Structure and Function of the Exocyst Complex

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DESCRIPTION (provided by applicant): Eukaryotic cells utilize small membrane-bound vesicles to transport cargo between subcellular organelles, and to the plasma membrane for secretion. The proper function and specificity of the vesicular transport and membrane fusion processes are crucial for maintenance of cellular integrity, growth, cellular movement and secretory events such as hormone release and neurotransmission. Vesicle transport and fusion at the plasma membrane require many essential proteins, including the SNARE proteins and Sec1p that are involved in the membrane fusion process, the Rab and Rho GTPases, and a large complex called the exocyst. The exocyst complex has been implicated in a number of different functions: selection of the site of exocytosis; physical tethering of secretory vesicles to these sites; communicating with cytoskeletal and cell cycle proteins; and regulating the specificity and assembly of the SNARE proteins. None of these are well understood at the molecular level. Our aim is to combine biochemical and biophysical techniques with genetics and cell biological methods in order to understand the molecular functions of the exocyst complex. We have chosen to study the exocyst proteins from the model organism Saccharomyces cerevisiae so as to take advantage of the wealth of genetic and cell biological techniques available. To accomplish this goal, we have purified the exocyst protein Sec6p and are i) Investigating the structure of Sec6p by biochemical, biophysical and x-ray crystallographic studies; ii) Analyzing the function of Sec6p in vivo through characterization of specific Sec6p mutants; and (iii) Testing the effect of Sec6p on SNARE complex assembly. 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.