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EXPLORING NEW PHENOTYPES OF SAACHROMYCES CEREVISIAE SEC6-46 MUTANT**

Abstract

Polarized protein secretion is essential for delivering proteins and lipids to specific membrane domains in eukaryotic cells and underlies fundamental processes such as cell growth, division, and maintenance of cell polarity. This process relies on multiple factors, including the exocyst, an evolutionarily conserved octameric protein complex that tethers secretory vesicles to the plasma membrane. Our work focuses on Sec6, a critical subunit of the exocyst. Previous studies have shown that mutations in specific surface-exposed residues of Sec6 result in temperature-sensitive yeast growth defects at 37 °C. Fluorescence microscopy shows that although the exocyst remains assembled in sec6-49 cells, the complex is mislocalized, suggesting a defect in membrane anchoring. Based on this known temperature-sensitive phenotype, we previously employed a multicopy plasmid-based genetic suppressor screen to identify putative membrane anchors of Sec6 that could restore growth at 37 °C. The screen yielded few candidate suppressors, but these were either previously known or failed validation. This outcome suggested that relying solely on temperature sensitivity may limit the identification of true suppressors. The focus of this study is to identify additional phenotypes of sec6-49 cells that could improve suppressor selection strategies. Because single mutations often produce pleiotropic effects, we examined sec6-49 growth under additional stress conditions that challenge cellular processes linked to membrane trafficking and secretion. In this work, we report preliminary growth testing under cold stress and in the presence of caffeine, diamide, ethanol, and cercosporamide. These assays were used to identify growth sensitivities rather than to perform suppressor screening. Identifying new stress-dependent growth phenotypes may enable future suppressor screens using alternative selection pressures and enhance our understanding of Sec6 function and exocyst complex biology. The exocyst is known to play diverse and conserved roles across multiple organisms, including humans, where defects in its function can disrupt normal cellular processes.

Acknowledgements

Georgia College & State University, Tri-Beta Student Research Funding

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