Molecular cloning and characterization of the noncatalytic subunit of the Rab3 subfamily-specific GTPase-activating protein.
We recently purified and characterized from rat brain a GTPase-activating protein (GAP) specific for the Rab3 small G protein subfamily implicated in Ca2+-dependent exocytosis. Rab3 GAP showed two bands with Mr of about 130,000 (p130) and 150,000 (p150) on SDS-polyacrylamide gel electrophoresis. p130, but not p150, showed the catalytic activity. Because p150 was likely the subunit of Rab3 GAP, here we cloned the cDNA of p150, determined its primary structure, and characterized it. The tissue and subcellular distribution patterns of p150 and p130 were similar, and both the proteins were enriched in the synaptic soluble fraction. p150 was co-immunoprecipitated with p130 from this fraction. Recombinant p150 formed a heterodimer with recombinant p130 as estimated by sucrose density gradient ultracentrifugation. Recombinant p150 neither showed the Rab3A GAP activity nor affected the activity of recombinant p130. When p150 and p130 were co-expressed in the cells, the subcellular localization of each protein did not change. These results indicate that p150 is the noncatalytic subunit of Rab3 GAP.
Nagano
F
F
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565-0871, Japan.
Sasaki
T
T
Fukui
K
K
Asakura
T
T
Imazumi
K
K
Takai
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013569:Synapses / Q000378:metabolism*; D020692:rab3 GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D017931:DNA Primers; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; C512588:Rab3 GTPase-activating protein p130, mouse; D020692:rab3 GTP-Binding Proteins