J-Protein Regulation of Yeast Prion Propagation

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A critical barrier to advancing the understanding of chaperone function in prion biology is that the fundamental chaperone requirements for most yeast prions remain unidentified. Existing knowledge is disjointed due to a lack of systematic evaluation that controls for variation in yeast strain background and prion structure. The continued existence of this barrier is an important problem because, until it is overcome, an understanding of how protein sequences give rise to amyloids with distinct patterns of chaperone interaction cannot be fully realized. The long- term goal is to utilize the highly tractable budding yeast, S. cerevisiae, to systematically decipher the complex relationships between amyloid-forming yeast prions and molecular chaperone proteins with a goal of better un- derstanding J-protein chaperone function and prion behavior. The objective of this particular application is to utilize genetic systems and biochemical assays to determine the functional elements involved in specific prion- chaperone interactions. The central hypothesis is that differences in amyloid structure, arising primarily from amino acid composition, create distinct challenges for prion transmission which are overcome by specific J- protein functions that buffer prions against loss during mitosis. The hypothesis has been formulated on the basis of data produced in the applicant's laboratory. The rationale for the proposed research is that unambiguous determinations of J-protein functional requirements are a necessary step toward understanding the mechanisms of J-protein function in amyloid biology. Using our own collection of well-studied, naturally-occurring prions, and the yeast cytosol as model systems, this hypothesis will be tested by pursuing two specific aims: 1) Determine the role of the Apj1 QS region in Hsp104-mediated prion elimination, and 2) Identify elements of higher eukaryotic Sis1 orthologs responsible for amyloid conformer-specific propagation. Proven yeast genetic manipulations, which have been established as feasible in the applicant's hands, will be the primary methods used to accomplish these aims. The approach is innovative because it represents a substantive departure from the status quo by placing emphasis on the ability to draw distinctions and make comparisons among multiple J-proteins and yeast prions as a way to broadly understand J-protein function. The contribution of the proposed research is expected to be the elucidation of the roles of distinct J-proteins in prion propagation and prion elimination. This contribution is significant because it is the next step in a continuum of research which is expected to contribute to the under- standing of the biochemical basis of J-protein·amyloid interactions. A molecular understanding of prion-chaper- one interactions has the potential to inform the development of interventions for protein misfolding disorders, including the increasing prevalent neurodegenerative disorders Alzheimer's and Parkinson's.