Fungal Hsp90: a Biological Transistor That Tunes Cellular Outputs to Thermal Inputs

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Fungal Hsp90: a biological transistor that tunes cellular outputs to thermal inputs

Michelle D. Leach1,2, Edda Klipp3, Leah E. Cowen1 and Alistair J. P. Brown2 Abstract | Heat shock protein 90 (HSP90) is an essential, abundant and ubiquitous eukaryotic chaperone that has crucial roles in protein folding and modulates the activities of key regulators. The fungal Hsp90 interactome, which includes numerous client proteins such as receptors, protein kinases and transcription factors, displays a surprisingly high degree of plasticity that depends on environmental conditions. Furthermore, although fungal Hsp90 levels increase following environmental challenges, Hsp90 activity is tightly controlled via post-translational regulation and an autoregulatory loop involving heat shock transcription factor 1 (Hsf1). In this Review, we discuss the roles and regulation of fungal Hsp90. We propose that Hsp90 acts as a biological transistor that modulates the activity of fungal signalling networks in response to environmental cues via this Hsf1–Hsp90 autoregulatory loop.

Heat shock protein 90 (HSP90) was first described divergence9. On this basis, one might have expected the among a defined set of HSPs that are rapidly induced in heat shock response to have diverged in fungi that inhabit fungal, plant and animal cells in response to acute ther- thermally buffered niches, such as the clinically important mal upshifts1–5. This HSP induction, which underpins the pathogen Candida albicans10, which is obligately associ- molecular adaptation to thermal insults, represents ated with warm-blooded animals11. Nevertheless, this the heat shock response that is ubiquitous across the response is strongly conserved in C. albicans, and heat bacterial, archaeal and eukaryotic domains1–5. HSPs have shock adaptation is essential for its virulence10,12. This been divided into families based on their molecular mass. reflects the fact that the heat shock apparatus is essential The HSP90–HtpG, HSP70–DnaK and HSP60–GroEL for cellular adaptation to the subtle or gradual thermal (also known as GroL) families tend to display strong transitions that organisms often experience in the wild, evolutionary conservation from bacteria to humans6,7. not just to the acute temperature upshifts that experimen- 13 1Department of Molecular Smaller HSPs, including Hsp42 and Hsp26 in the yeast talists tend to examine in the laboratory . In particular, Genetics, University of Saccharomyces cerevisiae, display greater evolutionary the strong conservation of Hsp90 attests to the fundamen- Toronto, Toronto, Ontario divergence5,8. Most of these HSPs are protein chaperones, tal importance of the cellular functions executed by this M5S 1A8, Canada. promoting folding and assembly of newly synthesized essential chaperone in fungal cells. Indeed, Hsp90 is essen- 2 School of Medical Sciences, proteins, and degradation or repair of damaged proteins tial for the growth and viability of evolutionarily divergent Institute of Medical Sciences, University of Aberdeen, that have become dissociated or have formed aggregates yeasts such as S. cerevisiae, Schizosaccharomyces pombe 14–16 Foresterhill, Aberdeen as a result of thermal or chemical stress. and C. albicans, even under normal growth conditions . AB25 2ZD, United Kingdom. The high extent of evolutionary conservation for the HSP90 is an essential component of the cytoplasmic 3Theoretische Biophysik, heat shock response across the fungal kingdom is intrigu- HSP90–HSP70 chaperone network that promotes pro- Institut für Biologie, Mathematisch- ing. This conservation might not seem surprising, as fungi tein folding and refolding in eukaryotic cells. HSP70 Naturwissenschaftliche occupy highly divergent environmental niches, where promotes the initial folding of certain nascent polypep- Fakultät I, Humboldt- they can be exposed to dramatic thermal fluctuations. tides as they emerge from the ribosome. Some of these Universität zu Berlin, However, other adaptive responses (to osmotic, oxidative proteins are then passed to HSP90, which facilitates the Invalidenstraße 42, and cell wall stresses, for example) have diverged substan- later stages of their folding and, in some cases, maintains 10115 Berlin, Germany. 9 17 Correspondence to A.J.P.B. tially across the fungal kingdom . Some key stress regula- them in a near-native conformational state . Indeed, e-mail: [email protected] tors have been conserved, but many upstream sensors and HSP90 has vital roles in the folding and maintenance doi:10.1038/nrmicro2875 downstream transcriptional regulators show considerable of a specific subset of proteins, termed ‘client proteins’.

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The Hsp90 chaperone cycle Proteotoxic stresses As a direct consequence of its role in maintaining Cellular stress conditions that the structural integrity of its client proteins, HSP90 is The structure of the chaperone Hsp90 and its conforma- prompt the accumulation of thought to generate ‘protein-folding reservoirs’ that can tional dynamics have been the subject of recent elegant unfolded or damaged proteins, buffer the phenotypic impact of mutations in client pro- reviews17,22,23. Briefly, Hsp90 has three domains and oper- or the formation of protein teins, thereby facilitating evolutionary change18. Hence, ates as a dimer24–26 (FIG. 1). The ~180‑residue carboxy- aggregates. HSP90 has acted as an evolutionary capacitor during terminal domain mediates constitutive dimerization, 19–21 Heat shock elements eukaryotic evolution . However, this Review focuses whereas the amino-terminal domain of ~215 residues Consensus sequences that on the impact of fungal Hsp90 over cellular, rather contains the ATP-binding domain. These domains are are present in the promoter than evolutionary, timescales. We focus on the effect of separated by an ~260‑residue central (middle) domain regions of heat shock genes and are bound by heat shock temperature on the interactions of fungal Hsp90 with its that mediates many Hsp90–client protein interactions. transcription factor 1 (Hsf1), client proteins. We suggest that, in addition to acting as The middle and N-terminal domains are connected by thereby activating the a capacitor over evolutionary timescales, Hsp90 acts as a a charged linker, mutations in which affect interactions expression of these genes. biological transistor over cellular timescales by modulat- with some client proteins and co‑chaperones. ing the activities of key signalling networks in response The flexible Hsp90 dimer undergoes major confor- to dynamic changes in environmental conditions. mational shifts during a dynamic chaperone cycle that is driven by ATP hydrolysis17,23–26 (FIG. 1). In the absence of ATP binding, the dimer takes up an open, V‑shaped Hsp90 ADP ADP conformation in which the two N-terminal domains are N N separated and the two subunits are held together via their C-terminal domains. ATP binding to the N-terminal M M COMPACT domain stimulates the closing of a lid over the nucleotide- binding pocket followed by the relatively slow formation of a closed form, in which the two N-terminal domains C C in the Hsp90 dimer associate closely together. Hsp90 has a weak intrinsic ATPase activity that is modulated by interactions with client proteins and co‑chaperones. After ATP hydrolysis, substantial remodelling occurs to regen- erate the open form of the protein27. The ATPase cycle ADP ADP is slow, with yeast Hsp90 hydrolysing an ATP molecule N N every 1–2 minutes28,29. This conformational cycle differs M M between species but remains crucial for the maturation of client proteins27,30. The classical pharmacological inhibitors of Hsp90, geldanamycin and radicicol, dock at ATP ATP P 2 ADP 17,31 i C C the ATP-binding site in the N-terminal domain , pro- N N N N viding useful tools for the dissection of Hsp90 function. Client M M M M Hsp90 is regulated at multiple levels Transcriptional control of HSP90. Hsp90 is naturally C C C C abundant in fungal cells and is induced to even greater levels by heat shock and other proteotoxic stresses10,15,32–35. CLOSED ATP ATP OPEN Hsp90 protein levels are regulated both transcriptionally 2 ATP N N and post-transcriptionally (FIG. 2). M M HSP90 transcription is controlled by heat shock transcription factor (HSF), a key regulator of the heat Hsp70 shock response36,37 that is evolutionarily conserved from C C Client S. cerevisiae (Hsf1) to mammals (HSF1 and HSF2). Hsf1 (unfolded) is essential for viability in yeasts10,38,39 and is required 10,39,40 Co-chaperone for the basal expression of HSP genes . It acts as a trimer, binding constitutively to heat shock elements Figure 1 | The Hsp90 chaperone cycle. The shape of the heat shock protein 90 (HSEs) in the promoters of HSP genes41–44. Hsf1 is acti- (Hsp90) dimer, illustrating the amino-terminal (N), middle (M) and carboxy-terminal (C) vated by hyperphosphorylation in response to specific domains; the shape of the Hsp90 monomer is adapted fromNature that Reviewsin REF. 26 | .Microbiology The Hsp90 environmental cues, which in some fungi include glu- 17,23 chaperone cycle has been reviewed recently . Hsp90 acts as a dimer that can take up cose starvation, the presence of superoxide, high oxygen various dynamic conformations in its ADP-bound state, in which the N-terminal concentrations and changes in membrane lipid composi- domains can be apart or closely associated (compact form). The Hsp90 dimer takes up tion, as well as heat shock10,39,45,46. Hsf1 activation drives an open state following release of ADP. The rapid association with ATP is linked with 10,47,48 interactions between Hsp90 and co‑chaperones, Hsp70 and unfolded or partially increased HSP gene transcription , primarily via the 12,49 folded client proteins. Different co‑chaperones can associate with different domains Hsf1 C-terminal activation domain , and this leads to 37 of the Hsp90 molecule and mediate interactions with distinct client proteins17,23. the accumulation of Hsp proteins . This is followed by the slow formation of a closed complex and the release of Hsp70 Notably, fungal Hsf1 is only temporarily activated and co‑chaperones. The folded client protein is then released, and the ATP is on thermal upshifts10. Hsf1 is rapidly phosphorylated hydrolysed to ADP. and then dephosphorylated after a 30–42 °C heat shock

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Both pharmacological inhibition and genetic deple- Heat shock tion of Hsp90 induces Hsf1 activation in C. albicans (M.D.L., A.J.P.B. and L.E.C., unpublished observations). P P Furthermore, a physical interaction between Hsp90 and Hsf1 Hsf1 has been confirmed by co‑immunoprecipitation HSP90 (M.D.L., A.J.P.B. and L.E.C., unpublished observations). HSE Therefore, following heat shock in fungi, Hsp90‑mediated Transcription Heat repression of Hsf1 is released, Hsf1 becomes activated shock and this leads to a transcriptionally mediated increase Autoregulatory in Hsp90 levels13. This response is then downregulated circuit HSP90 An when excess Hsp90 binds to Hsf1.

Heat Translation shock Post-transcriptional control of HSP90. In C. albicans, HSP90 transcript levels increase dramatically on thermal upshifts13, but Hsp90 levels do not increase to the Hsp90 same extent. Regulation of translation in heat-shocked 2 Heat Post-translational Degradation Heat Drosophila cells was reported three decades ago . shock modiﬁcations shock Translational regulation is not thought to occur in yeast following heat shock, as the patterns of protein synthe- Hsp90 Hsp90 Hsp90 sis generally reflect the dynamically changing mRNA 2 P SNO Ac populations during heat shock adaptation . However, most yeast heat shock mRNAs carry fairly unstructured 5ʹ leader regions that are less reliant on cap-dependent Hsp70 Co-chaperone mechanisms of translation initiation60. Hsp90 levels may also be regulated by protein turn Protein folding Client Client over. S. cerevisiae Hsp90 can be targeted for ubiquitin- mediated degradation by the cell cycle-regulated mitosis Figure 2 | Hsp90 levels and activity are regulated at multiple levels. The transcription inhibitor protein kinase, Swe1 (REF. 61). Hsp90 stability Nature Reviews | Microbiology of the heat shock protein 90 (HSP90) gene in yeast is regulated by heat shock is probably also regulated in C. albicans, as Hsp90 levels transcription factor 1 (Hsf1), the activation of which is negatively regulated by Hsp90 decline rapidly during adaptation to elevated tempera- via an autoregulatory loop13 that involves a physical interaction between Hsp90 and Hsf1 tures (M.D.L. and A.J.P.B, unpublished observations). (M.D.L., A.J.P.B. and L.E.C., unpublished observations). During heat shock, yeast HSP mRNAs Clearly, the amount of Hsp90 in the cell is tightly regulated might be preferentially translated, and Hsp90 turnover might also be modulated (see main at multiple levels. text for details). Hsp90 activity is controlled by post-translational modifications such as phosphorylation, S‑nitrosylation (SNO) and possibly acetylation. These changes influence the interaction of Hsp90 with specific co‑chaperones and, hence, affect the folding of Post-translational modifications of Hsp90. HSP90 activity specific subsets of client proteins. Furthermore, after proteotoxic stress, a change in Hsp90 is regulated by post-translational modifications. It has availability (mediated by altered HSP90 expression and changes in the amount of Hsp90 been known for some time that HSP90 is phosphoryl- associated with unfolded proteins) is predicted to affect the interaction of Hsp90 with ated in mammalian cells62,63. Similarly, phosphorylation client proteins13. of S. cerevisiae Hsp90 is thought to influence its activity and, hence, the rate of maturation of specific client proteins64,65. Yeast Hsp90 is phosphorylated by Swe1 and in C. albicans13, suggesting that this protein is regu- casein kinase 2 (CK2), and dephosphorylated by serine/ lated via a negative feedback loop. Almost three dec- threonine protein phosphatase T (Ppt1)61,64–66. CK2 ades ago, it was postulated that feedback components also phosphorylates Hsp90 in C. albicans67. However, downregulate the heat shock response2,50,51. Initially, the relationship between Hsp90 phosphorylation and HSP70 proteins were thought to be the key HSF repres- activity is complex. For example, Hsp90 hyperphos- sors in mammalian systems52–54, and Hsf1 is known to phorylation, induced by inactivation of Ppt1, leads to interact with Hsp70 family members in S. cerevisiae55. a reduction in the activity of the Hsp90 chaperone sys- However, yeast Sse1 (an Hsp70 protein) is required for tem64. Conversely, blocking Swe1‑mediated phosphory Hsp90‑dependent functions56, suggesting that Hsp90 is lation of Hsp90 at tyrosine 24, which normally occurs Hyperphosphorylation the Hsf1 repressor in fungi. in a cell cycle-dependent manner, inhibits Hsp90 from The phosphorylation of a Pharmacological inhibition of mammalian HSP90 interacting with a specific subset of co‑chaperones target protein at multiple correlates with HSF1 activation57. Furthermore, human (activator of Hsp90 ATPase 1 (Aha1)) and client pro- residues. HSF1 interacts physically with HSP90 complexes57,58, and teins (Ste11 and Slt2 (known as Mpk1 in C. albicans))61. S‑nitrosylated this is thought to repress HSF1 transcriptional activity. Also, phosphorylation at threonine 22 by CK2 modulates Containing a covalently No such physical interaction between Hsf1 and Hsp90 Hsp90 interactions with the co‑chaperones cell division attached nitrosyl group on the has been published for the fungal kingdom. However, cycle 37 (Cdc37; also known as p50 in mammals) and thiol moiety of one or more mutations that interfere with Hsp90 function dere- Aha1 (REF. 65). In addition, yeast Hsp90 is S‑nitrosylated, cysteine residues in a protein. 68 These nitrosyl groups are press the expression of Hsf1‑dependent reporter genes which affects its dimerization dynamics and activity , 59 69 added as a result of nitrosative in S. cerevisiae , and an Hsf1–Hsp90 autoregulatory and by analogy with its mammalian counterpart , yeast stress. loop has now been confirmed in C. albicans (FIG. 2). Hsp90 might also be acetylated.

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Synthetic genetic Co‑chaperone-mediated modulation of Hsp90 function. The Hsp90 interactome phenotypes Hsp90 activity is further regulated by its interactions with The global HSP90 interactome (the subset of cellular Phenotypes that are not various cofactors, or co‑chaperones, which influence the proteins that interact with HSP90) has been defined in apparent as a result of a single binding specificity of Hsp90 for particular client proteins. fungi and mammalian cells using a range of unbiased, perturbation alone, but are revealed by combining two An association with a specific co‑chaperone is thought genome-wide approaches. The elaboration of the fungal mutations or genetic and to fix Hsp90 in a specific open conformation that helps to Hsp90 interactome has combined the proteomic iden- pharmacological perturbations. establish the binding specificity for that Hsp90 mol- tification of components of tandem affinity-purified ecule and its chaperone complex23,70–73. A range of Hsp90 complexes with molecular two-hybrid-based screens for co‑chaperones have been identified in yeast, most of Hsp90‑interacting proteins, genetic screens for muta- which are conserved in mammals (TABLE 1). Yeast Hsp70 tions that confer hypersensitivity to Hsp90 inhibitors and Hsp40 proteins, the other members of the major such as geldanamycin and Macbecin II, and screens cytoplasmic chaperone network, have been described as for mutations that confer synthetic genetic phenotypes in Hsp90 co‑chaperones. Yeast Hsp70 associates with Hsp90 combination with a temperature-sensitive hsp90 muta- via the adaptor protein Sti1 (REF. 74) to form the minimal tion (hsp82G170D)67,78,91–93. The fungal interactome has been Hsp90 core complex75. Sti1 inhibits the ATPase activ- experimentally defined in S. cerevisiae78,92, S. pombe94 ity of Hsp90 (REF. 75), as does the co‑chaperone Cdc37 and C. albicans67. These data have been merged with (REF. 70). By contrast, Aha1 enhances the ATPase activity experimental HSP90 interaction data sets from plants, of Hsp90 (REFS 72,76), and another co-chaperone, Sba1 nematodes, flies and mammals to generate a conceptual (also known as p23 in mammals), couples this ATPase human HSP90 chaperone database, Hsp90Int.DB95. activity to polypeptide release77. The interactions of Sba1, The fungal Hsp90 interactome is large, comprising Cdc37 and Aha1 with Hsp90 are thought to be mutu- about 10% of the proteome (see FIG. 3 for examples). ally exclusive26. Additional yeast co‑chaperones include This is hardly surprising given that Hsp90 is a central Tah1 (REF. 78), Pih1 (REF. 78), Cns1 (REFS 79,80) and Sgt1 component of a large molecular machine, the role of (REF. 81), as well as Cpr6 and Cpr7 (also known as Cyp7), which is to promote the folding of numerous client pro- two homologues of mammalian cyclophilin 40 (CYP40; teins. Nevertheless, the evidence suggests that, despite also known as PPIase D or PPID)82. the breadth of the Hsp90 interactome, it comprises a The interaction of Hsp90 with these various specific subset of cellular proteins. For example, the co‑chaperones is thought to impose distinct architec- Hsp90 interaction network displays distinct topological tures on the Hsp90 complex, and this in turn drives features, such as its high density and connectivity, that interactions with different client proteins23,26. For reflect the underlying biological connections of this bio- example, Cdc37 mediates interactions with protein logical machine95. The consistent identification of HSP90 kinases in yeast23,26, and the same is true of CDC37 in co‑chaperones and cofactors in the various unbiased mammalian cells83,84. By contrast, Sgt1 contributes to global screens that have been carried out attests to the kinetochore assembly85, whereas Tah1 and Pih1 are validity of their biological outputs67,78,91–93. Furthermore, involved in chromatin remodelling and epigenetic the validity of numerous fungal Hsp90 client proteins regulation78,86. Inactivation of Tah1 and Pih1 has also identified in genome-wide screens has been confirmed been shown to impair the maturation of client proteins experimentally67,78,92. However, important differences do in S. cerevisiae78. This illustrates the breadth of cellular exist between the Hsp90 clients of S. cerevisiae and those processes that the HSP90 chaperone complex super- of C. albicans67, suggesting considerable evolutionary vises in eukaryotes87. Consequently, both mammalian plasticity in the fungal Hsp90 interactome. and fungal HSP90 proteins have become major foci for Members of the HSP90 interactome are divis- the development of anticancer and antifungal drugs88–90. ible into two main classes: HSP90 client proteins, and

Table 1 | Hsp90 co‑chaperones in fungi Co-chaperone* Essentiality‡ Function Sti1 (HOP) Non-essential Binds to the amino and carboxyl termini of Hsp90, preventing the amino‑terminal dimerization reaction that is required for ATP hydrolysis140 Aha1 (AHA1) Non-essential Enhances the ATPase activity of Hsp90 (REFS 72,76) Cdc37 (p50) Essential Links Hsp90 to client protein kinases70 and inhibits Hsp90 ATPase activity140 Pih1 Non-essential Interacts with Hsp90 and DNA helicases78 Cns1 (TTC4) Essential Interacts with the Hsp90 carboxyl terminus141 Tah1 Non-essential Interacts with the Hsp90 carboxyl terminus and DNA helicases78 Sgt1 (SGT1) Essential Binds non-ATP-bound forms of Hsp90, linking Hsp90 to client proteins81 Sba1 (p23) Non-essential Stabilizes the ATP-bound state of Hsp90 (REF. 77) Cpr6 and Cpr7 (CYP40) Non-essential Enhances the ATPase activity of Hsp90 (REF. 82) *The name of the mammalian orthologue is given in brackets. ‡Essentiality for viability in Saccharomyces cerevisiae. Aha1, activator of Hsp90 ATPase 1; Cdc37, cell division cycle 37; CYP40, cyclophilin 40; HOP, HSC70- and HSP90‑organizing protein;

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Kinases or phosphatases as clients. The protein phos- Hsp90 phatase calcineurin is an Hsp90 client protein in N N yeasts96,97. Calcineurin has a major role in calcium sig- M M nalling in eukaryotic systems. In yeasts, calcineurin signalling contributes to antifungal-drug tolerance, and Aha1 Cdc37 Cns1 Hsp90 calcineurin inhibitors such as cyclosporin and FK506 C C Cpr6 Cpr7 Pih1 Sba1 co-chaperones dramatically enhance the sensitivity of yeasts to anti- Sgt1 Sti1 Tah1 fungal drugs such as fluconazole and caspofungin98,99. Recently, genetic or pharmacological attenuation of 97,100 96,101 Environmental cues Exemplar Hsp90 clients Hsp90 function in S. cerevisiae and C. albicans was shown to block the activation of calcineurin. Cell growth Furthermore, a physical interaction between Hsp90 and Signalling 96,97 Cka1, Hog1, and division calcineurin has been demonstrated in both yeasts , Rim11, Slt2, Bem2, Cdc28, thereby confirming that this important regulatory mol- Tpk3, Yak1 Sla2, Ras2 ecule is an Hsp90 client protein. Calcineurin is stabilized by Hsp90 and becomes activated following its disso- Metabolism 97 Transcription Aat2, Erg2, ciation from the chaperone . This is important from a Cap1, Mig1, Msn4, Gat1, Hxk2 Cell Wall therapeutic point of view because Hsp90 inhibitors pro- Rim101, Tup1 Chs5, Ccw1, vide a potent mechanism for increasing the sensitivity Mnn9, Sun41 TF of pathogenic fungi, including C. albicans, Aspergillus fumigatus and Aspergillus terreus, to clinically important Chromatin antifungal drugs20,102. remodelling Intracellular The S. cerevisiae mitogen-activated protein kinase Ada2, Spt3, traﬃcking Swi1, Snf2 Kin2, Sec34, (MAPK) Slt2, which is an essential component of the cell Vma9, Vps28 integrity signalling pathway, is an Hsp90 client protein91. Hsp90 interacts with the phosphorylated, active form Figure 3 | The fungal Hsp90 chaperone machine and exemplar client proteins. of Slt2. This interaction is essential for Slt2‑mediated The specificity of a heat shock protein 90 (Hsp90) complexNature depends Reviews on which | Microbiology activation of the transcription factor Rlm1 (REF. 91), co‑chaperone it binds. Co‑chaperones can bind different faces of Hsp90. Examples which regulates key cell wall biosynthesis enzymes of Hsp90 client proteins that are representative of the major cellular processes that required for the maintenance of cell wall integrity when Hsp90 influences are listed in green boxes. C, carboxy-terminal domain; M, middle cells are exposed to cell wall stresses such as antifungal domain; N, amino-terminal domain; TF, transcription factor. The Hsp90 monomer drugs103,104. This chaperone–client relationship has been image is adapted from that in REF. 26. evolutionarily conserved in C. albicans. The C. albicans orthologue of Slt2, Mkc1, is also regulated by Hsp90, and depletion of the chaperone leads to destabilization of this the HSP90 co‑chaperones and cofactors that promote the crucial MAPK and inactivation of the cell integrity sig- conformational integrity of these client proteins. There nalling pathway101. Therefore, the increased sensitivity is considerable overlap between fungal and mamma- of yeasts to antifungal drugs that is imparted by Hsp90 lian cells with regard to the categories of HSP90 client inhibitors is mediated by downregulation of Mkc1 (or proteins that have been defined to date. The two main Slt2) and cell integrity signalling, as well as through inhi- categories, transcription factors and protein kinases or bition of calcineurin signalling. phosphatases, are clearly ubiquitous in eukaryotic sys- The highly conserved stress-activated MAPK Hog1 tems95 (see Supplementary information S1 (figure)). The (known as Sty1 in S. pombe) has crucial roles in stress third category is less well defined but is also conserved. adaptation in S. cerevisiae, S. pombe and C. albicans32,105–109, This category clusters together structurally unrelated and also contributes to virulence in C. albicans110. There mammalian HSP90 client proteins that include recep- fore, the fact that Hog1 has been identified as an Hsp90 tors involved in innate immunity and proteins involved client protein67,111 is important in terms of fungal stress in viral replication26. In fungal systems, this category adaptation and pathogenicity. It implies that Hsp90 probably includes Hsp90 clients involved in secre- modulates the activity of this key stress signalling path- tion and vesicular transport, as well as mitochondrial way. However, whereas Sty1 is activated by mild heat membrane components78,92. It is significant that fungal shocks in S. pombe112, Hog1 signalling decreases fol- Hsp90 client proteins are highly enriched for regula- lowing equivalent upshifts in C. albicans108. Therefore, tors that have crucial roles in the control of growth, cell Hsp90 might differentially modulate the activity of this division, environmental adaptation and development MAPK in these evolutionarily divergent yeasts. (FIG. 3). A more comprehensive listing of fungal Hsp90 Hsp90 also contributes to cell cycle regulation. The client proteins is provided in Supplementary informa- Hsp90 co‑chaperone Cdc37, as well as Hsp90 itself, tion S1 (figure), and examples of these three categories modulates the function of the crucial cell cycle regulator of Hsp90 client proteins are discussed below. Given the and cyclin-dependent kinase Cdc28 in S. cerevisiae61,113. focus of this Review on the modulation of cell signalling Furthermore, in C. albicans, Cdc28 interacts physically by Hsp90, our emphasis here is on protein kinases or with and is stabilized by Hsp90 (REF. 114). Importantly, phosphatases. cell cycle and morphogenesis are tightly regulated in

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C. albicans, and Hsp90 has been implicated for the functional categories that are substantially enriched thermal regulation of morphogenesis115, a key virulence in the outputs of these screens and by the fact that the factor in this pathogen. resultant Hsp90 interaction networks display topological features that clearly distinguish them from randomly Transcription factors as clients. The fungal Hsp90 selected networks95. However, the overlap between these interactome includes numerous transcription factors experimentally determined Hsp90 interaction networks and chromatin-remodelling components67,78,91–93,116 (see is limited when the growth conditions are changed. For Supplementary information S1 (figure)). Tah1 and Pih1 example, substantial differences are observed in the were identified through genome-wide screens as novel Hsp90 interactomes of C. albicans cells grown at 30 °C Hsp90 co‑chaperones in yeast, and their interactions or at 37 °C67, with more protein kinases appearing in the with Hsp90 have been confirmed experimentally78. In interactome when cells are grown at the higher tempera- yeast, Tah1 and Pih1 promote Hsp90‑mediated matura ture. Similarly, in S. cerevisiae, more proteins involved in tion of the glucocorticoid receptor (a model Hsp90 signal transduction, cell cycle, cytokinesis and budding substrate) and of Rvb1 and Rvb2 (essential components are observed in the 37 °C Hsp90 interactome than in of the yeast Ino80 chromatin-remodelling complex, the 30 °C interactome92. Other differences are observed which modulates the expression of ~ 5% of yeast genes), under different stress conditions. For example, cell wall as well as folding of subunits of the Swr1 chromatin- stresses strengthen the interactions between the MAPK remodelling complex (SWR-C; which controls gene Slt2 and Hsp90 in S. cerevisiae91 and encourage many expression close to heterochromatic regions)78. The list new proteins to become Hsp90 clients in C. albicans67. of Hsp90 clients also includes numerous S. cerevisiae Clearly, the Hsp90 interactome displays substantial and C. albicans transcription factors that are involved in environmental contingency67. Although considerable transcriptional reprogramming in response to environ energy has been devoted to the characterization of the mental cues (see Supplementary information S1 Hsp90 interactome, we suggest that much remains to (figure))67,78,91–93,116, reinforcing the view that Hsp90 be discovered about its environmental plasticity and, in underpins fungal adaptation and pathogenicity. particular, the temporal dynamics of this plasticity. For example, the under-representation of functions asso- Other types of client. The genome-wide screens for ciated with stress responses and protein folding in the Hsp90‑interacting proteins revealed other types of Hsp90 global Hsp90 interactome95 probably reflects the fact client protein, involved in processes such as secretion and that most of the genome-wide screens have been car- intracellular trafficking, cell wall synthesis and metabo- ried out essentially under steady-state conditions. The lism67,78,91–93,116 (FIG. 3 and see Supplementary informa- true plasticity of the Hsp90 interactome is likely to be tion S1 (figure)). Indeed, in a screen of S. cerevisiae best observed under transient conditions, when the deletion mutants, of the 25 mutants displaying the chaperone machine is contributing to dynamic cellular greatest sensitivity to the Hsp90 inhibitor Macbecin II adaptation in response to environmental insults. at 30˚C, 14 carried mutations in transport-related genes, including numerous vacuolar protein-sorting genes and Hsp90 circuitry — dynamic behaviours those encoding the four lobe B components of the bilobular Our understanding of the organization of cellular net- conserved oligomeric Golgi complex92. Consistent with works and their dynamic behaviours can be substantially this, Hsp90 inhibition was shown to reduce secretion improved by quantitative analyses and mathematical without affecting glycosylation, suggesting that Hsp90 modelling of these processes and of their responses to influences the functions of proteins involved in the external perturbations (BOX 1). Systems biology models secretory pathway92. These findings indicate that Hsp90 have increased our understanding of, for example, the has an impact on the fungal cell surface and thereby is dynamics of the S. cerevisiae cell cycle117; the highly likely to affect interactions between fungal pathogens complex response of osmotically stressed cells, involving and their hosts. cell signalling, gene regulation and metabolic adapta- tion118; and the impact of osmotic stress on cell cycle Plasticity of the Hsp90 interactome. Clearly, Hsp90 progression119. interacts specifically with a range of key cellular regula- Given its essentiality and ubiquity, the heat shock tors in fungi and modulates their activities under normal response is a good candidate for mathematical modelling. conditions (in the absence of an environmental stressor). At the network level, analysis of the effects of heat shock However, the various genomic characterizations of the on the organization of the yeast interactome revealed fungal Hsp90 interactome have highlighted a second that there is partial disintegration of the interactome important point: that this interactome displays input- in response to this stress120. In the absence of global dependent plasticity — that is, that it responds to and protein–protein interaction data sets generated under Chemical genomic screens varies with environmental conditions. heat shock conditions, transcriptomic data sets were Screens that combine The various proteomic, genetic and chemical genomic exploited to show that during heat shock there is a sub- small-molecule inhibitors or screens that have been carried out to characterize the stantial decrease in the connectivity between the mod- activators with genome-wide fungal Hsp90 interactome might give the impres- ules of the yeast interactome. For example, two central mutant collections to identify 67,78,91–93 mutations that confer sion that this interactome is robust . This view ribosomal modules, which reflect the major role of sensitivity or resistance to is reinforced by the reproducible identification of protein synthesis in unstressed growing yeast cells, dis- these molecules. key co‑chaperones in these screens, by the common play decreased community centrality in the interaction

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Box 1 | Modelling the structure and dynamics of regulatory networks but in the longer term, the chaperone system becomes overwhelmed, leading to an increased probability Systems biologists use different modelling approaches, depending on the specific of protein aggregation and cell death122. scientific question they wish to address, the particular system of interest, the nature More recently, an ODE (ordinary differential equa- and amount of available experimental data, and, simply, taste (for an introduction, see tion) model was used to investigate the dynamics of the REF. 138). Network-oriented approaches enumerate compounds such as proteins or 13 (FIG. 2) metabolites and analyse the type and frequency of their interactions. This allows the Hsp90–Hsf1 autoregulatory loop . The model identification of patterns or statistical correlations that are not obvious from analyses focuses on the Hsp90–Hsf1 interaction because the of the individual components. By contrast, dynamic models are usually based on the available data suggest that Hsp90 is the main chaperone characterization of the network and its interactions, describing their changes over that interacts with and represses Hsf1 (REFS 13,59). In this time. These changes might arise, for example, through inherently dynamic processes model, after heat shock, Hsp90 becomes sequestered in such as cell cycle or circadian rhythms, or as a result of external perturbation of complexes with unfolded and damaged proteins, and is the system, caused by factors such as environmental stresses or nutrient changes. therefore temporarily less available for complex forma- An important class of models describes the dynamics of the model components using tion with Hsf1. As a result, the released Hsf1 becomes systems of ODEs (ordinary differential equations) — that is, equations in which the activated and induces HSP90 transcription, thereby lead- concentrations of compounds and complexes are represented by (usually nonlinear) ing to the production of more Hsp90. Ultimately, excess functions describing the changes in concentrations over time, which can be represented as the sum of stoichiometric coefficients multiplied by the rates of the Hsp90 rebinds Hsf1, blocking further transcriptional 13 individual reactions. These reaction rates depend on the compound concentrations activation . Therefore, this model predicts that altera- at the time and on parameter values such as kinetic constants, binding constants, tions in ambient temperature lead to changes in the con- maximal rates or Hill coefficients. centration of free Hsp90 and influence the interactions Although our confidence about the wiring of networks is increased through, for of Hsp90 with one of its putative client proteins, Hsf1. example, high-throughput or dedicated protein–protein interaction studies or through Aspects of this model have been confirmed in vivo12, and metabolic reconstructions139, the precise form of rate expressions and the values Hsf1 has now been shown to be an Hsp90 client pro- of kinetic parameters are often elusive, and the choice of rate laws and respective tein (M.D.L., A.J.P.B. and L.E.C., unpublished observa- parameter values depends on the available experimental data and laborious parameter tions). Given the observed environmental plasticity of estimation exercises. Achieving the goal of models that are firmly based on quantitative the Hsp90 interactome67,92, thermal fluctuations are also data and that have predictive value is hindered by limitations in our ability to describe the whole cell and all its multifarious levels of regulation. Thus, to define the boundaries likely to influence the interactions of Hsp90 with other of the system being modelled, some assumptions must be made about certain client proteins. processes, and the system must be simplified to consider the major players relevant In principle, how might changes in ambient temper- to the scientific question. Descriptions are frequently restricted to compounds that ature affect the fungal Hsp90 interactome? As described are amenable to experimental quantification, and the reactions and regulatory steps above, the binding specificities and affinities of the between them are therefore often compressed into single equations. Hsp90 chaperone machine for its client proteins are driven by its co‑chaperones. However, for the purpose of this discussion, let us simplify things by considering merely that Hsp90 displays differing affinities for dif- network after heat shock. By contrast, the metabolism ferent client proteins. As described above, Hsp90 abun- module retains its central position in the network. dance increases following heat shock (and in response to Although the general connectivity of network modules other environmental insults). However, the abundance decreases during heat shock, HSPs contribute to the of free Hsp90 that is available for interactions with client integration of modules in this partially decoupled inter- proteins declines transiently as Hsp90 becomes seques- action network, reflecting their major influence on tered in complexes with unfolded proteins. Modelling of protein function under these conditions120. a simple conceptual Hsp90 interactome (FIG. 4), in which The kinetics of human HSP90–chaperone complex two client proteins (A and B, involved in pathway A and formation has also been analysed121. Mass spectrometry pathway B) are present at differing levels, is sufficient of complexes formed in vitro by recombinant human to demonstrate that changes in Hsp90 availability will HSP90, HSP70, HOP (HSC70- and HSP90‑organizing substantially affect its interactions with these client protein; also known as STIP1), and FKBP52 (52 kDa proteins (FIG. 4c). FK506‑binding protein; also known as FKBP4) allowed Let us, then, for the sake of discussion, make the likely the authors to construct an interaction network for these presumptions that Hsp90 displays differing affinities for factors and to predict dominant complexes that are formed different client proteins, and that changes in ambient during the HSP90 cycle. The similarity of the binding con- temperature affect the affinity of Hsp90 for some client stants for the protein complexes studied support the view proteins more than these changes affect its affinity for that the HSP90 chaperone machinery is complex and others. In this case, our model of this simple Hsp90 inter- dynamic121. actome shows that an increase in the affinity of Hsp90 The role of HSP70 and HSP90 in protein homeostasis for client protein B will result in a substantial increase in has also been modelled dynamically122. This study exam- the proportion of protein B that complexes with Hsp90 ined the relationships between HSP90, HSP70, JUN relative to the proportion of complexed client protein A amino-terminal kinase 1 (JNK1; also known as MAPK8) (FIG. 4d). If Hsp90 binding activates these client pro- and MAPK p38α (also known as MAPK14) in the con- teins, this increased affinity for protein B will result in text of neurodegeneration. The simulations suggest that a dramatic shift from a situation in which pathway A is following the imposition of a proteotoxic stress, pro- more active than pathway B, to a new situation in which tein homeostasis can be maintained for short periods, pathway B is more active than pathway A.

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a b Therefore, modelling of this simple Hsp90 inter- Dynamics A actome indicates that the kinetics of binding between

d • • Hsp90 and its client proteins is likely to be substan- [A Hsp90] = k1[A][Hsp90] – k–1[A Hsp90] A • Hsp90 dt tially affected by changes in Hsp90 abundance, bind- d [B • Hsp90] = k [B][Hsp90] – k [B • Hsp90] ing affinities, relative concentrations and on–off rates. Hsp90 dt 2 –2 Clearly, thermal fluctuations will exert dramatic effects B • Hsp90 Conservation relations on the Hsp90 interactome, having an important role in the dynamic competition between binding partners. [Hsp90 ] = [Hsp90] + [A • Hsp90] + [B • Hsp90] B total Three major aspects are relevant here: the relative affini- • [Atotal] = [A] + [A Hsp90] ties of client and unfolded proteins for Hsp90, the rela-

• tive abundances of these different ligands, and whether [Btotal] = [B] + [B Hsp90] specific client proteins are activated or inhibited through c their interactions with Hsp90. We suggest that Hsp90 5 essentially acts as a biological transistor, tuning the activ- 4.5 ities of key signalling pathways in response to thermal 4 inputs and other proteotoxic environmental cues. 3.5

3 B • Hsp90 Cellular consequences. What are the likely cellular consequences of Hsp90 using differential client interactions 2.5 to modulate the activity of global signalling networks in 2 B response to changes in ambient temperature? Thermal 1.5 Hsp90 modulation of Hsp90 availability promotes the activa- 1 tion of some signalling pathways in fungal cells. Hsp90 Concentration (arbitrary units) (arbitrary Concentration A • Hsp90 0.5 regulates morphogenesis in C. albicans by repressing A Ras1–protein kinase A (PKA) signalling at low tempera- 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 tures115. At elevated temperatures (~37 °C), this Hsp90 Total Hsp90 (arbitrary units) repression is relieved, leading to filamentation. Hence, through the Hsp90 transistor, changes in ambient tem- d perature influence cellular morphology, a key virulence determinant in this pathogen (FIG. 5). 2 Hsp90 also has evolutionarily conserved roles in cell cycle progression. Several key cell cycle regulators have Hsp90 1.5 Hsp90 been identified as Hsp90 interactors in S. cerevisiae, including Swe1, Cdc28, Cdc50 and Cdc60 (REFS 61,92,123). Furthermore, Hsp90 has recently been implicated in 1 cell cycle progression in C. albicans. Cell cycle arrest is B • Hsp90 believed to promote C. albicans filamentation in response A • Hsp90 0.5 to compromised Hsp90 function; Cdc28 has been identi- Concentration (arbitrary units) (arbitrary Concentration A fied as an Hsp90 client, and key morphogenesis effec- B tors that are induced by compromised Hsp90 or elevated 0 temperature include Pho85 and Pcl1, providing pos- 1 2 Aﬃnity for B (arbitrary units) sible links between Hsp90, cell cycle regulation and morphogenesis114,124. Hsp90 also has a profound impact on responses to Figure 4 | Alterations in Hsp90 concentrations are predictedNature Reviews to differentially | Microbiology affect interactions with specific client proteins. a | A simple conceptual network drug-induced cellular stress, in this case through acti- illustrating the interactions between heat shock protein 90 (Hsp90) and two distinct vation of other signalling pathways. For example, cell client proteins, A and B. b | Equations describing the dynamic relationships between integrity and Ca2+–calmodulin signalling are downregu- these individual proteins and the complexes in this conceptual network, and their lated following destabilization of Slt2 (REFS 91,101) and conservation in the network. In steady state, the two dynamic equations are set to calcineurin96, respectively, by Hsp90 depletion. These equal zero. [X] represents the concentration of X, and k and k are rate constants. n −n pathways play major parts in antifungal-drug tolerance, c | The impact of changing the total Hsp90 level on the concentrations of free and and the impact of Hsp90 on this phenotype has been well Hsp90‑bound client proteins A and B. In this case, the affinities of Hsp90 for A and B characterized in both C. albicans and S. cerevisiae. For are the same, but the total concentration of B is fivefold that of A. Clearly, Hsp90 example, Hsp90 depletion phenocopies the azole sensi- availability influences the proportions of A and B that are incorporated into Hsp90 96,101 complexes. d | The effects of altering the affinity of Hsp90 for B (from 0 to 2) while the tivity of calcineurin and protein kinase C mutants . affinity of Hsp90 for A remains constant (set to 1). In this scenario, p = k / k = 1 (in Importantly, elevated temperatures also recapitulate the 1 1 −1 antifungal-drug sensitivity caused by Hsp90 inhibition20. which p is the binding constant, or affinity); p2 = k2 / k-2 = 0…2; [Atotal] = 1; and [Btotal] = 1. As the affinity of Hsp90 for B increases, the concentration of the B–Hsp90 complex Therefore, thermal upshifts downregulate antifungal- (B ∙ Hsp90) increases and the levels of free B and free Hsp90 decrease. By contrast, drug resistance via the effects of the Hsp90 transistor the concentration of the A–Hsp90 complex (A ∙ Hsp90) remains relatively unaffected. on cell integrity and Ca2+–calmodulin signalling (FIG. 5). Hence, the proportions of A and B that are chaperoned by Hsp90 change as a result Given the impact of the cell integrity and Ca2+– of this altered affinity for one of the client proteins. calmodulin pathways on the C. albicans cell wall, a clear

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co‑chaperones are conserved across all eukaryotes87. Ambient Many features of the Hsp90 interactome are also highly temperature conserved from yeast to humans, despite there having been substantial network rewiring over evolutionary time. These conserved features include a preponderance Hsp90 of protein kinases and transcription factors as client pro- teins67,78,91–93. As a consequence of the extensive connectivity of the Hsp90 transistor in interaction networks and its profound impact on signal transducers, this transistor Cell wall stress Antifungals Morphogens protein tunes physiological responses to environmental conditions. Hsp90 also modulates the phenotypic effects of Hsp90 genetic variation, thereby influencing evolution. By gov- Hsp90 Hsp90 erning the activation of diverse cell signalling regulators, Hsp90 CDKs Hsp90 can influence the phenotypic effects of genetic Ras1–cAMP Slt2 variation in an environmentally responsive manner in Calcineurin at least two distinct ways. First, Hsp90 can buffer the effects of genetic or epigenetic variation, keeping it silent until the chaperone reservoir is compromised Cell wall Antifungal-drug Growth and biosynthesis tolerance morphogens in response to stress. This role as a capacitor for variation has been observed in diverse eukaryotes, including flies, plants and fungi18,19,129–134. Second, Hsp90 can enable the phenotypic effects of new mutations, either Immune Therapeutic Virulence recognition intervention by stabilizing mutant regulators or by stabilizing other, Figure 5 | The Hsp90 transistor could tune multiple cellular outputs in response non-mutant cellular proteins that are required for adaptation to environmental stress or to accommodate a to thermal input. Thermal fluctuations influence heat shockNature protein 90 Reviews | (Hsp90)Microbiology availability and probably the affinity of the Hsp90 chaperone machine for certain client mutation. This role as a potentiator for genetic varia- proteins (see main text for details). Hsp90 modulates the activity of many client proteins, tion has been observed in fungi as well as in mamma- including regulators in key signalling pathways. Hence, the Hsp90 chaperone machine is lian cancer cells20,88,135,136. A recent study exploiting the proposed to act like a transistor that modulates the activity of these signalling pathways genetic tractability of S. cerevisiae revealed that Hsp90 in response to thermal (and other proteotoxic) inputs. As a result, temperature modulates influences approximately 20% of natural genetic vari- cell division, adaptation, growth and morphogenesis through the Hsp90 transistor. ation, serving both to maintain phenotypic robustness cAMP, cyclic AMP; CDKs, cyclin-dependent kinases. and to promote diversification18. Furthermore, the link between Hsp90, Hsf1 and environmental stress has been reinforced by the recent observation that stimulation of prediction is that ambient temperature will also affect a stress response, either by heat shock or by increased the architecture of the cell surface and, hence, immune expression of HSF‑1, reduces the phenotypic effects of recognition by the host (FIG. 5). Indeed, C. albicans cells mutations in the nematode Caenorhabditis elegans137. grown at 37 °C contain longer mannans than those grown Thus, Hsp90 has a multitude of impacts on cellular at 25 °C125. Mannans from the C. albicans cell wall have circuitry: as a capacitor that buffers genetic variation the ability to stimulate cytokine production in the until this variation is released by stress, as a potentia- host126,127 and induce maturation of dendritic cells128. tor that enables the phenotypic effects of new mutations These effects of Hsp90 on cellular processes will not to be manifest and as a transistor that tunes cellular occur only during acute heat shocks. Mathematical mod- outputs to environmental inputs. These multiple roles elling strongly suggests that cells are constantly tuning illustrate the stunning complexity of ways in which the Hsp90 levels to subtle changes in the ambient tempera- stress response circuitry orchestrates adaptation on both ture. Hence, it is highly likely that the Hsp90 transis- physiological and evolutionary timescales. tor is constantly tuning the activity of the cell signalling An elegant combination of molecular, genetic and network to subtle thermal fluctuations. Furthermore, the genomic approaches has dramatically increased our Hsp90 transistor probably modulates fungal signalling understanding of Hsp90 as an evolutionary capaci- networks in response to other proteotoxic stresses that tor. However, these approaches have provided only influence Hsp90 availability. glimpses of the role of Hsp90 as a cellular transistor. Understanding this role represents a major challenge Conclusions for the future. How does the Hsp90 chaperone machine In conclusion, Hsp90 has a central role in tuning cell modulate the functions of the key cellular regulators ular outputs to thermal inputs, discussed here primar- that drive physiological adaptation during the minutes Filamentation ily in the context of yeast species. However, as Hsp90 is following an environmental challenge? To address The formation of cells with an one of the most ancient and highly conserved regulators this question, we need high-throughput biochemi- elongated morphology, such as hyphae, pseudohyphae and of cell signalling, this is likely to have broad relevance cal, biophysical and mathematical tools to define and yeast cells that have not across the eukaryotic domain. There is some plastic- understand the short-term dynamism of the Hsp90 undergone cell separation. ity in the Hsp90 chaperone machine, although key interactome as well as its long-term plasticity.

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137. Casanueva, M. O., Burga, A. & Lehner, B. Fitness Acknowledgements (grants 080088 and 097377) and the European Commission trade-offs and environmentally induced mutation The authors are grateful to numerous colleagues, and to J. (grants FINSysB, PITN‑GA‑2008‑214004 and STRIFE buffering in isogenic C. elegans. Science 335, 82–85 Heitman in particular, for insightful discussions. M.D.L. is a ERC‑2009‑AdG‑249793). (2012). Sir Henry Wellcome Postdoctoral Fellow (Wellcome Trust 138. Klipp, E. et al. (eds) Systems Biology: A Textbook grant 096072). E.K. is supported by grants from the German Competing interests statement (Wiley-VCH, 2009). Research Council (grants GRK 1772, SFB618 and SFB740), The authors declare no competing financial interests. 139. Herrgard, M. J. et al. A consensus yeast metabolic the German Ministry of Education and Research (grants network reconstruction obtained from a community 0315786A and 0315584B) and the European Commission FURTHER INFORMATION approach to systems biology. Nature Biotech. 26, (grants FINSysB, PITN‑GA‑2008‑214004 and Unicellsys Edda Klipp’s homepage: 1155–1160 (2008). HEALTH‑2007‑201142). L.E.C. is supported by a Career http://www2.hu-berlin.de/biologie/theorybp 140. Richter, K., Muschler, P., Hainzl, O., Reinstein, J. & Award in the Biomedical Sciences from the Burroughs Leah E. Cowen’s homepage: Buchner, J. Sti1 is a non-competitive inhibitor of the Wellcome Fund, a Canada Research Chair in Microbial http://individual.utoronto.ca/cowen/index.html Hsp90 ATPase. J. Biol. Chem. 278, 10328–10333 Genomics and Infectious Disease, a Ministry of Research and Alistair J. P. Brown’s homepage: (2003). Innovation (Ontario, Canada) Early Researcher Award, and by http://www.abdn.ac.uk/ims/staff/details.php?id=al.brown 141. Marsh, J. A., Kalton, H. M. & Gaber, R. F. Cns1 is grants from the Natural Sciences and Engineering Research Hsp90Int.DB: http://www.picard.ch/Hsp90Int an essential protein associated with the Hsp90 Council of Canada (Discovery Grant 355965) and the chaperone complex in Saccharomyces cerevisiae Canadian Institutes of Health Research (grants MOP‑86452 SUPPLEMENTARY INFORMATION that can restore cyclophilin 40‑dependent functions and MOP‑119520). A.J.P.B. is supported by grants from the See online article: S1 (figure) in cpr7Δ cells. Mol. Cell. Biol. 18, 7353–7359 UK Biotechnology and Biological Research Council (grants ALL LINKS ARE ACTIVE IN THE ONLINE PDF (1998). BB/D009308/1 and BB/F00513X/1), the Wellcome Trust

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