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GENETIC SUPPRESSOR SCREEN FOR INTERACTORS OF SEC6 TEMPERATURE SENSITIVEMUTANT SEC6-49 AND IN SACCHAROMYCES CEREVISIAE**

Abstract

Protein delivery at the cellular level is a vital process involving complex steps which require accuracy. Each step of vesicle budding, physical transport, tethering and fusion involves the recruitment and coordination of hundreds of unique proteins. The focus of this project is the Sec6 protein, a subunit of the octamer exocyst complex that mediates tethering of post-Golgi vesicles to the plasma membrane in Saccharomyces cerevisiae. The earlier study by Songer and Munson (2008) presented sec6-49, a SEC6 mutant that harbors mutations of several amino acids that form a distinct patch on the protein surface. These surface amino acid mutations result in severe growth and secretory defects at 37°C. Interestingly, upon further investigation of sec6-49, the exocyst complex appeared to be misplaced from the expected sites, but the complex assembly remains intact. This observation led us to hypothesize that the Sec6 protein is an integral part of the complex serving an important anchor that holds the assembled complex in place at the plasma membrane. We undertook a genetic approach with the goal of identifying the putative interactors that Sec6 binds for anchoring. The screen was initiated by transforming 2µ based genomic library into sec6-49 via electroporation. Transformants were then grown and selected for survival at 37°C , followed by a spotting assay to confirm the suppression of temperature sensitive defect. Selected yeast colonies were subjected to plasmid extraction and retransformed into E. coli for further isolation of unique plasmids. These unique plasmids were retransformed into sec6-49 one by one for testing growth, and sequencing of the plasmid DNA that suppressed temperature sensitivity defect allow us to identify candidate genes for suppression. Currently, we are in the process of sequencing three more unique plasmids that display growth defect suppression and plan on cloning candidate genes to test specific suppression effects.

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