The misfolding normal, cellular encoded proteins and their subsequent aggregation has emerged as a common mechanism underlying several neurodegenerative diseases in mammals, including Huntington's and the Transmissible Spongiform Encephalopathies (prion diseases). A key event in this process is the efficient amplification of misfolded form, which occurs when existing aggregates direct the conversion of normal protein to a like state. This autocatalytic misfolding in influenced by sequence elements within the implicated proteins, such as repeated elements that modulate both the appearance and persistence of the misfolded form. Despite their importance, the mechanism(s) by which these sequence elements influence the autocatalytic misfolding pathway is currently unknown. The goal of this proposal is to address this significant gap in knowledge. For this work, we will use the Sup35/[PSI+] prion system in the yeast S. cerevisiae. The Sup35 protein contains five and a half imperfect copies of an oligopeptide repeat. Deletion of one or more full repeats completely abolishes the autocatalytic misfolding of Sup35, while expansion of the repeat region increases the propensity of cells to adopt and propagate this form. We hypothesize that the oligopeptide repeats alter the efficiency of discrete steps in the Sup35 misfolding pathway and thereby the stability of the prion-associated phenotype by impacting the nature of Sup35 physical interactions. To test this hypothesis, we will identify the step(s) in the Sup35 autocatalytic misfolding pathway that are altered by variation in the number of repeats and impact of these variations on Sup35 interactions with itself and other cellular factors. By understanding how specific sequence elements modulate the protein folding environment in vivo, we will begin to understand how they contribute to both the normal cellular state and the corresponding disease state. Understanding how these sequence elements can modulate protein folding pathways within the context of the cellular environment will provide insight into how protein misfolding diseases are initiated and maintained.