An Hsp90 co-chaperone protein in yeast is functionally replaced by site-specific posttranslational modification in humans

Abstract

Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone. To properly chaperone its clientele, Hsp90 proceeds through an ATP-dependent conformational cycle influenced by posttranslational modifications (PTMs) and assisted by a number of co-chaperone proteins. Although Hsp90 conformational changes in solution have been well-studied, regulation of these complex dynamics in cells remains unclear. Phosphorylation of human Hsp90α at the highly conserved tyrosine 627 has previously been reported to reduce client interaction and Aha1 binding. Here we report that these effects are due to a long-range conformational impact inhibiting Hsp90α N-domain dimerization and involving a region of the middle domain/carboxy-terminal domain interface previously suggested to be a substrate binding site. Although Y627 is not phosphorylated in yeast, we demonstrate that the non-conserved yeast co-chaperone, Hch1, similarly affects yeast Hsp90 (Hsp82) conformation and function, raising the possibility that appearance of this PTM in higher eukaryotes represents an evolutionary substitution for HCH1.

Figure 1. Loss of HCH1 relieves the Hsp82-W585T growth phenotype.

See also Supplementary Fig. 1. (a) In the image on the left, one protomer of yeast Hsp82 is in green, the other in grey (PDB 2CG9). Residues 582QFGWSANME590 are in blue, with W585 in red and A587 in orange. The missing loop (which is not resolved in this crystal structure) containing Y606—598LRDSSMSSYMSSK610—is shown as a dotted line in magenta. The image on the right is a magnification of the middle domain/C-terminal domain interface of the Hsp82 protomer shown in the box in the left panel. (b) hsc82hsp82 and hch1hsc82hsp82 cells expressing a WT copy of HSC82 were transformed with either WT or W585T HSP82 constructs. Transformants were struck on to 5-FOA for two days, grown overnight in selective media, diluted 10-fold and grown on selective media plates at the indicated temperatures for two days. (c) hsc82hsp82 cells expressing HSP82 WT or W585T were transformed with vector or pRS416GPD-HCH1. Resulting colonies were grown as described in b.

Figure 2. Hch1 reduces client accumulation and activity.

(a) HCH1 (MR318) or hch1 (CLY7) cells expressing Hsp82 WT or W585T (MR923-MR926) were transformed with a plasmid encoding the mammalian steroid hormone receptor GR and the β-galactosidase reporter pUCΔSS-26X. Resultant colonies were grown to mid-log phase followed by the addition of synthetic hormone. Following a five-hour incubation, β-galactosidase activity was quantified (each sample was run in triplicate and error bars represent s.d. from the mean). These same strains were lysed and analysed using SDS–PAGE and the indicated antibodies. (b) The same strains used in a were transformed with the GAL-inducible Ste11ΔN construct as well as the β-galactosidase reporter PRE-lacZ (ref. 55). Transformants were grown to mid-log phase in selective media supplemented with raffinose followed by a 6-h galactose induction. Cells were then measured for β-galactosidase activity as in a. These strains were also lysed following galactose induction and run on a 4–20% SDS gel and probed with the indicated antibodies. (c) HCH1 or hch1 cells expressing either WT or W585T Hsp82 as indicated were transformed with pRS316GAL-v-SRC. Transformants were grown overnight in minimal media and then diluted 10-fold and grown for four days at 30° on minimal media supplemented with glucose (URA D.O.) or galactose (URA-GAL). (d) Cells from c were grown overnight in URA-RAF and switched to URA-GAL media overnight. Cells were lysed and analyzed as in b.

Figure 3. Hsp90-Y627 phosphorylation disrupts client interactions.

See also Supplementary Fig. 2. (a) 293 A cells were transfected with the pcDNA3-Flag vector or Flag-Hsp90α WT, Y627F or Y627E as indicated. Cells were collected, lysed and Flag complexes were isolated using anti-Flag resin and analysed using SDS–PAGE. (b) Transfections were performed and analysed as in a. Except a pLNCX-chick-v-SRC construct was co-transfected with each sample.

Figure 4. The Hsp82-Y606E mutation complements Hch1 in vivo activity.

See also Supplementary Fig. 3. (a) hsc82hsp82 cells were transformed with Hsp82 WT, Y606F or Y606E constructs. The transformed cells were then grown on 5-FOA to lose the WT copy of Hsp90 in those strains and transformed with either vector or pRS416GPD-HCH1. The second set of transformants were then grown overnight in selective media, diluted 10-fold and grown for 2 days at the indicated temperatures. (b) hsc82hsp82 cells were transformed with the indicated HSP82 mutants and grown on 5-FOA for 2 days. Colonies grown on 5-FOA were then grown overnight in minimal media, diluted 10-fold and grown on YPD or 5-FOA plates for two days at 30°. (c) hsc82hsp82 cells transformed with the indicated mutants as well as vector or WT pRS416GPD-HCH1 were grown as in a on selective media.

Figure 5. Hsp82-Y606E disrupts client protein expression and activity.

See also Supplementary Fig. 4. (a,b) hsc82hsp82 and hch1hsc82hsp82 cells expressing Hsp82 WT, Y606F or Y606E (MR923, MR925, CLY25, CLY29, CLY24 and CLY28 respectively) were transformed with plasmids containing either pG/N795-GR and pUCΔSS-26X (a) or pRS414GAL-Ste11ΔN and PRE-lacZ (b). Assays were performed in triplicate as indicated in the Fig. 2a,b. (c) HCH1 or hch1 cells expressing Hsp82 WT, Y606F or Y606E as indicated were transformed with pRS316GAL-v-SRC. Transformants were grown overnight in minimal media, diluted 10-fold and grown for 2 days at 30° on minimal media supplemented with glucose (URA D.O.) or galactose (URA-GAL). (d) Transformants from c. were grown overnight in Ura-RAF and switched to URA-GAL media overnight. Cells were lysed and analyzed as indicated.

Figure 6. A107N mutant rescues Hsp82-W585T and HCH1 phenotypes.

See also Supplementary Fig. 5. (a) hsc82hsp82 and hch1hsc82hsp82 cells expressing Hsp82 WT (MR923 and MR925), W585T (MR851 and MR858), A107N-W585T (MR852 and MR859) or A107N (AF3 and AF6) were transformed with vector or pRS414GPD-HCH1 (arrow pointing up) as well as a plasmids containing pG/N795-GR and pUCΔSS-26X. These cells were grown to mid-log phase followed by the addition of synthetic hormone. Following a 5-h incubation, β-galactosidase activity was quantified (each sample was run in triplicate and error bars represent s.d. from the mean). These strains were then lysed and analysed using SDS–PAGE and the indicated antibodies. (b) hsc82hsp82 (MR318) cells were transformed with either HSP82 WT, W585T or A107N-W585T constructs. Transformants were struck on to 5-FOA for 2 days, grown overnight in rich media, diluted 10-fold and grown on rich media plates containing DMSO or 25 μg ml⁻¹ of the Hsp90 inhibitor Radicicol and incubated at 30° for 2 days. (c,d) hsc82hsp82 and hch1hsc82hsp82 cells were transformed with the indicated His-tagged Hsp82 constructs as well as vector or pRS414GPD-HCH1 (arrow pointing up). Cells were grown overnight in minimal media and collected. These cells were then lysed and His-complexes were isolated using nickel resin beads in the presence (+) or absence (−) of AMP-PNP. Following SDS–PAGE, His-complexes were analyzed by Western blot with antibodies shown.

Figure 7. A107N restores function and conformation of Hsp82-Y606E.

See also Supplementary Fig. 6. (a) hsc82hsp82 cells expressing Hsp82 WT (MR923), Y606E (CLY24), A107N (AF3) and A107N-Y606E (MR1016) were transformed pG/N795-GR and pUCΔSS-26X. Resultant colonies were grown to mid-log phase followed by the addition of synthetic hormone. Following a 5-h incubation, β-galactosidase activity was quantified (each sample was run in triplicate and error bars represent s.d. from the mean). These same strains were lysed and analysed using SDS–PAGE and the indicated antibodies. (b,c) hsc82hsp82 cells were transformed with the indicated His-tagged Hsp82 constructs. Resultant colonies were grown overnight in rich media and cell pellets were collected. Collected cell pellets were lysed and His-complexes were isolated using nickel resin in the presence (+) or absence (−) of AMP-PNP. Following SDS–PAGE, His-complexes were analysed by Western blot as in Fig. 6.