Genetic Screening to identify putative interactors of Sec6 in Saccharomyces cerevisiae
Faculty Mentor(s) Name(s)
Ellen France
Abstract
Vesicle delivery within eukaryotic cells is a complex process involving initiation of vesicle budding, physical transport, tethering, and fusion of vesicles, all orchestrated by the recruitment and interaction of distinct proteins. Our research focuses on the Sec6 protein, a component of the octameric exocyst complex. Originally discovered in budding yeast, the exocyst is responsible for tethering post-Golgi vesicles to the plasma membrane. Songer and Munson (2008) generated two new mutant alleles of SEC6 by mutating specific amino acids that forms small patches on the Sec6 protein surface; both mutants are temperature-sensitive and inviable when grown at 37°C. Our investigation centers on one mutant allele, sec6-49. Further examination revealed misplacement of the exocyst complex, while the complex's overall assembly remained unaffected. We hypothesized that the Sec6 protein acts as an anchor through uncharacterized interactions with unknown proteins on the plasma membrane via these mutated amino acid patches, ensuring stable complex positioning at the plasma membrane. To identify these unknown protein interactors, we conducted a genetic screen using sec6-49 mutant cells. Following transformation with genomic library plasmids, individual mutant cells were screened at 37°C for survival, with surviving cells subjected to plasmid extraction for analysis. As yeast colonies may harbor multiple plasmids, we employed several steps to isolate individual plasmids and identify those conferring survival. Currently, we focus on one such transformant M8 that harbor four plasmids, aiming to test each on cell survival and sequence the genomic segment. Testing of transformant S2 progressed to isolation of plasmid S2-2, which was retested for growth and sequenced to reveal potential genes: LSB5, SRO9, FUS1, BIK1, and STE50. Highlighting individual gene testing in growth assays may lead to identification of putative interactors. Our study underscores the intricate mechanisms underlying vesicle delivery and the importance of Sec6 in cellular processes.
Start Date
27-3-2024 10:50 AM
End Date
27-3-2024 10:58 AM
Location
Arts and Sciences 2-70
Genetic Screening to identify putative interactors of Sec6 in Saccharomyces cerevisiae
Arts and Sciences 2-70
Vesicle delivery within eukaryotic cells is a complex process involving initiation of vesicle budding, physical transport, tethering, and fusion of vesicles, all orchestrated by the recruitment and interaction of distinct proteins. Our research focuses on the Sec6 protein, a component of the octameric exocyst complex. Originally discovered in budding yeast, the exocyst is responsible for tethering post-Golgi vesicles to the plasma membrane. Songer and Munson (2008) generated two new mutant alleles of SEC6 by mutating specific amino acids that forms small patches on the Sec6 protein surface; both mutants are temperature-sensitive and inviable when grown at 37°C. Our investigation centers on one mutant allele, sec6-49. Further examination revealed misplacement of the exocyst complex, while the complex's overall assembly remained unaffected. We hypothesized that the Sec6 protein acts as an anchor through uncharacterized interactions with unknown proteins on the plasma membrane via these mutated amino acid patches, ensuring stable complex positioning at the plasma membrane. To identify these unknown protein interactors, we conducted a genetic screen using sec6-49 mutant cells. Following transformation with genomic library plasmids, individual mutant cells were screened at 37°C for survival, with surviving cells subjected to plasmid extraction for analysis. As yeast colonies may harbor multiple plasmids, we employed several steps to isolate individual plasmids and identify those conferring survival. Currently, we focus on one such transformant M8 that harbor four plasmids, aiming to test each on cell survival and sequence the genomic segment. Testing of transformant S2 progressed to isolation of plasmid S2-2, which was retested for growth and sequenced to reveal potential genes: LSB5, SRO9, FUS1, BIK1, and STE50. Highlighting individual gene testing in growth assays may lead to identification of putative interactors. Our study underscores the intricate mechanisms underlying vesicle delivery and the importance of Sec6 in cellular processes.