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

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

1st Edition - January 25, 2001

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  • Editors: John Abelson, Melvin Simon
  • eBook ISBN: 9780080496849

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Description

Small GTPases play a key role in many aspects of contemporary cell biology: control of cell growth and differentiation; regulation of cell adhesion and cell movement; the organization of the actin cytoskeleton; and the regulation of intracellular vesicular transport.This volume and its companions (Volumes 255, 256, 257, and the forthcoming 325) cover all biochemical and biological assays currently in use for analyzing the role of small GTPases in these aspects of cell biology at the molecular level.

Readership

Biochemists, molecular biologists, cell biologists, pharmacologists, neurophysiologists, and neurochemists.

Table of Contents

  • 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

Product details

  • No. of pages: 553
  • Language: English
  • Copyright: © Academic Press 2001
  • Published: January 25, 2001
  • Imprint: Academic Press
  • eBook ISBN: 9780080496849

About the Serial Editors

John Abelson

Affiliations and Expertise

California Institute of Technology, Division of Biology, Pasadena, U.S.A.

Melvin Simon

Affiliations and Expertise

The Salk Institute, La Jolla, CA, USA

About the Serial Volume Editors

W. Balch

Affiliations and Expertise

The Scripps Research Institute, La Jolla, CA, USA

Channing Der

Affiliations and Expertise

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, USA

Alan Hall

Affiliations and Expertise

University College of London, U.K.

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