Haas, Kelaine C. Zimmerman2012-07-242012-07-242012-05https://hdl.handle.net/11299/128857University of Minnesota Ph.D. dissertation. May 2012. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Dr. Robin Wright. 1 computer file (PDF); v, 152 pages.Most organisms must possess molecular mechanisms that enable them to respond to both long-term and transient temperature fluctuations in their environment. As a first step in understanding these mechanisms, we focused on identifying molecular mechanisms that allow the budding yeast, Saccharomyces cerevisiae, to respond to low temperature. The goal of this study was to analyze the growth rate of S. cerevisiae mutant strains to identify genes required for growth at 10˚C. Within the S. cerevisiae homozygous deletion collection, we identified cold-sensitive strains representing 790 genes. The CS790 genes encode proteins that function in a wide range of biological processes, suggesting that genes required for S. cerevisiae growth at low temperatures represent complex networks of cellular and molecular functions. Two processes were statistically enriched among these 790 genes: genes required for threonine and tryptophan biosynthesis, and genes encoding components of the dynein/dynactin complex. These results provide a framework for future studies to identify the specific molecular mechanisms responsible for cold adaptation and growth of organisms. In addition to these enriched categories, previous work in our lab has also shown that three S. cerevisiae null mutants, ubc7∆, cue1∆, and doa10∆, are cold sensitive at 10˚C. UBC7, CUE1, and DOA10 encode proteins that function in the Ubc7-dependent ERAD pathway, which targets aberrant proteins in the ER lumen or membrane for ubiquitin-mediated proteasomal degradation. By introducing point mutations in the Ubc7 active site C89 residue, we created a catalytically inactive Ubc7 protein, and confirmed that the ubc7∆ strain is cold sensitive due to the loss of Ubc7 enzymatic activity. Therefore, the ability of Ubc7 to ubiquitinate appropriate target substrates is required for yeast growth at 10˚C. In an effort to identify relevant targets of this ERAD pathway, we used the Synthetic Genetic Array (SGA) protocol to create a complete set of haploid double mutants that carry both a deletion of UBC7 and a deletion of another gene. Genetic suppressors were identified by double mutant strains that no longer exhibited the cold-sensitive phenotype seen in ubc7∆ strains. This analysis identified 10 deletions that suppress the cold-sensitivity of ubc7∆, cue1∆, and doa10∆ cold-sensitivity, indicating that the suppression is not limited to the UBC7 gene, but instead affects the entire Ubc7-dependent ERAD pathway. Therefore, the proteins represented by these 10 deletion mutants are potential Ubc7-ERAD targets. Interestingly, four of these suppressors, PHO84, PHO81, SPL2 and YML122C, are members of the high-affinity phosphate transport pathway. Additional analysis of S. cerevisiae triple mutant strains with deletions in UBC7, SPL2, and the high-affinity Pi transporters, PHO84 or PHO89, shows that UBC7 may be working in a parallel pathway to SPL2. These results suggest that Ubc7 may directly or indirectly regulate the low-affinity branch of the phosphate transport pathway, via Pho87 and Pho90.en-USAmino acid biosynthesisColdDeletion CollectionPhosphateSynthetic Genetic ArrayYeastMolecular, Cellular, Developmental Biology and GeneticsThesis or Dissertation