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Papers In Press, published online ahead of print August 21, 2006
Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578
Corresponding Author: nori{at}mail.tains.tohoku.ac.jp
Small GTPase Rab27A plays a pivotal role in melanosome transport in melanocytes and in secretion by various secreting cells. Since the GTP- or GDP-locked mutant of Rab27A causes perinuclear aggregation of melanosomes, appropriate GTP-GDP cycling of Rab27A is essential for melanosome transport, and certain guanine nucleotide exchange factors and GTPase-activating proteins (GAPs) of Rab27A must be present in melanocytes. However, no such regulators of Rab27A have ever been identified. In this study we developed novel methods of rapidly screening 40 different TBC (Tre2/Bub2/Cdc16) proteins, putative Rab-GAPs, for Rab27A-GAP by: (i) searching for TBC proteins that induce melanosome aggregation in melanocytes; (ii) trapping GTP-Rab27A with a Rab27A effector domain (i.e., the SHD of Slac2-a) in cultured cells that express both Rab27A and TBC proteins; and (iii) measuring in vitro Rab27A-GAP activity. These methods allowed us to identify EPI64, previously characterized as an EBP50-binding protein that contains an orphan TBC domain, as a specific Rab27A-GAP. We further showed that mutations in the catalytic domain of EPI64 caused complete loss of its ability to induce melanosome aggregation. This is the first report of screening for Rab27A-GAP based on functional interactions, and our screening methods can be applied for other uncharacterized TBC proteins.
J. Biol. Chem, 10.1074/jbc.M603808200
Submitted on April 20, 2006
Revised on July 27, 2006
Accepted on August 21, 2006
Identification of EPI64 as a GTPase-activating protein specific for Rab27A
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