Cellubrevin is a resident protein of insulin-sensitive GLUT4 glucose transporter vesicles in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and fat cells by inducing translocation of GLUT4 glucose transporters from a storage site to the cell surface. The mechanism of this translocation and the identity of the storage site are unknown, but it has been hypothesized that transporters recycle between an insulin-sensitive pool, endosomes, and the cell surface. Upon cell homogenization and fractionation, the storage site migrates with light microsomes (LDM) separate from the plasma membrane fraction (PM). Cellubrevin is a recently identified endosomal protein that may be involved in the reexocytosis of recycling endosomes. Here we describe that cellubrevin is expressed in 3T3-L1 adipocytes and is more abundant in the LDM than in the PM. Cellubrevin was markedly induced during differentiation of 3T3-L1 fibroblasts into adipocytes, in parallel with GLUT4, and the development of insulin regulated traffic. In response to insulin, the cellubrevin content decreased in the LDM and increased in the PM, suggesting translocation akin to that of the GLUT4 glucose transporter. Vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin-II, a protein associated with regulated exocytosis in secretory cells, also redistributed in response to insulin. Both cellubrevin and VAMP-2 were susceptible to cleavage by tetanus toxin. Immunopurified GLUT4-containing vesicles contained cellubrevin and VAMP-2, and immunopurified cellubrevin-containing vesicles contained GLUT4 protein, but undiscernible amounts of VAMP-2. These observations suggest that cellubrevin and VAMP-2 are constituents of the insulin-regulated pathway of membrane traffic. These results are the first demonstration that cellubrevin is present in a regulated intracellular compartment. We hypothesize that cellubrevin and VAMP-2 may be present in different subsets of GLUT4-containing vesicles.