Acyl-coenzyme a dehydrogenases are localized on GLUT4-containing vesicles via association with insulin-regulated aminopeptidase in a manner dependent on its dileucine motif

Insulin-regulated aminopeptidase (TRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of TRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55-82) is reportedly involved in retention of GLUT4-containing vesicles. The region of TRAP fused with glutathione-S-transferase [GST-IRAP(55-82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with TRAP. The association was nearly abolished by mutation of the dileucine motif of TRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from TRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with TRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.