High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins
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.
Key wordsGTPase Rab GTPase Phosphate-binding protein Phosphate PBP-MDCC GTP hydrolysis GAP reaction High throughput GAP assay
This work was supported by an NIH grant GM056324 to DGL.
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