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Requirement for Hsp90 and a CyP-40-type Cyclophilin in Negative Regulation of the Heat Shock Response*

  • Andrea A. Duina
    Affiliations
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
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  • Helen M. Kalton
    Affiliations
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
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  • Richard F. Gaber
    Correspondence
    To whom correspondence should be addressed. Tel.: 847-491-5452; Fax: 847-467-1422;
    Affiliations
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
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  • Author Footnotes
    * This work was supported by National Science Foundation Grant MCB-9724050 (to R. F. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:July 24, 1998DOI:https://doi.org/10.1074/jbc.273.30.18974
      The heat shock response is a highly conserved mechanism that allows cells to withstand a variety of stress conditions. Activation of this response is characterized by increased synthesis of heat shock proteins (HSPs), which protect cellular proteins from stress-induced denaturation. Heat shock transcription factors (HSFs) are required for increased expression of HSPs during stress conditions and can be found in complexes containing components of the Hsp90 molecular chaperone machinery, raising the possibility that Hsp90 is involved in regulation of the heat shock response. To test this, we have assessed the effects of mutations that impair activity of the Hsp90 machinery on heat shock related events inSaccharomyces cerevisiae. Mutations that either reduce the level of Hsp90 protein or eliminate Cpr7, a CyP-40-type cyclophilin required for full Hsp90 function, resulted in increased HSF-dependent activities. Genetic tests also revealed that Hsp90 and Cpr7 function synergistically to repress gene expression from HSF-dependent promoters. Conditional loss of Hsp90 activity resulted in both increased HSF-dependent gene expression and acquisition of a thermotolerant phenotype. Our results reveal that Hsp90 and Cpr7 are required for negative regulation of the heat shock response under both stress and nonstress conditions and establish a specific endogenous role for the Hsp90 machinery in S. cerevisiae.
      All cells possess a defense mechanism known as the heat shock response, which allows them to survive exposure to otherwise lethal doses of certain stresses (
      • Craig E.A.
      ,
      • Lindquist S.
      ,
      • Morimoto R.I.
      • Sarge K.D.
      • Abravaya K.
      ). These stresses include environmental challenges, such as elevated temperatures, and pathophysiological states, such as viral infections (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ). The heat shock response is characterized by increased synthesis of a set of proteins collectively referred to as heat shock proteins (HSPs)
      The abbreviations used are: HSP, heat shock protein; HSF, heat shock transcription factor; HSE, heat shock element.
      1The abbreviations used are: HSP, heat shock protein; HSF, heat shock transcription factor; HSE, heat shock element.
      whose principal role is to assist target substrates in their synthesis, transport, and proper folding (
      • Parsell D.A.
      • Lindquist S.
      ,
      • Mager W.H.
      • Ferreira P.M.
      ,
      • Craig E.A.
      • Weissman J.S.
      • Horwich A.L.
      ). The requirement for chaperoning activities increases as cells are exposed to elevated temperatures or to other conditions that promote protein denaturation and aggregation. Because chaperoning activity is also crucial for the function of proteins not exposed to stress, HSPs play important roles for life under normal conditions as well.
      Expression of HSPs is under the control of heat shock transcription factors (HSFs) (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ,
      • Wu C.
      ,
      • Sorger P.K.
      ). In most eukaryotic systems, HSF is maintained as a monomer unable to bind DNA until activated by stress (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ,
      • Wu C.
      ). Activated HSF forms homotrimers capable of binding to heat shock elements (HSEs) present at promoters of genes encoding HSPs, ultimately leading to transcriptional activation (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ,
      • Wu C.
      ). The monomer to trimer transition is believed to be negatively regulated, at least in part, by Hsp70 (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ,
      • Wu C.
      ). The acquisition of transcriptional activity by HSF is correlated with increased phosphorylation (
      • Sorger P.K.
      • Pelham H.R.
      ); however, the functional relationship between phosphorylation and regulation of the heat shock response is still not fully understood.
      In contrast to most eukaryotic cells, in Saccharomyces cerevisiae HSF is bound to HSEs even in the absence of stress (
      • Jakobsen B.K.
      • Pelham H.R.B.
      ). This observation led to the proposal that yeast HSF bypasses the monomer to trimer and DNA binding regulatory steps. However, subsequent work has shown that heat shock treatment leads to increased HSE occupancy by HSF at the yeast HSP82 promoter (
      • Giardina C.
      • Lis J.T.
      ), suggesting that regulation of HSF activity in yeast is mediated in part by conversion of HSF from a non-DNA binding form to one competent to bind HSEs.
      Perhaps the best characterized aspect of HSF regulation in S. cerevisiae comes from investigation of the functional relationship between Hsp70 and the heat shock response. Mutations that decrease Hsp70 levels confer increased expression of several HSPs and constitutive thermotolerance (
      • Craig E.A.
      • Jacobsen K.
      ,
      • Werner-Washburne M.
      • Stone D.E.
      • Craig E.A.
      ). Furthermore, these mutants exhibit a slow growth phenotype that can be suppressed by a mutation inHSF that decreases function of the transcription factor (
      • Halladay J.T.
      • Craig E.A.
      ). These and other results from both mammalian and yeast systems, including the observation that the heat shock response is transient in nature, have led to the proposal for an autoregulatory loop in which Hsp70 normally represses HSF activity. According to this model, during heat shock Hsp70 dissociates from HSF, resulting in a net increase in synthesis of HSPs, including Hsp70 itself. Elevated levels of Hsp70 in turn lead to increased binding of the chaperone to HSF resulting in repression of HSF and subsequent down-regulation of the response (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ,
      • Wu C.
      ,
      • Craig E.A.
      • Gross C.A.
      ).
      In addition to Hsp70, other molecular chaperones have been proposed to be involved in the regulation of the heat shock response (
      • Sorger P.K.
      ). A recent study has shown that the mammalian cochaperone Hdj1 is involved in negative regulation of HSF1 activity (
      • Shi Y.
      • Mosser D.D.
      • Morimoto R.I.
      ). Because yeast and mammalian HSF can physically associate with Hsp90 (
      • Nair S.C.
      • Toran E.J.
      • Rimerman R.A.
      • Hjermstad S.
      • Smithgall T.E.
      • Smith D.F.
      ,
      • Nadeau K.
      • Das A.
      • Walsh C.T.
      ),
      S. Lindquist, personal communication.
      2S. Lindquist, personal communication.
      it is possible that the Hsp90 machinery also participates in regulation of the heat shock response. Furthermore, because Hsp70 and Hsp90 can be found together in the same protein complexes (
      • Pratt W.B.
      • Toft D.O.
      ), some of the functions ascribed to Hsp70 in regulation of HSF activity may actually reflect a joint effect with Hsp90.
      Hsp90 associates with several proteins, including the cyclophilin CyP-40, to form heterocomplexes that regulate the activity of a number of cellular factors, such as steroid receptors and oncogenic tyrosine kinases (
      • Pratt W.B.
      • Toft D.O.
      ,
      • Brugge J.S.
      ). More recently, mammalian Hsp90 has been shown to associate with endothelial nitric oxide synthase and to facilitate activation of the synthase in response to different signals (
      • Garcia-Cardena G.
      • Fan R.
      • Shah V.
      • Sorrentino R.
      • Cirino G.
      • Papapetropoulos A.
      • Sessa W.C.
      ). Although S. cerevisiae has been widely used to study the requirements for Hsp90 and Hsp90-associated proteins on activities of heterologous substrates (
      • Picard D.
      • Khursheed B.
      • Garabedian M.J.
      • Fortin M.G.
      • Lindquist S.
      • Yamamoto K.R.
      ,
      • Duina A.A.
      • Chang H.-C.J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ,
      • Chang H.-C.J.
      • Nathan D.F.
      • Lindquist S.
      ,
      • Xu Y.
      • Lindquist S.
      ), to date no endogenous role for Hsp90 has been identified in yeast. To test the possibility that the Hsp90 machinery is involved in regulation of the heat shock response, we have taken advantage of recently discovered mutations that significantly decrease the effectiveness of the Hsp90 machinery in S. cerevisiae and assessed their effects on HSF-dependent events. Our results show that Hsp90 and the yeast Cyp-40 homolog Cpr7 are required for negative regulation of the heat shock response in yeast.

      DISCUSSION

      The discovery that the yeast CyP-40 homolog Cpr7 is required for full Hsp90 activities in vivo (
      • Duina A.A.
      • Chang H.-C.J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ) provided us with a new genetic tool with which to analyze the role of the Hsp90 machinery in regulation of the heat shock response. Our results reveal that Cpr7 and Hsp90 negatively regulate HSF activity under both nonstress and stress conditions. The magnitude of increased expression of HSF-regulated genes in cpr7Δ cells showed that Cpr7 plays a major role in this regulation. Furthermore, our data demonstrate that Cpr7 and Hsp90 function to repress HSF-dependent gene expression in a synergistic manner, supporting the notion that the activities of Cpr7 and Hsp90 in regulation of the heat shock response are closely allied.
      Expression of HSF-responsive genes increases sharply when cells are exposed to environmental stresses. We have discovered that in Hsp90 mutant cells heat shock regimens that are normally sufficient to confer the maximal heat shock response result in the induction of HSF-dependent genes to levels significantly greater than those observed in wild-type cells under the same conditions. This suggests that even during heat shock Hsp90 functions to negatively regulate HSF activity.
      It is formally possible that increased expression of HSF-responsive genes in cells with reduced Hsp90 activity occurs through a mechanism independent of HSF. Because HSF is an essential gene in S. cerevisiae (
      • Sorger P.K.
      • Pelham H.R.
      ), this possibility can not be tested directly by assessing the effects of Hsp90 mutations in cells lacking HSF. However, the observation that three different HSF-responsive genes are each affected in a similar manner by mutations in Hsp90 and CPR7suggests that the increase in gene expression occurs via derepression of HSF activity. More importantly, the demonstration that an HSF-responsive reporter gene (HSE2::lacZ) that is made independent of HSF through point mutations that abolish HSF-binding (HSE12::lacZ) is no longer affected by Hsp90 mutations strongly supports the notion that Hsp90 and its associated proteins repress heat shock-related events by negatively regulating HSF activity.
      How might the Hsp90 machinery regulate HSF activity? It is possible that negative regulation of HSF by Hsp90 occurs independently of the ability to associate with each other. For example, Hsp90 could separately interact with and inhibit a positive regulator of the heat shock response upstream from HSF. Alternatively, consistent with proposals that protein denaturation can signal activation of the heat shock response (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ,
      • Wu C.
      ,
      • Sorger P.K.
      ), one or more proteins might induce the response simply by failing to achieve native conformations in the absence of the chaperone. This would raise the intriguing possibility that disruption of the interaction between Hsp90 and certain substrates is a specific mechanism by which cells sense stress. Such a signal would likely be mediated by one or a few key factors and not by a state of general protein denaturation, because recent results demonstrate that the in vivo substrates of Hsp90 are highly restricted (
      • Nathan D.F.
      • Harju Vos M.
      • Lindquist S.
      ). Because Hsp90 can interact with HSF in both mammalian (
      • Nair S.C.
      • Toran E.J.
      • Rimerman R.A.
      • Hjermstad S.
      • Smithgall T.E.
      • Smith D.F.
      ) and yeast cells (
      • Nadeau K.
      • Das A.
      • Walsh C.T.
      ),2 a particularly attractive model is one in which HSF is negatively regulated by physical association with Hsp90 under nonheat shock conditions. During heat shock, one or more components of the Hsp90 heterocomplex may dissociate from HSF in a temperature-dependent manner, thereby allowing HSF-mediated activation of transcription. Consistent with this proposal, Nairet al. (
      • Nair S.C.
      • Toran E.J.
      • Rimerman R.A.
      • Hjermstad S.
      • Smithgall T.E.
      • Smith D.F.
      ) have shown that the Hsp90-HSF1 interaction can be disrupted by moderately elevated temperatures. Regulation of HSF activity by the Hsp90 complex is reminiscent of the role postulated for Hsp70 in HSF regulation (
      • Morimoto R.I.
      • Jurivich D.A.
      • Kroeger P.E.
      • Mathur S.K.
      • Murphy S.P.
      • Nakai A.
      • Sarge K.D.
      • Abravaya K.
      • Sistonen L.T.
      ,
      • Wu C.
      ,
      • Craig E.A.
      • Gross C.A.
      ). Whether Hsp70 and Hsp90 function independently in this regard is unknown. Nevertheless, the discovery that Hsp90 and a member of the CyP-40 class of cyclophilins are required for negative regulation of the heat shock response establishes the first specific endogenous role for these molecules in S. cerevisiae.

      ACKNOWLEDGEMENTS

      We thank S. Lindquist for Hsp104 and L3 antibodies, and for the hsc82Δ hsp82Δ hsp82G170Dcells. We also thank D. Winge for the pHSE2-lacZ andpHSE12-lacZ reporter genes, E. A. Craig for thepZJHSE2–26 reporter gene, and D. Thiele and D. Gross for HSF antibodies.

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