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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… Read more
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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
WB
JA
MS
CD
AH