Project Title
Screening for novel interactors of Temperature sensitive mutant sec6-49 in Saccharomyces Cerevisiae
Faculty Mentor(s) Name(s)
Ellen France
Abstract
Protein delivery is a vital process on the cellular level involving complex steps for successful delivery. First vesicle budding, then trafficking, and finally tethering and fusion. Each step 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 involved in tethering vesicles arriving at the plasma membrane in the last step of protein delivery. The earlier Songer and Munson (2008) study included sec6-49, a temperature sensitive Saccharomyces cerevisiae Sec6 mutant with altered amino acid surface patches. The surface mutations resulted in severe growth and secretory defects at 37°C. Interestingly, upon further investigation, the exocyst complex is misplaced from its expected location but remains intact. From this study, it is currently hypothesized the Sec6 protein is an integral part of the complex, and it acts as an anchor, holding the complex in place in the plasma membrane. We undertook a genetic approach to discover Sec6’s direct and indirect protein interactions on the plasma membrane, to understand it’s vital role as a complex anchor. This process was initiated by transforming a sec6-49 2µ multicopy from the genomic library plasmids via electroporation. Transformants were then grown and selected at 37°C, followed by a spotting assay to verify temperature sensitive suppression. Selected yeast colonies were mini-prepped to extract their plasmids and then retransformed into E. coli for further isolation of unique plasmids. Currently, isolation of unique plasmids via bacterial mini prep and restriction digest are leading to the next steps of sequencing to identify possible proteins of interest on the insertion site, and eventually testing the mutation suppression of each individual gene via NeB HiFi Cloning.
Screening for novel interactors of Temperature sensitive mutant sec6-49 in Saccharomyces Cerevisiae
Protein delivery is a vital process on the cellular level involving complex steps for successful delivery. First vesicle budding, then trafficking, and finally tethering and fusion. Each step 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 involved in tethering vesicles arriving at the plasma membrane in the last step of protein delivery. The earlier Songer and Munson (2008) study included sec6-49, a temperature sensitive Saccharomyces cerevisiae Sec6 mutant with altered amino acid surface patches. The surface mutations resulted in severe growth and secretory defects at 37°C. Interestingly, upon further investigation, the exocyst complex is misplaced from its expected location but remains intact. From this study, it is currently hypothesized the Sec6 protein is an integral part of the complex, and it acts as an anchor, holding the complex in place in the plasma membrane. We undertook a genetic approach to discover Sec6’s direct and indirect protein interactions on the plasma membrane, to understand it’s vital role as a complex anchor. This process was initiated by transforming a sec6-49 2µ multicopy from the genomic library plasmids via electroporation. Transformants were then grown and selected at 37°C, followed by a spotting assay to verify temperature sensitive suppression. Selected yeast colonies were mini-prepped to extract their plasmids and then retransformed into E. coli for further isolation of unique plasmids. Currently, isolation of unique plasmids via bacterial mini prep and restriction digest are leading to the next steps of sequencing to identify possible proteins of interest on the insertion site, and eventually testing the mutation suppression of each individual gene via NeB HiFi Cloning.