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High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins

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Rab GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1298))

Abstract

Measurement of intrinsic as well as GTPase-Activating Protein (GAP)-catalyzed GTP hydrolysis is central to understanding the molecular mechanism and function of GTPases in diverse cellular processes. For the Rab GTPase family, which comprises at least 60 distinct proteins in humans, putative GAPs have been identified from both eukaryotic organisms and pathogenic bacteria. A major obstacle has involved identification of target substrates and determination of the specificity for the Rab family. Here, we describe a sensitive, high-throughput method to quantitatively profile GAP activity for Rab GTPases in microplate format based on detection of inorganic phosphate released after GTP hydrolysis. The method takes advantage of a well-characterized fluorescent phosphate sensor, requires relatively low protein concentrations, and can in principle be applied to any GAP-GTPase system.

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Acknowledgements

This work was supported by an NIH grant GM056324 to DGL.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, Program in Molecular Medicine, University of Massachusetts Medical School, Two Biotech, Suite 119, 373 Plantation Street, Worcester, MA, 01605, USA

    Ashwini K. Mishra & David G. Lambright

Authors
  1. Ashwini K. Mishra
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  2. David G. Lambright
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Corresponding author

Correspondence to David G. Lambright .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

    Guangpu Li

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Mishra, A.K., Lambright, D.G. (2015). High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins. In: Li, G. (eds) Rab GTPases. Methods in Molecular Biology, vol 1298. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2569-8_4

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  • DOI: https://doi.org/10.1007/978-1-4939-2569-8_4

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2568-1

  • Online ISBN: 978-1-4939-2569-8

  • eBook Packages: Springer Protocols

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