Characterization of degradative protein quality control mechanisms using model substrates derived from temperature sensitive alleles

The purpose of protein homeostasis (proteostasis) is to maintain proteome integrity, thereby promoting viability at both the cellular and organism levels. Exposure to a range of acute stresses often produces misfolded proteins, which present a challenge to maintaining proteostatic balance. The accumulation of misfolded proteins can lead to the formation of potentially toxic protein aggregates, which are characteristic of a number of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Therefore, a number of protein quality control pathways exist to promote protein folding by molecular chaperones or target terminally misfolded proteins for degradation via the ubiquitin proteasome system or autophagy. Within the cytosol the mechanisms responsible for targeting substrates for proteasomal degradation remain to be fully elucidated. In this thesis, we established and employed thermosensitive model substrates to screen for factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations in Saccharomyces cerevisiae. Using a genome- wide flow cytometry based screen we identified the prefoldin chaperone subunit Gim3 as well as the E3 ubiquitin ligase Ubr1. An absence of Gim3 leads to the accumulation of model substrates in cytosolic inclusions and their delayed degradation. We propose that Gim3 promotes degradation by maintaining substrate solubility. In the course of screening for factors involved in degradative protein quality control, we identified secondary mutations in the general stress response gene WHI2 among a number of E3 ligase deletion strains. We demonstrate that an absence of WHI2 is responsible for the observed impairment in the proteolytic degradation of Guk1-7. We propose a link between mutations in WHI2 to a deficiency in the Msn2/4 transcriptional response, thereby altering the cell’s capacity to degrade misfolded cytosolic proteins. Collectively, the data in this thesis generated with the Guk1-7 model substrate underscores how changes in the elaborate protein quality control network can perturb proteostasis. Given that proteostasis is altered in a number of diseases ranging from cancer to ageing, identifying the factors that mediate protein quality control and understanding the interplay between members of the proteostatic network are important not only for understanding the basic biological processes but also for potential therapeutic applications.Science, Faculty ofGraduat