Saccharomyces cerevisiae Genetic Screen: A Tale of SEC6
Abstract
Polarized protein secretion is a fundamental process for all eukaryotic cells involving many steps mediated by hundreds of proteins. Our interest lies on Sec6 protein, a component of an eight-subunit-complex named exocyst, which is required for the proper delivery of secretory vesicles to the plasma membrane. An earlier study published on temperature sensitive mutants of Sec6 (Sanger and Munson 2009) have altered amino acids on the protein surface, which resulted in severe growth and secretion defects at 37°C. Interestingly, analyses of exocyst assembly in these mutant backgrounds revealed that the complex was intact with all eight subunits at 37°C, yet the whole complex gets misplaced from the site they should locate. We currently hypothesize that Sec6 has an important anchoring function for the exocyst, and that the mislocalization of exocyst stems from disruption of Sec6’s surface interaction with unknown factor(s) on the plasma membrane. In order to identify the potential anchoring factors, we employed a genetic screen using a genomic library. The screen design allows us to identify genes that compensate the growth defect of sec6-49 cells at 37°C, allowing mutant cells to grow. We successfully isolated approximately 30 plasmids that allowed sec6-49 cells to survive the temperature shift. DNA sequencing of these plasmids was initiated to identify candidate genes, and we found several interesting genes. Shortly, we need to validate these individual candidate genes to discover new interacting proteins and explore the relationship with Sec6 further. Overall, the isolation and characterization of novel anchoring interactors 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.
Saccharomyces cerevisiae Genetic Screen: A Tale of SEC6
Polarized protein secretion is a fundamental process for all eukaryotic cells involving many steps mediated by hundreds of proteins. Our interest lies on Sec6 protein, a component of an eight-subunit-complex named exocyst, which is required for the proper delivery of secretory vesicles to the plasma membrane. An earlier study published on temperature sensitive mutants of Sec6 (Sanger and Munson 2009) have altered amino acids on the protein surface, which resulted in severe growth and secretion defects at 37°C. Interestingly, analyses of exocyst assembly in these mutant backgrounds revealed that the complex was intact with all eight subunits at 37°C, yet the whole complex gets misplaced from the site they should locate. We currently hypothesize that Sec6 has an important anchoring function for the exocyst, and that the mislocalization of exocyst stems from disruption of Sec6’s surface interaction with unknown factor(s) on the plasma membrane. In order to identify the potential anchoring factors, we employed a genetic screen using a genomic library. The screen design allows us to identify genes that compensate the growth defect of sec6-49 cells at 37°C, allowing mutant cells to grow. We successfully isolated approximately 30 plasmids that allowed sec6-49 cells to survive the temperature shift. DNA sequencing of these plasmids was initiated to identify candidate genes, and we found several interesting genes. Shortly, we need to validate these individual candidate genes to discover new interacting proteins and explore the relationship with Sec6 further. Overall, the isolation and characterization of novel anchoring interactors 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.