Glucose transport into fat and muscle, which is tightly regulated, is determined by the amount of the GLUT4 glucose transporter in the plasma membrane (PM). In the fasted state, when insulin levels are low, GLUT4 is actively sequestered intracellularly by rapid endocytosis and slow recycling. Elevated insulin in the postprandial state induces a redistribution of GLUT4 to the PM, predominantly by accelerating GLUT4 recycling. Increased levels of PM GLUT4 are actively maintained by rapid GLUT4 recycling during the postprandial period of elevated insulin. GLUT4 PM levels return to pre-stimulation amounts when circulating insulin levels decrease. Thus, the control of glucose uptake by fat and muscle cells is dependent upon regulation of GLUT4 trafficking between the interior and PM of cells. Compromised GLUT4 redistribution to the PM contributes to hyperglycemia associated with insulin-resistance and type 2 diabetes. Elucidating the molecular mechanism underlying GLUT4 traffic and its regulation by insulin will significantly impact our understanding of insulin resistance and thereby provide a foundation for the future development of therapeutic interventions. Although the phenomenon of GLUT4 trafficking is well described, there is much to be learned about the molecular mechanisms of the process. GLUT4 is largely distributed among 2 intracellular membrane compartments: insulin-responsive vesicles (IRVs) that are specialized for the traffic of GLUT4 and the GLUT4 TGN perinuclear compartment. IRVs have been intensively studied because they ferry GLUT4 to the PM in both unstimulated (basal) and insulin-stimulated cells. The perinuclear compartment has a pivotal role in the intracellular sequestration of GLUT4 in basal adipocytes and in the mobilization of GLUT4 to support the increased demand in insulin-stimulated cells. The molecular mechanisms controlling GLUT4 trafficking to and from the perinuclear site have not been described in detail. Here I propose to address that gap in knowledge. The workplan, building on my labs past accomplishments, addresses several key questions in the field of GLUT4 trafficking. The major objectives of the project are to: define the perinuclear compartment proteome, thereby identifying proteins that function in the regulation of GLUT4 traffic as well as identifying cargo proteins other than GLUT4 that are stored their; discover and characterize the protein machinery responsible for regulating flux of GLUT4 through this perinuclear compartment. To accomplish these objectives, we will use state-of-the-art mass spectrometry profiling methods, and a comprehensive battery of functional intact cell assays to characterize the molecular mechanism regulating trafficking to and from the perinuclear compartment.