Insulin facilitates glucose uptake into adipocytes and muscle cells by relocating glucose transporter 4 (GLUT4) from intracellular reservoirs to the plasma membrane. The translocation involves a vesicle fusion step that is mediated by three SNAREs - syntaxin 4, SNAP-23 and VAMP2 - and a number of regulatory proteins including Munc18c, synip and tomosyn. While the physiological importance of the SNARE regulatory proteins are clear, their molecular mechanisms of action and functional interactions among themselves are not known due to the complexity of the cellular environment. Recently, we reconstituted SNARE-mediated fusion in both liposome (synthetic bilayers) and "flipped" SNARE cell (native membranes) fusion systems. Here I propose to capitalize on these unique developments to ask key mechanistic questions about GLUT4 vesicle fusion, especially questions concerning how regulatory proteins act alone or in concert to control exocytosis at the molecular level. The specific hypothesis behind this proposed research is that regulatory proteins control different stages of the SNARE assembly cycle and contribute to the temporal and spatial regulation of GLUT4 vesicle fusion. Regulatory proteins will be added either as pure recombinant proteins or expressed as flipped proteins on the cell surface. Kinetic effects of each regulator can be assessed when it is added (alone or in combination) to the core fusion machinery of SNAREs. Three specific aims are proposed: 1) Define the effects of the Sec1/Munc18 (SM) protein Munc18c on each stage of complex assembly and fusion kinetics of GLUT4 exocytic SNAREs; 2) Establish the effects of exocytosis-specific regulators on SNAREpin assembly, Munc18c-SNARE complex formation and fusion kinetics; 3) Characterize the fusion pore dynamics and transition states in the SNARE fusion pathway. The long-term goal is to work our way up, protein by protein, until we can reconstitute the basic properties and fine-tuning of GLUT4 exocytosis. Insulin-regulated GLUT4 transport is crucial for glucose homeostasis and imbalances in this process may lead to type 2 diabetes. Knowledge of how the SNARE regulators work will likely identify novel targets for therapeutic intervention. Since many components of GLUT4 transport are conserved, our work can also shed light upon other exocytic pathways such as platelet and lung epithelial secretion.

K99DK080080

2007-09-20

2009-08-31