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. 2007 Jan;27(2):768-76.
doi: 10.1128/MCB.01034-06. Epub 2006 Nov 13.

Nucleotide-dependent interaction of Saccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1, Cpr6, and Sba1

Jill L Johnson  1 , Agnieszka Halas, Gary Flom
Affiliations

Nucleotide-dependent interaction of Saccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1, Cpr6, and Sba1

Jill L Johnson et al. Mol Cell Biol. 2007 Jan.

Abstract

The ATP-dependent molecular chaperone Hsp90 and partner cochaperone proteins are required for the folding and activity of diverse cellular client proteins, including steroid hormone receptors and multiple oncogenic kinases. Hsp90 undergoes nucleotide-dependent conformational changes, but little is known about how these changes are coupled to client protein activation. In order to clarify how nucleotides affect Hsp90 interactions with cochaperone proteins, we monitored assembly of wild-type and mutant Hsp90 with Sti1, Sba1, and Cpr6 in Saccharomyces cerevisiae cell extracts. Wild-type Hsp90 bound Sti1 in a nucleotide-independent manner, while Sba1 and Cpr6 specifically and independently interacted with Hsp90 in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. Alterations in Hsp90 residues that contribute to ATP binding or hydrolysis prevented or altered Sba1 and Cpr6 interaction; additional alterations affected the specificity of Cpr6 interaction. Some mutant forms of Hsp90 also displayed reduced Sti1 interaction in the presence of a nucleotide. These studies indicate that cycling of Hsp90 between the nucleotide-free, open conformation and the ATP-bound, closed conformation is influenced by residues both within and outside the N-terminal ATPase domain and that these conformational changes have dramatic effects on interaction with cochaperone proteins.

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Figures

FIG. 1.
FIG. 1.
Interaction of cochaperone proteins with WT His-tagged Hsc82 and His-Hsc82 containing alterations of residues required for ATP binding and hydrolysis. A. Cell extracts were prepared from cells expressing His-Hsc82 as the only Hsp90 protein in the cell and supplemented with no exogenous nucleotide (lanes 1 and 2), 5 mM ADP (lanes 3 and 4), 5 mM ATP (lanes 5 and 6), or AMP-PNP (lanes 7 and 8). His-Hsc82 complexes were isolated after a 5-min incubation on ice (odd-number lanes) or at 30°C (even-number lanes). L, whole-cell extract. B. Cell extracts were prepared from cells expressing His-Hsc82 WT, -E33A, or -D79N along with WT untagged Hsp82. His-Hsc82 complexes were isolated from lysates incubated for 5 min at 30°C in the presence of no exogenous nucleotide (lanes 1, 4, and 7), 5 mM AMP-PNP (lanes 2, 5, and 8), or 5 mM ATP plus an ATP-regenerating system (ATP+RS, lanes 3, 6, and 9). Lanes 1 to 3, WT His-Hsc82; lanes 4 to 6, His-Hsc82-E33A; lanes 7 to 9, His-Hsc82-D79N. Nickel resin-bound protein complexes were separated by SDS-PAGE followed by Coomassie blue staining or immunoblot analysis. The Coomassie blue-stained band corresponding to His-Hsc82 is shown in the upper panel, and the lower panels represent immunoblot analysis using antibodies against the indicated proteins.
FIG. 2.
FIG. 2.
Effect of mutations predicted to affect lid closure and N-terminal dimerization. A. Cell extracts were prepared from yeast expressing His-Hsc82 WT or His-Hsc82-A107N. His-Hsc82 complexes were isolated from lysates incubated for 5 min at 30°C in the presence of no exogenous nucleotide, 5 mM AMP-PNP, or 5 mM ATP plus an ATP-regenerating system as indicated. Nickel-bound protein complexes were separated by SDS-PAGE and analyzed as described in the legend to Fig. 1. B. As above, except that WT His-Hsc82, His-Hsc82-T22I, or His-T101I was isolated from cells coexpressing WT untagged Hsp82, since hsc82-T22I and hsc82-T101I confer a lethal phenotype.
FIG. 3.
FIG. 3.
Effect of alteration of residues in the catalytic loop. Cell lysates from strains expressing indicated His-Hsc82 mutants were isolated, supplemented with a nucleotide, and incubated as described for Fig. 1, except that the effect of 5 mM ADP was also monitored. His-Hsc82 mutants unable to support viability of an hsc82 hsp82 strain (E33A, R376A, and E377K mutants) were coexpressed along with untagged WT Hsp82. In the remaining cases (WT and N373A and Q380A mutants), His-Hsc82 was the only Hsp90 protein expressed in the cell. Nickel-bound protein complexes were separated by SDS-PAGE. Upper panels, His-Hsc82 present in a Coomassie blue-strained gel. Lower panels, immunoblots using antibodies specific for Sti1, Cpr6, or Sba1.
FIG. 4.
FIG. 4.
Effect of additional Hsc82 mutations on cochaperone interaction in the presence of AMP-PNP. WT and mutant His-Hsc82 complexes were isolated and analyzed as described in the legend to Fig. 3 except that lysate was supplemented with 5 mM AMP-PNP. In all cases, WT or mutant His-Hsc82 was the only Hsp90 protein present in the cell.
FIG. 5.
FIG. 5.
Interaction of mutant Hsc82 with Sti1, Sba1, and Cpr6 in the presence of ATP plus an ATP-regenerating system. His-Hsc82 complexes were isolated and analyzed as for Fig. 4, except that samples were supplemented with 5 mM ATP+RS. In all cases, WT or mutant His-Hsc82 was the only Hsp90 protein present in the cell.
FIG. 6.
FIG. 6.
Cpr6 interacts with His-Hsc82 in a strain lacking Sba1. Untagged Hsc82 (lanes 1 to 3) or His-Hsc82 (lanes 4 to 6) was expressed in strain JJ816 (hsc82 hsp82). In lanes 7 to 9, His-Hsc82 was expressed in strain JJ40 (hsc82 hsp82 sba1). Cell lysates were isolated and supplemented with a nucleotide as described in the legend to Fig. 1: lanes 1, 4, and 7, no exogenous nucleotide; lanes 2, 5 and 8, 5 mM AMP-PNP; lanes 3, 6, and 9, 5 mM ATP plus an ATP regenerating system. For the upper panel, nickel resin-bound protein complexes were separated by SDS-PAGE (7.5% acrylamide) followed by staining with Coomassie blue. For the lower panels, protein complexes were subjected to SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. In the panel marked “lysate,” whole-cell extract was separated by SDS-PAGE and immunoblotted with an antibody specific for Sba1 to confirm the lack of expression of Sba1 in the hsc82 hsp82 sba1 strain.
FIG. 7.
FIG. 7.
Model of Hsc82 interaction with Sba1 and Cpr6. Our results suggest the presence of two intermediate complexes during the ATPase cycle: Sba1 interaction prior to Cpr6 interaction (as observed with the E33A and E377K mutants) and Sba1 release prior to Cpr6 release (as observed with the A107N and W296A mutants). See the text for details.
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