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Saccharomyces cerevisiae Genetic Suppressor Screen Searching for Novel Sec6 Interacting Proteins

Bullington, M., Opara-Nadi, P., Burke, K., Reneker, B., and France, E.*, Department of Biological and Environmental Science, Georgia College, Milledgeville, GA, 31061 Munson, M., Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605 *Corresponding author

A fundamental process, polarized protein secretion, is essential to all eukaryotic cells involving many steps mediated by hundreds of proteins. Exocyst, an eight-subunit-complex, is essential for proper delivery of secretory vesicles to the plasma membrane; our interest lies specifically on the Sec6 subunit. Earlier work showed temperature sensitive mutants of SEC6, which have altered amino acid clusters on the protein’s surface, resulted in severe growth and secretion defects at 37°C (Songer and Munson, 2009). Interestingly, analyses of exocyst assembly in these mutant backgrounds revealed that the complex was fully assembled with all eight subunits at 37°C, yet the whole complex mislocalized from expected sites of secretion. We currently hypothesize that Sec6 has an important anchoring function for exocyst, and that mislocalization of the complex stems from disruption of Sec6’s surface interaction with unknown factor(s) on the plasma membrane. In order to identify potential anchoring factors, we employed a genetic screen using a genomic library. Our screen design allows for identification of genes which compensate the growth defect of sec6-49 cells at 37°C, suppressing the mutation and allowing mutant cells to grow. Previously, we have successfully isolated approximately 30 plasmids that allowed sec6-49 cells to survive the temperature shift, and DNA sequencing of these plasmids was initiated to identify these candidate suppressor genes. Through yeast genome database searches and literature review, we have found several putative suppressor candidates and are currently validating suppression of individual candidate genes. Once true suppressors are confirmed through extensive analysis, we plan to explore their functional relationship(s) with Sec6 further. Overall, isolation and characterization of novel interacting proteins will shed light on mechanistic details of Sec6 and exocyst function, which is critical for understanding mechanistic details of quality control in the secretory pathway in higher eukaryotes.

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