Rabconnectin-3, a novel protein that binds both GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family.
Rab3A, a member of the Rab3 small G protein family, regulates Ca(2+)-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). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M(r) of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.
Nagano
Fumiko
F
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.
Kawabe
Hiroshi
H
Nakanishi
Hiroyuki
H
Shinohara
Masahiko
M
Deguchi-Tawarada
Maki
M
Takeuchi
Masakazu
M
Sasaki
Takuya
T
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 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D064211:Connectin; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D014018:Tissue Distribution; D020692:rab3 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D064211:Connectin; C452848:DMXL2 protein, human; D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C577285:TTN protein, human; D005982:Glutathione Transferase; D011494:Protein Kinases; D020692:rab3 GTP-Binding Proteins; D002118:Calcium