Post on 20-Aug-2020
GTPases in Biology I
Contributors
K. Aktories, D.L. Altschuler, M.W. Anderson, C. Barlowe,E. Bergmann, J. Bian, L. Birnbaumer, G.M. Bokoch, H.R. Bourne,P. Brennwald, C.C. Burgess, E.S. Burstein, S.L. Campbell-Burk,M.-F. Carlier, L. Carnell, J. Cavallius, R.A. Cerione,P. Chardin, P.S. Charifson, R.A. Chavez, Y.-T. Chen, G.J. Clark,J. Coburn, T.A. Darden, M.A. De Matteis, C.J. Der, J^ Downward,P. Dupree, T. Evans, L.A. Feig, G. Fischer von Mollard,C.K. Foley, D. Gallwitz, T.K. Ghosh, J.B. Gibbs, D.L. Gill,R. Gilmore, B.D. Gomperts, R.S. Goody, A. Hall, M.J. Hart,J.S. Herskovits, L.A. Huber, A. Hwang, H. Itoh, R. Jahn,F. Jurnak, I. Just, R.A. Kahn, K. Kaibuchi, Y. Kaziro,A. Kikuchi, S.-H. Kim, E.G. Lapetina, D. Leonard, T.H.W. Lillie,A. Liitcke, A. Luini, I.G. Macara, K. Matsumoto, F. McCormick,W.C. Merrick, M.V. Milburn, S.G. Miller, H.-P.H. Moore, J. Moss,P. Novick, V.M. Olkkonen, E.F. Pai, D. Pantaloni, L.G. Pedersen,S.R. Pfeffer, G.G. Prive, P.J. Rapiejko, A.J. Ridley,T. Sasaki, R. Schekman, K. Shinjo, A.D. Short, K. Simons,D.W. Stacey, M. Strom, T.C. Sudhof, Y. Takai, K. Tanaka,A. Toh-e, M. Torti, R.B. Vallee, T.E. Van Aken, M. Vaughan,R.T. Waldron, R.A. Weinberg, M. Wessling-Resnick,A. Wittinghofer, Y.-A. Yoon, H. Yu, M. Zerial, Y. Zheng
Editors
Burton F. Dickey and Lutz Birnbaumer
Springer-VerlagBerlin Heidelberg New York London ParisTokyo Hong Kong Barcelona Budapest
Contents
Section I: Biological Importance of GTPase-Driven Switches
CHAPTER 1
GTPases Everywhere!H.R. BOURNE. With 5 Figures 3
A. Introduction 3B. The GTPase Cycle and the Molecular Switch 3C. Structure of the GTPase Switch 4D. Primary Structures Identify GTPases with Related Functions . . . . 6E. Uses of the GTPase Switch: Stoichiometric Activation 7F. Uses of the GTPase Switch: Assembling a Complex 9G. Other Potential Uses of the GTPase Switch : 12H. Cascades of GTPases 12I. Perspectives 14References 14
CHAPTER 2
Proofreading in the Elongation Cycle of Protein SynthesisE. BERGMANN and F. JURNAK. With 2 Figures 17
A. Introduction 17B. General Concepts 18
I. Specificity 18II. Proofreading 20
C. Parameters of Protein Biosynthesis 22D. EF-Tu-Dependent Kinetic Proofreading 23E. EF-Tu-Independent Error Correction Mechanisms 26
I. Peptidyl Transfer 26II. EF-G-Dependent Translocation 26
III. Allosteric Linkage Between A and E Sites 28F. Summary 28References 29
XVIII Contents
CHAPTER 3 <v
A New Look at Receptor-Mediated Activation of a G-ProteinL. BIRNBAUMER. With 1 Figure 31References 36
CHAPTER 4
Small GTPases and Vesicle Trafficking: Sec4p and its Interactionwith Up- and Downstream ElementsP. NOVICK and P. BRENNWALD. With 2 Figures 39
A. Introduction 39B. The Sec4 Cycle 43
I. A Cycle of Sec4 Localization 43II. Intrinsic Properties of Sec4 44
III. GTP Binding and Membrane Attachment Are Essential forSec4 Function 44
IV. GTP Hydrolysis Is Important for Sec4 Function 44C. Accessory Proteins in the Sec4 Cycle 45
I. A Specific Sec4 GAP Is Present in Yeast and MammalianCells 45
II. GDI from Bovine Brain and Yeast Solubilizes Sec4 in aNucleotide-Specific Fashion 46
III. Suppressors from Yeast and Rat Brain Encode NucleotideExchange Proteins 46
D. A Potential Downstream Effector of Sec4 Function: TheSec8/Secl5 Complex 47
References 48
CHAPTER 5
Cytoskeletal Assembly: The Actin and Tubulin NucleotidasesM.-F. CARLIER and D. PANTALONI. With 1 Figure 53
A. Introduction 53B. The Nucleotidase Cycle in the Polymerization of Actin and
Tubulin 53C. Elementary Steps in NTP Hydrolysis on Actin Filaments and
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 using
AlF^" and BeF^~, H2O 58F. Conclusions 59References 60
Contents XIX
CHAPTER 6
Dynamin, A Microtubule-Activated GTPase Involved in EndocytosisR.B. VALLEE, J.S. HERSKOVITS, and C.C. BURGESS. With 3 Figures . . 63
A. Introduction 63B. Structure and Enzymatic Properties : 63C. The Drosophila Shibire Gene 66D. Transfection of Dynamin into Cultured Mammalian Cells 68References 71
CHAPTER 7
Transmembrane Protein Translocation: Signal Recognition Particleand Its Receptor in the Endoplasmic ReticulumP.J. RAPIEJKO and R. GILMORE. With 3 Figures 75
A. Introduction 75B. The Signal Recognition Particle and Its Receptor 75C. Protein Translocation Across the Rough Endoplasmic Reticulum
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 Recognition
Particle are GTP Independent 80G. Current Models for GTP Function During Protein Translocation 81References 7 83
CHAPTER 8
GTPases and Actin as Targets for Bacterial ToxinsK. 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 89I. Introduction 89
II. Diphtheria Toxin 89III. Pseudomonas aeruginosa Exotoxin A 90IV. Functional Consequences of the ADP-Ribosylation of
Elongation Factor 2 91D. ADP-Ribosylation of G-Proteins 91
I. Introduction 91II. Cholera Toxin 91
III. Heat-Labile E. coli Enterotoxins 92
XX Contents
IV. Functional Consequences of the ADP-Ribosylation ofG-Proteins by Cholera- and Heat-Labile E. coliEnterotoxins 92
V. Pertussis Toxin 94VI. ADP-Ribosylation of Gi; Go, and Gt by Pertussis Toxin 94
E. ADP-Ribosylation of Small GTPases 95I. Introduction 95
II. C3-Like ADP-Ribosyltransferases 95III. Functional Consequences of the ADP-Ribosylation of Rho
Proteins 97IV. ADP-Ribosylation of Small GTPases by Pseudomonas
aeruginosa Exoenzyme S 99F. ADP-Ribosylation of Actin 99
I. Introduction 99II. Clostridium botulinum C2 Toxin 100
III. Other Actin-ADP-Ribosylating Toxins 100IV. Functional Consequences of the ADP-Ribosylation of Actin 101
G. Perspectives 102References 102
Section II. Structure of the GTPase Switches
CHAPTER 9
Eukaryotic Translation Factors Which Bind and Hydrolyze GTPJ. CAVALLIUS and W.C. MERRICK. With 2 Figures 115
A. GTPase Factors 115B. Consensus Sequences of GTPases Factors 117C. Evolution of EF-la 118D. The EF-Tu Family 122E. Structures of the EF-Tu Family 126References 128
CHAPTER 10
Heterotrimeric G-Proteins: a, fi, and y SubunitsH. ITOH and Y. KAZIRO. With 5 Figures 131
A. Introduction 131B. Mammalian G-Proteins 131
I. a Subunits 1311. Isolation of cDNAs and Genomic DNAs 131
a) Gsa 131b) Gia 134
Contents XXI
c) Goa 135d) G ta and Ggusta 135e) Gza 136f) Gqa and G12ct 137
2. Comparison of the Amino Acid Sequences 137a) P Region 137b) G' Region 137c) G Region 138d) G" Region 138e) Cholera Toxin ADP-Ribosylation Site 138
3. Sequence Conservation 1394. Evolutionary Tree 139
II. p y Subunits 139C. G-Proteins in Lower Eukaryotes 140
I. G-Proteins from Saccharomyces cerevisiae 1411. Two a Subunits, GPA1 and GPA2 1412. p and y Subunits 141
II. G-Proteins from Schizosaccharomyces pombe 144III. G-Proteins from Caenorhabditis elegans 144IV. G-Proteins from Plants 144
References 145
CHAPTER 11
Molecular Diversity in Signal Transducing G-ProteinsL. BIRNBAUMER. With 1-Figure 151
A. The a Subunits 151I. Molecular Diversity 151
II. a Subunit Functions 153B. The p y Dimers 155References 156
CHAPTER 12
Structural Conservation of Ras-Related Proteins and Its FunctionalImplicationsP. CHARDIN. With 2 Figures 159
A. Introduction: The Discovery of Ras and ^as-Related Genes 159B. Sequence Comparisons 163
I. The N-Terminal Extension 166II. The Phosphate-Binding Part 166
III. The Guanine-Binding Part 167IV. The C-Terminal Extension 168V. The CaaX Motif 168
XXII Contents
C. Evolutionary Relationships 168I. Construction of a Homology Tree 168
II. Insertions and Deletions 169III. Estimation of the Number of Ras-Related Proteins in
Mammals 170D. Discussion 171
I. Internal Residues 171II. External Residues and Potential Targets for Interacting
Proteins 171III. Relation to Other GTPase Families 172IV. Is There a Conserved Functional Mechanism for All
Ras-Related Proteins? 173References 173
CHAPTER 13
Conformational Switch and Structural Basis for Oncogenic Mutationsof Ras ProteinsS.-H. KIM, G.G. PRIVE, and M.V. MILBURN. With 5 Figures 177
A. Introduction 177B. Conformational Switch 178
I. Conformational Differences Between GDP- and GTP-BoundRas Proteins: Switch I and II Regions 179
II. Conformational Domino Effect and Frozen Dynamic States 183III. Small Conformational Changes in the Phosphate-Binding
Loop, LI 186C. Structural Basis for Oncogenic Mutations 186
I. Mutations at Gln-61 and the Stabilization of the TransitionState 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 /?-Turn for PhosphateBinding 189
IV. Mutation at Ala-59 and Switch II Conformation 191D. Discussion 192References 193
CHAPTER 14
Structural and Mechanistic Aspects of the GTPase Reaction ofH-ras p21A. WITTINGHOFER, E.F. PAI, and R.S. GOODY. With 3 Figures 195
A. Introduction 195B. The Structure of the p21-Triphosphate State 195C. The Structure and Biochemistry of p21 Mutants 198
Contents XXIII
D. The Kinetic Mechanism of the GTPase Reaction 199E. The Kinetic Mechanism of the GAP-stimulated GTPase 201F. GTPase Mechanism 203G. Arguments For and Against the Proposed Mechanism 204H. Role of GAP in the Chemical Mechanism 206I. Conclusion 208References 208
CHAPTER 15
Analysis of Ras Structure and Dynamics by Nuclear MagneticResonanceS.L. CAMPBELL-BURK and T.E. VAN AKEN. With 9 Figures 213
A. Introduction 213B. NMR Studies of Proteins 214
I. NMR Structure Determination 2141. NMR Methods: Larger Proteins 2142. NMR Resonance Assignments: Application to Ras 2143. Secondary Structure Determination: Application to Ras . . 2164. Tertiary Structure and Structure Refinement 219
II. Comparison of Solution and Crystal Structures 2191. Computer Simulation: RasGMPPNP Solution Structure . . 2202. Protein Dynamics 220
C. Comparison of Full length and Truncated Ras Proteins 221I. Protein Stability: Sample Preparation 221
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 RasGMPPCP 225
D. Comparison of Ras-GTP, Ras-GTPyS, Ras-GMPPCPand Ras-GDP 226I. Chemical Shift Differences 226
E. Kinetic Measurements 228I. Kinetic and Fluorescence Studies 228
II. 31P NMR: Ras-GTP Hydrolysis 228III. [XH-15N]-Edited NMR Spectroscopy: GTP Hydrolysis 230
F. Conclusion 230References 231
CHAPTER 16
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
XXIV Contents
A. Introduction 235B. Methods 236C. Results and Discussion 237
I. 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 Family
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 Tumor
Cells 260I. Biological Detection of Activating ras Genes 260
II. Direct Detection of ras Mutations in Tumor DNA and RNA 261III. Polymerase Chain Reaction Based Approaches to Screening
Tumors 261C. Frequent Occurrence of Mutated ras Genes in Human Tumors .. 262D. ras Activation is Associated with Experimentally Induced
Rodent Tumors 267E. Biological Activities of Oncogenic ras Proteins 268
I. Malignant Transformation of Established Rodent FibroblastCell Lines 268
II. ras Requires Cooperation with Other Oncogenes forTransformation of Primary Cells 269
III. Induction of Differentiation and Growth Inhibition byOncogenic ras 270
IV. Transgenic Mouse Studies Establish ras Oncogenicity 271F. Structural and Biochemical Consequences of Oncogenic
Mutations 271
Contents XXV
I. Activating Mutations at Residues 12, 13, or 61 PromoteActive, GTP-Complexed ras Formation 272
II. Other Activating Mutations Also Perturb the ras GDP-GTPCycle 272
G. Clinical Implications of Oncogenic ras for Diagnosis andTreatment 274I. Diagnostic and Prognostic Applications of ras Mutations 274
II. Protein Prenylation: Oncogenic ras Proteins as Targets ofTherapy 275
H. Future Questions 276References 277
CHAPTER 19
Dominant Inhibitory Ras Mutants: Tools for Elucidating Ras FunctionL.A. FEIG. With 1 Figure 289
A. Introduction 289B. Mechanism of Inhibitory Action 290C. Denning Biochemical Pathways Dependent upon Ras Function .. 293D. Some Surprises Revealed by Dominant Inhibitory Ras Mutants .. 296E. Conclusions 298References 298
CHAPTER 20
The Involvement of Cellular ras in Proliferative SignalingD.W. STACEY. With 3 Figures 301
A. Introduction 301B. The Relationship Between Tyrosine Kinase Oncogenes
and Cellular ras 301I. Neutralizing Anti-ras Antibody 301
II. Inhibition in the Late Gl Phase of the Cell Cycle 302III. ras and Other Oncogene Classes 302
C. A Model for Proliferative Signal Transduction 302I. Other Studies Which Support the Model 303
D. Lipids and the Control of ras Activity 304I. Dependence of Lipid Mitogens upon ras 304
II. Biochemical Effects of Lipids upon ras 305E. Biochemical Analyses of the Interaction Between ras and Lipids 306
I. Lipids and ras-Related Proteins 306II. Neurofibromin and Lipid Inhibition 307
III. Production of GAP-Inhibitory Lipids by MitogenStimulation 308
IV. Physical Association Between GAP and Lipids 308
XXVI Contents
V. Mutational Analysis of ras and the Lipid InhibitoryPhenotype 309
VI. Other Studies of Lipids and GAP Activity 310VII. Tyrosine Kinases and Lipid Metabolism 312
VIII. Model for the Control of Proliferation at the Level of rasActivity 313
F. Cellular Factors Affecting ras Activity 314I. N17 ras Interferes with the Activation of Cellular ras 314
II. RAST is Preferentially Inhibitory for Oncogenic ras 315III. 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 21
Regulation of Ras-Interacting Proteins in Saccharomyces cerevisiaeK. TANAKA, A. TOH-E, and K. MATSUMOTO. With 2 Figures 323
A. Introduction 323B. Regulation of Ras Activity by Guanine Nucleotides 324
I. Biochemical Properties of Ras 324II. The CDC25 Gene 325
III. IRA1 and IRA2 Genes 326C. Regulation of Adenylyl Cyclase by Ras 328D. Domains of Ras Interacting with Other Proteins 329E. Conclusions 330References 330
CHAPTER 22
Lipid Modifications of Proteins in the Ras SuperfamilyJ.B. GIBBS 335
A. Background 335B. Farnesylation 336
I. Farnesyl-Protein Transferase 336II. Function of Farnesylation 338
C. Geranylgeranylation 339D. Other Modifications 340
I. Proteolysis 340II. Methylation 341
III. Palmitoylation 341
Contents XXVII
E. Conclusions 342References 342
CHAPTER 23
GTPase Activating ProteinsF. MCCORMICK. With 4 Figures 345
A. Introduction 345B. GTPase Activating Proteins for ras p21 Proteins 346
I. GTPase Activating Proteins in Saccharomyces cerevisiae . . . . 346II. GTPase Activating Proteins in Schizosaccharomyces pombe 346
III. GTPase Activating Proteins in Drosophila melanogaster . . . . 346IV. GTPase Activating Proteins in Mammalian Cells 348
C. GTPase Activating Proteins for rap p21's 353D. GTPase Activating Proteins for rho-Like Proteins 354E. GTPase Activating Proteins for other small GTPases 355F. Concluding Remarks 355References 356
CHAPTER 24
Guanine Nucleotide Dissociation StimulatorsI.G. MACARA and E.S. BURSTEIN. With 3 Figures 361
A. Introduction 361B. Possible Mechanisms for conversion to the GTP-Bound State . . . . 361C. Nonspecific Guanine Nucleotide Dissociation Stimulators 363D. Ras-Specific Guanine Nucleotide Dissociation Stimulators 365
I. Mammalian Guanine Nucleotide Dissociation Stimulators . . . 365II. Yeast Guanine Nucleotide Dissociation Stimulators: CDC25,
SCD25 and ste6 '. 367III. A Ras-Specific Guanine Nucleotide Dissociation Stimulator
in Drosophila: SOS 368E. RAB3-Specific Guanine Nucleotide Dissociation Stimulator 369F. Other Guanine Nucleotide Dissociation Stimulators 370G. Conclusions 371References 372
CHAPTER 25
The Biology of RapG.M. BOKOCH. With 3 Figures 377
A. Introduction 377B. Cloning/Isolation of Rap(s) 377
XXVIII Contents
C. Posttranslational Modification of Rap Proteins 378I. Isoprenylation 378
II. Phosphorylation 379D. Rapl Regulatory Proteins 380
I. GTPase Activating Proteins 380II. GDP/GTP Dissociation Stimulator 380
E. Biological Activities of Rapl Protein 381I. Antagonism of Ras by Rapl 381
II. Interaction of Rapl A with the Phagocyte ReducedNicotinamide Adenine Dinucleotide Phosphate Oxidase 384
F. Conclusion 387References 388
B. Vesicle Transfer/Vesicle Fusion
CHAPTER 26
GTPases and Interacting Elements in Vesicle Budding andTargeting in YeastC. BARLOWE and R. SCHEKMAN. With 2 Figures 397
A. Introduction 397B. Isolation and Characterization of Secretion Defective Yeast
Strains 398C. Biochemical Analysis of Protein Transport from the Endoplasmic
Reticulum to the Golgi Apparatus 399D. Sarlp Function in Vesicle Formation from the Endoplasmic
Reticulum 401E. Concluding Remarks 404References 405
CHAPTER 27
Ypt Proteins in Yeast and Their Role in Intracellular TransportM. STROM and D. GALLWITZ. With 2 Figures 409
A. Introduction 409B. Ypt Proteins in Saccharomyces cerevisiae 411
I. Yptl Protein 411II. Sec4 Protein 412
III. Ypt3, Ypt6 and Ypt7 Proteins 413C. Ypt Protein Structure 414
I. Nucleotide Binding 414II. Effector Region 416
III. C Terminus 416D. GTPase Activating Proteins for YPT Family Members 417
Contents XXIX
E. Summary 418References 418
CHAPTER 28
Compartmentalization of rab Proteins in Mammalian CellsV.M. OLKKONEN, P. DUPREE, L.A. HUBER, A. LUTCKE, M. ZERIAL,and K. SIMONS. With 4 Figures 423
A. Subcellular Compartmentalization and Membrane Traffic 423I. Membrane Trafficking 424
1. Indications for a Role of Sec4/Yptl/rab GTPases 424B. Localization of rab Proteins on Subcellular Compartments 425
I. The rab Proteins Associated with the Biosynthetic Route . . . 4251. Endoplasmic Reticulum and Golgi Apparatus 4252. The rab3a Protein on Regulated Exocytic Vesicles 426
II. The rab Proteins on Endocytic Compartments 4271. The rab5 and rab4 Proteins on Early Endosomes 4272. The rab Proteins on Late Endocytic Compartments 427
III. The Molecular Basis of rab Compartmentalization 4281. The C-Terminal Modifications 4282. Role of the C-Terminal Variable Region 428
C. The Function of rab Proteins in Membrane Trafficking 429I. The Present Model for rab Function 429
II. Experimental Evidence for rab Function in MembraneTrafficking , 4321. The rabl, rab2, and rab9 Proteins are Involved in
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 435
D. The Novel rab Proteins 435I. Why Clone More rab Sequences? 435
II. Subcellular Localization 4361. Novel Proteins on the Biosynthetic Pathway 4362. Novel Proteins on Early Endocytic Compartments 436
III. Epithelial-Specific rab Proteins? 438E. Conclusion 439References 440
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
XXX Contents
B. In Vitro Assays to Analyze the Role of GTP inMembrane Traffic 447
I. Introduction 447II. Transport of Mannose 6-Phosphate Receptors From Late
Endosomes to the trans Golgi Network In Vitro 448III. GTPyS Inhibits Endosome-to-TGN Transport In Vitro 449IV. A GTPyS-Sensitive Transport Component Requires Late
Endosomes for Its Activity 450C. Role of rab Proteins in Endosome to trans Golgi Network
Transport 451D. A Model for rab Protein Function 453
I. Recruitment of rab Proteins onto Nascent TransportVesicles 4531. Newly Synthesized rab Proteins are Cytosolic 4532. Membrane Association 453
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 461
A. Introduction 461B. Development of ̂ Cell-Free Assays 461
I. Endosomal Fusion 462II. Early Endocytic Events: Formation, Invagination, and
Budding of Coated Vesicles 464III. Late Endocytic Events: Sorting, Processing, and Recycling . . 466
C. GTPases Implicated in Endocytic Traffic 468I. Evidence Supporting a Functional Role for GTPases 468
II. Rab Proteins 469III. Heterotrimeric G-Proteins 470IV. ADP-Ribosylation Factors 472
D. Future Prospectives 472References 473
CHAPTER 31
Synaptic Vesicle Membrane Traffic and the Cycle of Rab3G. FISCHER VON MOLLARD, T.C. SUDHOF, and R. JAHN. With 1 Figure 477
A. Membrane Traffic of Synaptic Vesicles in Neurons 477B. Rab3 Proteins: Structure, Posttranslational Modifications and
Subcellular Localization 478
Contents XXXI
C. The Cycle of Rab3A in Nerve Terminals 480References 483
CHAPTER 32
Regulated Exocytosis and Interorganelle Vesicular Traffic:A Comparative AnalysisA. LUINI and M.A. DE MATTEIS 487
A. Introduction 487B. GTPases in Membrane Traffic: Experimental Approaches 489C. GTPases in Constitutive Transport 489
I. Vesicle Formation 489II. Vesicle Targeting and Fusion 492
1. Rab Proteins 4922. ARF Proteins 4933. Heterotrimeric G-Proteins 493
D. GTPases in Regulated Exocytosis 494I. Granule Formation 494
II. Granule Targeting and Fusion 494E. Regulation of the Secretory Pathways by Transduction Systems .. 496
I. Regulated Exocytosis 496II. Constitutive Traffic 496
F. Conclusions 499References 500
CHAPTER 33
Regulated and Constitutive Secretion Studied In Vitro: Control byGTPases at Multiple LevelsH.-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 MembraneActivities 508
C. Controlling Passage Through the Regulated Secretory Pathway -Distinctions Between Constitutive and Regulated Secretion 511
I. Exocytosis 511II. Formation of Granules 512
III. Sorting of Contents 513D. Regulation of Traffic Through the Constitutive Pathway '. . .. 514E. GTPases and Intracellular Membrane Transport 515
I. SARI 516II. Trimeric G-proteins 517
XXXII Contents
III. The ADP-Ribosylation Factor Family 518IV. The rab Family 520
F. Conclusions 522References 523
CHAPTER 34
The Biology of ADP-Ribosylation FactorsR.A. KAHN. With 1 Figure 529
A. Introduction 529B. The ARF Family of Small GTPases 530
I. Structural Definition 530II. Functional Definition 530
C. ARF Functions in the Yeast, Saccharomyces cerevisiae 532I. Yeast ARF Genes and Proteins 532
II. Phenotypes of arf Mutants 532III. Evidence that ARF Is Required in the Secretory Pathway . . . 532
D. Biochemical Characterization of ARF Proteins 533I. ARF Purified from Mammalian Sources is Heterogeneous . . 533
II. ARF Cofactor Activity 533III. Guanine Nucleotide Binding 534IV. GTPase Activity 535V. The Role of Myristoylation 535
VI. Binding of ARF to Lipid Bilayers 536VII. Evidence that ARF is Required at Several Steps in the
Secretory and Endocytic Pathways 536E. Use of ARF Antibodies 537
I. 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 538I. Brefeldin A Causes Rapid Release of ARF from Golgi
Stacks 538II. An In Vitro Assay for ARF as Regulator of Coat Protein
Binding 538References 539
CHAPTER 35
Molecular Characterization of ADP-Ribosylation FactorsJ. Moss and M. VAUGHAN. With 4 Figures 543
A. Introduction 543B. Activation of Cholera Toxin by ADP-Ribosylation Factors 544
I. Mechanism of Activation of Cholera Toxin by ADP-Ribosylation Factors 544
Contents XXXIII
II. Guanine Nucleotide-Dependent Association of CholeraToxin with ADP-Ribosylation Factors 546
III. Activation of Escherichia coli Heat-Labile Enterotoxin byADP-Ribosylation Factor 546
C. Structure of ADP-Ribosylation Factors 547I. Deduced Amino Acid Sequences and Gene Structure of
ADP-Ribosylation Factors 547II. Expression of ADP-Ribosylation Factors in Eukaryotic
Species 549D. Hormonal and Developmental Regulation of ADP-Ribosylation
Factors 550E. Physiological Roles for ADP-Ribosylation Factors 551References 555
C. rho and rho-Like Proteins
CHAPTER 36
rho and rho-Related ProteinsA.J. RIDLEY and A. HALL. With 5 Figures 563
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 568I. Mammalian rho Proteins 568
II. The rac Proteins 5701. rac and the Actin Cytoskeleton 5702. rac and the Superoxide Production 5713. Other rho-Related Proteins • 572
F. Conclusions - 574References 574
CHAPTER 37
The Mammalian Homolog of the Yeast Cell-Division-Cycle Protein,CDC42: Evidence for the Involvement of a Rho-Subtype GTPase inCell 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
XXXIV Contents
B. Reconstitution of an Epidermal Growth Factor StimulatedPhosphorylation 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 YeastCell Division Cycle Protein CDC42Sc 583
D. Function of CDC42Sc in Saccharomyces cerevisiae 584E. Possible Involvement of CDC42Hs in Cell Growth Regulation . . . 585
I. cDNA Transfection Studies 585II. CDC42Hs Regulatory Proteins 586
1. CDC42Hs GTPase Activating Protein 5862. CDC42Hs Guanine Nucleotide Dissociation Stimulator . . . 5883. CDC42Hs Guanine Nucleotide Dissociation Inhibitor 591
References 593
D. Regulation of and by Small GTPases
CHAPTER 38
Role of RaplB 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 599B. Phosphorylation of Raplb 601
I. Structural Properties 6011. cAMP-dependent Phosphorylation of Raplb in Human
Platelets 6012. Phosphorylation of Raplb by a Neuronal Ca2+/
Calmodulin-dependent Protein Kinase, CaM Kinase Gr . . 6023. Mutational Analysis of the Protein Kinase A-dependent
Phosphorylation Site of Raplb 6034. Phosphorylation-dependent Activation of Raplb:
Role of Guanine Nucleotide Dissociation Stimulator 604II. Physiological Properties: The Platelet Model 604
1. Thrombin-induced Association of Raplb with Ras-GTPase Activating Protein: Effect of Phosphorylation . . . 605
2. Ras-GAP Associates with Phospholipase Cy-l in HumanPlatelets 606
III. A Working Model and Open Questions 607References 609
CHAPTER 39
GDP/GTP Exchange Proteins for Small GTP-Binding ProteinsY. TAKAI, K. KAIBUCHI, A. KIKUCHI, and T. SASAKI. With 5 Figures.. 613
Contents XXXV
A. Introduction 613B. Physical Properties of GDP/GTP Exchange Protein 614C. Two Actions of GDP/GTP Exchange Protein and Requirement
of the Posttranslational Processing of Small GTPases forGDP/GTP Exchange Protein Actions 614
D. Substrate Specificity of GDP/GTP Exchange Protein andFunctional Cooperation Between Guanine NucleotideDissociation Stimulator and Guanine Nucleotide DissociationInhibitor 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 RegulatingSuperoxide Generation 618
H. The Function of smg Guanine Nucleotide DissociationStimulator, rho, and rho Guanine Nucleotide DissociationInhibitor in Regulating the Actomyosin System 618
I. The Function of smg p25 Guanine Nucleotide DissociationInhibitor in Regulating Intracellular Vesicle Transport 620
J. Conclusions 621References 622
CHAPTER 40
GTP-Mediated Communication Between Intracellular Calcium PoolsD.L. GILL, T.K. GHOSH, A.D. SHORT, J. BIAN, and R.T. WALDRON.
With 8 Figures 625
A. Intracellular Ca2+ Signaling Pools 625I. Nature of Intracellular Ca2 + Pools 625
II. Movements of Ca2+ Induced by Inositol Phosphates 626III. Intracellular Ca2+ Channels 627IV. Significance of Ca2 + Within the InsP3-Sensitive Ca2+ Poo l . . . 629
B. Ca2+ Movements Activated by Guanine Nucleotides 630I. GTP-Induced Ca2+ Fluxes 630
II. Ca2+ Compartments Sensitive to GTP and InsP3 631III. Distinctions Between GTP- and InsP3-Induced Ca2+
Transport 632IV. Rationale for the Action of GTP 632
C. Interorganelle Translocation of Ca2+ 634I. Model for GTP-Activated Ca2+ Translocation 634
II. GTP-Activated Ca2+ Transfer into the InsP3-Sensitive Ca2+
Pool 637III. Isolation of InsP3-Releasable and InsP3-Recruitable Pools . . . 637IV. Functional Organization of Ca2+-Regulatory Organelles . . . . 639
XXXVI Contents
D. G-Proteins and Interorganelle Transfer of Ca2+ 642I. 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 651
A. Introduction 651B. Receptors and Intracellular Signals that Regulate p21 ras 651
I. Activation of p21 ras in Cells Other than T Lymphocytes 651II. Activation of p21 ras in T Lymphocytes 653
C. GTPase Activating Proteins Regulate p21 r a s in T Lymphocytes... 654D. Mechanisms of Regulation of ras GTPase Activating Proteins in
T Cells 655E. Function of p21 r a s in T Lymphocytes 656References 657
CHAPTER 42
GTPases as Regulators of Regulated SecretionT.H.W. LILLIE and B.D. GOMPERTS. With 7 Figures 661
A. GTP: A Sine Qua Non for Exocytosis 661I. Ca2+-Dependent Secretion in Myeloid Granulocytes 661
B. Probing Exocytosis: Permeabilised Cells 662I. GTPyS-Induced, Ca2+-Independent Exocytosis 664
II. Ca2+-Induced, GTP-Dependent Exocytosis 664III. One or Two Effectors? 665
1. Chloride Suppresses and Glutamate Enhances GuanineNucleotide Sensitivity of Exocytosis 666
IV. Kinetics of Exocytosis 6681. Mg2+ Permits Abrupt Onset 6692. Mg2+ Deprivation Causes Onset Delays 670
V. GTPases Regulate and Modulate Exocytosis in Many Cellsand Tissues 670
C. On the Nature of GE 671I. The Example of G s 671
II. The Example of the Monomeric GTPases 671D. Single Cell Analysis of GTPyS-Induced Exocytosis 672E. Two GTPases in Regulated Exocytosis? 674References 675
Contents XXXVII
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