CELL GROWTH AND DEVELOPMENT

Role of HSP90 in Salt Stress Tolerance via Stabilization and Regulation of Calcineurin

Jun Imai, Ichiro Yahara
DOI: 10.1128/MCB.20.24.9262-9270.2000

ABSTRACT

The role of HSP90 in stress tolerance was investigated inSaccharomyces cerevisiae. Cells showing 20-fold overexpression of Hsc82, an HSP90 homologue in yeast, were hypersensitive to high-NaCl or H-LiCl stresses. Hsc82-overexpressing cells appeared similar to calcineurin-defective cells in salt sensitivity and showed reduced levels of calcineurin-dependent gene expression. Co-overexpression of Cna2, the catalytic subunit of calcineurin, suppressed the hypersensitivity. Cna2 and Hsc82 coimmunoprecipitated from control cells grown under normal conditions but not from stressed cells. In contrast, coimmunoprecipitation was detected with Hsc82-overexpressing cells even after exposure to stresses. Cna2 immune complexes from stressed control cells showed a significant level of calcineurin activity, whereas those from stressed Hsc82-overexpressing cells did not. Treatment of extracts from Hsc82-overexpressing cells with Ca2+-calmodulin increased the calcineurin activity associated with Cna2 immune complexes. Geldanamycin, an inhibitor of HSP90 abolished the coimmunoprecipitation but did not activate calcineurin. When the expression level of Hsc82 decreased to below 30% of the normal level, cells also became hypersensitive to salt stress. In these cells, the amount of Cna2 was reduced, likely as a result of degradation. The present results showed that Hsc82 binds to and stabilizes Cna2 and that dissociation of Cna2 from Hsc82 is necessary for its activation.

Subfamilies of heat shock proteins that are constitutively expressed in cytosol, such as HSP70, function as molecular chaperones under both normal and stressful conditions in mediating the folding, oligomeric assembly, and intracellular transport of proteins (14). The 90-kDa heat shock protein, HSP90, is the most abundant protein in cytosols of eucaryotic cells. HSP90 is a major molecular chaperone which is distributed ubiquitously among all living organisms and which is indispensable for yeast cells to survive even under nonstressful conditions (2). In vitro, HSP90 prevents the aggregation of chemically denatured or heat-denatured proteins (16, 47, 50) and inherently unstable proteins such as casein kinase II (30). Nonnative proteins associated with HSP90 were released from the complexes and then refolded with the aid of reticulocyte lysates (50) or a combination of HSP70 and HSP40 (10, 29). Consistent with the results of these in vitro studies, overexpression of HSP90 confers a tolerance to stress in mammalian and yeast cells (2, 49). On the other hand, in vivo studies have revealed that HSP90 functions as a molecular chaperone for specific substrate proteins, most of which are involved in signal transduction (33, 38).

The budding yeast Saccharomyces cerevisiae has two genes,HSP82 and HSC82, that encode homologues of HSP90 (2). In cells grown under normal conditions, Hsc82 is constitutively expressed, while the expression of Hsp82 is very low. When cells are exposed to stress, the expression of Hsp82 is markedly enhanced. Since the induction and accumulation of Hsp82 in stressed cells take 1 h or longer, HSC82 rather thanHSP82 must be biologically active in the protection of normally growing yeast cells from acute stress. In the course of carrying out this study, we made the unexpected finding that cells overexpressing Hsc82 are hypersensitive to various stresses including high concentrations of NaCl. The phenotypes associated with the sensitivity to salt stress of Hsc82-overexpressing cells are similar to those of calcineurin-defective mutants. This finding led us to investigate the genetic and physical interactions between Hsp82 and Hsc82 and calcineurin.

Calcineurin is a serine/threonine protein phosphatase activated by calcium and the Ca2+-calmodulin complex which is involved in a variety of signal transduction processes (21, 45). Purified calcineurin from tissues is a heterodimer consisting of a catalytic subunit and a Ca2+-binding regulatory subunit. InS. cerevisiae, genes CNA1 and CNA2encode the catalytic subunit, while CNB1 encodes the regulatory subunit (8, 9, 22, 24). While calcineurin is dispensable in yeast cells for growth under normal conditions, it is essential for growth under salt stress conditions (27, 32). Recently a substrate phosphoprotein for calcineurin, Tcn1/Crz1, was identified in yeast (44). Calcineurin activity is required for the transcriptional activation by transcriptional factor Tcn1/Crz1 (26, 43, 44) of several genes including PMR2,PMC1, and FKS2 (7, 11).

Ca2+-calmodulin activates calcineurin by binding to the catalytic subunit (13). Calcineurin binds to anchoring proteins such as FKBP12 (4, 5, 17, 46) and AKAP79 (20). Since the calcineurin catalytic subunit has an unstable conformation when present alone (19), it is not surprising that calcineurin interacts with a variety of proteins. Calmodulin is the only substance to bind to the catalytic subunit in vitro and protect it from proteolysis (25). We report that Hsc82 protects Cna2 from degradation in vivo when expressed to an appropriate extent but interferes with its activation when overexpressed.

MATERIALS AND METHODS

Microbiological techniques.Yeast transformations were performed according to the method of Ito et al. (15). Rich medium containing glucose (YPD) and synthetic minimal medium (SD), as described elsewhere, were used (40). SC is SD supplemented with 0.5% Casamino Acids (Difco) and 100 mg of uracil, adenine sulfate, and tryptophan/liter. YPGal, SDGal, and SCGal are YPD, SD, and SC, respectively, in which 2% glucose is replaced with 2% galactose and 0.2% sucrose. SC-U and SCGal-U are SC and SCGal, respectively, lacking uracil. SC-U,W is SC lacking tryptophan and uracil. Medium was buffered to pH 5.5 via the addition of 5 mM succinic acid and supplemented with CaCl2, NaCl, or LiCl. HGal-L,H,W is SDGal supplemented with 20 mg each of uracil, arginine, and methionine, 30 mg each of tyrosine, isoleucine, and lysine, 150 mg of valine, 60 mg of phenylalanine, and 50 mg of adenine/liter and 10 mM KPB buffer (pH 7.5). HGal-L,H,W,U is HGal-L,H,W lacking uracil. A 2% mixture of galactose and glucose with 0.2% sucrose was used in medium as a carbon source. HS-U is SD supplemented with 20 mg each of argentine, tryptophan, and methionine, 30 mg each of tyrosine, leucine, histidine, isoleucine, and lysine, 150 mg of valine, 60 mg of phenylalanine, and 50 mg of adenine/liter, and was buffered to pH 5.0 via the addition of 5 mM succinic acid. HS-U,W is HS-U lacking tryptophan. HS-L,H,W,U is HS-U lacking tryptophan, leucine, and histidine. Medium was sometimes supplemented with CaCl2. Yeast cells were cultured at 30°C, unless otherwise indicated.

Antibodies and reagents.A mouse monoclonal antihemagglutinin (anti-HA) antibody (16B12) was obtained from Berkeley Antibody Company. Rabbit polyclonal anti-calcineurin B antibodies (PA3-025) were obtained from Affinity Bioreagents, Inc. Canavanine was obtained from Sigma, and geldanamycin was obtained from Gibco BRL. FK506 was obtained from Fujisawa Co. Ltd. Bovine calmodulin was obtained from BIOMOL Research Laboratories, Inc., as was the BIOMOL GREEN calcineurin assay kit. Protein concentration was determined using BCA protein assay reagent (Pierce Chemical Co.) with bovine serum albumin (Sigma) as the standard.

Gene replacements.A 1.1-kbpSmaI-PvuII fragment from pJJ281 containingTRP1 was inserted into the EcoRV site of pRS90 (18) to produce KS-HSP82. KS-HSP82 was digested bySalI and SacI to linearize thehsp82::TRP1 fragment and transformed into YPH500 using one-step gene replacement, resulting inJH82D. Gene disruption was confirmed by Western blotting.

Strains and plasmids.The yeast strains used are listed in Table 1. A Δhsc82 mutant (Y499-1H) was crossed with a Δhsp82 mutant (YJH82D/pRS315-1GAL10-HSC82) and then sporulated and dissected to produce a Δhsc82 Δhsp82/pGAL1:HSC82 double mutant (YJH82DD). Plasmids pYO324-HSC82 and pYO326-HSC82 carry the 4.2-kbp SpeI-SpeI fragment of HSC82 in anXbaI site of 2 μm plasmids pYO324 and pYO326 (34), respectively. Plasmid pYO326-CNA2 was constructed in the following manner. The complete CNA2 coding region was amplified from the yeast genome by PCR using convergent primers 5′-GCGGGATCCGATGTCTTCAGACGCTAT and 5′-GCGGGATCCCTATTTCGTATCATTCTTT. The amplified fragments were inserted into the KpnI and SacI sites of pRS304 after blunting to construct pRS304-CNA2. pRS304-CNA2 was digested with SalI and integrated at the CNA2locus of the yeast genome to create YJC2-W. The genome DNA from YJC2-W was digested with BglII and self-ligated to produce pRS304-CNA2G. Next, 1.5- and 3-kbpBglII-SalI fragments from pRS304-CNA2G were inserted into the BamHI-SalI sites of pYO326 and pUC119, respectively, to produce pYO326-CNA2N and pUC119-CNA2C. A 3-kbpSalI-KpnI fragment from pUC119-CNA2C was inserted into the SalI-KpnI site of pYO326-CNA2N to produce pYO326-CNA2. Plasmid pSM102-CNA2 was constructed in the following manner and used for expression of triple-HA epitope-tagged product of CNA2. The SphI site of pSM102 was changed into a BamHI site via an insertion of aBamHI linker (Takara Syuzo Co.) after blunting to produce pSM102BamHI. The complete CNA2 coding region was amplified from pYO326-Cna2 by PCR using convergent primers 5′-GCGGGATCCGATGTCTTCAGACGCTAT and 5′-GCGGGATCCCTATTTCGTATCATTCTTT. The amplified fragments were then inserted into the BamHI site of pSM102BamHI to construct pSM102-CNA2. A 2-kbp BglII (just before ATG of the triple-HA coding sequence)-SacI fragment of pSM102-CNA2 was inserted in frame into the BamHI-SacI site of pUCGPD to produce pRS316-pGPD-ha-CNA2. A 0.5-kbpBamHI-BamHI fragment from pMC1871 was inserted into the BamHI site of p2UGPD to produce pYO326-pGPD-lacZ. A 0.5-kbp XhoI-SacI fragment from pRS316-1GAL10 was inserted into the XhoI-SacI site of pRS315 to produce pRS315-1GAL10. A 2-kbp ScfI fragment of pYO326-HSC82 was inserted into the BamHI site of pRS315-1GAL10 after blunting to produce pRS315-1GAL10-HSC82. The complete CNB1coding region was amplified from the yeast genome by PCR using convergent primers 5′-GCGAGATCTGATGGTGCTGCTCCTTC and 5′-GCGAGATCTTTACACATCGTATTGCAA. The amplified fragments were then inserted into the BamHI site of pUCGPD to construct pRS316-pGPD-CNB1. Plasmid pYO326-cna2H105Q was constructed in the following manner. A 600-bp fragment of the N terminus-encoding region of CNA2 was amplified from pYO326-CNA2 by PCR using convergent primers 5′-GCGGGATCCGATGTCTTCAGACGCTAT and 5′-CGCCCGCGGAGTAGCCAGAAATGGTC. The amplified fragments were then inserted into the EcoRV site of pT7Blue to produce pT7-Cna2DPP. A 1.2-kbp fragment of the C terminus-encoding region ofCNA2 was amplified from pYO326-CNA2 by PCR using convergent primers 5′-TTCTGGCTACTCCGCGGTAACCAAGAATGTAAGCAT and 5′-GCGGGATCCCTATTTCGTATCATTCTTT. The amplified fragments were then inserted into the EcoRV site of pT7Blue to produce pT7-Cna2DPPC. A 600-bp SacI-SacII fragment of pT7-Cna2DPPN was inserted into the SacI-SacII site of pT7-Cna2DPPC to produce pT7-Cna2DPP. A 0.2-kbpSalI-XbaI fragment of pT7-Cna2DPP was inserted into the SalI-XbaI site of pYO326-CNA2 to produce pYO326-Cna2H105Q. For construction of pRS314-pCNA2-ha-CNA2 and pYO324-pCNA2-ha-CNA2, the complete CNA2 promoter region was amplified from pYO326-Cna2 by PCR using convergent primers 5′-CGCGAGCTCAAAACGTGCC and 5′-CGCAGATCTTGGCGTTGAGAGTGT. The amplified fragments were then inserted into theEcoRV-SacI site of pRS314 to construct pRS314-CNA2p. A 2-kbp BglII-XhoI fragment of pSM102-CNA2 was inserted into the BglII-SalI site of pUCGPD to produce pRS314-pCNA2-ha-CNA2. A 2-kbpSacI-SacI fragment of pRS314-pCNA2-ha-CNA2 was inserted into the SacI site of pYO324 to produce pYO324-pCNA2-ha-CNA2. Plasmid pSM102 was a gift from S. Matsumoto (Tokyo Metropolitan Institute of Medical Science). Plasmids p2UGPD (GPD, glyceraldehyde-3-phosphate dehydrogenase) (31), pUCGPD, and pRS316-1GAL10 were gifts from Y. Kimura (Tokyo Metropolitan Institute of Medical Science). Plasmids pAMS363 and pAMS366 were gifts from M. S. Cyert (Stanford University).

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Table 1.

Yeast strains used in this study

PAGE.To resolve isoforms Hsp82 and Hsc82, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a 4 to 20% polyacrylamide gradient gel (Daiichi Pure Chemicals). In this system, no SDS was added in either the upper or lower electrode buffers.

Immunoblotting.Yeast cells in the exponential-growth phase were harvested and then disrupted with glass beads in the presence of 1% SDS containing 1 mM phenylmethylsulfonyl fluoride and (PMSF), 1 mg of leupeptin, 1 mg of antipain, 1 mg of aprotinin, and 1 mg of pepstatin A/ml. Protease inhibitors were purchased from Sigma. After unbroken cells and glass beads were removed by brief centrifugation at 800 × g, the supernatant was used as the lysate. Lysate was resolved by SDS-PAGE, transferred to a membrane, and subjected to Western blotting with various antibodies.

Immunoprecipitation.Yeast cells in the exponential-growth phase were harvested and then disrupted with glass beads in 200 μl of radioimmunoprecipitation assay (RIPA)-Mo buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, [pH 8.0] 10 mM sodium molybdate, 10 mM MgCl2) containing 1 mM PMSF and 1 mg of leupeptin, antipain, aprotinin, and pepstatin A/ml. All protease inhibitors were purchased from Sigma. After unbroken cells and glass beads were removed by brief centrifugation at 800 × g, the lysates were clarified twice by centrifugation at 15,000 × g at 4°C for 10 min. The protein concentration of each lysate was determined using BCA protein assay reagent, and the lysates were diluted to the same concentration with RIPA-Mo buffer with protease inhibitors. Protein G beads (Sigma) (50% [vol/vol]) were added to aliquots of lysates containing equivalent amounts of the cell lysates, after which the mixtures were kept at 4°C for 1 h with gentle rotation. The beads were removed by centrifugation. Antibodies were added to the preabsorbed lysates and the mixtures were incubated at 4°C for 2 h with gentle rotation. Subsequently, protein G beads (50% [vol/vol]) were added to the mixtures, which were then incubated at 4°C for 2 h with gentle rotation. The beads were then collected by centrifugation and washed four times with RIPA-Mo buffer. Proteins were eluted by boiling the beads in sample buffer (50 mM Tris [pH 6.8], 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol). They were then resolved by SDS-PAGE, transferred to a membrane, and subjected to Western blotting with various antibodies.

β-Galactosidase activity.Cells were grown overnight in an appropriate medium to mid-log phase and incubated for an additional 6 h in the presence of 0.2 mM CaCl2. FK506 or geldanamycin was added to the incubation medium. β-galactosidase activity was determined at room temperature using chloroform-SDS-permeabilized cells as described previously and normalized by cell numbers (12).

Calcineurin phosphatase assay.Phosphatase activities of immune complexes were determined using the BIOMOL GREEN calcineurin assay kit (BIOMOL Research Laboratories, Inc.). Immune complex-bound beads were prepared by immunoprecipitation. The immune complex-bound beads (10 μl) were added to 50 μl of reaction cocktail containing 50 mM Tris, pH 8.0, 100 mM NaCl, 6 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mg of bovine serum albumin/ml, 0.1 mM CaCl2, and 0.3 mM RII phosphopeptide substrate. The mixtures were incubated at 30°C for 10 min. The supernatants of the mixtures were collected using a spin filter. Amounts of the released free phosphate in the supernatants were determined by the classic malachite green assay according to the supplier's protocol, after which calcineurin activity was calculated by subtracting background phosphate.

RESULTS

Hypersensitivity of Hsc82-overexpressing cells to various stresses.To investigate the role of Hsp82 and Hsc82 in stress tolerance using S. cerevisiae, a wild-type strain was transfected with a multicopy plasmid harboring HSC82including its own promoter, creating a strain overexpressing Hsc82. Western blot analysis with the anti-Hsp82/Hsc82 antibody revealed that the strain overexpressed Hsc82 approximately 20-fold under normal conditions (Fig. 1A). Hsc82-overexpressing cells were examined for resistance to various stresses including heat, canavanine, and salt. The expression levels of Hsp82 and Hsc82 in Hsc82-overexpressing cells did not change significantly when these cells were continuously exposed to stresses over a period of a few days (Fig. 1A). Unexpectedly, Hsc82-overexpressing cells were found more sensitive to stresses than cells expressing Hsp82 and Hsc82 at normal levels (Fig. 1B). For instance, growth of Hsc82-overexpressing cells was severely restricted on plates containing 100 μg of canavanine/ml, 1 M NaCl, or 0.2 M LiCl. Experiments using liquid cultures showed that these cells were also hypersensitive to heat, 15% (vol/vol) ethanol, and 2.5 M NaCl (Fig. 2). When Hsc82-overexpressing cells were incubated at a nonlethal high temperature, the cells acquired the same level of heat resistance as control cells (HSC82 HSP82), but the preincubation did not affect the hypersensitivity to 2.5 M NaCl (Fig. 2). The hypersensitivity to 1 M NaCl was ascribed to a high concentration of Na+ and not to the concentration of Cl− or to a high osmolarity given that Hsc82-overexpressing cells are hypersensitive to 1 M sodium acetate (data not shown) but not to 1 M KCl (Fig. 1B), 0.5 M MgCl2(data not shown), or 2 M sorbitol (Fig. 1B). The deficient growth in media containing 1 M NaCl or 0.2 M LiCl was suppressed via the addition of 0.2 M KCl (Fig. 1B). Hsc82-overexpressing cells are also sensitive to 6.0 mM MnCl2 (data not shown). On the other hand, Hsc82-overexpressing cells are more resistant to 0.6 M CaCl2 than control cells (Fig. 1B). The salt sensitivities of a Δcnb1 strain of MCY3-1C cells were not affected by Hsc82 overexpression (data not shown). Taken together, the above findings indicate that the phenotypes of Hsc82-overexpressing cells as to sensitivity to high-salt media and concomitant calcium tolerance are similar to those of calcineurin-defective mutants (27, 32).

Fig. 1.

Effects of overexpression of Hsc82 on stress sensitivity. (A) Quantification of Hsp82 and Hsc82 expression in control (YPH500/pYO326) and Hsc82-overexpressing (YPH500/pYO326-HSC82) cells. Lanes 1 and 2, expression levels of Hsp82 and Hsc82 in control cells incubated with or without canavanine (100 μg/ml), respectively; lanes 3 and 4, expression levels of Hsp82 and Hsc82 in Hsc82-overexpressing cells incubated with or without canavanine (100 μg/ml), respectively. Lysates were prepared from control and Hsc82-overexpressing cells that had been cultured in SC-U medium with or without canavanine for 3 days at 22°C. Samples equivalent to proteins from 107 cells were resolved by SDS-PAGE and Western blotted with an anti-Hsp82/Hsc82 antibody. (B) Hypersensitivity of Hsc82-overexpressing cells to various stresses. Control and Hsc82-overexpressing cells were grown overnight in SC-U medium, appropriately diluted, and spotted on SC-U plates with or without the indicated reagents. The plates were incubated at 18°C for 4 days (control and 100 μg canavanine/ml), at 30°C for 2 days after being exposed to 50°C for 30 min (heat shock) and at 36°C for 2 days (others).

Fig. 2.
Fig. 2.

Hypersensitivity of Hsc82-overexpressing cells to various stresses determined in liquid cultures. Control (YPH500/pYO326; solid squares) and Hsc82-overexpressing (YPH500/pYO326-HSC82; solid circles) cells were grown overnight in SC-U medium, appropriately diluted, and then incubated at 50°C in SC-U (A), in SC-U containing 15% (vol/vol) ethanol (Et-OH) at 30°C (B), or in SC-U containing 2.5 M NaCl at 30°C (C) for the indicated periods. Both control cells (open squares) and Hsc82-overexpressing cells (open circles) were pretreated at 37°C for 1 h and exposed to stress for the indicated periods. CFUs were then determined by plating on YPD plates at 30°C.

Suppression of hypersensitivity of Hsc82-overexpressing cells to salt stress by co-overexpression of the catalytic subunit of calcineurin, Cna2.The phenotypic similarity between Hsc82-overexpressing cells and calcineurin-defective cells prompted us to examine both the genetic and physical interactions between Hsc82 and calcineurin. Given that the expression of CNA2, encoding the catalytic subunit of calcineurin, suppresses phenotypes associated with calcineurin-defective strains, we examined whether or not an overexpression of Cna2 affects the phenotypes of Hsc82-overexpressing cells. We observed that the overexpression of Cna2 under the control of its own promoter suppressed the hypersensitivity of Hsc82-overexpressing cells to 1.0 M NaCl (data not shown) or 0.2 M LiCl (Fig. 3). However, the overexpression of Cna2 neither lowered the expression level of Hsc82 (data not shown) nor compensated for the hypersensitivity to canavanine or heat (Fig. 3). When cna2 H105Q, a mutant encoding an amino acid substitution near the metal-binding site (28), was used instead of CNA2, no suppression of hypersensitivity was observed (Fig. 3). Overexpression of the regulatory subunit of calcineurin, Cnb1, did not suppress the hypersensitivity (data not shown).

Fig. 3.
Fig. 3.

Suppression of the hypersensitivity of Hsc82-overexpressing cells to LiCl by co-overexpression of the catalytic subunit of calcineurin. Shown are the effects of co-overexpression of Cna2. Four strains, YPH500/pYO324 pYO326 (two-vector plasmids), YPH500/pYO324-HSC82 (Hsc82 overexpression plasmid) pYO326, YPH500/pYO324-HSC82 pYO326-CNA2 (Cna2 overexpression plasmid), andYPH500/pYO324-HSC82 pYO326-cna2H105Q (a Cna2 mutant overexpression plasmid), were used. Cells of these strains were grown overnight in SC-U,W medium, appropriately diluted, and spotted on SC-U,W plates with or without the indicated reagents (Can., canavanine) and incubated at 36°C for 2 days (control and 0.2 M LiCl) or 18°C for 4 days (100 μg of canavanine/ml).

Reduction in calcineurin-dependent gene expression by overexpression of Hsc82.The in vivo activity of calcineurin can be assessed by determining calcineurin-dependent gene expression. Repeated CDRE (calcineurin-dependent response element) ofFKS2, a calcineurin-dependent gene, was fused to the 5′ end of LacZ and expressed in the cells to be examined. Plasmids, pAMS363 (two copies of CDRE) (43) and pAMS366 (four copies of CDRE) (43) were separately transfected into wild-type and Hsc82-overexpressing cells, and the transfected cells were exposed to 0.2 M CaCl2. Calcineurin-dependent gene expression, as indicated by the activity of β-galactosidase, was significantly reduced by the overexpression of Hsc82, whereas the expression ofpGPD-LacZ was not affected (Fig.4). No β-galactosidase activity was detected when the plasmids (pAMS363 and pAMS366) were not transfected (data not shown). CDRE-dependent gene expression was abolished in the presence of FK506, a calcineurin inhibitor (Fig. 4). These results suggest that the catalytic subunit of calcineurin, Cna2, might be bound to and sequestered by an excess of Hsc82.

Fig. 4.
Fig. 4.

Effects of Hsc82 overexpression on calcineurin-dependent gene (two and four copies of CDRE) expression. Control (YPH500/pYO324) and Hsc82-overexpressing (YPH500/pYO324-HSC82) cells were transformed with pAMS363 (two copies of CDRE) or pAMS366 (four copies of CDRE) and grown to mid-log phase in H-U,W medium. The cells were appropriately diluted and incubated at 30°C for 6 h in HS-U,W (pH 5.0) medium with or without CaCl2 (0.2 M) or FK506 (1 μg/ml). β-Galactosidase was also expressed under the control of theGPD promoter in wild-type and Hsc82-overexpressing cells. The cells were exposed to CaCl2 and FK506 (1 μg/ml), and the β-galactosidase activity was determined as described above.

Association of Cna2 with Hsp82 and Hsc82.To examine the physical association between Cna2 and Hsp82 and Hsc82 in vivo, HA-CNA2 was constructed and expressed in the cells to be examined. Cells arrested in a high-salt medium regained growth by simultaneous overexpression of Cna2 or HA-Cna2 and Cnb1, indicating that HA-Cna2 is as functional in cells as Cna2 (data not shown). HA-CNA2 was transfected into control and Hsc82-overexpressing cells. The overexpression of Hsc82 did not reduce the expression of HA-Cna2 (data not shown). Cells of the transfectants were cultured and then exposed to 1 M NaCl stress, after which cell extracts were prepared. Cna2 was immunoprecipitated from cell extracts with an anti-HA antibody. Immunoprecipitates were then resolved by SDS-PAGE and blotted with anti-HA and anti-Hsp82 antibodies. The results showed that Hsp82 and Hsc82 were coimmunoprecipitated with Cna2 from cell extracts of normally growing control cells transfected with HA-CNA2, whereas coimmunoprecipitation from extracts of cells incubated in 0.75 M NaCl was decreased (Fig.5A). No coimmunoprecipitation of Hsp82 and Hsc82 was seen by using the anti-HA antibody from cell extracts of normally growing control cells transfected with the control plasmid, which did not contain HA-Cna2 (data not shown). In contrast, Hsc82 was coimmunoprecipitated with Cna2 from extracts of both NaCl-stressed and nonstressed Hsc82-overexpressing cells that had been transfected with HA-CNA2 (Fig. 5A). Essentially the same results were obtained when immunoprecipitation was performed with the anti-Hsp82/Hsc82 antibody instead of the anti-HA antibody (Fig. 5B). The coimmunoprecipitation of Cna2 and Hsp82 or Hsc82 was not detected in extracts of either control or Hsc82-overexpressing cells when these cells were treated with geldanamycin (Fig. 5B). Treatment of cells with geldanamycin reduced calcineurin-dependent gene expression, however (Fig. 5C), indicating that dissociation of Cna2 from Hsc82 did not always activate Cna2. We found that the amount of Cna2 was decreased upon treatment of cells with geldanamycin (Fig. 5D). Although calcineurin-dependent gene expression was inhibited by FK506 (Fig. 4B), treatment of cells with FK506 did not affect the results of coimmunoprecipitation using any strain (data not shown). The above results support the notion that Cna2 forms a complex with Hsp82 and Hsc82 in normally growing control cells but that the association is disrupted when these cells are exposed to NaCl stress. For Hsc82-overexpressing cells, the association between Cna2 and Hsc82 is not disrupted even when these cells are exposed to stress, probably as a result of the excess of Hsc82 present in cells. Treatment of cell extracts from either control or Hsc82-overexpressing cells with an excess amount of calmodulin in the presence of 2 mM CaCl2decreased the coimmunoprecipitation of Cna2 and Hsc82 (Fig. 5E).

Fig. 5.
Fig. 5.

Coimmunoprecipitation of Hsp82 and Hsc82 with HA-Cna2. (A) Coimmunoprecipitation disappeared in extracts from control cells exposed to NaCl stress, whereas it was still observed in extracts from Hsc82-overexpressing cells exposed to stress. Control (YPH500/pYO324 pRS316-pGPD-HA-CNA2) and Hsc82-overexpressing (YPH500/pYO324-HSC82 pRS316-pGPD-HA-CNA2) cells were grown to mid-log phase in SC-U,W medium and appropriately diluted in SC-U,W medium with or without NaCl (0.75 M) at 30°C for 4 h. Lysates were prepared from these cells and subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates were resolved by SDS-PAGE and blotted with anti-HA and anti-Hsp82/Hsc82 antibodies. (B) Disappearance of the coimmunoprecipitation by treatment of cells with geldanamycin. Control cells (see panel A) were grown to mid-log phase in SC-U,W medium and treated with various concentrations of geldanamycin (GA) in SC-U,W medium for 1 h at 30°C. Lysates were prepared from these cells and subjected to immunoprecipitation with the anti-Hsp82/Hsc82 antibody. Immunoprecipitates were resolved by SDS-PAGE and blotted with anti-HA and anti-Hsp82/Hsc82 antibodies. Only data for control cells are shown, but the results were essentially the same for Hsc82-overexpressing cells. (C) Effects of geldanamycin on calcineurin-dependent gene expression. Wild-type cells (YPH499/pAMS363 or YPH499/pAMS366) were grown in HS-U medium overnight at 30°C, appropriately diluted, and incubated for 6 h at 30°C in HS-U (pH 5.0) medium containing 0.2 M CaCl2 in the presence or absence of geldanamycin (18 μM). The β-galactosidase activity of these cells was determined as described in Materials and Methods. (D) Decrease in the quantity of HA-Cna2 by treatment of cells with geldanamycin. The cell lysates, equivalent to proteins in panel B, were resolved by SDS-PAGE and blotted with the anti-HA antibody. (E) Abolishment of the coimmunoprecipitation by treating cell lysate with bovine calmodulin (CaM). Control cells (see panel A) were grown to mid-log phase in SC-U,W medium at 30°C, after which lysates were prepared from these cells and subjected to immunoprecipitation with the anti-HA antibody. CaCl2, to a final concentration of 2 mM, and, when indicated, calmodulin were added to the preabsorbed lysates 1 h after the addition of anti-HA antibody. The mixtures were incubated at 4°C for 1 h, after which immune complexes were collected. Concentrations of calmodulin were 0 (lane 2), 0.4 (lane 3), and 4 μg/ml (lane 4). Immunoprecipitates were resolved by SDS-PAGE and blotted with anti-HA and anti-Hsp82/Hsc82 antibodies.

Next, calcineurin activity in immune complexes was directly determined. Plasmids pRS314-pCNA2-ha-CNA2, a single-copy plasmid harboring HA-CNA2 under the control of its own promoter, and pYO324-pCNA2-ha-CNA2, a multicopy plasmid harboring HA-CNA2under the control of its own promoter, were separately transfected into control and Hsc82-overexpressing strains. The activity was proportional to the expression level of HA-Cna2 and was abolished by treatment with EGTA or EDTA (data not shown), indicating that the phosphatase activity determined in this system was elicited by HA-calcineurin (Fig.6A). Calcineurin activity was significantly reduced in immune complexes from Hsc82-overexpressing cells compared to the activity in those from control cells (Fig. 6A). Co-overexpression of HA-Cna2 together with Hsc82 increased the activity of the associated immune complexes (Fig. 6A). In accordance with the effect on the physical association between Cna2 and Hsc82 (Fig. 5E), treatment of the immune complexes from Hsc82-overexpressing cells with calmodulin in the presence of Ca2+ increased the activity (Fig. 6B).

Fig. 6.
Fig. 6.

(A) Reduction of calcineurin activity of cells expressing HA-Cna2 by overexpression of Hsc82 and its suppression by further co-overexpression of Cna2. HA-Cna2 (YPH499/pRS314-pCNA2-ha-CNA2) cells, HA-Cna2 cells overexpressing Hsc82 (YPH499/pRS314-pCNA2-ha-CNA2 pYO326-HSC82), and cells co-overexpressing HA-Cna2 and Hsc82 (YPH499/pYO324-pCNA2-ha-CNA2 pYO326-HSC82) were grown to mid-log phase in SC-U,W medium. The cells were appropriately diluted and incubated at 30°C for 4 h in SC-U,W (pH 5.5) medium with 0.75 M NaCl. From these cells, cell lysates were prepared. Complexes containing HA-Cna2 were immunoprecipitated by anti-HA antibodies from the cell lysates, after which phosphatase activities associated with the immune complexes were determined as described in Materials and Methods with or without EGTA (5 mM) and presented as averages of three independent determinations (± standard deviations). The amount of HA-Cna2 was determined by Western blotting. (B) Suppression by Ca2+-calmodulin of the inhibitory effect of excess Hsc82 on calcineurin activity. Cell lysates were prepared from control and Hsc82-overexpressing cells as for panel A, in which HA and calcineurin were coexpressed, and incubated for 1 h with 4 μg of calmodulin (CaM)/ml. Immunoprecipitation was performed as described for panel A. Calcineurin activities associated with the immune complexes were determined as described in Materials and Methods.

Requirement of Hsp82 and Hsc82 for activation of calcineurin.Although steroid hormone receptors are inactive as transcription factors in complexes with HSP90, they have high-affinity ligand-binding sites only in the complexes, and thus HSP90 is necessary for steroid hormone receptor functions (36). The above results strongly suggest that calcineurin in Hsp82 complexes is as inactive as steroid hormone receptors. We next examined whether Hsp82 and Hsc82 are required for calcineurin functions in S. cerevisiae. A correlation between the activity of calcineurin and the expression levels of Hsp82 and Hsc82 was determined using the following experimental system. We constructed a strain in which the expression of Hsc82 is controlled by the Gal1 promoter and both endogenousHSP82 and HSC82 were disrupted (JH82DD/pRS315-1GAL10-HSC82). Then, 2% (wt/vol) mixtures of galactose and glucose were used as carbon sources in culture media. In these cells, the GAL1 promoter is activated as a function of galactose concentrations, and, therefore, the intracellular concentration of Hsc82 decreases as the concentration of galactose in media is decreased. The expression of Hsc82 was greater than 60% of the normal expression level in HSP82 HSC82 cells growing in SC medium when 1.8% galactose plus 0.2% glucose were used as a carbon source mixture; it was reduced as the fraction of galactose was further decreased (Fig. 7A). The cells expressed approximately 1/17 of the Hsc82 expressed by wild-type cells and grew very slowly in medium containing 1% galactose and 1% glucose (Fig. 7B). Cells of a wild-type strain grown in media containing different galactose/glucose ratios did not show different sensitivities to LiCl, suggesting that the sensitivity was not affected by catabolite repression (Fig. 7B). These results clearly demonstrated that cells expressing lower levels of Hsc82 than wild-type cells are more sensitive to 0.2 M LiCl (Fig. 7B) or 1 M NaCl (data not shown).

Fig. 7.
Fig. 7.

Requirement of Hsp82 and Hsc82 for resistance against LiCl stress and calcineurin-dependent gene expression. (A) Galactose-dependent expression of Hsc82 in Δhsp82 Δhsc82/pGAL1:HSC82 cells (YJH82DD/pRS315-1GAL10-HSC82). YJH82DD cells were grown overnight in H-L,H,W medium containing various ratios of galactose and glucose, appropriately diluted, and cultured in the same medium for 6 h. Lysates equivalent to proteins from these cells were resolved by SDS-PAGE and blotted with the anti-Hsp82/Hsc82 antibody. The amount of Hsc82 expressed in these cells is expressed as a percentage of Hsp82 and Hsc82 expressed in wild-type cells grown in SC medium. (B) Hypersensitivity to LiCl stress of cells expressing Hsc82 at low levels. YPH499 (HSP82 HSC82/pRS313 pRS314 pRS315-1GAL10) and YJH82DD (Δhsp82 Δhsc82/pRS315-1GAL10-HSC82) cells were separately cultured overnight in HGal-L,H,W medium, diluted, and spotted on YP plates containing various ratios of galactose and glucose with or without LiCl (0.2 M). The plates were incubated at 30°C for 2 days. (C) Requirement of Hsc82 for calcineurin-dependent gene expression.YJH82DD cells were transformed with pAMS363 (two copies of CDRE; open squares), pAMS366 (four copies of CDRE; solid squares) and pYO326-pGPD-lacZ (pGPD-lacZ; open circles). The transformants were grown overnight in HS-L,H,W,U medium containing various ratios of galactose and glucose and diluted in HS-L,H,W,U medium (pH 5.0) containing various ratios of galactose and glucose with 0.2 M CaCl2 for 6 h at 30°C. Then the β-galactosidase activity was determined as described in Materials and Methods. The relative amount of β-galactosidase activity (percentage of that for control cells in the same medium) was plotted against the amount of Hsc82 (percentage of Hsp82 plus Hsc82 in wild-type cells).

We examined whether lower expression levels of Hsc82 than the level in wild-type cells would affect calcineurin-dependent gene expression. The CaCl2-induced LacZ expression level inGAL1-HSC82 cells harboring pAMS363 or pAMS366 incubated in 2.0% galactose medium was almost the same as the endogenous promoter-driven Hsp82 and Hsc82 expression levels in wild-type cells. The calcineurin-dependent expression of LacZ was reduced as the expression level of Hsc82 was decreased (Fig. 7C). In contrast, reduced expression levels of Hsc82 did not affect the expression ofpGPD-LacZ (Fig. 7C).

We next examined whether temperature-sensitive (ts−) mutations of hsp82 affect the sensitivity to high salt stress. ts− mutants hsp82 G170D andhsp82 S485Y (18) and wild-typeHSP82 on the genomic background of a Δhsc82strain were examined. The expression levels of the Hsp82 protein in anHSP82 Δhsc82 strain and these ts− hsp82 mutant strains were almost the same but were approximately 25% those of Hsp82 and Hsc82 in a wild-type (HSP82 HSC82) strain (data not shown). As seen in Fig.8A, bothhsp82 G170D and hsp82 S485Ycells were hypersensitive to 0.2 M LiCl, whereas HSP82 cells were comparably resistant even at the nonrestrictive temperature of 25°C. Essentially the same results were obtained when cells were exposed to 1 M NaCl (data not shown). On the other hand, both of these ts− mutant cells were more resistant to 0.5 M CaCl2 than control cells (Fig. 8A). However, overexpression of Cna2 did not suppress the salt-hypersensitive phenotype of ts− mutants (data not shown).hsp82 G170D and hsp82 S485Ystrains transfected with pAMS363 or pAMS366 were created and examined for calcineurin-dependent gene expression. Wild-type cells responded well to 0.2 M CaCl2 and expressed a calcineurin-dependent gene (Fig. 8B). CaCl2-induced, calcineurin-dependent gene expression levels were markedly reduced inhsp82 G170D and hsp82 S485Ycells even at 25°C (Fig. 8B).

Fig. 8.
Fig. 8.

Effects of ts− mutations of Hsp82 on salt sensitivity. (A) Hypersensitivity to 0.2 N LiCl and tolerance of 0.5 M CaCl2 of ts− hsp82 mutants. ts− hsp82 G170D Δhsc82(YOK5H) and hsp82 S485Y Δhsc82 (YOK9H) mutants were examined. Cells of the ts− mutants were grown in YPD medium overnight at 25°C, diluted, spotted on YPD plates with or without LiCl (0.2 M) or CaCl2 (0.5 M), and incubated at 25°C for 3 days. (B) Reduction in calcineurin-dependent gene expression in a ts− hsp82 mutant. Wild-type (YPH500; bars 1), HSP82 Δhsc82 (Y500-1H; bars 2),hsp82 G170D Δhsc82(YOK5H; bars 3), and hsp82 S485Y Δhsc82 (YOK9H; bars 4) cells were transformed with plasmid pAMS363 (two copies of CDRE) or pAMS366 (four copies of CDRE). Cells of each transformant were grown in HS-U medium overnight at 25°C, appropriately diluted, and incubated for 6 h at 25°C in HS-U (pH 5.0) medium with or without CaCl2 (0.2 M). The β-galactosidase activity of these cells was determined as described in Materials and Methods.

Instability of the calcineurin catalytic subunit, Cna2, in cells expressing reduced levels of Hsc82.As shown above, the activity of calcineurin, as determined by calcineurin-dependent gene expression, was greatly reduced in cells expressing low levels of Hsc82. This may be the consequence of a reduced quantity of Cna2 in these cells. Alternatively, Cna2 may be rendered inactive by forming complexes with proteins other than Hsc82, and furthermore the association may not be disrupted upon receipt of activation signals. To examine these possibilities, the amount of Cna2 in cells expressing a reduced level of Hsc82 was determined and compared with that in cells expressing a normal level of Hsc82. HA-Cna2 was expressed under the control ofGPD promoter in a Δhsp82 Δhsc82/pGAL1:HSC82 strain (YJH82DD/pRS315-1GAL10-HSC82). Cells of this strain were separately cultured in media containing 2% mixtures of galactose and glucose. Extracts were prepared from these cells, resolved by SDS-PAGE, and blotted with the anti-HA antibody (Fig.9A). A significant amount of Cna2 was detected in extracts from cells cultured in 2% galactose medium, whereas only a trace of Cna2 was detected in extracts from those cultured in 1.6% galactose and 0.4% glucose medium. Exposure of these cells to 1 M NaCl stress did not alter the results.

Fig. 9.
Fig. 9.

Effects of Hsp82 on stability of Cna2 and Cnb1. (A) Destabilization of Cna2 in cells expressing reduced levels of Hsc82.Δhsp82 Δhsc82/pGAL1:HSC82 cells (YJH82DD/pRS315-p1GAL10-HSC82 pRS316-pGPD-ha-CNA2) were grown overnight in H-L,H,W,U medium containing various ratios of galactose and glucose, appropriately diluted, and cultured in the same medium for 6 h at 30°C. A cell lysate equivalent to proteins from each culture was resolved by SDS-PAGE and blotted with anti-HA and anti-Hsp82/Hsc82 antibodies for determination of the amounts of Cna2 and Hsc82, respectively. (B) Destabilization of Cna2 in ts− hsp82 cells. Lysates equivalent to proteins of Δhsp82 (Y500-1H/pRS316-pGPD-ha-CNA2; lane 1) and ts− (YOK5H/pRS316-pGPD-ha-CNA2; lane 2) cells grown to mid-log phase in SC-U medium at 25°C were resolved by SDS-PAGE and blotted with anti-Hsp82/Hsc82 and anti-HA antibodies, respectively. (C) No effect of ts− hsp82 on Cnb1 was observed. Lysates equivalent to proteins ofΔhsp82 (Y500-1H/316-pGPD-CNB1; lane 1) and ts− (YOK5H/316-pGPD-CNB1; lane 2) cells grown to mid-log phase in SC-U medium at 25°C were resolved by SDS-PAGE and blotted with the anti-Cnb1 antibody.

An hsp82 G170D Δhsc82 strain transfected with pRS316-pGPD-ha-CNA2 was created and subjected to analysis of the Cna2 protein. Lysates were prepared from control and ts− hsp82 cells that had been grown at 25°C. We found that the amount of Cna2 was also decreased in cell extracts of the ts− hsp82 mutant strain compared to that inΔhsc82 cell extracts (Fig. 9B). The regulatory subunit of calcineurin, Cnb1, was also overexpressed in both Δhsc82and ts− hsp82 strains by transfection with pRS316-pGPD-CNB1. In contrast to what was found for Cna2, the amount of Cnb1 was not reduced in cells of ts− hsp82mutant strains (Fig. 9C). Co-overexpression of Cnb1 together with HA-Cna2 did not stabilize HA-Cna2 in these ts− mutant cells (data now shown).

DISCUSSION

HSP90 is a major cytosolic molecular chaperone that has been shown to protect denatured proteins from irreversible aggregation in vitro (16, 47). Higher concentrations of HSP90 (Hsp82 and Hsc82) are required for growth of yeast cells at higher temperatures (2). A CHO variant which expresses HSP90 at a level severalfold higher than normal is relatively heat resistant compared to the parental strain (49). It is plausible that HSP90 functions like HSP70 in vivo by protecting cells from damage caused by various stresses including elevated temperatures. For this reason, we expected that overexpression of either Hsp82 or Hsc82 in S. cerevisiae might confer stress tolerance on this organism. On the contrary, as was shown above, a strain overexpressing Hsc82 approximately 20-fold was hypersensitive to various stresses including canavanine, heat shock, and high salt. These results are consistent in part with the report by Cheng et al. (6), wherein overexpression of Hsp82 in yeast caused strain-dependent reductions in growth at 37.5°C and in thermotolerance. We observed that preconditioned Hsc82-overexpressing cells acquired thermotolerance in the same manner as wild-type cells did, indicating that the induced stress tolerance mechanism suppressed the hypersensitivity. These preheated, Hsc82-overexpressing cells were still hypersensitive to high salt stress, however, suggesting that the molecular mechanism operating in salt stress differs at least in part from that operating in thermotolerance.

Hsc82-overexpressing cells are also hypersensitive to 1 M NaCl and 0.2 M LiCl, but not to 1 M KCl or 1 M NaCl plus 0.2 M KCl. These results and those for the sensitivity to 2 M sorbitol and 0.6 M CaCl2 suggest that Hsc82-overexpressing cells are similar in their phenotypes to a calcineurin-defective mutant (27, 32). In fact, we observed that calcineurin-dependent gene expression was reduced. We found that co-overexpression of the catalytic subunit of calcineurin, Cna2, in Hsc82-overexpressing cells at least partially suppresses the hypersensitivity. This raised the possibility that Cna2 not only genetically but also physically interacts with Hsc82, which turned out to be the case. Coimmunoprecipitation experiments clearly demonstrated that, when cells are exposed to salt stress, Cna2 is dissociated from complexes of Cna2 and Hsc82 in wild-type cells but that Cna2 still exists as a complex with Hsc82 in Hsc82-overexpressing cells. As the complexes of steroid receptors and a complex of pp60v-srcwith HSP90 are inactive as transcription factors and as a tyrosine protein kinase, respectively (23), Cna2 in a complex with Hsc82 was shown in vitro to be inactive as a protein phosphatase. Thus, the overexpression of Hsc82 caused hypersensitivity to stresses in yeast at least in part by sequestering Cna2 from its activation process. This interpretation was supported by the fact that the hypersensitivity of Δcnb1 cells to salt stresses was not affected by overexpression of Hsc82. Co-overexpression of Cna2 in Hsc82-overexpressing cells suppressed the hypersensitivity to salt stress but not to canavanine or heat shock. This suggests that calcineurin is involved in the protection of cells from salt stress, but not from canavanine or heat shock, and that other Hsc82 substrates, whose functions are necessary for survival under other types of stress, are also sequestered by an excess of Hsc82. Consistent with these results, Δcna1 Δcna2 cells are not hypersensitive to heat shock or canavanine (unpublished results). Although yeast cells treated with the immunosuppressing drug FK506 have salt-sensitive phenotypes similar to those of calcineurin-deficient strains (32), our results showed that FK506 did not affect the complex formation of Cna2 and Hsc82 but reduced calcineurin-dependent gene expression. Therefore, the drug is likely to affect calcineurin after it is dissociated from Hsp82 and Hsc82.

HSP90 was initially thought to simply suppress the function of steroid receptors (23, 37) and pp60v-src (3, 23). Genetic analysis of yeast (1, 35, 48) and reconstitution experiments in vitro (39) have demonstrated that HSP90 is also a positive regulator of steroid receptors and pp60v-src. Thus, we examined whether Hsc82 is simply a negative regulator of calcineurin or, alternatively, whether the complex formation of Cna2 with Hsc82 is necessary for activation of Cna2 if Hsc82 is appropriately expressed. We demonstrated that underexpression of Hsc82 increased susceptibility to stress and reduced NaCl-induced, calcineurin-dependent gene expression. The present results also suggested that Cna2 was unstable and susceptible to proteolytic degradation in Hsc82-deficient cells. Similarly, when pp60v-src was expressed in yeast cells, the expression of the protein was significantly reduced by lowering the levels of Hsp82 and Hsc82 expression (48). Furthermore, ts− hsp82 mutant strains were hypersensitive to stress and Cna2 was decreased in these mutants even under nonstressful conditions. NaCl-induced, calcineurin-dependent gene expression was also reduced in a ts− hsp82 mutant even at the permissive temperature. This effect of the hsp82 mutation appeared to be more severe on calcineurin than on glucocorticoid receptors and pp60v-src, given that the effects on the last were not detected at the permissive temperature (18, 33). Geldanamycin, an HSP90-specific inhibitor, dissociates Cna2 from Hsc82 and Hsp82 but does not activate it because Cna2 is destabilized as a consequence of the dysfunction of Hsc82 and Hsp82. Taken as a whole, the above findings suggested that, like steroid receptors and pp60v-src, newly translated Cna2 binds to and forms a complex with Hsp82 and Hsc82 likely because it has a partially unstable structure when present alone (19). As revealed by coimmunoprecipitation experiments, Hsp82 and Hsc82 form complexes with and stabilize Cna2 until cells are exposed to stress. The results also demonstrated that dissociation of the complexes leading to the activation of calcineurin involves mobilization of Ca2+ to activate calmodulin. An excess of Hsc82 probably competitively interferes with the binding of Ca2+-calmodulin to Cna2, thus inhibiting the activation of calcineurin. In addition, stress must mobilize preexisting Hsp82 and Hsc82, which prevents harmful aggregation of denatured proteins, causing a shift in the equilibrium that dissociates the complexes. However, this equilibrium shift is not sufficient for the activation of calcineurin because neither canavanine nor heat shock activated calcineurin (unpublished observations). The present finding is consistent in part with the report by Someren et al. (42), in that Hsp90 interacts with the catalytic subunit of calcineurin in vivo. However, as described above, we observed that HSP90 stabilizes, but does not activate, calcineurin; this is inconsistent with the notion that HSP90 increases calcineurin activity in a dose-dependent manner (42). The discrepancy remains to be elucidated.

ACKNOWLEDGMENTS

This work was conducted as a CREST research project.

We thank M. S. Cyert (Stanford University) for the gift of plasmids pAMS363 and pAMS366 and Δcnb1 strainMCY3-1C.

FOOTNOTES

    • Received 27 April 2000.
    • Returned for modification 12 June 2000.
    • Accepted 28 September 2000.

REFERENCES

  1. 1.↵
    1. Bohen S. P.,
    2. Yamamoto K. R.
    Modulation of steroid-receptor signal transduction by heat shock proteins The biology of heat shock proteins and molecular chaperones. Morimoto R. I., Tissiers A., Georgopoulos C. 1993 313 334 Cold Spring Harbor Laboratory Press Cold Spring Harbor, N.Y
  2. 2.↵
    1. Borkovich K. A.,
    2. Farrelly F. W.,
    3. Finkelstein D. B.,
    4. Taulien J.,
    5. Lindquist S.
    HSP82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.Mol. Cell. Biol. 9 1989 3919 3930
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Brugge J. S.
    Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 an pp90.Curr. Top. Microbiol. Immunol. 123 1986 1 22
    OpenUrlPubMedWeb of Science
  4. 4.↵
    1. Cameron A. M.,
    2. Steiner J. P.,
    3. Roskams A. J.,
    4. Ali S. M.,
    5. Ronnett G. V.,
    6. Snyder S. H.
    Calcineurin associated with the inositol 1,4,5-triphosphate receptor FKBP12 complex modulates Ca2+ flux.Cell 83 1995 463 472
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Cameron A. M.,
    2. Nucifora F. C. Jr.,
    3. Fung E. T.,
    4. Livingston D. J.,
    5. Aldape R. A.,
    6. Ross C. A.,
    7. Snyder S. H.
    FKBP12 binds the inositol 1,4,5-triphosphate receptor at leucine-proline (1400–1401) and anchors calcineurin to this FK506-like domain.J. Biol. Chem. 272 1997 27582 27588
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Cheng L.,
    2. Hirst K.,
    3. Piper P. W.
    Authentic temperature-regulation of a heat shock gene inserted into yeast on a high copy number vector. Influences of overexpression of HSP90 protein on high temperature growth and thermotolerance.Biochem. Biophys. Acta 1132 1992 26 34
    OpenUrlPubMed
  7. 7.↵
    1. Cunningham K. W.,
    2. Fink G. R.
    Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases.J. Cell Biol. 124 1994 351 363
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Cyert M. S.,
    2. Thorner J.
    Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.Mol. Cell. Biol. 12 1992 3460 3469
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Cyert M. S.,
    2. Kunisaw R.,
    3. Kaim D.,
    4. Thorner J.
    Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase.Proc. Natl. Acad. Sci. USA 88 1991 7376 7380
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Freeman B. C.,
    2. Morimoto R. I.
    The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding.EMBO J. 15 1996 2969 2979
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    1. Garciadeblas B.,
    2. Rubio F.,
    3. Quintero F. J.,
    4. Banuelos M. A.,
    5. Haro R.,
    6. Rodriguez-Navarro A.
    Differential expression of two genes encoding isoform of the ATPase involved in sodium efflux in Saccharomyces cerevisiae.Mol. Gen. Genet. 236 1993 363 368
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    1. Guarente L.
    Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast.Methods Enzymol. 101 1983 181 191
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    1. Hashimoto Y.,
    2. Perrino B. A.,
    3. Soderling T. R.
    Identification of an autoinhibitory domain in calcineurin.J. Biol. Chem. 265 1990 1924 1927
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Hendrick J. P.,
    2. Hartl F. U.
    Molecular chaperone functions of heat-shock proteins.Annu. Rev. Biochem. 62 1993 349 384
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    1. Ito H.,
    2. Fukuda Y.,
    3. Murata K.,
    4. Kimura A.
    Transformation of intact yeast cells treated with alkali cations.J. Bacteriol. 153 1983 163 168
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Jakob U.,
    2. Lilie H.,
    3. Meyer I.,
    4. Buchner J.
    Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo.J. Biol. Chem. 270 1995 7288 7294
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Jayaraman T.,
    2. Brillantes A. M.,
    3. Timerman A. P.,
    4. Fleisher S.,
    5. Erdjument-Bromage H.,
    6. Tempst P.,
    7. Marks A. R.
    FK506 binding protein associated with the calcium release channel (ryanodine receptor).J. Biol. Chem. 267 1992 9474 9477
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Kimura Y.,
    2. Matsumoto S.,
    3. Yahara I.
    Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae.Mol. Gen. Genet. 242 1994 517 527
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    1. Kissinger C. R.,
    2. Parge H. E.,
    3. Knighton D. R.,
    4. Lewis C. T.,
    5. Pelletier L. A.,
    6. Tempczyk A.,
    7. Kalish V. J.,
    8. Tucker K. D.,
    9. Showalter R. E.,
    10. Moomaw E. W.,
    11. et al.
    Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.Nature 378 1995 641 644
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    1. Klauck T. M.,
    2. Faux M. C.,
    3. Labudda K.,
    4. Langeberg L. K.,
    5. Jaken S.,
    6. Scott J. D.
    Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein.Science 271 1996 1589 1592
    OpenUrlAbstract
  21. 21.↵
    1. Klee C. B.,
    2. Draetta G. F.,
    3. Hubbard M. J.
    Calcineurin.Adv. Enzymol. Relat. Areas Mol. Biol. 61 1988 149 200
    OpenUrlPubMedWeb of Science
  22. 22.↵
    1. Kuno T.,
    2. Tanaka H.,
    3. Mukai H.,
    4. Chang C. D.,
    5. Hiraga K.,
    6. Miyakawa T.,
    7. Tanaka C.
    cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae.Biochem. Biophys. Res. Commun. 180 1991 1159 1163
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    1. Lindquist S.,
    2. Craig E. A.
    The heat-shock proteins.Annu. Rev. Genet. 22 1988 631 677
    OpenUrlCrossRefPubMedWeb of Science
  24. 24.↵
    1. Liu Y.,
    2. Ishii S.,
    3. Tokai M.,
    4. Tsutsumi H.,
    5. Ohki O.,
    6. Akada R.,
    7. Tanaka K.,
    8. Tsuchiya E.,
    9. Fukui S.,
    10. Miyakawa T.
    The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B.Mol. Gen. Genet. 227 1991 52 59
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    1. Manalan A. S.,
    2. Klee C. B.
    Activation of calcineurin by limited proteolysis.Proc. Natl. Acad. Sci. USA 80 1983 4291 4295
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Matheos D. P.,
    2. Kingsbury T. J.,
    3. Ahsan U. S.,
    4. Cunningham K. W.
    Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae.Genes Dev. 11 1997 3445 3458
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Mendoza I.,
    2. Rubio F.,
    3. Rodriguez-Navarro A.,
    4. Pardo J. M.
    The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae.J. Biol. Chem. 269 1994 8792 8796
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Mertz P.,
    2. Yu L.,
    3. Sikkink R.,
    4. Rusna F.
    Kinetic and spectroscopic analyses of mutants of a conserved histidine in the metallophosphatases calcineurin and lambda protein phosphatase.J. Biol. Chem. 272 1997 21296 21302
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Minami Y.,
    2. Kawasaki H.,
    3. Minami M.,
    4. Tanahashi N.,
    5. Tanaka K.,
    6. Yahara I.
    A critical role for the proteasome activator PA28 in the Hsp90-dependent protein refolding.J. Biol. Chem. 275 2000 9055 9061
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Miyata Y.,
    2. Yahara I.
    The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity.J. Biol. Chem. 267 1992 7042 7047
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Mumberg D.,
    2. Muller R.,
    3. Funk M.
    Yeast vectors for the controlled expression of heterologous protein in different genetic backgrounds.Gene 156 1995 119 122
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    1. Nakamura T.,
    2. Liu Y.,
    3. Hirata D.,
    4. Namba H.,
    5. Harada S.,
    6. Hirokawa T.,
    7. Miyakawa T.
    Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions.EMBO J. 12 1993 4063 4071
    OpenUrlPubMedWeb of Science
  33. 33.↵
    1. Nathan D. F.,
    2. Vos M. H.,
    3. Lindquist S.
    In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone.Proc. Natl. Acad. Sci. USA. 94 1997 12949 12956
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Ohya Y.,
    2. Goebl M.,
    3. Goodman L. E.,
    4. Petersen-Bjorn S.,
    5. Friesen J. D.,
    6. Tamanoi F.,
    7. Anraku Y.
    Yeast CAL1 is a structural and functional homologue to the DPR1(RAM) gene involved in ras processing.J. Biol. Chem. 266 1991 12356 12360
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Picard D.,
    2. Khursheed B.,
    3. Garabedian M. J.,
    4. Fortin M. G.,
    5. Lindquist S.,
    6. Yamamoto K. R.
    Reduced levels of hsp90 compromise steroid receptor action in vivo.Nature 348 1990 166 168
    OpenUrlCrossRefPubMedWeb of Science
  36. 36.↵
    1. Pratt W. B.
    The role of the hsp90-based chaperone system in signal transduction by nuclear receptors and receptor signaling via MAP kinase.Annu. Rev. Pharmacol. Toxicol. 37 1997 297 326
    OpenUrlCrossRefPubMedWeb of Science
  37. 37.↵
    1. Pratt W. B.,
    2. Sanchez E. R.,
    3. Bresnick E. H.,
    4. Meshinchi S.,
    5. Scherrer L. C.,
    6. Dalman F. C.,
    7. Welsh M. J.
    Interaction of the glucocorticoid receptor with the Mr 90,000 heat shock protein: an evolving model of ligand-mediated receptor transformation and translocation.Cancer Res. 15 1989 2222s 2229s
    OpenUrl
  38. 38.↵
    1. Rutherford S. L.,
    2. Zuker C. S.
    Protein folding and the regulation of signaling pathways.Cell 79 1994 1129 1132
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    1. Scherrer L. C.,
    2. Dalman F. C.,
    3. Massa E.,
    4. Meshinchi S.,
    5. Pratt W. B.
    Structural and functional reconstitution of the glucocorticoid receptor-hsp90 complex.J. Biol. Chem. 265 1990 21397 21400
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Sherman F.,
    2. Fink G. R.,
    3. Hicks J. B.
    Methods in yeast genetics. 1986 Cold Spring Harbor Laboratory Press Cold Spring Harbor, N.Y
  41. 41.
    1. Sikorski R. S.,
    2. Hieter P.
    A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.Genetics 122 1989 19 27
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    1. Someren J. S.,
    2. Faber L. E.,
    3. Klein J. D.,
    4. Tumlin J. A.
    Heat shock proteins 70 and 90 increase calcineurin activity in vitro through calmodulin-dependent and independent mechanisms.Biochem. Biophys. Res. Commun. 260 1999 619 625
    OpenUrlCrossRefPubMedWeb of Science
  43. 43.↵
    1. Stathopoulos A. M.,
    2. Cyert M. S.
    Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast.Genes Dev. 11 1997 3432 3444
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Stathopoulos-Gerontides A.,
    2. Guo J. J.,
    3. Cyert M. S.
    Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation.Genes Dev. 13 1999 798 803
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    1. Stewart A. A.,
    2. Ingebristen T. S.,
    3. Manalan A.,
    4. Klee C. B.,
    5. Cohen P.
    Discovery of a Ca2+- and calmodulin-dependent protein phosphatase: probable identity with calcineurin (CaM-BP80).FEBS Lett. 137 1982 80 84
    OpenUrlCrossRefPubMedWeb of Science
  46. 46.↵
    1. Timerman A. P.,
    2. Ogunbumni E.,
    3. Freund E.,
    4. Wiederrecht G.,
    5. Marks A. R.,
    6. Fleischer S.
    The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein. Dissociation and reconstitution of FKBP-12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum.J. Biol. Chem. 268 1993 22992 22999
    OpenUrlAbstract/FREE Full Text
  47. 47.↵
    1. Wiech H.,
    2. Buchner J.,
    3. Zimmermann R.,
    4. Jakob U.
    Hsp90 chaperones protein folding in vitro.Nature 358 1992 169 170
    OpenUrlCrossRefPubMedWeb of Science
  48. 48.↵
    1. Xu Y.,
    2. Lindquist S.
    Heat-shock protein hsp90 governs the activity of pp60v-src kinase.Proc. Natl. Acad. Sci. USA 90 1993 7074 7078
    OpenUrlAbstract/FREE Full Text
  49. 49.↵
    1. Yahara I.,
    2. Iida H.,
    3. Koyasu S.
    A heat shock-resistant variant of Chinese hamster cell line constitutively expressing heat shock protein of Mr 90,000 at high level.Cell Struct. Funct. 11 1986 65 73
    OpenUrlCrossRefPubMed
  50. 50.↵
    1. Yonehara M.,
    2. Minami Y.,
    3. Kawata Y.,
    4. Nagai J.,
    5. Yahara I.
    Heat-induced chaperone activity of HSP90.J. Biol. Chem. 271 1996 2641 2645
    OpenUrlAbstract/FREE Full Text
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KEYWORDS

calcineurin
Calcium-Binding Proteins
HSP90 Heat-Shock Proteins
Heat-Shock Proteins
Phosphoprotein Phosphatases
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
Salts

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