Genetic suppressor screen in search of novel Sec6 interacting proteins in Saccharomyces cerevisiae
Abstract
Polarized protein secretion is a fundamental process for all eukaryotic cells involving many steps mediated by hundreds of proteins. The eight-subunit-complex, named Exocyst, is essential for the proper delivery of secretory vesicles to the plasma membrane, and our interest lies specifically on the Sec6 subunit. Earlier work showed temperature sensitive mutants of SEC6, which have altered amino acid clusters on the protein surface, resulted in severe growth and secretion defects at 37°C (Songer and Munson, 2009). Interestingly, analyses of exocyst assembly in these mutant backgrounds revealed that the complex was fully assembled with all eight subunits at 37°C, yet the whole complex mislocalized from the expected sites of secretion. We currently hypothesize that Sec6 has an important anchoring function for Exocyst, and that the mislocalization of the complex 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, suppressing the mutation and allowing mutant cells to grow. We successfully isolated approximately 30 plasmids that allowed sec6-49 cells to survive the temperature shift, and DNA sequencing of these plasmids was initiated to identify candidate genes. We found several putative suppressor candidates and are currently validating the suppression of individual candidate genes. Once we confirm the true suppressors, we plan to explore the functional relationship with Sec6 further. Overall, the isolation and characterization of novel anchoring proteins 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 in higher eukaryotes.
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Genetic suppressor screen in search of novel Sec6 interacting proteins in Saccharomyces cerevisiae
Polarized protein secretion is a fundamental process for all eukaryotic cells involving many steps mediated by hundreds of proteins. The eight-subunit-complex, named Exocyst, is essential for the proper delivery of secretory vesicles to the plasma membrane, and our interest lies specifically on the Sec6 subunit. Earlier work showed temperature sensitive mutants of SEC6, which have altered amino acid clusters on the protein surface, resulted in severe growth and secretion defects at 37°C (Songer and Munson, 2009). Interestingly, analyses of exocyst assembly in these mutant backgrounds revealed that the complex was fully assembled with all eight subunits at 37°C, yet the whole complex mislocalized from the expected sites of secretion. We currently hypothesize that Sec6 has an important anchoring function for Exocyst, and that the mislocalization of the complex 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, suppressing the mutation and allowing mutant cells to grow. We successfully isolated approximately 30 plasmids that allowed sec6-49 cells to survive the temperature shift, and DNA sequencing of these plasmids was initiated to identify candidate genes. We found several putative suppressor candidates and are currently validating the suppression of individual candidate genes. Once we confirm the true suppressors, we plan to explore the functional relationship with Sec6 further. Overall, the isolation and characterization of novel anchoring proteins 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 in higher eukaryotes.