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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.

Original publication

DOI

10.1002/0471143030.cb1518s57

Type

Journal article

Journal

Curr Protoc Cell Biol

Publication Date

12/2012

Volume

Chapter 15

Pages

Unit - 15.18

Keywords

Animals, Cell Extracts, Cell Membrane, DNA-Binding Proteins, Escherichia coli, Humans, Mass Spectrometry, Protein Interaction Maps, Protein Transport, Saccharomyces cerevisiae, Transcription Factors, Two-Hybrid System Techniques, rab GTP-Binding Proteins