Characterization of a protein responsible for the protein-based inheritance of [PSI(+)] in yeast

The [PSI+] genetic element in the yeast Saccharomyces cerevisae is an altered form of Sup35p, a subunit of the translation termination factor. When Sup35p in the prion-like [ PSI+] state, translation termination activity is reduced and ribosomes frequently read through nonsense codons, conferring upon cells a suppression phenotype on all three stop codons. The mechanism by which the conformational switch occurs in Sup35p is still unknown. Studies of [ PSI+] are of interest for two reasons. (1) It is a new mechanism of genetic inheritance. (2) It might provide cure for several neurodegenerative diseases, especially for the transmissible spongiform encephylapathies (TSE) caused by the prion protein PrP.;This work has been directed at (1) providing physical evidence that [PSI+] is indeed the prion form of Sup35p and (2) identifying structurally and functionally important regions in Sup35p to understand how it self assembles into the higher-order structure of prion. I first demonstrated that Sup35p can exist in different physical states by visualizing the distinct distribution patterns of GFP-tagged Sup35p and its N-terminal fragment in isogenic [PSI+] and [psi--] strains. I then investigated the role of the oligopeptide-repeat region of Sup35p (aa57--93) in the formation and propagation of [PSI+]. Deletion of the repeat region abolished the ability of Sup35p to form and propagate [PSI+], whereas expansion of the repeats greatly increased the spontaneous appearance of [PSI+], the first mutation of its type amongst these newly described elements of inheritance. Next, I showed that In vitro, fully denatured repeat-expansion protein formed beta-sheet rich, higher-ordered structures much more rapidly than wild-type protein. These data provide insight on the nature of the conformational changes underlying protein-based mechanisms of inheritance and strengthen the link between this process and those producing neurodegenerative prion diseases in mammals. I also characterized the function of the Sup35p middle region (M, aa124--253). Although the DeltaM mutant was still able to form and propagate [PSI+], it was aggregation-prone and mitotically unstable. From these data I conclude that M is involved in stabilizing the conformational states of Sup35p. Finally, I have shown that the Hsp104 curing effect on [PSI +] does not require M.