Isolation and Characterization of a GTPase Activating Protein Specific for the Rab3 Subfamily of Small G Proteins*
- Koji Fukui‡,
- Takuya Sasaki‡,
- Katsunori Imazumi‡§,
- Yoshiharu Matsuura¶,
- Hiroyuki Nakanishi‡∥ and
- Yoshimi Takai‡**
- ‡ Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Osaka, Japan and
- the ¶ Department of Virology II, National Institute of Health, Tokyo 162, Japan
- ** To whom correspondence should be addressed. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp
Abstract
The Rab small G protein family, consisting of nearly 30 members, is implicated in intracellular vesicle trafficking. They cycle between the GDP-bound and GTP-bound forms, and the latter is converted to the former by the action of a GTPase activating protein (GAP). No GAP specific for each Rab family member or Rab subfamily has been isolated in mammal. Here we purified a GAP with Rab3A as a substrate from rat brain. The purified protein was specifically active on the Rab3 subfamily members (Rab3A, -B, -C, and -D). Of this subfamily, Rab3A and -C are implicated in Ca2+-dependent exocytosis, particularly in neurotransmitter release. This GAP, named Rab3 GAP, was active on the lipid-modified form, but not on the lipid-unmodified form. Rab3 GAP showed a minimum molecular mass of about 130 kDa on SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a human brain cDNA library, and the isolated cDNA encoded a protein with a Mr of 110,521 and 981 amino acids, which showed no homology to any known protein. The recombinant protein exhibited GAP activity toward the Rab3 subfamily members, and the catalytic domain was located at the C-terminal region. Northern blot analysis indicated that Rab3 GAP was ubiquitously expressed.
Footnotes
-
↵* This work was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, Sports, and Culture, Japan (1995, 1996), by grants-in-aid for abnormalities in hormone receptor mechanisms and for aging and health from the Ministry of Health and Welfare, Japan (1995, 1996), and by grants from the Human Frontier Science Program (1995, 1996) and the Uehara Memorial Foundation (1995, 1996). 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.
-
↵1 The abbreviations used are:
- GDI
-
GDP dissociation inhibitor
- GEP
-
GDP/GTP exchange protein
- GAP
-
GTPase activating protein
- Sf9 cells
-
Spodoptera frugiperda cells
- DTT
-
dithiothreitol
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- PAGE
-
polyacrylamide gel electrophoresis
- SS
-
synaptic soluble
- bp
-
base pair(s).
-
↵2 Wada, M., Nakanishi, H., Satoh, A., Hirano, H., Obaishi, H., Matsuura, Y., and Takai, Y. (1997) J. Biol. Chem. 272, 3875-3878.
-
- Received November 11, 1996.
- Revision received December 16, 1996.
- © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
