The role of the Exocyst complex in insulin regulated glucose transport

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DESCRIPTION (provided by applicant): The regulation of glucose transport by insulin represents the rate limiting step in glucose utilization and storage, and is known to represent a primary lesion in patients with insulin resistance. The exocyst complex was first identified in yeast as a tethering site for targeted exocytosis, and later found in our laboratory to play a role in the regulation of glucose transport, targeting Glut4 vesicles to sites of docking and fusion in fat cells. The overall goal of this proposal is to explore the molecular details of exocyst assembly and its role in insulin-stimulated glucose transport. Our preliminary data suggest that, contrary to what was originally conceived, the exocyst may be assembled in two subcomplexes, a target (t-) complex that comes together at the plasma membrane, and a vesicular (v-) complex that assembles at Glut4 vesicles. We hypothesize that the assembly of each sub-complex is controlled by distinct G proteins activated by a different process, and then unified prior to vesicle docking. We will explore this hypothesis by dissecting the molecular components in adipocytes. We will first study the assembly of the t-exocyst, characterizing the molecular interactions of these proteins in vitro and in cells, examine the order of assembly and the role of lipid rafts as an assembly site. We will then characterize the v-exocyst that assembles on Glut4 vesicles, evaluating the regulation of the formation of this complex. Finally, we will explore the role of the G-protein RalA in this process, examining how it is activated by insulin, and its molecular effectors that mediate its ability to unify the entire complex upon vesicle trafficking to the plasma membrane. We anticipate that these data will present a clearer picture of the regulation of Glut4 trafficking, docking and fusion.