Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes

A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes, The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting, Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation, These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4, When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface, This observation provides direct evidence for the functional consequence of vp165 translocation.