Effect of mutation of the tetratricopeptide repeat and asparatate-proline 2 domains of stil on Hsp90 signaling and interaction in Saccharomyces cerevisiae

Through simultaneous interactions with Hsp70 and Hsp90 via separate tetratricopeptide repeat (TPR) domains, the cochaperone protein Hop/Sti1 has been proposed to play a critical role in the transfer of client proteins from Hsp70 to Hsp90. However, no prior mutational analysis demonstrating a critical in vivo role for the TPR domains of Stil has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Stil function. A single amino acid alteration in TPR2A disrupted Hsp90 interaction in vivo but did not significantly affect function. However, deletion of a conserved residue in TPR2A or mutations in the carboxy-terminal DP2 domain completely disrupted Stil function. Surprisingly, mutations in TPR1, previously shown to interact with Hsp70, were not sufficient to disrupt in vivo functions unless combined with mutations in TPR2B, suggesting that TPR1 and TPR2B have redundant or overlapping in vivo functions. We further examined the genetic and physical interaction of Stil with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Stil in regulating Hsp90 function.