GTPases in Biology I

GTPases in Biology I

Burton F. Dickey Lutz Birnbaumer

Section I: Biological Importance of GTPase-Driven Switches

CHAPTER 1

GTPases Everywhere !H .R. BOURNE . With 5 Figures 3

A. Introduction 3

B. The GTPase Cycle and the Molecular Switch 3

C. Structure of the GTPase Switch 4

D. Primary Structures Identify GTPases with Related Functions 6E. Uses of the GTPase Switch : Stoichiometric Activation 7

F. Uses of the GTPase Switch : Assembling a Complex 9

G. Other Potential Uses of the GTPase Switch 1 2

H. Cascades of GTPases 1 2

1 . Perspectives 1 4References 1 4

CHAPTER 2

Proofreading in the Elongation Cycle of Protein SynthesisE . BERGMANN and F . JURNAK . With 2 Figures 1 7

A. Introduction 1 7B . General Concepts 1 8

1 . Specificity 1 8II . Proofreading 20

C. Parameters of Protein Biosynthesis 22D . EF-Tu-Dependent Kinetic Proofreading 23E. EF-Tu-Independent Error Correction Mechanisms 26

1 . Peptidyl Transfer 26II. EF-G-Dependent Translocation 26

III. Allosteric Linkage Between A and E Sites 2 8F. Summary 2 8References 29

CHAPTER 3

A New Look at Receptor-Mediated Activation of a G-ProteinL. BIRNBAUMER . With 1 Figure 3 1References 3 6

CHAPTER 4

Small GTPases and Vesicle Trafficking : Sec4p and its Interactionwith Up- and Downstream Element sP. NOVICK and P . BRENNWALD . With 2 Figures 3 9

A. Introduction 3 9B. The Sec4 Cycle 4 3

1 . A Cycle of Sec4 Localization 4 3I1 . Intrinsic Properties of Sec4 4 4

1I1 . GTP Binding and Membrane Attachment Are Essential fo rSec4 Function 4 4

IV. GTP Hydrolysis Is Important for Sec4 Function 4 4C. Accessory Proteins in the Sec4 Cycle 45

1 . A Specific Sec4 GAP Is Present in Yeast and Mammalia nCells 45

11 . GDI from Bovine Brain and Yeast Solubilizes Sec4 in aNucleotide-Specific Fashion 46

III . Suppressors from Yeast and Rat Brain Encode Nucleotid eExchange Proteins 46

D. A Potential Downstream Effector of Sec4 Function : TheSec8/Sec15 Complex 47

References 48

CHAPTER 5

Cytoskeletal Assembly : The Actin and Tubulin NucleotidasesM.-F . CARLIER and D . PANTALONI . With 1 Figure 5 3

A. Introduction 5 3B. The Nucleotidase Cycle in the Polymerization of Actin and

Tubulin 5 3C. Elementary Steps in NTP Hydrolysis on Actin Filaments an d

Microtubules : The Regulation of Polymer Assembly 55D. Nucleotide and Metal Ion Binding to Actin and Tubulin 57E. Probing the Nucleotidase Mechanism of Actin and Tubulin usin g

A1F4 and BeF3, H 2 O 58F. Conclusions 59References 60

CHAPTER 6

Dynamin, A Microtubule-Activated GTPase Involved in Endocytosi sR.B . VALLEE, J .S . HERSKOVITS, and C.C . BURGESS. With 3 Figures 6 3

A. Introduction 6 3B. Structure and Enzymatic Properties 63C. The Drosophila Shibire Gene 66D. Transfection of Dynamin into Cultured Mammalian Cells 68References 7 1

CHAPTER 7

Transmembrane Protein Translocation : Signal Recognition Particleand Its Receptor in the Endoplasmic Reticulu mP .J . RAPIE.IKO and R . GILMORE . With 3 Figures 75

A. Introduction 75B. The Signal Recognition Particle and Its Receptor 75C. Protein Translocation Across the Rough Endoplasmic Reticulu m

Requires GTP 76D. Binding and Hydrolysis of Guanine Ribonucleotides by Signal

Recognition Particle and Its Receptor 78E. Site-Directed Mutagenesis of SRa 79F. The Sorting and Targeting Functions of Signal Recognitio n

Particle are GTP Independent 80G. Current Models for GTP Function During Protein Translocation

8 1References 83

CHAPTER 8

GTPases and Actin as Targets for Bacterial Toxin sK. AKTORIES and I . JUST 87

A. Introduction 87B . General Features of ADP-Ribosylating Toxins 87C. ADP-Ribosylation of Elongation Factor 2 by Diphtheria Toxin

and Pseudomonas aeruginosa Exotoxin A 8 91 . Introduction 8 9

II. Diphtheria Toxin 8 9III. Pseudomonas aeruginosa Exotoxin A 9 0IV. Functional Consequences of the ADP-Ribosylation o f

Elongation Factor2 9 1D. ADP-Ribosylation of G-Proteins 9 1

I. Introduction 9 1II. Cholera Toxin 9 1

III. Heat-Labile E . coli Enterotoxins 92

IV. Functional Consequences of the ADP-Ribosylation o fG-Proteins by Cholera- and Heat-Labile E . coliEnterotoxins 92

V. Pertussis Toxin 94VI. ADP-Ribosylation of Gi , G o , and Gt by Pertussis Toxin 94

E . ADP-Ribosylation of Small GTPases 9 5I . Introduction 95

11 . C3-Like ADP-Ribosyltransferases 9 5III. Functional Consequences of the ADP-Ribosylation of Rh o

Proteins 97IV. ADP-Ribosylation of Small GTPases by Pseudomonas

aeruginosa Exoenzyme S 9 9F. ADP-Ribosylation of Actin 9 9

1 . Introduction 9 9II . Clostridium botulinum C2 Toxin 10 0

III . Other Actin-ADP-Ribosylating Toxins 10 0IV. Functional Consequences of the ADP-Ribosylation of Actin

10 1G . Perspectives 102References 102

Section H. Structure of the GTPase Switches

CHAPTER 9

Eukaryotic Translation Factors Which Bind and Hydrolyze GT PJ . CavaLLius and W .C . MERRICK . With 2 Figures 115

A. GTPase Factors 11 5B. Consensus Sequences of GTPases Factors 117C. Evolution of EF-1a 11 8D. The EF-Tu Family 12 2E. Structures of the EF-Tu Family 12 6References 12 8

CHAPTER 10

Heterotrimeric G-Proteins: a, ß, and y SubunitsH. ITOH and Y . KAZIRO . With 5 Figures 13 1

A. Introduction 13 1B. Mammalian G-Proteins 13 1

1 . a Subunits 13 11 . Isolation of cDNAs and Genomic DNAs 13 1

a) G s 13 1b) Gi 134

c) Goa 13 5

d) G,, ,and Ggust a 13 5

e) GZa 136

f) Gqa and G 12 a 137

2. Comparison of the Amino Acid Sequences 137

a) P Region 137

b) G' Region 137

c) G Region 13 8

d) G" Region 13 8

e) Cholera Toxin ADP-Ribosylation Site 13 8

3 . Sequence Conservation 13 9

4 . Evolutionary Tree 13 9

II . fly Subunits 13 9

C. G-Proteins in Lower Eukaryotes 140

1 . G-Proteins from Saccharomyces cerevisiae 14 1

1. Two a Subunits, GPA1 and GPA2 14 1

2. /land y Subunits 14 1

II . G-Proteins from Schizosaccharomyces pombe 144

III . G-Proteins from Caenorhabditis elegans 144

IV . G-Proteins from Plants 144

References ` 14 5

CHAPTER 1 1

Molecular Diversity in Signal Transducing G-Protein sL . BIRNBauMER . With 1 Figure 15 1

A. The a Subunits 15 11 . Molecular Diversity 15 1

11 . a Subunit Functions 15 3B. The # y Dimers 15 5References 156

CHAPTER 12

Structural Conservation of Ras-Related Proteins and Its Functiona lImplication sP . CxaRDIN. With 2 Figures 15 9

A. Introduction : The Discovery of Ras and Ras-Related Genes 159B. Sequence Comparisons 163

1 . The N-Terminal Extension 16 611 . The Phosphate-Binding Part 16 6

1II . The Guanine-Binding Part 16 7IV. The C-Terminal Extension 16 8

V. The CaaX Motif 168

C. Evolutionary Relationships 16 81 . Construction of a Homology Tree 16 8

II. Insertions and Deletions 16 9III. Estimation of the Number of Ras-Related Proteins i n

Mammals 17 0D . Discussion 17 1

1 . Internal Residues 17 1II. External Residues and Potential Targets for Interactin g

Proteins 17 1III. Relation to Other GTPase Families 172IV. Is There a Conserved Functional Mechanism for Al l

Ras-Related Proteins? 173References 173

CHAPTER 1 3

Conformational Switch and Structural Basis for Oncogenic Mutation sof Ras ProteinsS .-H . KIM, G.G. PANE, and M .V . MILBURN . With 5 Figures 177

A . Introduction 17 7B . Conformational Switch 17 8

I. Conformational Differences Between GDP- and GTP-BoundRas Proteins : Switch I and II Regions 17 9

II. Conformational Domino Effect and Frozen Dynamic States

18 3III. Small Conformational Changes in the Phosphate-Binding

Loop, Ll 186C. Structural Basis for Oncogenic Mutations 18 6

1 . Mutations at Gln-61 and the Stabilization of the Transitio nState of the y-Phosphate of GTP 186

II. Mutations at Gly-12 and the Stabilization of the TransitionState of the y-Phosphate of GTP 189

III. Residues 12 and 13 Form a Type II fl-Turn for PhosphateBinding 189

IV. Mutation at Ala-59 and Switch II Conformation 19 1D. Discussion 19 2References 19 3

CHAPTER 14

Structural and Mechanistic Aspects of the GTPase Reaction o fH-ras p2 1A . WITTINGHOFER, E.F . PAI, and R .S . GOODY . With 3 Figures 19 5

A. Introduction 195B. The Structure of the p21-Triphosphate State 19 5C. The Structure and Biochemistry of p21 Mutants 198

D. The Kinetic Mechanism of the GTPase Reaction 199

E. The Kinetic Mechanism of the GAP-stimulated GTPase 20 1

F. GTPase Mechanism 203

G. Arguments For and Against the Proposed Mechanism 204

H. Role of GAP in the Chemical Mechanism 206

I. Conclusion 20 8

References 20 8

CHAPTER 1 5

Analysis of Ras Structure and Dynamics by Nuclear Magneti c

ResonanceS.L. CAMPBELL-BURK and T.E. VAN AKEN . With 9 Figures 21 3

A. Introduction 21 3

B. NMR Studies of Proteins 21 4

1 . NMR Structure Determination 21 4

1. NMR Methods : Larger Proteins 21 4

2. NMR Resonance Assignments : Application to Ras 21 4

3. Secondary Structure Determination : Application to Ras 21 6

4. Tertiary Structure and Structure Refinement 21 9II . , Comparison of Solution and Crystal . Structures 219

1. Computer Simulation : Ras•GMPPNP Solution Structure 2202. Protein Dynamics 220

C. Comparison of Full length and Truncated Ras Proteins 22 1L Protein Stability : Sample Preparation 22 1

II. Chemical Shift Differences 222III. Selective Isotope Enrichment Studies: Site Specific Probes 223

1. Identification of C-Terminal Peaks 2232. Internal Dynamics 2233. Comparison of Intact Ras-GDP and Ras•GMPPCP 225

D . Comparison of Ras•GTP, Ras•GTPyS, Ras•GMPPC Pand Ras-GDP 2261 . Chemical Shift Differences 226

E. Kinetic Measurements 2281 . Kinetic and Fluorescence Studies 228

II. 31P NMR: Ras•GTP Hydrolysis 228III. [ 111- 15 N]-Edited NMR Spectroscopy : GTP Hydrolysis 230

F. Conclusion 230References 23 1

CHAPTER 1 6

Molecular Dynamics Studies of H-ras p21-GTPC.K . FOLEY, L.G . PEDERSEN, T.A . DARDEN, P .S . CHARIFSON ,A. WITTINGHOFER, and M.W . ANDERSON . With 3 Figures 235

A. Introduction 23 5B. Methods 236C. Results and Discussion 237

1 . General Features of the Wild-Type Simulations 2371. RMS 2372. Protein-GTP Contacts 2383. Secondary Structure 239

II . Mechanism of Hydrolysis 239References 244

Section III : Small Ras - Related GTPases

A. Control of Growth and Differentiation by the Ras Famil y

CHAPTER 17

The Discovery of Ras and Its Biological ImportanceR .A . WEINBERG 249References 256

CHAPTER 18

Oncogenic Activation of ras ProteinsG .J . CLARK and C.J . DER . With 1 Figure 259

A. Introduction 259B . Oncogenic Versions of Cellular ras Genes Detected in Tumo r

Cells 26 01 . Biological Detection of Activating ras Genes 26 0

II. Direct Detection of ras Mutations in Tumor DNA and RNA

26 1III. Polymerase Chain Reaction Based Approaches to Screenin g

Tumors 26 1C. Frequent Occurrence of Mutated ras Genes in Human Tumors 262D . ras Activation is Associated with Experimentally Induce d

_Rodent Tumors 267

E. Biological Activities of Oncogenic ras Proteins 268

1 . Malignant Transformation of Established Rodent Fibroblas t

Cell Lines 268II. ras Requires Cooperation with Other Oncogenes fo r

Transformation of Primary Cells 26 9III. Induction of Differentiation and Growth Inhibition by

Oncogenic ras 27 0

IV. Transgenic Mouse Studies Establish ras Oncogenicity 27 1F. Structural and Biochemical Consequences of Oncogeni c

Mutations 271

I . Activating Mutations at Residues 12, 13, or 61 Promote

Active, GTP-Complexed ras Formation 27 2

II . Other Activating Mutations Also Perturb the ras GDP-GTPCycle 272

G. Clinical Implications of Oncogenic ras for Diagnosis an d

Treatment 274

1 . Diagnostic and Prognostic Applications of ras Mutations 274

II. Protein Prenylation : Oncogenic ras Proteins as Targets of

Therapy 275

H. Future Questions 27 6

References 277

CHAPTER 1 9

Dominant Inhibitory Ras Mutants: Tools for Elucidating Ras Function

L .A. FEiG. With 1 Figure 289


B. Mechanism of .Inhibitory Action 29 0

C. Defining Biochemical Pathways Dependent upon Ras Function 29 3

D. Some Surprises' Revealed by Dominant Inhibitory Ras Mutants 296

E. Cgnclusions 29 8

References 298

CHAPTER 2 0

The Involvement of Cellular ras in Proliferative SignalingD .W . STACEY . With 3 Figures 30 1

A. Introduction 30 1B. The Relationship Between Tyrosine Kinase Oncogene s

and Cellular ras 30 11 . Neutralizing Anti-ras Antibody 30 1

II . Inhibition in the Late G1 Phase of the Cell Cycle 30 2III . ras and Other Oncogene Classes 30 2

C. A Model for Proliferative Signal Transduction 30 21 . Other Studies Which Support the Model 30 3

D. Lipids and the Control of ras Activity 30 41 . Dependence of Lipid Mitogens upon ras 304

II . Biochemical Effects of Lipids upon ras 305E. Biochemical Analyses of the Interaction Between ras and Lipids

3061 . Lipids and ras-Related Proteins 306

II. Neurofibromin and Lipid Inhibition 307III. Production of GAP-Inhibitory Lipids by Mitoge n

Stimulation 308IV. Physical Association Between GAP and Lipids 308

V. Mutational Analysis of ras and the Lipid InhibitoryPhenotype 309

VI. Other Studies of Lipids and GAP Activity 310VII. Tyrosine Kinases and Lipid Metabolism 31 2

VIII. Model for the Control of Proliferation at the Level of rasActivity 31 3

F. Cellular Factors Affecting ras Activity 3141 . N17 ras Interferes with the Activation of Cellular ras 314

II. RAST is Preferentially Inhibitory for Oncogenic ras 31 5III. Model for Inhibition of ras Activity by Dominant Inhibitory

Mutants 316IV. Biochemical Support for the Idea that RAST Binds an

Effector 317G. Summary 318References 319

CHAPTER 2 1

Regulation of Ras-Interacting Proteins in Saccharomyces cerevisiae

K . TANAKA, A. Tox-E, and K . MATSUMOTO . With 2 Figures 323

A. Introduction 323B . Regulation of Ras Activity by Guanine Nucleotides 324

1 . Biochemical Properties of Ras 324II. The CDC25 Gene 325

III. IRAI and IRA2 Genes 326C. Regulation of Adenylyl Cyclase by Ras 328D. Domains of Ras Interacting with Other Proteins 329E. Conclusions 330References 330

CHAPTER 2 2

Lipid Modifications of Proteins in the Ras Superfamil yJ.B . GIBBS 33 5

A. Background 33 5B. Farnesylation 33 6

I . Farnesyl-Protein Transferase 33 6I1 . Function of Farnesylation 33 8

C. Geranylgeranylation 33 9D. Other Modifications 34 0

I. Proteolysis 340II. Methylation 34 1

111 . Palmitoylation 341

E. Conclusions 342

References 34 2

CHAPTER 23

GTPase Activating Protein sF. McCoRMICK . With 4 Figures 345

A. Introduction 345

B . GTPase Activating Proteins for ras p21 Proteins 346

1 . GTPase Activating Proteins in Saccharomyces cerevisiae 346

II. GTPase Activating Proteins in Schizosaccharomyces pombe

346

III. GTPase Activating Proteins in Drosophila melanogaster 34 6

IV. GTPase Activating Proteins in Mammalian Cells 34 8

C. GTPase Activating Proteins for rap p21's 35 3

D. GTPase Activating Proteins for rho-Like Proteins 35 4

E. GTPase Activating Proteins for other small GTPases 35 5

F. Concluding Remarks 35 5

References 356

CHAPTER 2 4f

Guanine Nucleotide Dissociation Stimulator sI .G. MACARA and E .S . BURSTEIN . With 3 Figures 36 1

A . Introduction 36 1B. Possible Mechanisms for conversion to the GTP-Bound State 36 1C. Nonspecific Guanine Nucleotide Dissociation Stimulators 363D . Ras-Specific Guanine Nucleotide Dissociation Stimulators 365

1 . Mammalian Guanine Nucleotide Dissociation Stimulators 365II. Yeast Guanine Nucleotide Dissociation Stimulators : CDC25 ,

SCD25 and ste6 367III. A Ras-Specific Guanine Nucleotide Dissociation Stimulato r

in Drosophila : SOS 36 8E. RAB3-Specific Guanine Nucleotide Dissociation Stimulator 36 9F. Other Guanine Nucleotide Dissociation Stimulators 37 0G. Conclusions 37 1References 37 2

CHAPTER 25

The Biology of RapG .M. BOKOCH . With 3 Figures 37 7

A. Introduction 377B. Cloning/Isolation of Rap(s) 377

C. Posttranslational Modification of Rap Proteins 3781 . Isoprenylation 378

I1 . Phosphorylation 379D. Rapt Regulatory Proteins 380

1 . GTPase Activating Proteins 380

II . GDP/GTP Dissociation Stimulator 380

E. Biological Activities of Rapl Protein 38 1

1 . Antagonism of Ras by Rapt 38 1

II . Interaction of Rap1A with the Phagocyte ReducedNicotinamide Adenine Dinucleotide Phosphate Oxidase 38 4

F. Conclusion 38 7

References 388

B. Vesicle Transfer/Vesicle Fusion

CHAPTER 2 6

GTPases and Interacting Elements in Vesicle Budding andTargeting in YeastC. BARLOWE and R . SCHEKMAN . With 2 Figures 397

A. Introduction 39 7B. Isolation and Characterization of Secretion Defective Yeas t

Strains 39 8C. Biochemical Analysis of Protein Transport from the Endoplasmi c

Reticulum to the Golgi Apparatus 399

D. Sarlp Function in Vesicle Formation from the Endoplasmi c

Reticulum 40 1

E. Concluding Remarks 404

References 40 5

CHAPTER 27

Ypt Proteins in Yeast and Their Role in Intracellular Transpor tM . STROM and D. GALLWITZ . With 2 Figures 409

A . Introduction 409B . Ypt Proteins in Saccharomyces c&evisiae 41 1

1 . Yptl Protein 41 1II. Sec4 Protein 41 2

III. Ypt3, Ypt6 and Ypt7 Proteins 41 3

C. Ypt Protein Structure 41 4

1 . Nucleotide Binding 414

II. Effector Region 416

III. C Terminus 416

D . GTPase Activating Proteins for YPT Family Members 417

.E . Summary 41 8

References 41 8

CHAPTER 28

Compartmentalization of rab Proteins in Mammalian Cell sV.M. OLKKONEN, P . DUPREE, L .A. HUBER, A. LÜTCKE, M . ZERIAL ,

and K. SIMONS . With 4 Figures 423

A. Subcellular Compartmentalization and Membrane Traffic 423

1 . Membrane Trafficking 424

1. Indications for a Role of Sec4/Yptl/rab GTPases 424

B . Localization of rab Proteins on Subcellular Compartments 42 5

1 . The rab Proteins Associated with the Biosynthetic Route 42 5

1. Endoplasmic Reticulum and Golgi Apparatus 42 5

2. The rab3a Protein on Regulated Exocytic Vesicles 42 6

II . The rab Proteins on Endocytic Compartments 42 7

1. The rab5 and rab4 Proteins on Early Endosomes 427

2. The rab Proteins on Late Endocytic Compartments 427

III . The Molecular Basis of rab Compartmentalization 428

1. The C-Terminal Modifications 428

2. Role of the C-Terminal Variable Region 428

C. The Function of rab Proteins in Membrane Trafficking 4291 . The Present Model for rab Function 429

IL Experimental Evidence for rab Function in Membran eTrafficking 4321. The rab1, rab2, and rab9 Proteins are Involved i n

Transport Steps on the Biosynthetic Route 4322. The rab3a Protein and Regulated Secretion 4333. Functional Studies on rab5 and rab4 4334. Conclusion from the Functional Data 43 5

D . The Novel rab Proteins 43 51 . Why Clone More rab Sequences? 43 5

II . Subcellular Localization 43 61. Novel Proteins on the Biosynthetic Pathway 43 62. Novel Proteins on Early Endocytic Compartments 43 6

III . Epithelial-Specific rab Proteins? 43 8E. Conclusion 43 9References 44 0

CHAPTER 29

GTPases in Transport Between Late Endosomes andthe Trans Golgi NetworkS .R . PFEFFER . With 3 Figures 447

A. Small GTPases in Membrane Traffic 447

B . In Vitro Assays to Analyze the Role of GTP inMembrane Traffic 44 7

1 . Introduction 44 7II. Transport of Mannose 6-Phosphate Receptors From Lat e

Endosomes to the trans Golgi Network In Vitro 44 8III. GTPyS Inhibits Endosome-to-TGN Transport In Vitro 44 9IV. A GTPyS-Sensitive Transport Component Requires Lat e

Endosomes for Its Activity 45 0C. Role of rab Proteins in Endosome to trans Golgi Network

Transport 45 1D. A Model for rab Protein Function

45 31 . Recruitment of rab Proteins onto Nascent Transpor t

Vesicles 45 31. Newly Synthesized rab Proteins are Cytosolic 45 32. Membrane Association 45 3

II . Action of rab Proteins After Transport Vesicle Formation 454E. Future Perspectives 456References 456

CHAPTER 30

Endocytic Function in Cell-Free SystemM. WESSLING-RESNICK . With 1 Figure

46 1

A . Introduction 46 1B . Development of Cell-Free Assays 46 1

1 . Endosomal Fusion 46 2

II. Early Endocytic Events : Formation, Invagination, andBudding of Coated Vesicles 46 4

III. Late Endocytic Events : Sorting, Processing, and Recycling 46 6C. GTPases Implicated in Endocytic Traffic 46 8

1 . Evidence Supporting a Functional Role for GTPases 468II. Rab Proteins 469

III. Heterotrimeric G-Proteins 470IV. ADP-Ribosylation Factors 472

D . Future Prospectives 472References 473

CHAPTER 3 1

Synaptic Vesicle Membrane Traffic and the Cycle of Rab3G . FISCHER VON MOLLARD, T .C . SÜDHOF, and R . JAHN . With 1 Figure 477

A. Membrane Traffic of Synaptic Vesicles in Neurons 47 7

B. Rab3 Proteins : Structure, Posttranslational Modifications andSubcellular Localization 478

C. The Cycle of Rab3A in Nerve Terminals 480

References 483

CHAPTER 32

Regulated Exocytosis and Interorganelle Vesicular Traffic :A Comparative AnalysisA. LuiNi and M .A . DE MATTEIS 487


B.' GTPases in Membrane Traffic : Experimental Approaches 48 9

C. GTPases in Constitutive Transport 48 9

1 . Vesicle Formation 48 9

IT. Vesicle Targeting and Fusion 49 2- 1 . Rab Proteins 49 2

2. ARF Proteins 49 33. Heterotrimeric G-Proteins 49 3

D. GTPases in Regulated Exocytosis 494I. Granule Formation 494

II . Granule Targeting and Fusion 494E. Regulation of the Secretory Pathways by Transduction Systems 496

1 . Regulated Exocytosis 496II . Constitutive Traffic 496

F. Conclusions 499References 500

CHAPTER 33

Regulated and Constitutive Secretion Studied In Vitro : Control b yGTPases at Multiple Level sH.-P.H . MOORE, L. CARNELL, R .A. CHAVEZ, Y .-T. CHEN ,A . HWANG, S. G . MILLER, Y .-A. YooN, and H . Yu. With 2 Figures 507

A. Introduction 507B. The Regulated Secretory Pathway : A General Mechanism for the

Control of Cell-Cell Communication and Plasma Membran eActivities 508

C. Controlling Passage Through the Regulated Secretory Pathway -Distinctions Between Constitutive and Regulated Secretion 51 1

1 . Exocytosis 51 1II . Formation of Granules 512

III . Sorting of Contents 51 3D. Regulation of Traffic Through the Constitutive Pathway 514E. GTPases and Intracellular Membrane Transport 51 5

I. SAR1 516II. Trimeric G-proteins 517

III. The ADP-Ribosylation Factor Family 518IV. The rab Family 520


CHAPTER 34

The Biology of ADP-Ribosylation FactorsR . A. KAHN . With 1 Figure 529

A. Introduction 529B. The ARF Family of Small GTPases 530

1 . Structural Definition 530II . Functional Definition 530

C. ARF Functions in the Yeast, Saccharomyces cerevisiae 532I. Yeast ARF Genes and Proteins 532

II. Phenotypes of arfMutants 532III. Evidence that ARF Is Required in the Secretory Pathway 532

D . Biochemical Characterization of ARF Proteins 53 31 . ARF Purified from Mammalian Sources is Heterogeneous 53 3

II. ARF Cofactor Activity 53 3III. Guanine Nucleotide Binding 534IV. GTPase Activity 53 5V. The Role of Myristoylation 53 5

VI. Binding of ARF to Lipid Bilayers 536VII. Evidence that ARF is Required at Several Steps in th e

Secretory and Endocytic Pathways 536E. Use of ARF Antibodies 537

1 . Abundance of Different ARF Proteins is Quite Variable 537II . Localization of ARF Proteins in Animal Cells 537

F. ARF as a Regulator of Coat Protein Binding to Membranes

5381 . Brefeldin A Causes Rapid Release of ARF from Golg i

Stacks 538II . An In Vitro Assay for ARF as Regulator of Coat Protei n

Binding 53 8References 53 9

CHAPTER 35

Molecular Characterization of ADP-Ribosylation FactorsJ . Moss and M . VAUGHAN . With 4 Figures 54 3

A. Introduction 54 3B . Activation of Cholera Toxin by ADP-Ribosylation Factors

54 41 . Mechanism of Activation of Cholera Toxin by ADP -

Ribosylation Factors 544

II. Guanine Nucleotide-Dependent Association of Choler aToxin with ADP-Ribosylation Factors 546

III. Activation of Escherichia coli Heat-Labile Enterotoxin b y

ADP -Ribosylation Factor 546

C. Structure of ADP-Ribosylation Factors 547

1 . Deduced Amino Acid Sequences and Gene Structure of

ADP -Ribosylation Factors 547

II . Expression of ADP-Ribosylation Factors in Eukaryoti c

Species 54 9

D. - Hormonal and Developmental Regulation of ADP-Ribosylatio n

Factors 55 0

E. Physiological Roles for ADP-Ribosylation Factors 55 1

References 55 5

C. rho and rho-Like Proteins

CHAPTER 36

rho and rho-Related ProteinsA .J . RIDLEY and A. HALL . With 5 Figures 563

t

A. Introduction 563B. Sequence and Structure 563C. Expression and Localisation 565D. Upstream Regulation of rho-Like Proteins 566

I. Nucleotide Exchange 566II. GTP Hydrolysis 566

E. Downstream Functions of rho-Like Proteins 5681 . Mammalian rho Proteins 568

II . The rac Proteins 5701. rac and the Actin Cytoskeleton 5702. rac and the Superoxide Production 57 13. Other rho-Related Proteins 572


CHAPTER 37

The Mammalian Homolog of the Yeast Cell-Division-Cycle Protein ,CDC42: Evidence for the Involvement of a Rho-Subtype GTPase i nCell Growth RegulationM .J . HART, D . LEONARD, Y . ZHENG, K . SHINJO, T . EVANS, andR .A. CERIONE . With 4 Figures 579

A. Growth Factor-Coupled Signal Transduction 579

B. Reconstitution of an Epidermal Growth Factor Stimulate dPhosphorylation of a 22-kDa GTPase 580

C. Molecular Cloning of the Human Gp/G25K Protein :Identification of this Protein as the Human Homolog of the Yeast

Cell Division Cycle Protein CDC42Sc 583

D. Function of CDC42Sc in Saccharomyces cerevisiae 58 4

E. Possible Involvement of CDC42Hs in Cell Growth Regulation 58 5

1 . cDNA Transfection Studies 58 5

II . CDC42Hs Regulatory Proteins 58 6

1. CDC42Hs GTPase Activating Protein 586

2. CDC42Hs Guanine Nucleotide Dissociation Stimulator 588

3. CDC42Hs Guanine Nucleotide Dissociation Inhibitor 59 1

References 593

D. Regulation of and by Small GTPases

CHAPTER 38

Role of Rap1B and Its Phosphorylation in Cellular Function :A Working ModelD.L. ALTSCHULER, M . ToRTi, and E .G. LAPETINA . With 4 Figures 599

A. Introduction : The Rap Family of Proteins 599

B. Phosphorylation of Raplb 60 1

1 . Structural Properties 60 1

1. cAMP-dependent Phosphorylation of Raplb in Human

Platelets 60 1

2. Phosphorylation of Raplb by a Neuronal Ca2+/

Calmodulin-dependent Protein Kinase, CaM Kinase Gr 602

3. Mutational Analysis of the Protein Kinase A-dependent

Phosphorylation Site of Raplb 60 3

4. Phosphorylation-dependent Activation of Raplb :Role of Guanine Nucleotide Dissociation Stimulator 60 4

Il . Physiological Properties : The Platelet Model 60 4

1. Thrombin-induced Association of Raplb with Ras -

GTPase Activating Protein : Effect of Phosphorylation 605

2. Ras-GAP Associates with Phospholipase Cy-1 in Huma n

Platelets 60 6

I11 . A Working Model and Open Questions 60 7

References 60 9

CHAPTER 39

GDP/GTP Exchange Proteins for Small GTP-Binding Protein sY. TAKAi, K . KAiBUCHi, A . KIKUCHI, and T . SASAKI . With 5 Figures . .

613

A. Introduction 613B. Physical Properties of GDP/GTP Exchange Protein 614C. Two Actions of GDP/GTP Exchange Protein and Requiremen t

of the Posttranslational Processing of Small GTPases fo rGDP/GTP Exchange Protein Actions 614

D. Substrate Specificity of GDP/GTP Exchange Protein an dFunctional Cooperation Between Guanine Nucleotid eDissociation Stimulator and Guanine Nucleotide Dissociatio nInhibitor 615

E. Activation of smg p21 by Protein Kinases A and G 616F. The Function of smg Guanine Nucleotide Dissociation Stimulator

in Regulating Gene Expression and Cell Poliferation 616G. The Function of smg Guanine Nucleotide Dissociation Stimulator

and rho Guanine Nucleotide Dissociation Inhibitor in Regulatin gSuperoxide Generation 618

H. The Function of smg Guanine Nucleotide Dissociatio nStimulator, rho, and rho Guanine Nucleotide DissociationInhibitor in Regulating the Actomyosin System 618

1 . The Function of smg p25 Guanine Nucleotide Dissociatio nInhibitor in Regulating Intracellular Vesicle Transport 620

J. Conclusions 62 1References 622

CHAPTER 40

GTP-Mediated Communication Between Intracellular Calcium Pool sD.L . GILL, T.K. GHOSH, A.D . SHORT, J . BIAN, and R.T . WALDRON .

With 8 Figures 625

A. Intracellular Ca 2+ Signaling Pools 6251 . Nature of Intracellular Ca 2+ Pools 625

II. Movements of Ca2+ Induced by Inositol Phosphates 62 6III. Intracellular Ca2+ Channels 627IV. Significance of Ca2+ Within the InsP 3-Sensitive Ca2+ Pool 62 9

B. Ca2+ Movements Activated by Guanine Nucleotides 6301 . GTP-Induced Ca 2+ Fluxes 63 0

II. Ca2+ Compartments Sensitive to GTP and InsP 3 63 1III. Distinctions Between GTP- and InsP3-Induced Ca2 +

Transport 63 2IV. Rationale for the Action of GTP 63 2

C. Interorganelle Translocation of Ca Z+ 63 41 . Model for GTP-Activated Ca2+ Translocation 63 4

II. GTP-Activated Ca2+ Transfer into the InsP3-Sensitive Ca2 +

Pool 63 7III. Isolation of InsP3 -Releasable and InsP 3 -Recruitable Pools 63 7IV. Functional Organization of Ca 2+-Regulatory Organelles 639

D . G-Proteins and Interorganelle Transfer of Ca Z+

6421 . Identification of Possible G-Protein Mediators of Ca2 +

Transfer 642II . Conclusions on the Role of G-Proteins 645

References 647

CHAPTER 41

Coupling of ras to the T Cell Antigen ReceptorJ . DOWNWARD 65 1

A. Introduction 65 1B . Receptors and Intracellular Signals that Regulate p2l ras 65 1

I. Activation of p2l ras in Cells Other than T Lymphocytes 65 1II. Activation of p2l ras in T Lymphocytes 65 3

C. GTPase Activating Proteins Regulate p2l ras in T Lymphocytes 65 4D . Mechanisms of Regulation of ras GTPase Activating Proteins in

T Cells 65 5E. Function of p2l ras in T Lymphocytes 656References 65 7

CHAPTER 42

GTPases as Regulators of Regulated SecretionT.H.W . LILLIE and B .D. GOMPERTS . With 7 Figures 66 1

A. GTP : A Sine Qua Non for Exocytosis 66 11 . CaZ+-Dependent Secretion in Myeloid Granulocytes 66 1

B . Probing Exocytosis : Permeabilised Cells 66 21 . GTPyS-Induced, Ca Z+ -Independent Exocytosis 66 4

11 . CaZ+-Induced, GTP-Dependent Exocytosis 66 4111 . One or Two Effectors?

66 51 . Chloride Suppresses and Glutamate Enhances Guanin e

Nucleotide Sensitivity of Exocytosis 66 6IV. Kinetics of Exocytosis 66 8

1. Mg2+ Permits Abrupt Onset 66 92. Mg2+ Deprivation Causes Onset Delays 67 0

V. GTPases Regulate and Modulate Exocytosis in Many Cell sand Tissues 67 0

C. On the Nature of GE 67 11 . The Example of Gs 67 1

II . The Example of the Monomeric GTPases 67 1D . Single Cell Analysis of GTPyS-Induced Exocytosis 67 2E . Two GTPases in Regulated Exocytosis? 67 4References 675

CHAPTER 43

ADP-Ribosylation of Small GTPases by Clostridium botulinumExoenzyme C3 and Pseudomonas aeruginosa Exoenzyme SJ . COBURN 679

A. Introduction 679B. Small GTPases 680C. Clostridium botulinum Exoenzyme C3 680D. Pseudomonas aeruginosa Exoenzyme S 682E. Conclusions 684References 685

Subject Index 689

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GTPases in Biology I