Earlier we showed that altering the expression of wild type Hsp40s (J-proteins), nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain proteins can either enhance or impair propagation of different prions in both wild type and Hsp70 mutant cells. We also identified several mutant alleles of Hsp40s and TPR proteins that impair or enhance propagation of different prions. Our further work pointed to the fact that many, if not all, of the observed effects that these co-chaperones have on prions are mediated by the various ways they can regulate activities of Hsp70. Hsp70 has a major role in cooperating with Hsp90 in a protein folding pathway that is important for maturation of many "client" proteins in the cell, among which are many signaling proteins and transcription factors.The Hsp70 TPR co-chaperone Sti1 interacts with both Hsp70 and Hsp90 to facilitate the transfer of substrates between them. We showed earlier that the two TPR regions of Sti1 can act separately to regulate the functions of Hsp70 and Hsp90 independently. However, we provided the first direct evidence that the client folding pathway requires a pysical bridging of Hsp70 and Hsp90 by an intact Sti1 protein. We also found that Sti1 has a critical role in the curing of PSI+ prions when Hsp104 is overexpressed and that Sti1 links Hsp90 to this curing process. Work by another group on the E. coli Hsp90 homolog HtpG identified single amino acid alterations in HtpG that impair its function. Further study suggested the residues identified participate in the binding of HtpG to client substrates. To gain more insight into physiological role and degree of functional conservation of these residues we assessed the effects of making homologous alterations in the yeast Hsp90 (Hsp82). We found the mutations had effects in various cellular functions including folding of different clients, such as transcription factors and kinases, and ability to support growth at optimal and non-optimal temperature. Effects of the mutations ranged from only slight to abolishing Hsp90's ability to support growth. Although the results with mutations in Hsp82 confirm the importance of the identified residues for Hsp90 function, they did not provide direct evidence that the same residues are as critical for substrate interactions with Hsp82 as they are with HtpG. Since Hsp82 function is known to be regulated by more co-chaperones than HtpG, this difference could be related to a more complex situation for binding of substrates by Hsp82. Continuing our work on these mutations we found that none of them affected propagation of PSI+ prions, but some of them did interfere with the ability of overexpressed Hsp104 to cure cells of PSI+ prions. These results are consistent with our assertion over the years that the way Hsp104 functions in prion replication differs from the way it acts in curing cells of prions. They also are in line with our earlier work that uncovered a role for Hsp90 in the mechanism of this Hsp104 curing, and imply that continued work with these mutants will provide insight into that mechanism.
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