REVIEWS 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’. NATURE REVIEWS | MICROBIOLOGY VOLUME 10 | OCTOBER 2012 | 693 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS 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