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Comparative Study
. 2006 Jan 25;25(2):367-76.
doi: 10.1038/sj.emboj.7600930. Epub 2006 Jan 12.

The phosphatase Ppt1 is a dedicated regulator of the molecular chaperone Hsp90

Sebastian K Wandinger  1 , Michael H Suhre, Harald Wegele, Johannes Buchner
Affiliations
Comparative Study

The phosphatase Ppt1 is a dedicated regulator of the molecular chaperone Hsp90

Sebastian K Wandinger et al. EMBO J. .

Abstract

Ppt1 is the yeast member of a novel family of protein phosphatases, which is characterized by the presence of a tetratricopeptide repeat (TPR) domain. Ppt1 is known to bind to Hsp90, a molecular chaperone that performs essential functions in the folding and activation of a large number of client proteins. The function of Ppt1 in the Hsp90 chaperone cycle remained unknown. Here, we analyzed the function of Ppt1 in vivo and in vitro. We show that purified Ppt1 specifically dephosphorylates Hsp90. This activity requires Hsp90 to be directly attached to Ppt1 via its TPR domain. Deletion of the ppt1 gene leads to hyperphosphorylation of Hsp90 in vivo and an apparent decrease in the efficiency of the Hsp90 chaperone system. Interestingly, several Hsp90 client proteins were affected in a distinct manner. Our findings indicate that the Hsp90 multichaperone cycle is more complex than was previously thought. Besides its regulation via the Hsp90 ATPase activity and the sequential binding and release of cochaperones, with Ppt1, a specific phosphatase exists, which positively modulates the maturation of Hsp90 client proteins.

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Figures

Figure 1
Figure 1
Interaction of Ppt1 and yHsp90. (A) Calorimetric analysis of the Ppt1–yHsp90 interaction. ITC was performed at 25°C. Data were analyzed using the Origin software package provided by the manufacturer. The upper panel shows the change in heat upon injection of Ppt1 (273 μM in the syringe) to yHsp90 (16 μM) in the cell. The lower panel shows the derived binding curve. The filled squares correspond to the integration of the peaks of the upper panel. (B) Schematic representation of the Ppt1 and yHsp90 fragments used. Ppt1 consists of two domains, the TPR (amino acids 12–113), and the catalytic domain (amino acids 188–513). The C-terminal subdomain is denoted as C. The TPR fragment ranges from amino acids 1 to 170, the phosphatase fragment from amino acids 171–513. (C) ELISA-based analysis of the interaction of the full-length Ppt1 and its fragments with the full-length yHsp90 (top panel) and the C-terminal yHsp90 fragment 530C (bottom panel). Proteins denoted in the top line were coated on the ELISA plate as described in Materials and methods. FITC-labeled yHsp90/530C was employed to detect interactions by recording the FITC fluorescence. BSA and Sti1 served as negative and positive control, respectively.
Figure 2
Figure 2
Analysis of the enzymatic properties of Ppt1 for the substrate pNPP. (A) Titration of Ppt1 with increasing concentrations of pNPP. The substrate titration was performed at the optimal pH value of 7.8 as described in Materials and methods. pNPP was added up to 150 mM. The symbols in (A) correspond to increasing concentrations of pNPP: open hexagon: 0.75 mM pNPP; filled hexagon: 1.5 mM pNPP; open triangle up: 3.75 mM pNPP; filled triangle up: 7.5 mM pNPP; open diamond: 15 mM pNPP; filled diamond: 22.5 mM pNPP; open square: 37.5 mM pNPP; filled square 67.5 mM pNPP; open triangle down: 75 mM pNPP; filled triangle down: 97.5 mM pNPP; open circle: 135 mM pNPP; and filled circle: 148 mM pNPP. This kind of substrate titration was used to obtain the reaction kinetics of Ppt1 as shown in (B). (B) Reaction kinetics of Ppt1 (1 μM) with pNPP. Shown is the average of a triple experiment (▴) together with the standard error (bars). The black curve corresponds to the regression of the Michaelis–Menten equation. The formation of pNP was recorded photometrically at 410 nm to obtain the reaction velocity as described in Materials and methods. (C) Comparison of the reaction kinetics of Ppt1 (▴) and the phosphatase fragment (•). Both titrations were performed as in (A). The kcat of the phosphatase fragment (∼47 min−1) is approximately seven-fold higher than that of the full-length Ppt1 (∼7 min−1).
Figure 3
Figure 3
Analysis of the activity of Ppt1 towards phosphorylated proteins of the yHsp90 system. (A) Activity of Ppt1 (1 μM) towards yHsp90 (1 μM). Hsp90 was prepared using CKII and [γ-32P]ATP as described in Materials and methods. After labeling, apyrase was added to hydrolyze the remaining ATP before Ppt1 was added. Ppt1 addition was omitted in the experiment shown in the top panel of (A) to demonstrate the stability of the phosphorylation. (B) Activity of Ppt1 towards partner proteins of yHsp90. Hsp90 partner proteins were prepared as in (A). (C) Activity of Ppt1 towards Hsp104TRAP. The assay was performed as in (A). (D) Activity of Ppt1 towards partner proteins of yHsp90 in the presence of yHsp90. The assay was performed as in (B), except that prior to addition of Ppt1, yHsp90 was added. (E) Activity of Ppt1 towards casein and MBP. Proteins were labeled as described in (A). Ppt1 addition was omitted in the experiments shown in the top panels (denoted as −Ppt1). As a comparison, the yHsp90 dephosphorylation is shown in the lowest panel.
Figure 4
Figure 4
Investigation of the dependence of the TPR interaction in the yHsp90 dephosphorylation. (A) Activity of Ppt1 (1 μM) towards phosphorylated ΔMEEVD-yHsp90 (1 μM), a deletion mutant of the chaperone, lacking the C-terminal MEEVD residues necessary for binding of TPR domains. Phosphorylated proteins are always listed at the first position in the legend. (B) Competition of Ppt1 and Sti1 in binding to the common TPR acceptor site of yHsp90. The assay was performed as in (A). Prior to the addition of Ppt1 to phosphorylated yHsp90, Sti1 (10-fold excess) was added as indicated. (C) Inhibition of the phosphatase activity of Ppt1 in the presence of the TPR fragment of Ppt1 in different stoichiometries. In the experiment of the second panel, the TPR fragment was used in five-fold excess over Ppt1, while it was employed equimolar in the third panel. (D) Inhibition of the yHsp90 dephosphorylation by Ppt1 in the presence of an EEVD-containing peptide. Labeling of yHsp90 was performed as described in (A). Prior to addition of Ppt1, peptides were added in excess over yHsp90. Peptide sequences were PPAPEAEGPTVEEVD (EEVD peptide) and EVGLKRVVTKAMSSR (control peptide). (E) Analysis of the yHsp90 dephosphorylation by the phosphatase fragment of Ppt1 in the absence and presence of the TPR fragment.
Figure 5
Figure 5
Analysis of the in vivo effect of ppt1Δ. (A) Immunoblot analysis for Ppt1 expression. Lysate of the ppt1Δ yeast strain (left) and the wt yeast (right) was investigated using a polyclonal rabbit serum directed against purified Ppt1 (α-Ppt1). The arrow indicates the position of Ppt1. The scanning electron microscopic images compare ppt1Δ yeast cells and wt yeast cells. (B) Radioactive phosphorylation of identical amounts of yHsp90 derived from a ppt1Δ yeast strain and the corresponding wt strain. Identical amounts of both proteins were treated with CKII and [γ-32P]ATP to reveal the availability of phosphorylation sites. After the phosphorylation reaction was allowed to take place for 2 h, yHsp90 was separated from free ATP by SDS–PAGE. Aliquots of different protein quantities were analyzed and the relative amount of radioactivity was calculated. The graph shows the mean value corrected for the respective protein concentration. Three independent experiments gave similar results.
Figure 6
Figure 6
In vivo activation of yHsp90 client proteins. (A) In vivo activation of GR in ppt1Δ and wt yeast. The indicated yeast strains harboring a plasmid with the GR reporter β-galactosidase construct were grown in their logarithmic growth phase and identical cell numbers were induced with the GR ligand DOC (20 μM) for 90 min. Subsequently, cells were harvested and lysed. The β-galactosidase activity in the lysate, which corresponds to the GR activity, was determined with a chemoluminescence assay system as described in Materials and methods. The data shown represent the mean of five independent samples. The GR activity in the ppt1Δ yeast is reduced to ∼55% of the wt level and to ∼50% in ppt1Δ yeast cells expressing Ppt1 (H311A). (B) Analysis of luciferase activity in ppt1Δ and wt yeast cells. Identical numbers of yeast cells expressing firefly luciferase for 3 h were lysed and the lysate was supplemented with luciferin in a 96-well plate. Chemoluminescence was recorded using a Tecan Genios plate reader and normalized for the total protein concentration as determined by a Bradford assay (Bradford, 1976). Data shown are representative of multiple independent experiments. (C) Immunoblot analysis of v-Src activation in ppt1Δ, Ppt1 (H311A) and wt yeast. (Upper box) Immunoblot for autophosphorylated v-Src with a monoclonal antibody specific for phosphotyrosine residues (α-(P)-Tyr). The gel was loaded with identical quantities of cell lysate as indicated. The arrow points to the position of activated v-Src. (Lower box) Immunoblot of the lysates as shown above using a polyclonal serum directed against Ppt1. Data shown are representative of multiple independent experiments. (D) Growth behavior of ppt1Δ and wt yeast cells in response to the α-mating factor. Shown is a dilution series of identical numbers of yeast cells in the absence (−) and presence (+) of 5 μM α-factor in the media. (E) Analysis of eGFP production in ppt1Δ and wt yeast cells. Lysate of identical amounts of logarithmically grown yeast cells expressing eGFP constitutively or inducibly were prepared as described in Materials and methods. Induction of eGFP production was performed for 3 h. After eGFP oxidation, the fluorescence of the indicated cell lysates was recorded and normalized for the total protein concentration as determined by a Bradford assay (Bradford, 1976). Data shown are representative of multiple independent experiments.
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