Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates.

Rab GTPases are key regulators of membrane trafficking. When GTP-bound, or "active," Rabs are anchored to membranes and recruit effector proteins that mediate vesicle formation, transport, and fusion. Rabs are inactivated by GTPase-activating proteins (Rab-GAPs), which catalyze GTP hydrolysis, rendering Rabs cytosolic. In vivo, C-terminal prenylation modifications link activated Rabs to organelle and vesicle membranes, yet historically, in vitro Rab-GAP activity assays have been performed in the absence of membranes. We have developed a method for assaying Rab-GAP activity in a physiological context, with dissociation of the Rab from the membrane serving as a readout for Rab-GAP activity. Given that membrane-binding status is a key consequence of Rab activation state, this assay will be useful for the study of a wide range of Rab/Rab-GAP pairs. © 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

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Carolyn M Highland, Laura L Thomas, J Christopher Fromme. Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates. Methods in molecular biology (Clifton, N.J.). 2023;2557:507-518

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PMID: 36512233

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