The overall goal of this proposal is to determine the mechanism of protein-conformation-based inheritance. [PSI+] is a prion-like element of Saccharomyces cerevisiae that causes reduced translational fidelity. [PSI+] is believed to represent a self-perpetuating conformational change in Sup35, the yeast translation termination factor eRF3, that leads to its aggregation and to the translational defect. Surprisingly, these aberrant Sup35 conformations are inherited in a dominant, non-Mendelian manner, indicating that protein conformation can direct heritable changes in phenotype independent of an altered genome. Variants of [PSI+] analogous to strains of mammalian prion exist in yeast. In vitro, purified Sup35 forms highly ordered amyloid-like fibers that vary in shape, texture and dimension. These fibers have been proposed to be the molecular basis of variants of [PSI+]. The specific goal of this proposal is to identify the structural parameters of Sup35 that lead to different [PSI+] variants in S. cerevisiae and to test whether these parameters lead to heritable traits, independent from a nucleic acid component. Pre-existing, well characterized [PSI+] variants as well as new variants generated by mutagenesis of Sup35 will be examined. Sup35 aggregates will be isolated from these variants and their structures will be analyzed by electron microscopy, atomic fore microscopy and circular dichroism. Finally, a seeding assay will be used to determine if specific Sup35 conformations can be inherited. Because of the substantial similarity of [PSI+] to mammalian prions, the work initiated here may ultimately lead to novel therapeutic approaches to treat human neurological diseases caused by prions. In addition, this work may provide insight into other epigenetic phenomena which have defied explanation so far.
