Isolation and Characterization of a GDP/GTP Exchange Protein Specific for the Rab3 Subfamily Small G Proteins

doi:10.1074/jbc.272.7.3875

Isolation and Characterization of a GDP/GTP Exchange Protein Specific for the Rab3 Subfamily Small G Proteins*

From the ^‡ Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-22, Japan, the
^§ Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan, and the
^∥ Department of Virology II, National Institute of Health, Tokyo 162, Japan

** To whom correspondence should be addressed:
Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Osaka, Japan.
Tel.: 81-6-879-3410; Fax: 81-6-879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp

↵^¶ Present address: Otsuka GEN Research Inst., Otsuka Pharmaceutical Co., Ltd., Tokushima 771-01, Japan.

Abstract

The Rab small G protein family, consisting of nearly 30 members, is implicated in intracellular vesicle trafficking. They cycle between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of a GDP/GTP exchange protein (GEP). No GEP specific for each Rab family member or Rab subfamily has been isolated. Here we purified a GEP from rat brain with lipid-modified Rab3A as a substrate. The purified protein was specifically active on Rab3A, Rab3C, and Rab3D of the Rab3 subfamily. Of these subfamily members, Rab3A and Rab3C are implicated in Ca²⁺-dependent exocytosis, particularly in neurotransmitter release. This GEP (Rab3 GEP) was active on the lipid-modified form, but not on the lipid-unmodified form. Rab3 GEP showed a minimum molecular mass of about 200 kDa on SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and determined its primary structure. The isolated cDNA encoded a protein with a M_r of 177,982 and 1,602 amino acids, which showed no homology to any known protein. The recombinant protein exhibited GEP activity toward Rab3A, Rab3C, and Rab3D. Northern blot and Western blot analyses indicated that Rab3 GEP was expressed in all the rat tissues examined with the highest expression in brain.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U72995[GenBank].
↵¹ The abbreviations used are:

GEP

GDP/GTP exchange protein

GDI

GDP dissociation inhibitor

GDF

GDI displacement factor

GAP

GTPase-activating protein

Sf9 cells

Spodoptera frugiperda cells

DTT

dithiothreitol

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

GTPγS

guanosine 5′-(3-O-thio)triphosphate

PAGE

polyacrylamide gel electrophoresis.
↵² H. Shirataki, H. Kawabe, K. Nakano, and Y. Takai, manuscript in preparation.
- Received November 25, 1996.