A possible target protein for smg-25A/rab3A small GTP-binding protein.
The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.
Shirataki
H
H
Department of Biochemistry, Kobe University School of Medicine, Japan.
Kaibuchi
K
K
Yamaguchi
T
T
Wada
K
K
Horiuchi
H
H
Takai
Y
Y
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014461:Ultracentrifugation; D020692:rab3 GTP-Binding Proteins
D003432:Cross-Linking Reagents; D009419:Nerve Tissue Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins