Characterizing cytosolic protein quality control pathways in saccharomyces cerevisiae using temperature-sensitive model substrates

A large pool of proteins localizes in the cytosol of eukaryotic cells. Proteins in the cell can be misfolded due to cellular stress, mutations, and transcriptional and translational errors. Several E3 ubiquitin ligases have been shown to target misfolded cytosolic proteins for degradation by the proteasome. In this study, we characterized a panel of thermosensitive mutant proteins in Saccharomyces cerevisiae from six essential genes. The wild-type alleles of these thermosensitive proteins are stable at restrictive temperature. In contrast, we found that roughly half the tested alleles are significantly degraded at non-permissive temperature in a proteasome-dependent manner. These unstable alleles thus display hallmarks of protein quality control substrates. We found that degradation of mutant protein Pro3-1p, one of the unstable alleles, is dependent on the dual action of Ubr1 and San1 ubiquitin ligases. The single deletions of the ligases do not affect the stability of Pro3-1p. In contrast, double deletion of UBR1 and SAN1 leads to significant stabilization of the mutant protein. The increase in stability of Pro3-1 is associated with suppression of thermosensitive phenotype at restrictive temperature, suggesting that the depletion of the essential protein due to its degradation leads to the loss of viability at restrictive temperature.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat