Functional and physical interaction between yeast Hsp90 and Hsp70

Abstract

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.

The Hsp70/Hsp90 Co-Chaperone Hop/Stip1 Shifts the Proteostatic Balance from Folding Towards Degradation

“…Our quantitative Hsp90-IP-MS experiments show that the steady-state association of Hsp70 and Hsp90 is less prominent. This is compatible with previously reported Kd values for the interactions that are relevant in this context; compared to the Kd for the direct interaction of yeast Hsp70 and Hsp90 of 14 µM (Kravats et al, 2018), the values for the yeast or human pairs Hop-Hsp70…”

“…These results led us to speculate that human Hsp70 and Hsp90 could directly interact as they do in bacteria (Genest et al, 2015;Kravats et al, 2017) and in ∆sti1 yeast (Kravats et al, 2018). We tested this notion using an in vitro association assay with recombinant proteins.…”

Comprehensive fitness maps of Hsp90 show widespread environmental dependence

“…Hsp90 positions with environmentally responsive selection coefficients were enriched in binding contacts with clients, co-chaperones and intramolecular Hsp90 contacts involved in transient conformational changes ( Figure 3D and S3B). About 65% of the environmentally responsive residues have been identified either structurally or genetically as interacting with binding partners [42][43][44][45][46][47][48][49][50][51][52][53][54][55], compared to about 15% of positions that were not responsive to stress conditions. While ATP binding and hydrolysis are the main structural determinants that constrain fitness in standard growth conditions, client and co-chaperone interactions have a larger impact on experimental fitness under stress conditions.…”

Growth-regulated Hsp70 phosphorylation regulates stress responses and prion maintenance

“…HSP82 (yeast HSP90), known to bind directly to Ssa1, is also present at higher levels in S151D-expressing cells ( Fig. S1) (Kravats et al, 2018). Combined with the observation that HSF1 is hyperactive in cells expressing Ssa1 S151D, these observations suggest that Ssa1 phosphorylation may have broad effects on many clients through altered co-chaperone binding properties.…”