Effect of Mutation of the Tetratricopeptide Repeat and Asparatate-Proline 2 Domains of Sti1 on Hsp90 Signaling and Interaction in Saccharomyces cerevisiae

Abstract

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 Sti1 has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Sti1 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 Sti1 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 Sti1 with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Sti1 in regulating Hsp90 function.

Proteomic analysis of stress-related proteins in rice seeds during the desiccation phase of grain filling

“…A protein identified as a putative stress-induced protein, sti1 (spot 5), contains two heat shock chaperonin-binding motifs (STI1) and three tetratricopeptide repeat. The function of tetratricopeptide repeat-containing proteins is mediated through protein-protein interaction to modulate diverse cellular processes, including HSP 90 signaling and interaction (Flom et al 2006). A 20 kDa putative chaperonin (spot 11) and chaperonin 21 precursor (spot 12) are presumably chloroplast co-chaperonins, composed of two chaperonin 10-like domains, that are involved in heat shock stress (Viitanen et al 1995).…”

Chaperoning the Chaperone: A Role for the Co-chaperone Cpr7 in Modulating Hsp90 Function in Saccharomyces cerevisiae

“…a-Flag monoclonal antibody, G418, and 59-adenylylimidodiphosphate (AMP-PNP) were obtained from Sigma. Polyclonal antibodies against Hsc82/Hsp82 have been described (Flom et al 2006). Polyclonal antisera was raised against keyhole limpet hemocyanin-conjugated peptides corresponding to amino acids 81-96 of Cpr6, 211-225 of Cpr7, 118-131 of Sba1, and 492-509 of Ppt1.…”

“…Strains JJ816 (hsc82:: LEU2 hsp82::LEU2/YEp24-HSP82) and JJ832 (sti1::MET2 hsc82::LEU2 hsp82::LEU2/YEp24-HSP82) have been described (Flom et al 2006). Additional yeast strains were constructed by transforming the appropriate gene disruption cassette from the yeast genome collection (Open Biosystems) into a hsc82::LEU2 hsp82::LEU2/YEp24-HSP82 diploid (JJ928).…”

Proteomic responses of rice young panicles to salinity

“…It is believed that the function of TPR-containing proteins is mediated through protein-protein interaction to modulate diverse cellular processes, including Hsp90 signaling and interaction [29], protein transport across mitochondria [30], regulation of meristem cellular organization [31], and gibberellin signaling [32]. The up-regulation of this regulatory protein may decrease the sterility of pollen during anther development [33].…”