The co-chaperone Hch1 regulates Hsp90 function differently than its homologue Aha1 and confers sensitivity to yeast to the Hsp90 inhibitor NVP-AUY922

Abstract

Hsp90 is a dimeric ATPase responsible for the activation or maturation of a specific set of substrate proteins termed 'clients'. This molecular chaperone acts in the context of a structurally dynamic and highly regulated cycle involving ATP, co-chaperone proteins and clients. Co-chaperone proteins regulate conformational transitions that may be impaired in mutant forms of Hsp90. We report here that the in vivo impairment of commonly studied Hsp90 variants harbouring the G313S or A587T mutation are exacerbated by the co-chaperone Hch1p. Deletion of HCH1, but not AHA1, mitigates the temperature sensitive phenotype and high sensitivity to Hsp90 inhibitor drugs observed in Saccharomyces cerevisiae that express either of these two Hsp90 variants. Moreover, the deletion of HCH1 results in high resistance to Hsp90 inhibitors in yeast that express wildtype Hsp90. Conversely, the overexpression of Hch1p greatly increases sensitivity to Hsp90 inhibition in yeast expressing wildtype Hsp90. We conclude that despite the similarity between these two co-chaperones, Hch1p and Aha1p regulate Hsp90 function in distinct ways and likely independent of their roles as ATPase stimulators. We further conclude that Hch1p plays a critical role in regulating Hsp90 inhibitor drug sensitivity in yeast.

Figure 1. Structure and sequence of Aha1p and Hch1p.

A. Domain structure of the 350 amino acid Aha1p and the 153 amino acid Hch1p. B. Alignment of Aha1p and Hch1p.

Figure 2. Deletion of HCH1, but not AHA1, alleviates temperature sensitivity in S. cerevisiae expressing Hsp82p^G313S or Hsp82p^A587T as the sole source of Hsp90.

Cells were grown overnight in YPD at 30°C and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD-agar plates. A. Viability of mutant strains (expressing untagged Hsp82p) grown at 30, 35.5 and 37°C on YPD agar plates; B. Viability of mutant strains (expressing His-tagged Hsp82p) grown at 30, 35.5 and 37°C on YPD agar plates. C. Western analysis of HisHsp82p expression versus actin in mutant strains.

Figure 3. Overexpression of C-terminally myc-tagged Hch1p impairs growth of yeast expressing Hsp82p^A587T but not yeast expressing wildtype Hsp82p.

A. Cells were grown overnight in YPD supplemented with 200 mg/L G418 and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD agar plates supplemented with 200 mg/L G418 and grown for 2 days at 30, 35.5 and 37°C. B. Western analysis of yeast shown in 3A with anti-myc tag, anti-His tag and anti-actin antibodies. Levels of mycHch1p (lanes 2, 4, 6, and 8) in strains expressing Hsp82p (lanes 1–4), or Hsp82p^A587T (lanes 5–8) with (lanes 3,4, 7, 8) or without (lanes 1, 2, 5, 6) HCH1 deletion. (Anti-myc antibody 9E10).

Figure 4. Hch1p regulates Hsp90 inhibitor drug sensitivity in yeast.

A. Deletion of HCH1 confers resistance to Hsp90 inhibitor NVP-AUY922 in yeast expressing Hsp82p, Hsp82p^G313S or Hsp82p^A587T. Cells were grown overnight in YPD and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD agar plates supplemented with indicated concentrations of NVP-AUY922. B. Overexpression of myc-tagged Hch1p results in hypersensitivity to Hsp90 inhibitor NVP-AUY922 in yeast. Cells were grown overnight in YPD supplemented with 200 mg/L G418 and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD agar plates supplemented with 200 mg/L G418 and indicated concentrations of NVP-AUY922.

Figure 5. ATPase stimulation of Hsp82p, Hsp82p^G313S and Hsp82p^A587T.

A. Stimulation of the ATPase activity of wildtype Hsp82p (circles), Hsp82p^G313S (squares), and Hsp82p^A587T (triangles) by increasing concentrations of Aha1p. B. Stimulation of the ATPase activity of wildtype Hsp82p (black bars), and Hsp82p^A587T (grey bars) by Hch1p. ATPase rate shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min). Reactions contained 2 µM Hsp82p and indicated concentration of Aha1p or Hch1p.

Figure 6. Inhibition of Aha1p-stimulated ATPase activity of Hsp82p, Hsp82p^G313S and Hsp82p^A587T.

A. Inhibition of Aha1p-stimulated ATPase activity of wildtype Hsp82p (circles), Hsp82p^G313S (squares), and Hsp82p^A587T (triangles) by increasing concentrations of Sba1p. ATPase rate for wildtype Hsp82p and Hsp82p^A587T shown on left axis (Reactions contained 2 µM Hsp82p, 10 µM Aha1p and indicated concentrations of Sba1p). ATPase rate for Hsp82p^G313S shown on right axis (reactions contained 5 µM Hsp82p, 10 µM Aha1p and indicated concentrations of Sba1p). B. Inhibition of Hch1p-stimulated ATPase activity of wildtype Hsp82p (circles), and Hsp82p^A587T (triangles) by increasing concentrations of Sba1p. Reactions contained 2 µM Hsp82p, 10 µM Hch1p and indicated concentrations of Sba1p. C. Inhibition of Aha1p-stimulated ATPase activity of wildtype Hsp82p (black bars) and Hsp82p^A587T (white bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. D. Inhibition of Aha1p-stimulated ATPase activity of Hsp82p^G313S (grey bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. E. Inhibition of Hch1p-stimulated ATPase activity of wildtype Hsp82p (black bars) and Hsp82p^A587T (white bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. ATPase rates shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min).

Figure 7. Co-chaperone competition in Hsp82p ATPase reactions.

A. Stimulation of the ATPase activity of wildtype Hsp82p (2 µM) by increasing concentrations of Hch1p in the presence (squares) or absence (circles) of Sba1p (8 µM). ATPase rate shown as a fold increase over intrinsic rate of Hsp82p alone. B. Inhibition of Aha1p-stimulated ATPase rate by increasing concentrations of Sba1p (triangles) and Sti1p (squares). Dashed line represents intrinsic Hsp82p ATPase rate. Reactions contain 2 µM Hsp82p, 4 µM Aha1p and indicated concentrations of Sba1p or Sti1p. ATPase rate shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min). C. Effect of increasing concentrations of Hch1p on the Aha1p-stimulated ATPase activity of wildtype Hsp82p (2 µM) in the presence (squares) or absence (circles) of Sba1p (8 µM). ATPase rate shown as a fold increase over intrinsic rate of Hsp82p alone. Reactions contained 2 µM Hsp82p and indicated concentration of Aha1p or Hch1p.

Figure 8. Analysis of complexes formed with Hsp82p, Hsp82p^G313S and Hsp82p^A587T.

A. Ni-NTA pulldown of His-tagged Hsp82p from lysates from yeast expressing either untagged wildtype Hsp82p (Lane 1), HisHsp82p (Lanes 2–4), Hsp82p^A587T (Lanes 5–7) or Hsp82p^G313S (Lanes 8–10). Isolated complexes were analyzed by western blotting with antibodies to the indicated proteins. Genetic background of each strain is indicated beneath each lane. B. 5 µM Hsp82p or Hsp82p^A587T was incubated with 5 µM of the indicated His-myc tagged co-chaperone and either 1 mM ADP (odd lanes) or 1 mM AMPPnP (even lanes). Complexes were isolated with beads coupled to anti-myc monoclonal antibody 9E10, run on SDS-PAGE and analyzed by coomassie blue staining (CB). C. 5 µM Hsp82p^G313S was incubated with 5 µM of the indicated His-myc tagged co-chaperone and either 1 mM ADP (odd lanes) or 1 mM AMPPnP (even lanes). Complexes were isolated with beads coupled to anti-myc monoclonal antibody 9E10, run on SDS-PAGE and analyzed by western blotting with anti-His tag antibodies and anti-myc tag antibodies. *indicates degradation products and light chain from the beads.