Prion disease is a type of amyloidosis, like Type 2 diabetes and Alzheimer's disease, but with the important distinction of being infectious. In these diseases abnormal folding of a cellular protein leads to its aggregation into fibrillar structures (amyloid) whose accumulation is associated with tissue pathology. Our research using yeast as a model system is aimed at better understanding amyloid formation and replication in vivo, and focuses on how cellular factors influence its propagation and transmission. The yeast [PSI] prion is a cytoplasmic amyloid of the translation termination factor Sup35p. When [PSI] is present much of the Sup35p is sequestered into aggregates believed to be amyloid. Prion aggregates or "seeds" replicate in the cytoplasm, presumably being broken into more numerous pieces by the disaggregating activity of the protein chaperone Hsp104. In vitro, Hsp70 and Hsp40 are required for resolubilization of proteins from aggregates by Hsp104. Hsp70 assists protein folding through regulated binding and release of partially folded proteins, and its activity is regulated by Hsp40. We earlier isolated a mutant allele of SSA1 (SSA1-21), an Hsp70 protein chaperone, that considerably impairs propagation of the yeast [PSI] prion. Complementation analysis has revealed that disregulation of the Hsp70 substrate binding cyle by the SSA1-21 mutation might exert its effects by altering the functioning of the Hsp104/70/40 chaperone machinery. Characterization of several second-site mutations in SSA1-21 that restored [PSI] propagation also suggested that some of them potentially altered Hsp70 interaction with other co-chaperones that regulate its activity. Using a candidate gene approach to identify such potential co-chaperones, we discovered that a subset of Hsp70 co-chaperones was involved in [PSI] propagation through their ability to regulate Hsp70 function. Our characterization of the effects of alterations in these co-chaperones provided the first functional evidence for their specific activities in vivo. Among these co-chaperones are some that were previously thought to be dedicated to the Hsp90 chaperone machinery. Hsp90 is an essential protein chaperone with many cellular roles and it functions with Hsp70 in a protein folding pathway for several "client" proteins, among which include signaling and transcription factors. We showed, however, that Hsp90 is not involved in [PSI] propagation. Our work was the first to demonstrate that these co-chaperones can regulate Hsp70 independently of Hsp90, and is prompting reconsideration of how Hsp70 and Hsp90 chaperone machinery functions.