Independent regulation of Hsp70 and Hsp90 chaperones by Hsp70/Hsp90-organizing protein Sti1 (Hop1)

Abstract

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many "client" proteins. The co-chaperone Sti1p coordinates functions of Hsp70 and Hsp90 in this pathway. Sti1p has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a binds Hsp90, and the ligand for TPR2b is unknown. Although Sti1p is thought to be dedicated to the client folding pathway, we earlier showed that Sti1p regulated Hsp70, independently of Hsp90, in a way that impairs yeast [PSI+] prion propagation. Using this prion system to monitor Sti1p regulation of Hsp70 and an Hsp90-inhibiting compound to monitor Hsp90 regulation, we identified Sti1p mutations that separately affect Hsp70 and Hsp90. TPR1 mutations impaired Sti1p regulation of Hsp70, but deletion of TPR2a and TPR2b did not. Conversely, TPR2a and TPR2b mutations impaired Sti1p regulation of Hsp90, but deletion of TPR1 did not. All Sti1p mutations variously impaired the client folding pathway, which requires both Hsp70 and Hsp90. Thus, Sti1p regulated Hsp70 and Hsp90 separately, Hsp90 is implicated as a TPR2b ligand, and mutations separately affecting regulation of either chaperone impair a pathway that is dependent upon both. We further demonstrate that client folding depended upon bridging of Hsp70 and Hsp90 by Sti1p and find conservation of the independent regulation of Hsp70 and Hsp90 by human Hop1.

FIGURE 1. Sti1p domain structure and location of mutations that impair regulation of Hsp70 and Hsp90

TPR repeat and aspartate-proline (DP) repeat regions are indicated. Amino acid residue numbers at the borders of the domains are shown along the top of the diagram (scale is approximate). Amino acid substitutions impairing the ability of Sti1p to regulate Hsp70 are indicated above the diagram of the intact gene, and those that impair Hsp90 regulation are shown below. Alleles lacking specific domains are shown with the deleted domain absent or spanned by a dotted line.

FIGURE 2. [PSI+] phenotypes of SSA1–21 sti1Δ cells expressing various Sti1 and Hop1 proteins

Cells with plasmids carrying the STI1 or HOP1 alleles were streaked for colonies onto plates maintaining selection for the plasmid and containing limiting adenine. To the left of the line, the top panels show cells expressing Sti1p mutants selected as affecting Hsp70 regulation, the middle panels are those selected as affecting Hsp90 regulation, and the bottom panels are those with engineered deletions (see Fig. 1). To the right of the line, the top and middle panels show cells expressing Hop1 variants in place of Sti1p, and the bottom panels show Sti1p wild type (STI1) and null (sti1Δ) controls. Weakening of [PSI⁺] by Ssa1-21p requires Sti1p, so sti1Δ cells are white and uniformly [PSI⁺], whereas STI1 cells are pink and have spontaneously arising [psi⁻] colonies (red) in the streak. The degree to which Sti1p mutations affect the ability of Sti1p to weaken [PSI⁺] propagation can be estimated by the extent to which the phenotypes resemble these controls. For example, Hop1 regulates Ssa1-21p essentially as well as Sti1p with regard to [PSI⁺] propagation, whereas Sti1ΔTPR1 (lower left) is essentially unable to regulate Ssa1-21p in a way that weakens [PSI⁺], and ΔDP2 has partial activity in this regard.

FIGURE 3. Relative ability of Sti1p and Hop1 variants to weaken [PSI+] as measured by nonsense suppression

A read-through of nonsense codons caused by [PSI⁺] was quantified using a bicistronic mRNA encoding different forms of luciferase separated by a linker, which contains or lacks a stop codon. In SSA1-21 sti1Δ [PSI⁺] cells expressing the transcript with the intervening stop codon, the ratio of expression of the luciferase downstream of the linker to that of the luciferase upstream of the linker provides a measurement of stop codon read-through, which is related to [PSI⁺] “strength.” This ratio is then normalized to the same ratio from the same cells expressing the mRNA without the stop codon. The height of the bars reflects the extent to which the Sti1 proteins retain the ability to regulate Ssa1-21p in a way that weakens [PSI⁺], relative to wild type Sti1p, which is set at 100. Data for the randomly isolated mutations are plotted as black bars; those for the Sti1p deletions and Hop1 derivatives are plotted as gray bars. Below the graph is a linear diagram of the Sti1p domain structure (not to scale) aligned to show the region of the protein where the indicated mutations are located.

FIGURE 4. Sensitivity of SSA1–21 sti1Δ [psi⁻] cells expressing mutant Sti1 proteins to the Hsp90 inhibitor radicicol

Upper panel, 5-fold serial dilutions of cells expressing Sti1 or Hop1 proteins (indicated above the panel) were grown on rich medium containing 25 μg/ml radicicol for 7 days at 33 °C. Lower panel, identical aliquots of the three highest dilutions of the same cells were grown on similar plates without radicicol for 2 days at 30 °C followed by 2 days at 25 °C. Between the panels is the Sti1p diagram described in the legend for Fig. 3.

FIGURE 5. Stimulation of GR activity by Sti1 and Hop1 proteins

A, deoxycorticosterone (DOC) was added to one-half of the split cultures of SSA1-21 sti1Δ [psi⁻] cells expressing GR and carrying a GR-regulated β-galactosidase gene. The β-galactosidase activities of the cultures were measured and normalized to that from cells expressing wild type Sti1p, which was set at 100. B, the β-galactosidase activities of deoxycorticosterone-treated cultures expressing the various Sti1p and Hop1 proteins, normalized as in A, are shown. Below the plot is the Sti1p diagram described in the legend for Fig. 3.

FIGURE 6. Sti1Δ TPR1 and Sti1Δ TPR2 independently regulate Hsp70 and Hsp90 in the same cell, but GR activation requires intact Sti1p

Upper panels, SSA1-21 sti1Δ [PSI⁺] cells lacking Sti1p (sti1Δ) or expressing wild type (STI1) or truncated forms of Sti1p as indicated were streaked onto medium with limiting adenine and grown as described in the legend for Fig. 2. Lower panels, a dilution series of the cells shown in A were grown as described for Fig. 4 with (+) or without (−) radicicol. Relative levels of GR activation for the same transformants, measured as described in the legend for Fig. 5 and normalized to wild type set at 100, are indicated at the bottom.

FIGURE 7. Relative expression of Sti proteins and protein chaperones

A, whole cell lysates of cells expressing the various Sti1 proteins, indicated at the top, were subjected to Western analysis using antibody to a Sti1p epitope spanning amino acid residues 530–544. B, abundance of mRNA from cells expressing indicated Sti1p variants that lack the epitope for the Sti1p antibody was quantified by RT-PCR and compared with mRNA from cells expressing wild type Sti1p (WT) and lacking Sti1p (sti1Δ). Abundance of actin mRNA was measured as a control in all reactions. Lane M shows molecular mass markers of 750 and 500 bp. The expected sizes of PCR products are 567 bp for actin and 496 bp for STI1. C, lysates of cells used for A and B were subjected to Western analysis probing for Hsp70, Hsp90, and Hsp104 as indicated. For A and C, portions of membranes stained by Amido Black are shown as loading and transfer controls (Input).