023 pinching off and,599-500,5991, G00f in retroviruses, 614 from trans-Golgi,599-600, 599f in vesicularrransporr,599-600,599f, 600f, 6L3. Seealso Vesicular

rransporr clathrin/AP-coated, 393, 586-589,587t, 589t,598-600,598f-500f i n o r g a n e l lpeu r i f i c a r i o n 3 .9 3 - 3 9 4 3. 9 3 f pinchingoff of. 59e-o00.5q9f, 600f, 1023 dynaminin, 1023 in receptor-mediated endocytosis, 606-607,607f uncoatingof, 600 COPI,s86-589, 587,587r,589t, 595 C O P I I ,5 8 6 - 5 8 9 5, 8 7 f , 5 8 7 t 5 , 8 8 f ,5 8 9 t , 592-593, 592f-594f, 595 fusionof, 535, 585, 585f,589-591,590f in exocytosis,414f, 580, 591 synaptic,1022 GoIgt,376-377, 377f secretory,373f, 376-377, 376f, 580, 602-603 constitutive,602 regulated,602-603 synaptic, 1019-1022, 1020f, 1021f. See a/soSynapticvesicles rransporr,376-377, 376f, 579-606. See a/soVesiculartransport uncoatingof, 500 Vesicularglutamatetransporters(VGLUTs),1020 Vesicular transport,376-)77, 549-606.See a/soCell movement/migration anterograde, 580, 58lf, 592-593,592f, 595-597,596f cargoproteinsin, 580, 588-589,593 in cls-Golgi,592-597, 592f-596f cisternalmaturationin, 580, 596, 597, 597f clathrin/AP-coated vesiclesin, 393, 586-589,587t,598-600,598f-600f pinchingoff of, 599-600,599f,600f uncoatingof, 500 coat assembly in, 587-588,588f endocytosisin, 414f, 581 endoplasmicreticulumto Golgi, 592-593, 592f-594f enerBysources for. 587-588,5ql exocytosisrn, 414f, 580, 591 experimentalstudiesof, 580-586 future researchdirectionsfor, 61.7 intercompaftmental k i n e s i nisn , t a l - 7 - 2 , 7 7 l f lysosomal,600-602, 600f, 601f, 608-610, 609f,612-616 mannose6-phosphatein, 600-602,500f, 601.f mechanismsof, 586-592 membrane fusionin, 474f, 535,585, 586f, 5 8 9 - 5 9 1 5, 9 0 f microtubules in, 593, 769-770,770f mitosis-promoting factor in, 866 overviewof, 580, 581f proteintargetingin, 588-591,600-602. SeealsoProteintargeringl Sorring signals to apicalor basolateral membrane, 504-60s,605f transcytosis in, 605 proteolyticprocessingin, 503-604, 603f, 604f Rab proteinsin, 589-591,590f retrograde, 580, 581f, 592, 594-595,596, 596f in secretorypathway,533-535, 534f, 579-606. Seealso Secretorypathway in early stages,592-597 in later stages,597-606 of secretoryproteins, 602-605 signalingin, 589, 589t,594-595,594f, 600-602. Seealso Sortingsignals SNARE proteins/complexes in, 586, 586f,

591 in trans-Golgi,597-606, 598f-601.f, 603f, 604f vesiclebuddingin, 585-587, 599-600, 599f, 600f, 613. Seea/soVesicles, buddingof vesicledockingin, 589-590, 590f vesicletargetingin, 589-591 i n y e a s t5, 8 4 ,5 8 4 f , 5 9 1 . , 5 9 6 , 5 9 7 f Vibrio cholerae,639-640, 816 Villi, intestinal, 471,916, 976f Vimentin,793t,794 Viral capsids,154-155,155f Viral DNA. replication of. 142-143,142(.l43f Viral envelope,154, 155f Viral infections time courseof, 1101f vaccinesfor, 1,1.01.-71.02 Viral oncoproteins,1121.-1723,11,22f, 1.1.28-L1.29, L1.29f Virions, 154 b u d d i n go f , 1 5 8 , 1 5 8 f progeny,1.56,1.56f,158, 158f,159f Virus(es),6, 154-1.59.Seealso Retrovirus(es) animal,154 buddingof,5l4, 675f cancer-causing, 1.1.21-11.23, 11.22f, L128-1.L29,1.L29f cloningof, 155 definitionof, 154 DNA, 154 oncogenic,1122-1123 DNA replicationin, 142-143, 1,42f,l43f enveloped, 154-155,155f retroviruses as, 158, 159f as experimentalorganisms,25 genomeof, 154 helical,154, 155f host rangeof, 154 icosahedral, 154, 155f infectivemechanisms of, 1057 integrinsand, 838 membraneinvasionby, 409, 409f mRNA transportin, 346-347, 346f oncogenic,1121-11,23 phage,154, 158 plant,154 plaqueassayfor, 155, 156f quantificationof, 155 replication of,1.057 lytic, 156-158,155f,1.57f nonlytic,158, 159f in plaqueassay,155 reproduction of,5,5f RNA, 154 structureof,6,6f tumor,158-159 veclor, b Virus-associatedRNA, in translation, 356 Viscometrg in actin polymerizatron, 7 19 Vision, 1027-1031. See also Eye acuity of, 1028 alternative splicing in, 340 bipolar cells in, 1.027f, 1029-7030 color, 7027f, 1028 evolution of, 1028-L029 image interpretation in, 1029-1031, 70301 integrative processesin, 1029-7037, 1.029f-1.03If neuronal organization in, 1,029-1031,, 1,029f p a t t e r n r e c o g n i t i o ni n , 1 0 2 9 - 1 0 3 1 , 1 0 3 1 f primary cilia in, 780 receptive fields in, 1028-7030, L029f signal transduction tn, 641-645, 642f-645f s p a t i a l p r o c e s s r n gi n , 1 0 3 1 , 1 0 3 1 f Visual adaptation, 644-645 INDEX

t-51

Visualcortex, 1028 Visualpathways,1002f Vitamin C deficiency,825 V-,* (maximalvelocity),80, 80f Voltage,electrochemical gradientand, 439, 464,465f Voltage-gated ion channels.SeeIon channels Von Behring,Emil, 1062-1063 Von \Willebrandfactor,834 VSV G protein transport,582-583, 582f Wall-associated kinases(\7AKs),84L-842 WASpprotein,725-726, 725f Water.Seea/so Solutions membranetransportof, 444445, 444f446f Water-channel proteins(aquaporins), 423424, 444445, 444f446f, 445 in diabetesinsipidus,445 structureof, 424, 424f, 446f Watermolecule,dipole natureof, 34, 35f Watson-Crickbasepairs, 114-715, 11.5f Watson,James,114 Weeprotein,in mitosis,860,862,862f Weel kinase,886t Weel protein-tyrosine kinase,852 Westernblotting, 98, 99f White blood cells.SeeLeukocyte(s) Whole genomeshotgunsequencing, 187 Whoopingcough,640 Wiechaus,Eric, 17l, 999-1000 Wiesel,Torsten,1031 \fild type organisms,166, 167l Wilkins, Maurice, 114 Wilms' tumoq 290,290f Wilson,H.V., 805 wingless, 699,702 Wnt pathway,667f, 697-698, 699-700 in axon guidance,1046-1047 in body segmentation, 909,914-915 in cancer,1125 in limb development,992-994,993f in stemcell differentiation,909,91,4-91,5 Wnt receptors,666f Wobbteposition,nonstandardbasepairing at, 130-131,131f Wound healing,dendriticepidermalT cellsin, 91,s 'Wounded-cell monolayerassay,748, 7 50f

l-52

.

INDEX

X chromosome, 1.3,1.3f,1,9,1.9f,955 dosagecompensation and, 253, 9 58-959, 9 59f inactivationof, 253, 958-959, 959f in heterochromatin formation,253-254 X-inactivationcenter,958, 959f X-linked recessive inheritance,200, 200f X-ray crystallography,22, 37, 38f Xenopuslaeuis mitosisregulationin, 856-858,856f,857f, 867-869, 868f neurogenesis in, 929, 989-990 oocytematurationin, 854-856, 854f, 855f n4ttPtnrno

rn

anterior-posteri or, 965-966, 966f 963-965 dorsal-ventral, 42, 1'1,42t Xerodermapigmentosum,148-1,49, 1.'1. Xist protein,222t,253 in genomicimprinting,958-959 XMAP2157 , 8 5 ,7 8 5 f Xolloid,965,966f

Y c h r o m o s o m e , 1 , 3 , 7139f ,, 1 , 9 f , 9 5 5S. e ea l s o Sexchromosomes Yeast,5. Seealso Ftngi in. autonomously replicating sequences 261,262f buddingin,872,873f c d cm u t a t i o nisn , 1 7 0 - 1 -l , l - 0 f cell-cycleregulationin, 851-852, 852f, 853t, 859-853, 860f-863f cell-typespecificationtn, 921-924, 922f-924f in, 261-263, 262f, 263f centromeres cytoplasmicinheritancein, 237-238, 2 3 7 - 2 3 82. 3 8 f . 2 3 8 1 as diploid vs. haploid organism,166, 169f 1.69-1.70, DNA replication, 877-879, 878f as experimentalorganisms,26 functionsof, 5 geneinactivationin, 205, 206f meiosisin, 895 mitochondrialDNA in, 237, 238f myosinV motors in, 743-744,743f nucleartransportin, 343-344, 344f

protein translocationin, 540-541, 542f pyruvatesynthesisin, 485 reproductionin, 7,71 in, 350-363 rRNA processing pathwayin, 584,584f secretory telomeresin, 261.,2621,263 transcription repressionin, 299-301.,300f, 301f transfectionexperimentsrn, 261-263, 262f vesiculartransportin, 584, 584f, 591, 596, 597f Yeastgenomiclibraries,180-181, 182-184, 18 3 f Yeastmatingsystems,MAP kinasesignaling in, 691.-692,692{ Yeastmating type(sl, 7, 7f alu, 169,170f in asymmetriccell division,930-93L, 9311 cell-typespecificationin, 922-923, 9221,923f 299-300, Yeastmating type silencersequences, 300f YeastSR proteins,343-344,344f Yeasttwo-hybrid systems,310, 311f YeastTy elements,232,232f YXXO sortingsignal,589, 589t,609-610

Z d\sk, 7 39, 739f, 7 40, 741.f desminand, 794 z DNA, 115, 115f Zebrafish as experimentalorganisms,26 transportproteinsin, 469, 469f Zellweggersyndrome,568 Zig-zagribbon, in chromatin,249,249f Zinc-I\nger motif, 69-7 0, 70I, 29 1',29 1'f. See a/soNuclearreceptor(s) 95 5-957,9 56f Zona pellucida, Zone of polarizingactivity,991, 99lf in fertilization,956f, 957 ZP glycoproteins, ZwtllelPinhead,920 Zwitterions, 52 Zygotes,S, 854, 950 of, 950, 960-96L,960f cleavage as stemcells,907 Zyklon B, 498499 Zymogens,9L