Regulators and Effectors of Small GTPases, Part E: GTPases Involved in Vesicular Traffic

Regulators and Effectors of Small GTPases

Contributors to Volume 329

Preface

Methods in Enzymology

Section I. Rab GTPases

1: Structural Basis for Rab Function: An Overview

2: Expression of Wild-Type and Mutant Green Fluorescent Protein-Rabl for Fluorescence Microscopy Analysis

Introduction

Preparation of Recombinant Expression Vectors

Infection and Transfection

Fixation and Mounting

Fluorescence Microscopy

Conclusion

3: Fluorescence Methods for Monitoring Interactions of Rab Proteins with Nucleotides, Rab Escort Protein, and Geranylgeranyltransferase

Introduction

Fluorescent Nucleotides

Generation of Fluorescent Proteins by Cysteine Labeling

Use of Intein and in Vitro Ligation Technologies to Introduce Fluorescent Groups into Rab7

Conclusion

Appendix

Acknowledgments

4: Prenylation of Rab Proteins in Vitro by Geranylgeranyltransferases

Introduction

Methods

Comments

5: Antibody and Oligonucleotide Probes to Distinguish Intracellular Expression and Localization Patterns of Rab GDP-Dissociation Inhibitor Isoforms

Introduction

Distinguishing between the Expression Pattern of GDI-1 and GDI-2 at the Protein Level

Distinguishing Expression Pattern between GDI-2 and GDIβ at mRNA Level

6: Expression, Purification, and Biochemical Properties of Ypt/Rab GTPase-Activating Proteins of Gyp Family

Introduction

Cloning and Expression of GYP Genes and of Fragments Encoding Their Catalytic Domains

Analysis of Ypt/Rab GAP Activity

Acknowledgments

7: Purification and Properties of Rab3 GDP/GTP Exchange Protein

Introduction

Materials

Methods

Comments

8: Purification and Properties of Rab3 GTPase-Activating Protein

Introduction

Materials

Methods

Comments

9: Rabphilin-3: A Target Molecule for Rab3 Small G Proteins

Introduction

Materials

Methods

Comments

10: Doc2α as Modulator of Ca2+-Dependent Exocytosis

Introduction

Materials

Methods

Comments

11: Purification and Properties of a GTPase-Activating Protein for Yeast Rab GTPases

Introduction

Construction of Expression Plasmids

Expression and Purification of His6-Tagged Proteins from Bacteria

Measuring GTPγS Binding Capacity of Rab Proteins

Guanine Nucleotide Analysis of GTPase Reaction

GAP Assay

Summary of Results

Acknowledgment

12: Purification and Characterization of Yeast Exocyst Complex

Introduction

Preliminary Characterization of Exocyst Complex

Exocyst Complex Purification

Structural Characterization of Exocyst Complex

Acknowledgments

13: Expression and Properties of Rab4 and Its Effector Rabaptin-4 in Endocytic Recycling

Introduction

Methods

Acknowledgment

14: Purification of EEA1 from Bovine Brain Cytosol Using Rab5 Affinity Chromatography and Activity Assays

Introduction

Purification of Native EEA1 from Bovine Brain

Functional Assays of Purified EEA1

Acknowledgments

15: Expression, Purification, and Characterization of Rab5 Effector Complex, Rabaptin-5/Rabex-5

Introduction

Description of Method

Acknowledgments

16: Measurement of Rab5 Protein Kinase B/akt and Regulation of Ras-Activated Endocytosis

Introduction

Experimental Procedure

Confocal Microsocopy and Video Analysis

Acknowledgments

17: Expression, Purification, and Biochemical Properties of Rabkinesin-6 Domains and Their Interactions with Rab6A

Introduction

Methods

18: Expression and Functional Analyses of Rab8 and Rablla in Exocytic Transport from trans-Golgi Network

Introduction

Methods

Conclusion

Acknowledgments

19: Expression and Properties of Rab7 in Endosome Function

Introduction

Methods and Assays

Conclusion

Acknowledgments

20: Expression, Purification, and Properties of Rab8 Function in Actin Cortical Skeleton Organization and Polarized Transport

Introduction

Construction of Bacterial Expression Vectors

Cloning and Mutagenesis of Mouse Rab8b

Production of Rab8b Hypervariable Peptide for Immunization

Affinity Purification of Antisera

Cell Transfection

Analysis of Rab8 by Confocal and Immunofluorescence Microscopy

21: Properties of Rab13 Interaction with Rod cGMP Phosphodiesterase δ Subunit

Introduction

Two-Hybrid Screen

In Vitro Interaction of Human δPDE with Rab13

Rab 13-Coupled Transcription/Translation in Vitro

Solubilization Assay of Membrane-Bound Rab13

Immunofluorescence Localization Studies

22: Subcellular Localization of Rab17 by Cryo-Immunogold Electron Microscopy in Epithelial Cells Grown on Polycarbonate Filters

Introduction

Fixation of Cell Monolayers on Transwell Polycarbonate Filters for Immunogold Labeling

Embedding Cells for Immunogold Labeling

Cryosectioning

Immunogold Labeling of Ultrathin Cryosections with Epithelial Cells on Transwell Polycarbonate Filters

Preparation of Carbon-and Formvar-Coated Copper Grids

Reagents and Solutions

Troubleshooting

Results

Acknowledgments

23: Expression and Properties of Rab25 in Polarized Madin-Darby Canine Kidney Cells

Introduction

Procedures

Results

Concluding Remarks

Acknowledgments

24: Purification of TRAPP from Saccharomyces cerevisiae and Identification of Its Mammalian Counterpart

Introduction

Construction of Bet3p-Protein A Fusion Used to Purify TRAPP

Large-Scale Purification of TRAPP and Identification of TRAPP Subunits

Small-Scale Precipitation of TRAPP

Identification of Mammalian TRAPP from HeLa Lysates

Section II. ADP-Ribosylation Factor (ARF) GTPases

25: Structural and Functional Organization of ADP-Ribosylation Factor (ARF) Proteins

26: Expression and Properties of ADP-Ribosylation Factor (ARF6) in Endocytic Pathways

Introduction

Expression of Wild-Type ARF6 and Mutants in Mammalian Cells

ARF6 in Endocytosis in HeLa Cells

Internalization and Recycling of Anti-Tac Antibodies to Monitor Tac Trafficking along ARF6 Endocytic Pathway

Concluding Remarks

27: Expression and Analysis of ARNO and ARNO Mutants and Their Effects on ADP-Ribosylation Factor (ARF)-Mediated Actin Cytoskeletal Rearrangements

Introduction

Constructs

Generation of ARNO Mutants

Culture of HeLa cells

Transient Transfection Using Lipofection

Analysis of ARF Nucleotide Exchange in Vivo

Thin-Layer Chromatography (TLC) Analysis of Bound Nucleotides

Morphologic Analysis of Cells Expressing ARNO and ARNO Mutants

Effects of Wild-Type and Mutant ARNO on Actin Cytoskeleton

28: Expression, Purification, and Measurements of Activity of ARNO1, a Guanine Nucleotide Exchange Factor for ADP-Ribosylation Factor 1 (ARF1)

Introduction

Methods

Acknowledgments

29: Expression, Purification, and Biochemical Properties of EFA6, a Sec7 Domain-Containing Guanine Exchange Factor for ADP-Ribosylation Factor 6 (ARF6)

Introduction

Production of Recombinant Proteins in Escherichia coli

Analysis of Bound Nucleotides on Recombinant ARF6

Analysis of Myristoylated ARF6

[35S]GTPγS Binding Assay

Intracellular Localization of EFA6 in Mammalian Cells

Acknowledgments

30: Isolation and Properties of GRP1, an ADP-Ribosylation Factor (ARF)-Guanine Nucleotide Exchange Protein Regulated by Phosphatidylinositol 3, 4, 5-Trisphosphate

Introduction

Isolation of GRP1

ARF Exchange by GRP1 Sec7 Homology Domain

Polyphosphoinositide Binding by GRP1 PH Domain

Acknowledgment

31: Functional Analysis of ADP-Ribosylation Factor (ARF) Guanine Nucleotide Exchange Factors Gea1p and Gea2p in Yeast

Introduction

Purification of His6-Gea1p from Saccharomyces cerevisiae

Expression and Purification of Myristoylated Yeast ARF2

Assay of GTPγS Binding to ARF

Analysis of Gealp and Gea2p Function in Vivo

32: Isolation, Cloning, and Characterization of Brefeldin A-Inhibited Guanine Nucleotide-Exchange Protein for ADP-Ribosylation Factor

Introduction

BIG1 Cloning and Preparation of Recombinant Protein

Structure and Function of BIG1

33: Expression, Purification, and Properties of ADP-Ribosylation Factor (ARF) GTPase Activating Protein-1

Introduction

Purification of GAP1 from Rat Liver

Expression and Purification of His6-GAP1(1-257) from Escherichia coli

Expression and Purification of Full-Length GAP1 from SF9 Cells

Properties of Recombinant GAP

Interaction of GAP1(1–257) with Liposomes

Acknowledgments

34: Expression, Analysis, and Properties of Yeast ADP-Ribosylation Factor (ARF) GTPase Activating Proteins (GAPs) Gcs1 and Glo3

Introduction

Production of Recombinant Yeast ARF-GAP Proteins

Genetic Analysis of Yeast ARF-GAP Function

Expression of ARF-GAP Proteins in Yeast

New ARF-GAP Mutations by PCR Mutagenesis

Acknowledgments

35: Purification and Properties of ARD1, An ADP-Ribosylation Factor (ARF)-Related Protein with GTPase-Activating Domain

Introduction

ARD1, A Unique Member of ARF Family

Methods

Conclusion

36: Purification and Characterization of GIT Family of ADP-Ribosylation Factor (ARF) GTPase-Activating Proteins

Introduction

Methods

37: Assay and Purification of Phosphoinositide-Dependent ADP-Ribosylation Factor (ARF) GTPase Activating Proteins

Introduction

ARF GAP Assay

Purification

38: Biological Properties and Measurement of Phospholipase D Activation by ADP-Ribosylation Factor (ARF)

Introduction

Role of Phospholipase D in Regulated Secretion

Assays of PLD Activity and Secretion in Intact and Permeabilized RBL-2H3 Mast Cells

Reconstitution of Secretory Function and Phospholipase D Activity in Cytosol-Depleted RBL Mast Cells

Expression of Recombinant Phospholipase D in Continuous Cell Lines

Establishing Cell Lines with Inducible Phospholipase D Activity

Assay of Phospholipase D Activity in Intact Cells

Assay of Phospholipase D Activity in Isolated Membrane Preparations Using Exogenous Substrates

Assay of Phospholipase D Activity in Immunoprecipitates Using Exogenous Substrates

39: Use of Aminoglycoside Antibiotics and Related Compounds to Study ADP-Ribosylation Factor (ARF) /Coatomer Function in Golgi Traffic

Introduction

Inhibition by CBM of Neomycin-Induced Precipitation of Coatomer

Inhibition of Coatomer Binding to Golgi Complex of Intact Cells with CBM

Acknowledgments

40: Adaptor Protein 1-Dependent Clathrin Coat Assembly on Synthetic Liposomes and Golgi Membranes

Introduction

Preparation of Liposomes

Preparation of Cytosols

Preparation of Clathrin-Coated Vesicles and Clathrin Coat Proteins

Preparation of Golgi Membranes

Coat Recruitment Assays and Morphologic Studies by Electron Microscopy

Lipid Composition Modulating ARF and AP-1 Binding

Conclusions

41: Receptor-Dependent Formation of COPI-Coated Vesicles from Chemically Defined Donor Liposomes

Introduction

Preparation of Various Components Used in Liposome-Based COPI Budding Assay

Concluding Remarks

Acknowledgments

42: ADP-Ribosylation Factor (ARF) as Regulator of Spectrin Assembly at Golgi Complex

Introduction

Construction of Plasmids for Expression of Spectrin Fusion Proteins

Protein Expression in Escherichia coli and in Cultured Cells

Antibodies and Immunodetection Procedures

Immunofluorescence Analysis In Permeabilized Cells

In Vitro Binding Assays on Isolated Golgi Membranes

Direct Analysis of Spectrin Domains Involved in Golgi Membrane Binding

Acknowledgments

43: Purification, Properties, and Analysis of yARL3

Introduction

Purification of Bacterially Expressed yARL3

Assay of yARL3

Acknowledgments

44: Preparation and Assay of Recombinant ADP-Ribosylation Factor-Like Protein-1 (ARL1)

Introduction

Preparation of Recombinant hARL1

Assays for ARL1

GTPγs-Binding Assay

Section III. Sar GTPases

45: Purification and Properties of Rat Liver Sec23–Sec24 Complex

Introduction

Methods

Summary

46: Purification of Functional Sec13p–Sec31p Complex, A Subunit of COPII Coat

Introduction

Strains, Materials, and Definitions

Growth, Lysis, and Preparation of Soluble Protein Extract

Purification of Sec13p–Sec31p Complex

Assay of Functional Sec13p–Sec31p Complex

Section IV. Dynamin GTPases

47: Expression, Purification, and Functional Assays for Self-Association of Dynamin-1

Introduction

Expression and Purification of Recombinant Dynamin-1

Velocity Sedimentation as Measure of Dynamin Assembly

GTPase Assay for Dynamin Function

GED-Stimulated GTPase Activity

48: Analysis of Phosphoinositide Binding by Pleckstrin Homology Domain from Dynamin

Introduction

Materials and Reagents

Methods

Interpretation of Results

Requirement for Oligomerization in Dyn1-PH Binding to Phosphoinositides

Importance of Phosphoinositide Binding by Dyn1-PH Studied in Vivo

49: Mapping Dynamin Interdomain Interactions with Yeast Two-Hybrid and Glutathione S-Transferase Pulldown Experiments

Introduction

Preparation of Yeast Two-Hybrid Expression Plasmids

Transformation of Yeast

Filter Assay for β-Galactosidase Activity

Transformation of Yeast with Two-Hybrid cDNA Library

Constructs for Protein Expression with Baculovirus System

Production of Recombinant Virus for Baculovirus Expression

Expression and Purification of Recombinant Protein

Expression of Radiolabeled Proteins with Reticulocyte Lysates

Protein Binding Experiments

Cross-Linking of Dynamin Assembly Domain

Acknowledgments

50: Interactions of Dynamin and Amphiphysin with Liposomes

Introduction

Methods

51: Activation of Dynamin GTPase Activity by Phosphoinositides and SH3 Domain-Containing Proteins

Introduction

Purification of Dynamin

Enzyme Assay

Activation by Phospholipids

Activation by Proteins Containing Src Homology 3 (SH3) Domains

Buffer Considerations

Acknowledgments

Section V. Septin GTPases

52: Expression and Analysis of Properties of Septin CDCrel-1 in Exocytosis

Introduction

Determination of Expression Patterns and Subcellular Distribution of CDCrel-1 Proteins

Expression and Purification of Wild-Type and Mutant CDCrel-1

Measurement of GTP Binding Properties of CDCrel-1

CDCrel-1–Syntaxin Binding Assays

Secretion Assay

Use of C-Terminal GFP-Tagged CDCrel-1 Fusion Protein to Monitor Septin Accumulation in vivo

Overexpression of CDCrel-1 Proteins in Mammalian Cells by Transfection

Conclusions

Author Index

Subject Index