Analysis of Rab GTPases

Lars Langemeyer1, Francis A. Barr1

1 Department of Biochemistry, University of Oxford, Oxford, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.18
DOI:  10.1002/0471143030.cb1518s57
Online Posting Date:  December, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes methods to identify proteins interacting with, and regulating, Rab GTPases. Rabs form the largest subgroup of Ras superfamily GTPases, and act as molecular switches controlling the specificity of membrane trafficking. The regulation and the signal readout of Rabs are mediated by four groups of proteins, the GDP‐GTP exchange factors (GEFs), GTPase activating proteins (GAPs), Rab chaperones, and effector proteins. Rabs are activated at the membrane surface by specific GEFs that promote the exchange of bound GDP to GTP. Effector proteins then bind to the activated Rab GTPase and mediate the cellular response at this membrane surface. Finally, a Rab‐specific GAP‐protein inactivates the GTPase by catalyzing the hydrolysis of bound GTP to GDP, thus terminating the cellular response. The methods described here are valuable for characterizing these different types of activity, and assigning which Rab they act on. Curr. Protoc. Cell Biol. 57:15.18.1‐15.18.17. © 2012 by John Wiley & Sons, Inc.

Keywords: Rab; GTPase; GEF; GAP; effector‐protein

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Yeast‐2‐Hybrid Screen for Interacting Proteins
  • Alternate Protocol 1: Yeast‐2‐Hybrid Screen for Interacting Proteins
  • Basic Protocol 2: Rab Effector Binding Assay
  • Basic Protocol 3: GAP Assay
  • Basic Protocol 4: GEF Assay
  • Support Protocol 1: Producing Electrocompetent E. coli
  • Support Protocol 2: Producing Competent Yeast Cells
  • Support Protocol 3: Yeast Transformation
  • Support Protocol 4: DNA Mini‐Prep from Yeast Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Yeast‐2‐Hybrid Screen for Interacting Proteins

  Materials
  • Competent Saccharomyces cerevisiae PJ69‐4a/a (Y2H reporter strain for screening; see protocol 7)
  • Bait vectors: pFBT9 (Kan/Trp; see protocol 8)
  • SC −Trp plates
  • Library/prey vectors: pACT2 (Amp/Leu)
  • DDO plates (SC –Leu/–Trp)
  • QDO plates (SC –Leu/Trp/His/Ade; 10‐cm and 15‐cm diameter plates)
  • Electrocompetent E. coli (see protocol 6)
  • 30°C incubator

Alternate Protocol 1: Yeast‐2‐Hybrid Screen for Interacting Proteins

  Materials
  • Competent Saccharomyces cerevisiae PJ69‐4a/a (Y2H reporter strain for screening; see protocol 7)
  • Bait vectors: pFBT9 (Kan/Trp)
  • Library/prey vectors: pACT2 (Amp/Leu)
  • DDO plates (SC –Leu/–Trp)
  • QDO plates (SC –Leu/Trp/His/Ade; 10–cm plates)
  • 30°C incubator

Basic Protocol 2: Rab Effector Binding Assay

  Materials
  • Proteins (wild type, GDP, and GTP‐locked mutant Rab GTPases tagged with GST)
  • Glutathione‐Sepharose (GSH‐beads)
  • Nucleotide exchange buffer (NE100; see recipe)
  • Nucleotide loading buffer (NL100; see recipe)
  • Binding buffer (NB0/100/200; see recipe)
  • Nucleotide stocks (10 mM GDP, GTP, and GTPγS, in NL100 buffer)
  • Elution buffer (NE200; see recipe)
  • 72% (w/v) trichloroacetic acid (TCA)
  • Acetone, −20°C
  • 1.5× sample loading buffer (prepare from 3× buffer; see recipe)
  • Centrifuge
  • 4°C roller incubator
  • Microcentrifuge
  • 95°C heating block
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1)

Basic Protocol 3: GAP Assay

  Materials
  • Activated charcoal slurry: 5 g finely powdered activated charcoal dissolved in 100 ml of 50 mM NaH 2PO 4
  • 10× assay buffer (see recipe)
  • 0.5 M and 10 mM EDTA, pH 8.0 ( appendix 2A)
  • 10 mM Mg‐GTP (see recipe)
  • [γ‐32P]GTP (10 mCi/ml; 5000 Ci/mmol; PerkinElmer)
  • Proteins:
    • Wild‐type Rab GTPases tagged with GST
    • Potential GAP‐proteins, not GST‐tagged
  • Scintillation fluid (Ultima Gold Pro, PerkinElmer)
  • 1.5‐ml snap‐cap microcentrifuge tubes
  • Scintillation vials
  • Scintillation counter
  • Microcentrifuge

Basic Protocol 4: GEF Assay

  Materials
  • 10× assay buffer (see recipe)
  • 2.5 mM EDTA, pH 8.0 ( appendix 2A)
  • 10 mM Mg‐GDP (see recipe)
  • 5 µCi [3H]‐GDP (1‐mCi/ml; 25‐50 Ci/mmol, Perkin Elmer)
  • Proteins:
    • Wild‐type Rab GTPases, GST‐tagged
    • Potential GEF‐proteins, not GST‐tagged
  • Scintillation liquid (Ultima Gold Pro, PerkinElmer)
  • 10× wash buffer (see recipe), ice cold
  • GSH‐Sepharose
  • 1.5‐ml snap‐cap microcentrifuge tubes
  • 30°C incubator
  • Centrifuge
  • Scintillation counter

Support Protocol 1: Producing Electrocompetent E. coli

  Materials
  • E. coli
  • LB medium ( appendix 2A)
  • Sterile water, ice cold
  • 10% (v/v) glycerol, ice‐cold
  • Yeast miniprep DNA or plasmid DNA
  • 37°C incubator with shaking
  • 2‐liter flask
  • Spectrophotometer
  • 50‐ml centrifuge tubes
  • Refrigerated centrifuge
  • Electroporation cuvettes
  • Electroporator
  • 1.5‐ml sterile microcentrifuge tubes (Eppendorf)

Support Protocol 2: Producing Competent Yeast Cells

  Materials
  • Competent Saccharomyces cerevisiae cells
  • YPAD: YPD medium (see recipe) containing 100 mg/ml adenine
  • Sterile water
  • LiSorb (see recipe)
  • Carrier DNA: 10 mg/ml sheared herring or salmon sperm DNA
  • Spectrophotometer
  • 30°C incubator
  • Centrifuge

Support Protocol 3: Yeast Transformation

  Materials
  • Frozen competent Saccharomyces cerevisiae cells (see protocol 7)
  • pFBT9 (bait vector)
  • pACT2 (prey vector)
  • LiPEG (see recipe)
  • DMSO
  • Sterile water
  • Selective plates (e.g., for yeast‐2‐hybrid screen, SC –Leu/Trp plates)
  • Vortex
  • 42°C water bath
  • Microcentrifuge
  • 30°C incubator

Support Protocol 4: DNA Mini‐Prep from Yeast Cells

  Materials
  • Liquid or plated cultured yeast cells
  • Sterile water
  • Buffer S (see recipe)
  • Lysis buffer (see recipe)
  • 3 M potassium acetate, pH 5.5 ( appendix 2A)
  • Absolute ethanol (−20°C)
  • 70% ethanol (room temperature)
  • Sterile 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Microcentrifuge
  • 37°C incubator
  • 65°C incubator with shaking
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Albert, S., Will, E., and Gallwitz, D. 1999. Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase‐activating proteins specific for Ypt/Rab transport GTPases. EMBO J. 18:5216‐5225.
   Barr, F. and Lambright, D.G. 2010. Rab GEFs and GAPs. Curr. Opin. Cell Biol. 22:461‐470.
   Fuchs, E., Short, B., and Barr, F. 2005. Assay and properties of Rab6 interaction with dynein‐dynactin complexes. Meth. Enzymol. 403:607‐618.
   Fuchs, E., Haas, A.K., Spooner, R.A., Yoshimura, S.‐i., Lord, J.M., and Barr, F.A. 2007. Specific Rab GTPase‐activating proteins define the Shiga toxin and epidermal growth factor uptake pathways. J. Cell Biol. 177:1133‐1143.
   Golemis, E.A., Serebriiskii, I., Finley, R.L., Kolonin, M.G., Gyuris, J., and Brent, R. 2008. Interaction trap/two‐hybrid system to identify interacting proteins. Curr. Protoc. Mol. Biol. 82:20.1.1‐20.1.35.
   Grosshans, B.L., Ortiz, D., and Novick, P. 2006. Rabs and their effectors: Achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. U.S.A. 103:11821‐11827.
   Haas, A.K., Fuchs, E., Kopajtich, R., and Barr, F.A. 2005. A GTPase‐activating protein controls Rab5 function in endocytic trafficking. Nat. Cell Biol. 7:887‐893.
   Pfeffer, S. and Aivazian, D. 2004. Targeting RAB GTPases to distinct membrane compartments. Nat. Rev. Mol. Cell Biol. 5:886‐896.
   Schoebel, S., Oesterlin, L.K., Blankenfeldt, W., Goody, R.S., and Itzen, A. 2009. RabGDI displacement by DrrA from Legionella is a consequence of tts guanine nucleotide exchange activity. Mol. Cell 36:1060‐1072.
   Suh, H.Y., Lee, D.W., Lee, K.H., Ku, B., Choi, S.J., Woo, J.S., Kim, Y.G., and Oh, B.H. 2010. Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA. EMBO J. 29:496‐504.
   Yoshimura, S.‐i., Haas, A.K., and Barr, F.A. 2008. Analysis of Rab GTPase and GTPase‐activating protein function at primary cilia. Meth. Enzymol. 439:353‐364.
   Yoshimura, S.‐I., Gerondopoulos, A., Linford, A., Rigden, D.J., and Barr, F.A. 2010. Family‐wide characterization of the DENN domain Rab GDP‐GTP exchange factors. J. Cell Biol. 191:367‐381.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library