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Originally published In Press as doi:10.1074/jbc.M603808200 on August 21, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31823-31831, October 20, 2006
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Identification of EPI64 as a GTPase-activating Protein Specific for Rab27A*Formula

Takashi Itoh{ddagger}1 and Mitsunori Fukuda{ddagger}§2

From the {ddagger}Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198 and the §Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan

Small GTPase Rab27A plays a pivotal role in melanosome transport in melanocytes and in secretion by various secreting cells. Because 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.


Received for publication, April 20, 2006 , and in revised form, July 27, 2006.

* This work was supported in part by Ministry of Education, Culture, Sports, and Technology of Japan Grants 17657067, 18022048, 18050038, 18057026, and 18207015 (to M. F.), the Kato Memorial Bioscience Foundation (to M. F.), the Sumitomo Foundation (to M. F.), and the Nakatomi Foundation (to M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

1 Supported by the Special Postdoctoral Researchers Program of RIKEN.

2 To whom correspondence should be addressed: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan. Tel.: 81-22-795-7731; Fax: 81-22-795-7733; E-mail: nori{at}mail.tains.tohoku.ac.jp.


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