Genetic Factors Involved In Yeast Prion Propagation.

Diabetes, Digestive, Kidney Diseases

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Abstract

Prion diseases are amyloidoses, like Type 2 diabetes and Alzheimer's disease, but with the important distinction of being infectious. In these diseases accumulation of amyloid fibers of misfolded protein is associated with tissue pathology. We use yeast as a model system to study how cellular factors influence amyloid propagation.[unreadable] [unreadable] The yeast [PSI] prion is a cytoplasmic amyloid of the essential translation termination factor Sup35p. We previously isolated an Hsp70 mutant (Ssa1-21p) that impairs [PSI] propagation. More recently we constructed the first Sup35-GFP fusion protein that functions normally in both translation termination and prion propagation. Using this system we showed that [PSI]-containing cells expressing Ssa1-21p have larger Sup35p aggregates than wild type cells, suggesting that Ssa1-21p is less able to restrict prion protein aggregation or interferes with disaggregation of prion aggregates. Our data from these studies suggested that much aggregated Sup35p does not function as prion seed or that current methods for estimating seed numbers are unreliable. We developed this system further and are currently using it to measure differences in prion protein aggregation with better resolution both in vivo and in vitro.[unreadable] [unreadable] To begin to analyze functions of mammalian chaperones we developed a yeast system in which we can replace cytosolic Hsp70 with isoforms from any other species. Using it we found that both plant and mammalian Hsc70 (constitutively expressed) support yeast growth but that Hsp70 (stress-inducible) counterparts do not, which uncovered considerable functional differences between these nearly identical isoforms. We also identified differences in how Hsp70 isoforms influence prion propagation, and the Hsp70 subdomains responsible for these functional differences. We thus have valuable tractable system we are using to define functions of mammalian chaperones in both growth and prion propagation.[unreadable] [unreadable] The disaggregating activity of the protein chaperone Hsp104, which is thought to break prion aggregates or "seeds" into more numerous pieces, is required for yeast prion propagation. Over a decade ago it was shown that overproduced Hsp104 eliminates [PSI], although the mechanism underlying this effect is still unknown. Protein disaggregation by Hsp104 requires assistance of Hsp70 and its co-chaperone Hsp40. Reasoning that Ssa1-21p's effect on prion seed numbers was related to the seed-generating capacity of Hsp104, we screened for and identified Hsp104 mutations that restored normal prion propagation in SSA1-21 mutants. This work showed that altering Hsp70/Hsp104 cooperation in a particular way considerably affects prion propagation, and that a conserved subdomain of Hsp104, whose function remains unknown, is dispensable for normal Hsp104 functions, including prion propagation, but is required for Hsp104 to eliminate prions when overexpressed. We thus showed that excess resolubilizing activity of Hsp104 alone does not eliminate [PSI] and have a system we can exploit to determine the mechanism by which excess Hsp104 causes elimination of [PSI].