Analyses of the co-localization of cellubrevin and the GLUT4 glucose transporter in rat and human insulin-responsive tissues

We have investigated the subcellular distribution and association of cellubrevin, a low molecular weight protein implicated in the process of membrane fusion, with intracellular membranes containing the insulin-sensitive GLUT4 glucose transporter from rat adipocytes, rat skeletal muscle and human skeletal muscle. SDS-PAGE and immunoblot analyses of subcellular fractions of adipocytes and skeletal muscle indicated a positive correlation between the distribution of GLUT4 and cellubrevin in intracellular membrane fractions tested from all tissues. The identity of the polypeptide reacting with antiserum against cellubrevin was further confirmed on the basis of its susceptibility to proteolysis by tetanus toxin. Immunoisolation of GLUT4-containing vesicles from a microsomal fraction enriched with GLUT4 and cellubrevin revealed that cellubrevin could be coprecipitated with GLUT4 vesicles from adipocytes. In contrast, intracellular GLUT4 vesicles isolated from both rat and human skeletal muscle were devoid of any detectable immunoreactivity towards cellubrevin. The observation that cellubrevin does not colocalise with intracellular GLUT4 in skeletal muscle from two different species, rat and human, would strongly suggest that it is unlikely to participate in the insulininduced delivery of GLUT4 to the cell surface in skeletal muscle.