Hsp104 interacts with Hsp90 cochaperones in respiring yeast

Abstract

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi(+)] prions in the budding yeast Saccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.

FIG. 1

Hsp104 forms complexes with Cpr7 and Cns1. (A) Recombinant GST.Cpr7 retains Hsp104 from a total yeast extract. GST or GST.Cpr7 purified from bacteria (4 μg) was bound to beads and incubated with 3 mg of yeast extract. Arrows on the right of the silver-stained SDS-PAGE gel point to identified bands. (B) GST.Cpr7 expressed in yeast pulls down Hsp104. The upper panel represents the Ponceau red-stained nitrocellulose filter of the anti-Hsp104 immunoblot in the lower panel. Lane M, molecular size marker proteins. (C) Flag-tagged Cns1 coprecipitates with Hsp104. Cells were grown either with glucose (−; no expression of Flag.Cns1) or in galactose (+; induced expression). Following immunoprecipitation (IP) with an anti- Flag antibody, Flag.Cns1 and endogenous Hsp104 were revealed by immunoblotting with anti-Flag and anti-Hsp104 antibodies, respectively. Input designates a small aliquot of the extract prior to immunoprecipitation.

FIG. 2

Hsp104 associates with endogenous Sti1 and exogenous GST.Cpr7 at high culture density. (A) Endogenous Sti1 is associated with endogenous Hsp104 at high (H) but not at low (L) cell culture density. Immunoprecipitation (IP) experiment with an antibody against Sti1 (anti-Sti1). IgH, antibody heavy-chain band. (B) GST.Cpr7 expressed in yeast is associated with Hsp104 at high density. GST pull-down experiment as in Fig. 1B. The upper panels represent the Ponceau red-stained nitrocellulose filters of the anti-Hsp104 immunoblots in the lower panels.

FIG. 3

Hsp90 and Hsp104 compete for interaction with Sti1 and Cpr7 in vivo. The association of Hsp104 with endogenous Sti1 (A) and GST.Cpr7 (B) is favored in a strain with an Hsp82 variant lacking the C-terminal pentapeptide that mediates interaction with TPR proteins. Extracts were prepared from cells grown to low density. wt and 704, strains HH1a-p2HG/Hsp82 and HH1a-p2HG/Hsp82(1-704), with wild-type Hsp82 and mutant Hsp82 lacking amino acids 705 to 709, respectively. In panel A, Hsp104 was expressed with a Flag epitope and revealed by immunoblotting with an anti-Flag antibody. Note that Flag.Hsp104 complements a Δhsp104 strain (data not shown). The upper panel in A represents the Ponceau red-stained nitrocellulose filter of the anti-Flag immunoblot in the lower panel.

FIG. 4

Hsp104 and Hsp90 associate with different domains of Sti1. (A) Schematic representation of the domain structure of Sti1 and of the galactose-inducible Flag-tagged derivatives used in the experiment in panel B. TPR clusters and the DP domain are indicated. The latter comprises the C-terminal sequences DPEV and DPVM and contributes with TPR1 to Hsp70 binding (10). The Flag epitope is represented by the black boxes. (B) Hsp104 and Hsp90 binding to Sti1 requires TPR1 and TPR2, respectively. Hsp82 wt and Hsp82(1-704), strains with wild-type Hsp82 and the C-terminal truncation mutant lacking the last five amino acids, respectively. The black arrowheads indicate the bands corresponding to T1, T2, and FL. Note that cells were grown to high density to favor interaction between Sti1 and Hsp104. IgL, antibody light-chain band.

FIG. 5

Interaction of Hsp104 with Sti1 and Cpr7 is direct. (A) Schematic representation of the very C-terminal sequences of Hsp104 and Hsp82 and their corresponding truncation mutants. ∗, C-terminal end. (B) In vitro interaction of purified Hsp104 and Sti1, dependent on C-terminal tail of Hsp104; 0.2 μg of input proteins was loaded for comparison. Note that there is unequal recognition of different His₆-tagged proteins by the anti-His₆ antibody. (C) Direct interaction of Hsp104 with Cpr7 fused to GST and competition by Hsp90 (Hsp82). The immunoblot of the upper panel represents aliquots of the input proteins prior to the GST pull-down. The Ponceau red-stained filter shows that equivalent amounts of GST.Cpr7 and GST were pulled down (data not shown).

FIG. 6

Respiratory growth conditions induce the association of Hsp104 with Sti1. (A) Increasing cell density coinciding with a switch to respiration induces the association. Immunoprecipitation (IP) experiment assessing coprecipitation of endogenous proteins with endogenous Sti1. Note that the total amounts of coprecipitated Sti1, Hsp90 (Hsc82/Hsp82), and Hsp70 (Ssa protein family) do not change significantly. OD₆₀₀, optical density as a measure of cell density. The levels of Hsp104 in the input extracts can be seen in Fig. 2, where low and high culture densities correspond to an OD₆₀₀ of 0.4 to 0.5 and 20, respectively. (B) Nonfermentable carbon sources (ethanol and glycerol) induce the association of Sti1 with Hsp104. A representative immunoblot experiment is shown.