Dissecting the Molecular Mechanisms of Exocytic Vesicle Tethering and Fusion

PROJECT SUMMARY Eukaryotic cells transport cargo between subcellular organelles, and to the plasma membrane for secretion, using small membrane-bound vesicles are carriers. The regulation of vesicular transport and membrane fusion processes are crucial for cellular morphology, growth, movement and secretion, including hormone release and neurotransmission. Many essential proteins are required for these processes, including the SNARE proteins and Sec1 that are involved in the membrane fusion process, the Rab and Rho GTPases, and the exocyst octameric tethering complex. Exocyst has been implicated in a number of different functions involved in recognition, tethering and quality control of SNARE assembly and fusion, but none of these are well understood at the molecular level. We use a multidisciplinary strategy of biochemical, structural and biophysical techniques, combined with genetics and cell biological methods, to understand the molecular architecture and function of the exocyst complex, Sec1 and regulation of SNARE complex assembly and fusion. We study the exocyst proteins from the model organism Saccharomyces cerevisiae to take advantage of the wealth of genetic, cell biological and biochemical techniques available, but have expanded to exocyst from other organisms. Our studies aim to address the following fundamental questions: How is the specificity of vesicle targeting and fusion achieved? How do exocyst and Sec1 function to regulate SNAREs? What are the roles for different partner proteins and lipids? What happens when exocyst and Sec1 functions are dysregulated? Because these proteins are conserved from yeast to human neurons, this research will advance our knowledge of how secretion and growth are regulated in all eukaryotic cells. We will also illuminate the molecular consequences of disease-associated dysfunction.