GTPases regulating membrane targeting and fusion

00 430mie_0121828085.jpg......Page 1
01 fm.pdf......Page 2
02 preface.pdf......Page 3
03 toc.pdf......Page 5
04 contributors.pdf......Page 11
Exploring Trafficking GTPase Function by mRNA Expression Profiling: Use of the SymAtlas Web-Application and the Membrome Data......Page 19
Introduction......Page 20
Use of the SymAtlas Web-Application......Page 21
Further Notes on the Use of Tissue mRNA Profiling Data......Page 25
References......Page 27
Introduction......Page 29
Algorithm Method......Page 30
Comments and Cautions......Page 35
References......Page 37
Application of Phylogenetic Algorithms to Assess Rab Functional Relationships......Page 39
Generation of Protein Sequence Alignment......Page 40
Principal Components Analysis of Alignment Data......Page 41
Scope and Comments......Page 47
References......Page 48
Introduction......Page 49
Principle......Page 50
Peptide Synthesis......Page 51
Unprenylated Peptides......Page 52
In Vitro Prenylation of Rab7C-K(Dans)-S-C-S-C and Purication of the Rab7C-K(Dans)-S-C(GG )-S-C(GG): REP-1 Complex......Page 53
Choice of Expression Vector and Generation of Thioester Tagged Rab GTPases......Page 54
Construction of Fluorescent Rab7CK(Dans)SC(GG)SC(GG):REP-1 Complex Using a Combination of Protein Ligation and In Vitro Preny......Page 55
Construction of Fluorescent Monoprenylated and Diprenylated Rab7 GTPase and Development of a Fluorescence-Based Rab Prenylati......Page 57
References......Page 61
Assay and Functional Properties of PrBP(PDEdelta), a Prenyl-Binding Protein Interacting with Multiple Partners......Page 63
PrBP(PDEdelta) Is a 17-kDa Prenyl-Binding Protein Closely Related to unc119 and RhoGDI......Page 64
PrBP(PDEdelta) Has a Wide Tissue Distribution......Page 65
PrBP(PDEdelta) Interacting Proteins Identified by Yeast Two-Hybrid Screening......Page 66
Construction of PrBP(PDEdelta) Bait and Library Screening......Page 67
Immunoblot Detection of PrBP(PDEdelta) in Retinal Homogenates and Rod Outer Segments (ROSs)......Page 68
Immunoblot Analysis......Page 69
Bovine and Amphibian......Page 70
Immobilized PrBP(PDEdelta) Pull-Down Assays for Identifying Binding Partners and Characterizing the Determinants of High-Affinity Binding to PrBP(PDEdelta)......Page 71
In Vitro Expression of GRK1/GRK7 for PrBP(PDEdelta) Interaction Assay......Page 73
Conclusion: Model for PrBP(PDEdelta) Involvement in Vectorial Protein Transport in Photoreceptors......Page 74
Acknowledgments......Page 75
References......Page 76
Introduction......Page 78
Determination of the Subcellular Localization of Rab Effectors......Page 79
Measurement of the Interaction of Rab Effectors with Members of the Rab GTPase Family......Page 81
Functional Assessment of the Involvement in Dense Core Granule Exocytosis......Page 84
Conclusion......Page 90
References......Page 91
Analysis of Rab1 Recruitment to Vacuoles Containing Legionella pneumophila......Page 93
Introduction......Page 94
Reagents......Page 95
Preparation of Murine Bone Marrow-Derived Macrophages......Page 96
Cell Fixation and Staining Procedures......Page 97
Expected Results and Variations......Page 98
L. pneumophila Infection of CHO FcalphaRII Cells......Page 99
General Comments......Page 100
Intracellular Growth Assays......Page 101
References......Page 102
Reconstitution of Rab4-Dependent Vesicle Formation In Vitro......Page 104
Overview......Page 105
Buffer Solutions......Page 107
Permeabilization and Cytosol Removal......Page 108
Experimental Results......Page 109
Immunodepletion......Page 111
Experimental Results......Page 112
Conclusions......Page 113
References......Page 114
Assay of Rab4-Dependent Trafficking on Microtubules......Page 116
Preparation of Fluorescent Endocytic Ligand......Page 117
Preparation of ASOR Endosomes from Rat Liver......Page 118
Polymerizing Fluorescent Microtubules......Page 119
Microscope System......Page 122
Protocol to Measure the Microtubule-Based Motility of Rab4-Containing Endosomes......Page 123
Frequency of Movement......Page 124
Fission......Page 125
Protein Colocalization......Page 126
Conclusion......Page 129
References......Page 130
CD2AP, Rabip4, and Rabip4prime: Analysis of Interaction with Rab4a and Regulation of Endosomes Morphology......Page 132
Yeast Expression Vector and Yeast Strain......Page 133
Yeast Cotransformation Procedure......Page 134
Measurement of beta-Galactosidase Activity......Page 135
Expression Constructs......Page 136
Purification of GST-Rab4a......Page 137
Loading of GST-Rab4a with Guanine Nucleotides (GDP or GTPgammaS) and Incubation with Products of the In Vitro Transcription– Translation Reaction......Page 138
Expression Constructs......Page 139
Comments......Page 140
Analysis of Cellular Localization of Rab4a and Its Effectors by Fluorescence Confocal Analysis......Page 141
Acknowledgments......Page 142
References......Page 143
Introduction......Page 144
Expression Constructs......Page 145
EEA.1-Based Sensor......Page 147
Choice of Cell Type, Transfections, and Imaging......Page 148
FRET Measurements and Calculations......Page 149
Detection of Rab5 Interaction with R5BD by FRET......Page 152
Detection of Rab5 Interaction with EEA.1sh by FRET......Page 153
FRET Measurements and Calculations......Page 155
References......Page 158
Introduction......Page 160
Preparation of the Target GTPase......Page 161
Phage Display Screen......Page 162
Methods......Page 164
Methods......Page 165
Materials......Page 166
Methods......Page 167
Isolation of Positive Clones......Page 169
Methods......Page 170
Materials......Page 171
Immunofluorescence......Page 172
Materials......Page 173
Materials......Page 174
In Vivo Imaging with Fluorescent scFvs......Page 175
Methods......Page 176
References......Page 177
Analysis of Rab Protein Function in Neurotransmitter Receptor Trafficking at Hippocampal Synapses......Page 179
Ultrastructural Studies of Rab Proteins at Synaptic Terminals......Page 180
Analysis......Page 181
Cloning and Expression of Recombinant Rab Proteins in Hippocampal Neurons......Page 182
Effect of Recombinant Rab Proteins on Basal Synaptic Transmission......Page 183
Electrophysiological Tagging......Page 184
Method......Page 186
Analysis of Local Dendritic Spine Trafficking......Page 187
References......Page 190
Introduction......Page 193
General Approach for Studying the Itinerary of Lipid Analogs or Lipid-Binding Toxins Using Mutant Rab Proteins......Page 194
Fluorescent Sphingolipid Analogs......Page 196
Transfection of Human Skin Fibroblasts (HSFs) with the Rab Constructs......Page 198
Roles of Rab4 and Rab11 in Membrane Recycling of BODIPY-LacCer......Page 199
Role of Rab9 in BODIPY-LacCer Transport from the PM to the Golgi Apparatus......Page 201
Methods to Elevate Cellular Cholesterol......Page 203
Preparation of mbetaCD/Cholesterol Complex......Page 204
Preparation of Endosome-Enriched Fraction......Page 205
Extraction of Rab4 from Endosome-Enriched Membranes by GST-alphaGDI......Page 206
References......Page 207
Assay of Rab13 in Regulating Epithelial Tight Junction Assembly......Page 210
Introduction......Page 211
Cloning and Mutagenesis of Human Rab13......Page 212
Cell Culture and Generation of Stable MDCK Cells Expressing Wild-Type and Rab13 Mutants......Page 213
Localization of Tight Junction Proteins by Indirect Immunofluorescence......Page 214
Assessment of Monolayer Integrity......Page 216
Synthesis of BODI PY R6G-Sphingomyelin......Page 218
Paracellular Flux Assay......Page 219
Freeze-Fracture Immunolabeling (FL)......Page 220
References......Page 221
Introduction......Page 222
Expression of the Rab Protein and Detection of Tyrosine Phosphorylation by Western Blot Analysis......Page 223
Immunoprecipitation of the Rab Protein and Detection of Tyrosine Phosphorylation by Western Blot Analysis......Page 225
Tyrosine Kinase Inhibitors......Page 226
Subcellular Distribution of the Phosphorylated Rab Protein......Page 227
References......Page 228
Assay of Rab25 Function in Ovarian and Breast Cancers......Page 231
Introduction......Page 232
Preparation of RAB25 Expression Constructs......Page 234
Transfection and Clonal Selection......Page 235
Apoptosis Assays......Page 236
RNA Interference......Page 237
Comment......Page 238
References......Page 243
Introduction......Page 245
Anti-granuphilin Antibodies......Page 247
Immunohistochemistry of Granuphilin, Rab27a, and Syntaxin-1a in Mouse Pancreatic Islets......Page 248
Interaction of In Vitro Translated Granuphilin with Bacterially Expressed GST-Fused Proteins......Page 249
Pull-Down Assay Using GST-Fused Proteins in Cell Extracts......Page 250
Coimmunoprecipitation in Cultured Cells......Page 251
Coimmunoprecipitation of Endogenous Proteins in Pancreatic Islets......Page 252
Targeting Assay......Page 253
Morphological Assay......Page 254
Comments......Page 256
References......Page 257
Assays for Functional Properties of Rab34 in Macropinosome Formation......Page 259
Introduction......Page 260
Introduction of Point Mutations into the cDNA......Page 261
Expression and Preparation of Recombinant Proteins......Page 263
GTP Binding/GTPase Assay by Thin Layer Chromatography......Page 264
GTP Binding/GTPase Assay In Vitro......Page 265
GTP Binding/GTPase Assay In Vivo......Page 266
Localization by Epitope Tagging and Enhanced Green Fluorescent Protein (EGFP) Tagging......Page 268
Epitope Tagging and EGFP Tagging......Page 269
Analyses of the Role of Rab34 in Macropinosome Formation......Page 270
Assay for Rab34 Function in PDGF or PMA-Treated Cells......Page 271
References......Page 272
Introduction......Page 274
Identification of the Rab Proteins Localized on Non-Clathrin-Derived Endosomes......Page 275
General Method to Measure Internalization of MHCI and Tfn......Page 277
Identification of the Endocytic Step Where Rab22a Associates with the Non-Clathrin-Derived Endosomes......Page 278
Time‐Lapse Imaging of MHCI-Internalization in GFP-Rab22a Expressing Cells......Page 279
Overexpression of Rab22a and Rab11 Mutants and siRNA-Mediated Depletion of Rab22a......Page 280
Effect of the Overexpression of Rab22a and Rab11 Mutants and siRNA-Mediated Depletion of Rab22a on the Recycling to.........Page 281
Assay to Measure Recycling of MHCI to the PM......Page 282
Concluding Remarks......Page 283
References......Page 284
Introduction......Page 285
Materials......Page 287
Mono Q PC 1.6/5 Column Chromatography......Page 288
Buffers for the GH Release Assay......Page 289
References......Page 290
Introduction......Page 293
Purification of Alsin and Its Individual Domains......Page 295
Purification of GTPases......Page 297
In Vivo Yeast Two-Hybrid Assays......Page 298
In Vitro Binding Assays......Page 299
In Vitro Rab Nucleotide Exchange Assay......Page 300
In Vivo Rac1 Nucleotide Exchange Assay......Page 301
Fractionation Procedure......Page 303
Treatment of P3 Fraction with Agents to Determine the Nature of Alsin's Association......Page 306
References......Page 307
Purification and Analysis of RIN Family-Novel Rab5 GEFs......Page 309
Construction and Selection of RIN-Containing Baculovirus......Page 310
Purification of the RIN Family from Sf9 Cells......Page 311
GDP-Dissociation Reaction......Page 312
Guanine-Nucleotide Binding Reaction......Page 313
Purification of the FLAG-RIN Family from COS7 Cells......Page 314
Gel Filtration Analysis of the RIN Family......Page 315
References......Page 316
Introduction......Page 317
Procedure......Page 320
Procedure......Page 322
Protein Expression and Purification......Page 323
GDP/GTP Exchange Assays......Page 324
Constructs......Page 325
References......Page 326
Introduction......Page 329
Buffers......Page 331
Recombinant Proteins......Page 332
Growing and Harvesting of Salmonella......Page 333
Preparation of Salmonella-Containing Phagosomes......Page 334
Recruitment of Rab5 Depends on the Presence of Viable Salmonella in the Phagosomes......Page 335
Identification of SopE, a Salmonella Effector Protein, as a Rab5 Binding Protein......Page 336
Determination of Binding of SopE with Rab5......Page 338
Conclusion......Page 340
References......Page 341
Introduction......Page 344
Expression Constructs......Page 345
Partial Purification of FLAG-Tagged ALS2/ALS2CL by Immunoprecipitation......Page 346
Expression and Purification of Rab5A......Page 348
GXP Loading......Page 349
GDP Dissociation Assay......Page 350
GTP-Binding Assay......Page 351
Characterization of Oliogomerized ALS2 by Gel Filtration......Page 352
Discussion......Page 353
References......Page 354
Introduction......Page 356
Cargo Recognition and Sorting......Page 357
New Approach for Expression Protein Complexes......Page 358
Construction of Vps22, Vps25, and Vps36 Cassettes......Page 359
Transformation of the Vps22, Vps25, and Vps36 Cassettes into pST39 Expression Vector......Page 362
Concluding Remarks......Page 363
References......Page 364
Introduction......Page 367
Tagging of Yip1p......Page 368
Genetic Analysis of yip1 Mutant Alleles......Page 369
References......Page 372
Introduction......Page 374
Recombinant Protein Purification......Page 376
Reaction buffer......Page 377
10/18 buffer......Page 378
Recombinant His6‐alphaGDI protein......Page 379
Comments......Page 380
References......Page 381
Discovery of GDF......Page 383
Yip3 Is a GDF for Endosomal Rabs......Page 385
Buffers and Solutions......Page 386
Purification......Page 387
Assay for GDI Displacement Factor Activity......Page 388
Method A: GDF Assay......Page 389
Acknowledgment......Page 390
References......Page 391
TIP47 in Mannose 6-Phosphate Receptor Transport......Page 392
Purification of Native TIP47 from Bovine Kidney......Page 395
Method......Page 396
Materials......Page 397
Cellular Depletion of TIP47 in Living Cells......Page 399
Acknowledgment......Page 400
References......Page 401
Introduction......Page 402
Reagents......Page 406
Homogenization and Preparation of a Postnuclear Supernatant (PNS)......Page 408
Endosome Fractionation by Flotation in a Sucrose Gradient......Page 409
Procedure......Page 410
Procedure B: Sequential......Page 411
Activation of p38 MAPK Modulates Endocytosis In Vivo......Page 412
Comments......Page 413
References......Page 414
Rab2 Purification and Interaction with Protein Kinase C iota/lambda and Glyceraldehyde-3-Phosphate Dehydrogenase......Page 417
Introduction......Page 418
Purification of Recombinant Rab2 from BL21 Transformed Bacteria......Page 419
Analysis of GDP-GTP Binding Properties of Recombinant Rab2......Page 420
Coimmunoprecipitation of Rab2 and Effectors......Page 421
Mammalian Two-Hybrid Assay......Page 422
Purification of His6-aPKCiota /lambda from Vaccinia Infected/pcDNA3-aPKCiota /lambda Transfected HeLa Cells......Page 423
ELISA to Map Rab2 Interacting Domains with aPKCiota/lambda and GAPDH......Page 424
Quantitative Membrane Binding Assay......Page 425
References......Page 426
Introduction......Page 428
Isolation of Rat Liver Golgi Membranes......Page 429
Preparation of Affinity Columns......Page 430
Affinity Purification of Proteins from Golgi Membranes......Page 431
Yeast Growth Media......Page 432
Small-Scale Transformations for Directed Two-Hybrid Tests......Page 433
Rab-Effector Binding Assays......Page 434
Preparation of Immobilized Rab Beads......Page 435
Preparation of Golgi Extracts for Rab-Binding Assays......Page 436
References......Page 437
Purification and Properties of Rabconnectin-3......Page 439
Introduction......Page 440
Preparation of Crude Synaptic Vesicle Fraction from Rat Brain......Page 441
Immunoprecipitation......Page 442
GH Release Experiments......Page 443
Comments......Page 444
References......Page 445
Introduction......Page 446
Materials......Page 447
Media......Page 448
Measurement of beta-Galactosidase Activity by Liquid Culture......Page 449
Purification......Page 451
In Vitro Binding Assay......Page 452
Infection of Pancreatic Islets Isolated from Noc2-/- Mice with Wild-Type Noc2 or Noc2AAA cDNA......Page 454
Concluding Remarks......Page 455
References......Page 456
Introduction......Page 458
Culture of Melan-a Mouse Melanocytes and Transfection......Page 460
Immunofluorescence Analysis of Endogenous Rab27A and Myosin Va in Melan-a Cells......Page 462
Assay for Interaction between Slac2-a and Rab27A, Myosin Va, and Actin In Vivo......Page 463
Assay for the Function of PEST-Like Sequences in the C-terminus of Slac2-a......Page 465
Assay for Depletion of Endogenous Slac2-a in Melan-a Cells by Specific siRNA......Page 466
Concluding Remarks......Page 467
References......Page 468
Identification and Biochemical Analysis of Slac2-c/MyRIP as a Rab27A-, Myos in Va/VIIa-, and Actin-Binding Protein......Page 471
Introduction......Page 472
Expression and Purification of GST-Slac2-c-SHD......Page 473
Assay for Interaction between GST-Slac2-c -SHD and FLAG- Rab Proteins......Page 475
Slac2-c Directly Interacts with Myosin Va and Myosin VIIa......Page 476
Assay for Direct Interaction between GST-Slac2-c-deltaSHD and the Globular Tail of Myosin Va......Page 477
Assay for In Vitro Formation of a Tripartite Protein Complex of Rab27A, Slac2-a/c, and Myosin Va from Purified Components......Page 478
Assay for Determination of the Actin-Binding Domain of Slac2-c......Page 479
Localization of the Slac2-c ABD on Actin Filaments in PC12 Cells......Page 481
Concluding Remarks......Page 482
References......Page 483
Introduction......Page 485
Biochemical Characterization of the Rab-Binding Partners of  Slp4-a In Vitro......Page 486
Expression of FLAG-Rab1–40 in COS-7 Cells and Generation of FLAG-Rab Panels......Page 487
Assay for Interaction between T7-Slp4-a and FLAG-Rabs In Vitro......Page 488
GST (Glutathione S-Transferase)-Fusion Protein Pull-Down Assay: Assay for Interaction between GST-Slp4-a-SHD and FLAG-Rabs......Page 489
Antibody Production......Page 490
Assay for Interaction between GST-Slp4-a and Endogenous Rab27A in PC12 Cells......Page 491
Inhibition of Dense-Core Vesicle Exocytosis by Slp4-a through Specific Interaction with Rab27A in PC12 Cells......Page 492
NPY Release Assay......Page 493
References......Page 495
Assay and Functional Interactions of Rim2 with Rab3......Page 498
Introduction......Page 499
Characterization of the Rab3A-Binding Activity of the Long Forms and  Short Forms of Rim2 RBD In Vitro......Page 500
Methods of Identification of Alternative Splicing Isoforms of Rim1 and Rim2......Page 501
Cotransfection Assay in COS-7 Cells......Page 502
Assay for Competition Experiments......Page 503
Assay for Comparison of Rab3A-Binding Affinity by Long Forms and Short Forms of Rim2 RBD In Vitro......Page 504
Immunofluorescence Analysis of GFP-Tagged Rim2 RBDs......Page 506
Summary of Results......Page 507
References......Page 508
Introduction......Page 510
Rab-Binding Domain of Rabphilin and Noc2 Interacts with Three Distinct Rab Species In Vitro and in PC12 Cells......Page 511
Assay for Interaction between T7-rabphilin or T7-Noc2 and FLAG-Rabs In Vitro......Page 512
Assay for Competition Experiments......Page 513
Assay for Interaction between GST-Rabphilin-RBD (or GST-Noc2-RBD) and Endogenous Rab27A in PC12 Cells......Page 515
Rabphilin and Noc2 Are Recruited to Dense-Core Vesicles and Regulate Their Exocytosis through Interaction with Rab27A in PC12 Cells......Page 516
Immunoelectron Microscopic Analysis......Page 518
NPY Release Assay......Page 519
References......Page 520
Functional Properties of the Rab-Binding Domain of Rab Coupling Protein......Page 523
Introduction......Page 524
Site-Directed Mutagenesis......Page 525
Transformation......Page 528
Interaction Assay......Page 529
Intracellular Localization of Mutants......Page 530
Subcellular Fractionation......Page 531
References......Page 532
Purification and Functional Properties of Rab11-FIP2......Page 534
Introduction......Page 535
Batch Purification of 6timesHis-Rab11 FIP2......Page 537
Fluorescent-Based Recycling Assay......Page 538
Quantification of Transferrin Internalization and Recycling......Page 540
References......Page 541
Purification and Functional Properties of Rab11-FIP3......Page 543
Expression and Purification of Recombinant Rab11-FIP3......Page 544
Hexahistidine-Fused Recombinant Protein Purification......Page 545
GST Recombinant Protein Purification......Page 546
Epitope Prediction......Page 547
Immunofluorescence Microscopy......Page 549
Mitotic Synchronization and Rab11-FIP 3 Subcellular Localization......Page 550
Plasmid Construction......Page 551
Analysis of Exogenous Expression of Rab11-FIP3......Page 553
Analysis of the Functional Consequences of Rab11-FIP3 Mutant Expression......Page 554
References......Page 555
Class I FIPs, Rab11-Binding Proteins That Regulate Endocytic Sorting and Recycling......Page 557
Introduction......Page 558
Identification of the Rab11 Family Interacting Proteins (FIPs)......Page 559
Expression and Purification of the Recombinant Rab11 and FIPs......Page 561
Additional Comments......Page 562
Identification and Characterization of Rab11-Binding Domain (RBD)......Page 563
Role of RCP and Rip11 in TfR Recycling and Degradation......Page 564
Knock-Down of FIPs by RNA-Interference (RNAi)......Page 565
Flow Cytometry-Based Transport Assays......Page 567
References......Page 569
Introduction......Page 571
Protocol......Page 572
Reagents......Page 574
Preparation of Pig Brain Extract and Binding Assay with GST-Ear Adaptins......Page 575
Preparation of HeLa Cell Lysate and Binding Assay......Page 576
Assay for Rabaptin-5alpha and gamma1-Adaptin Colocalization on Rab4-Containing Endosomes......Page 577
Isolation of GST-Rab Fusion Protein and Guanine Nucleotide Loading......Page 578
Pig Brain Cytosol Preparation and Binding Assay......Page 580
Pulse–Chase Protocol for Fluorescently Labeled Transferrin......Page 581
Concluding Remarks......Page 583
References......Page 584
Introduction......Page 586
GST Protein......Page 587
p85 Wild Type, and Mutants p85DeltaBH, p85R151A, and p85R274A......Page 588
General......Page 590
Comments......Page 592
Preparation of Immobilized GST Fusion Proteins......Page 593
GST Pull-Down Assay......Page 594
References......Page 596
Assay and Stimulation of the Rab5 GTPase by the p85alpha Subunit of Phosphatidylinositol 3-Kinase......Page 598
Rab5 Wild Type......Page 599
p85 Wild-Type and the p85R274A Mutant......Page 601
Steady-State GAP Assay......Page 602
Separation......Page 603
Single Turnover GAP Assay......Page 605
References......Page 606
Ubiquitin Regulation of the Rab5 Family GEF Vps9p......Page 608
Introduction......Page 609
Analysis of VPS9 Function in the Biosynthetic Pathway......Page 610
Procedure......Page 613
Buffer/Materials......Page 615
Procedure......Page 616
Vps9p Expression and Purification......Page 617
Buffer/Materials......Page 618
Procedure......Page 619
Analysis of Vps9p-Stimulated Vps21p Nucleotide Release......Page 621
Procedure......Page 622
Buffer/Materials......Page 623
Analysis of Vps9p Ubiquitylation In Vivo......Page 624
Procedure......Page 625
Buffer/Materials......Page 626
Procedure......Page 627
References......Page 628
Introduction......Page 631
Materials......Page 632
Expression of GGA GAT Domain Fragments......Page 633
Construction, Expression, and Purification of Rabaptin-5 551–862 Fragment......Page 635
Construction and Expression of Rabaptin-5 551–661 Variants......Page 636
Competition Analysis Emphasizing the Interaction between GGA1 GAT Domain and Full-Length Rabaptin-5......Page 637
Cocrystallization of the GGA1– Rabaptin-5 Complex......Page 638
References......Page 639
Introduction......Page 641
Pull-Down Experiment......Page 642
Gel Filtration......Page 645
References......Page 646
Introduction......Page 648
Procedure......Page 650
Procedure......Page 651
Procedure......Page 652
Specificity......Page 653
Cytosol Preparation......Page 654
References......Page 655
Assay and Properties of Rab6 Interaction with.Dynein-Dynactin Complexes......Page 657
Introduction......Page 658
Yeast Growth Media......Page 659
Preparation of Frozen Competent Yeast Cells......Page 661
Biochemical Analysis of Rab6-Binding Partners......Page 662
Expression and Purification of Recombinant Rab6......Page 663
Rab6 Effector-Binding Assays with Purified Proteins......Page 666
References......Page 667
Introduction......Page 669
Purification of MKlp2 and Plk1 from Insect Cells......Page 670
Purification of Proteins from Bacteria......Page 671
Interaction of MKlp2 with Microtubules......Page 672
Microtubule-Binding and -Bundling Assays......Page 674
Phosphorylation of MKlp2 by Plk1......Page 675
Studying MKlp2 Function in Cytokinesis......Page 676
References......Page 678
Introduction......Page 680
Constructs......Page 682
PtdIns 3-Kinase Activity Assay......Page 684
Rabbit Anti-hVPS34 Antibody......Page 686
Immunofluorescence......Page 690
Immunoelectron Microscopy......Page 691
Background......Page 692
Coimmunoprecipitation Experiments to Document Protein–Protein Interactions......Page 693
GST-Rab7 Fusion Protein Expression and Purification......Page 694
Monitoring GTP-Binding Activity of GST-Rab7......Page 695
GST-Rab7/p150-Binding Assay......Page 696
Flow Cytometry-Based Glutathione Bead Assay......Page 697
Conclusions......Page 698
References......Page 699
Introduction......Page 702
Rabbit Anti-XAPC7 Antibody......Page 704
Immunofluorescence......Page 705
Background......Page 706
EGFR Degradation Assay......Page 707
Metabolically Labeled Lysates......Page 708
Background......Page 709
Proteasome Assembly Assay......Page 710
XAPC7 Function Measured by Phagocytosis......Page 712
References......Page 714
Introduction......Page 716
Construction of Recombinant Expression Vectors......Page 717
Cell Culture and Transient Transfection......Page 718
Molecular Interaction of RILP......Page 719
Confocal Fluorescence Microscopy......Page 721
Estimation of [125I]LDL Degradation......Page 722
RILP Promotes Centripetal Migration of Rab7-Positive Organelles......Page 723
RILP Is Required for Transport to Lysosomes......Page 724
References......Page 725
Introduction......Page 728
Expression Constructs and Mutagenesis......Page 729
Generation and Purification of Rabbit Polyclonal Antibody against Rab34......Page 730
GTP Overlay Assay......Page 732
Yeast Two-Hybrid Interaction Assay......Page 733
GST Pull-Down Experiment and Immunoblotting Analysis......Page 734
Rab34 Regulates Lysosome Positioning by Interacting with RILP......Page 736
References......Page 739
Further Reading......Page 740
Rabring7: A Target Protein for Rab7 Small G Protein......Page 741
Introduction......Page 742
Materials......Page 743
Strain and Media......Page 744
Culture......Page 745
Immunoprecipitation......Page 746
Assay for Recruitment of Rabring7 to Late Endosome/Lysosome by the GTP-bound Form of Rab7......Page 747
Comments......Page 748
References......Page 749
Introduction......Page 751
Construction of pGEX5X-1-p40 cDNA......Page 754
Quality and Quantity Check for GST-Fusion Protein Expression......Page 755
Preparing and Drying the Lipid Mixture......Page 756
Liposome-Binding Assay......Page 757
References......Page 759
Introduction......Page 761
Recombinant Protein Production......Page 763
Analysis of Rab11a Binding......Page 764
Analysis of Rab11-FIP2 Binding to Rab11-FIP2......Page 766
References......Page 768
Introduction......Page 771
Constructs......Page 772
beta-Galactosidase Assay......Page 773
Assay of Myosin Vb Tail Interactions with Rab11 Family Members in Transfected Madin-Darby Canine Kidney (MDCK) Cells......Page 774
EGFP-Rab Constructs and Cell Lines......Page 776
Immunofluorescence......Page 777
References......Page 778
Properties of Rab13 Interaction with Protein Kinase A......Page 780
Introduction......Page 781
Calcium Switch Experiments......Page 782
Immunofluorescence Microscopy......Page 783
Immunoblotting......Page 784
Immunoprecipitation of VASP......Page 785
GST Pull-Down Assay......Page 786
In Vitro Kinase Assay......Page 787
References......Page 788
Functional Properties of Rab15 Effector Protein in Endocytic Recycling......Page 790
Cell Surface Biotinylation Assays to Measure TfR Recycling......Page 791
Buffers and Reagents......Page 792
Assay Protocol......Page 794
Additional Comments......Page 795
Buffers and Reagents......Page 796
Assay Protocol......Page 797
Additional Comments......Page 798
References......Page 800
Assays for Interaction between Rab7 and Oxysterol Binding Protein Related Protein 1L (ORP1L)......Page 802
Introduction......Page 803
Colocalization of ORP1L with Rab7 Assessed by Confocal Immunofluorescence Microscopy......Page 804
Approaches to Studying the Physical Interaction of ORP1L and Rab7......Page 806
Immunoprecipitation......Page 807
Two-Hybrid Luciferase Assay......Page 808
Pull-Down Assay......Page 811
Acknowledgments......Page 815
References......Page 816
Introduction......Page 818
Primer Design......Page 820
Reagents......Page 823
Cloning of Rab23 for Transient Expression......Page 824
Protocol......Page 826
Protocol......Page 827
Protocol......Page 828
Reagents......Page 829
Transferrin Internalization......Page 830
Protocol......Page 831
Protocol......Page 832
Immunolabeling......Page 834
References......Page 835
Introduction......Page 837
Solution......Page 838
Preparation of GST-Rab27a......Page 839
Expression and Purification of Recombinant Munc13-4......Page 840
Solution......Page 841
Purification of His6-Munc13-4......Page 842
Functional Analysis of Munc13-4 Using Permeabilized Platelets......Page 843
Labeling of Dense Granules with [3H]serotonin......Page 844
Serotonin Secretion Assay......Page 845
References......Page 846
Introduction......Page 848
Buffers......Page 849
Indirect Fluorescence Staining of hVps15 and hVps34 in Fixed Cells......Page 850
Comments......Page 851
Buffers......Page 852
Preparation of Inactive and Active Forms of GST-Rab5 by Nucleotide Exchange......Page 854
Coupled In Vitro Transcription/Translation of hVps15 Mutants......Page 855
GST-Rab5/hVps15 In Vitro Binding Assay......Page 856
References......Page 857
Further Reading......Page 858
Purification and Functional Properties of Prenylated Rab Acceptor 2......Page 859
Introduction......Page 860
Solutions......Page 861
Purification of Recombinant GST-VAP-33......Page 862
Effect of Boiling on Bacterial- and Mammalian-Expressed PRA2......Page 863
Coimmunoprecipitation of VAP-33 with PRA2......Page 865
Comments......Page 866
References......Page 867
x subject index.pdf......Page 868
xx author index.pdf......Page 889
xxx mie index.pdf......Page 922