The continuing overall objective of this research proposal is to determine the molecular mechanisms accounting for the intracellular trafficking and insulin-stimulated translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the plasma membrane. Over the past several years, substantial progress has been made in our understanding of the insulin signaling pathways and GLUT4 trafficking compartments involved in the translocation process. However, our understanding is still largely incomplete and there remain numerous questions and processes that remain enigmatic. Recently, we have found that entry of the newly synthesized GLUT4 protein in adipocytes acquires insulin responsiveness by direct entry into the insulin-responsive compartments without transiting the plasma membrane or undergoing endocytosis. This has created a new paradigm to examine the targeting machinery and vesicle trafficking events that function at the different membrane transport steps in the GLUT4 lifecycle. To determine the molecular basis for initial GLUT4 cargo selection, trafficking through the secretory pathway and subsequent acquisition of insulin responsiveness, we propose to: 1) to define the specific GLUT4 targeting motifs, the role of known interacting partners and signals that are responsible for the direct transport from the Golgi to the insulin-responsive GLUT4 compartment (GSC) and those responsible from plasma membrane endocytosis to the GSC; 2) to identify the specific role of GGA and PACS1 in the anterograde and retrograde transport of GLUT4 into the GSC; 3) to use recently established Golgi/TGN in vitro vesicle budding assay to identify specific elements in GLUT4 responsible for GGA selectivity for proteomic analysis of donor, total released and GLUT4 specific vesicle-associated proteins; and 4) to determine the role of reversible GLUT4 oligomerization in the retention and release of intracellular GLUT4 and/or its role in the apparent enhancement of GLUT4 transport activity following plasma membrane translocation.