Glut4 palmitoylation at Cys223 plays a critical role in Glut4 membrane trafficking.

Recently, we identified Glut4 as a palmitoylated protein in adipocytes. To understand the role of Glut4 palmitoylation in Glut4 membrane trafficking, a process that is essential for maintenance of whole body glucose homeostasis, we have characterized Glut4 palmitoylation. We found that Glut4 is palmitoylated at Cys223 and Glut4 palmitoylation at Cys223 is essential for insulin dependent Glut4 membrane translocation as substitution of Cys223 with a serine residue in Glut4 (C223S Glut4) diminished Glut4 responsiveness to insulin in membrane translocation in both adipocytes and CHO-IR cells. We have examined C223S Glut4 subcellular localization and observed that it was absence from tubular-vesicle structure, where insulin responsive Glut4 vesicles were presented. Together, our studies uncover a novel mechanism under which Glut4 palmitoylation regulates Glut4 sorting to insulin responsive vesicles, thereby insulin-dependent Glut4 membrane translocation.