ASSOCIATION OF ACYL-COA SYNTHETASE-1 WITH GLUT4-CONTAINING VESICLES

GLUT4, the glucose transporter present in insulin-sensitive tissues, r esides in intracellular vesicular structures and translocates to the c ell surface in response to insulin, In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from ra t adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 an tibody, We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein ban d, long chain acyl-CoA synthetase-l, specifically present in immunoads orbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimm une serum. Acyl-CoA synthetase activity detected in GLUT4-enriched ves icles prepared by gradient centrifugation from insulin-treated adipocy tes was decreased to about the same extent as GLUT4 protein. Additiona lly, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylati on of proteins when incubated with labeled palmitate, a pathway that r equires palmitate esterification with CoA. These data indicate that th e insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-l and its product fatty acyl-CoA, shown previously to be required for budding and fusion in me mbrane trafficking processes.