A novel rabconnectin-3-binding protein that directly binds a GDP/GTP exchange protein for Rab3A small G protein implicated in Ca(2+)-dependent exocytosis of neurotransmitter.
BACKGROUND
Rab3A, a member of the Rab3 small G protein family, regulates Ca2+-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP) and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). We have recently found a novel protein, named rabconnectin-3, which is co-immunoprecipitated with Rab3 GEP or GAP from the extract of the crude synaptic vesicle (CSV) fraction of rat brain. Rabconnectin-3 is abundantly expressed in the brain where it is associated with synaptic vesicles. We have found that two more proteins are co-immunoprecipitated with Rab3 GEP from the CSV fraction of rat brain. We attempted here to isolate and characterize one of them.


RESULTS
We determined its partial amino acid sequence, cloned its cDNA from a human cDNA library, and determined its primary structure. The protein consisted of 1490 amino acids (aa) and showed a calculated molecular weight of 163808. The protein had 7 WD domains. The protein was abundantly expressed in the brain where it co-localized with rabconnectin-3 on synaptic vesicles. The protein formed a stable complex with rabconnectin-3. We named this protein rabconnectin-3beta and renamed rabconnectin-3 rabconnectin-3alpha. Rabconnectin-3beta, but not rabconnectin-3alpha, directly bound Rab3 GEP. Neither rabconnectin-3alpha nor -3beta directly bound Rab3 GAP.


CONCLUSIONS
These results indicate that rabconnectin-3 consists of the alpha and beta subunits and binds directly Rab3 GEP through the beta subunit and indirectly Rab3 GAP through an unidentified molecule(s).
Kawabe
Hiroshi
H
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.
Sakisaka
Toshiaki
T
Yasumi
Masato
M
Shingai
Tatsushi
T
Izumi
Genkichi
G
Nagano
Fumiko
F
Deguchi-Tawarada
Maki
M
Takeuchi
Masakazu
M
Nakanishi
Hiroyuki
H
Takai
Yoshimi
Y
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005089:Exocytosis; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D015723:Gene Library; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions; D013572:Synaptic Vesicles; D014018:Tissue Distribution; D020692:rab3 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C452848:DMXL2 protein, human; D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D020692:rab3 GTP-Binding Proteins; D002118:Calcium