Sensitivity to Hsp90-targeting drugs can arise with mutation to the Hsp90 chaperone, cochaperones and plasma membrane ATP binding cassette transporters of yeast Peter W. Piper 1 , Stefan H. Millson 1 , Mehdi Mollapour 1 , Barry Panaretou 2 , Giuliano Siligardi 3 , Laurence H. Pearl 4 and Chrisostomos Prodromou 4 1 Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, UK; 2 Division of Life Sciences and 3 Pharmaceutical Optical Spectroscopy Centre, Department of Pharmacy, King’s College London, Franklin-Wilkins Building, London, UK; 4 Section for Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, London, UK The Hsp90 molecular chaperone catalyses the final activa- tion step of many of the most important regulatory proteins of eukaryotic cells. The antibiotics geldanamycin and rad- icicol act as highly selective inhibitors of in vivo Hsp90 function through their ability to bind within the ADP/ATP binding pocket of the chaperone. Drugs based on these compounds are now being developed as anticancer agents, their administration having the potential to inactivate sim- ultaneously several of the targets critical for counteracting multistep carcinogenesis. This investigation used yeast to show that cells can be rendered hypersensitive to Hsp90 inhibitors by mutation to Hsp90 itself (within the Hsp82 isoform of yeast Hsp90, the point mutations T101I and A587T); with certain cochaperone defects and through the loss of specific plasma membrane ATP binding cassette transporters (Pdr5p, and to a lesser extent, Snq2p). The T101I hsp82 and A587T hsp82 mutations do not cause higher drug affinity for purified Hsp90 but may render the in vivo chaperone cycle more sensitive to drug inhibition. It is shown that these mutations render at least one Hsp90- dependent process (deactivation of heat-induced heat shock factor activity) more sensitive to drug inhibition in vivo. Keywords: Hsp90 inhibitor resistance; Hsp90 mutants; Sti1p; ATP binding cassette transporters; yeast. The Hsp90 molecular chaperone catalyses the final activa- tion step of many of the most important regulatory proteins of eukaryotic cells [1–3]. Hsp90 is also a natural antibiotic target, such that its activity can be inhibited with a high degree of selectivity in vivo with the administration of the antibiotics geldanamycin (GA; a benzoquinone ansamycin produced by Streptomyces hygroscopicus [4]) and radicicol (RD; a macrolactone produced by certain mycopathogenic fungi [5]). GA and RD bind within the Hsp90 ADP/ATP binding site, thereby inhibiting the ATP binding step of the Hsp90 chaperone cycle [6–9]. Interest in Hsp90-targeting drugs as possible anticancer agents was triggered initially with the identification of GA and RD as compounds that could reverse the pheno- type of p60 v–src -transformed cells in culture [10,11]. GA and RD act upon Hsp90, whose action is needed for the p60 v–src tyrosine kinase to achieve an active state [12]. In a variety of cell culture systems, GA administration leads to a marked destabilization of several of the most oncolog- ically relevant proteins such as p53, Erb-b, Raf-1 and steroid receptors [12–15]. Hsp90 inhibition can therefore simultaneously destabilize several of the key components of multistep carcinogenesis [16]. This destabilization is probably a result of these Hsp90 ÔclientÕ proteins being unable to progress through the chaperone cycle. Cells that lose Hsp90 function, as with GA/RD treatment, rapidly lose the ability to activate many signalling proteins and undergo retinoblastoma protein-dependent cell cycle arrest [17]. Antitumour effects of Hsp90 drugs have now been demonstrated using several animal model systems, the 17-allylamino derivative of GA (17-AAG) being more effective and less hepatotoxic in vivo than the parent GA [18]. Although 17-AAG is now in clinical trials, its insolubility causes problems in administration. It is also potentially a redox-cycling drug. There is, therefore, an urgent need to identify or develop inhibitors of Hsp90 that are more selective and more soluble than 17-AAG [16,19]. It will be necessary to understand the factors that contribute to susceptibility or resistance to Hsp90 inhibitory compounds. To this end, we have investigated various mutants of the Hsp90 chaperone and pleiotropic drug resistance (PDR) systems of yeast, to help identify the factors that contribute to sensitivity to Hsp90 inhibitor drugs. Correspondence to P.W. Piper, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK. Fax: + 44 114 222 2850, Tel.: + 44 114 222 2851, E-mail: peter.piper@sheffield.ac.uk Abbreviations: GA, geldanamycin; RD, radicicol; ts, temperature- sensitive. (Received 4 August 2003, revised 28 September 2003, accepted 3 October 2003) Eur. J. Biochem. 270, 4689–4695 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03866.x Materials and methods Strains and growth media The Saccharomyces cerevisiae strains used for this study are listed in Table 1. Deletion of the SBA1 and STI1 open reading frames in W303-1a utilized PCR-generated kan- MX4 or HIS3MX6 [20] cassettes, respectively, these dele- tions being confirmed by colony PCR [21]. Cultures were grown on YPDA medium (2% glucose, 2% bactopeptone, 1% yeast extract, 20 mgÆL )1 adenine). GA was a gift from the National Cancer Institute (Bethesda, MD, USA). RD was purchased from Sigma. Drug sensitivity assays Cells were streaked on to 5 cm diameter YPDA plates containing the indicated level of drug [22]. Western blot analysis Total protein extracts were prepared and Western blots prepared as described previously [23] using rabbit polyclonal antisera raised against the bacterially expressed Hsp82 and Sba1p of yeast. Hsp90 ATPase assays Hsp90 ATPase assays used a regeneration coupled enzyme assay [24], each 1 mL of assay using 2 l M of purified recombinant Hsp82 as described previously [23]. Assays of HSF induction Heat shock factor activity was measured using cells transformed with a URA3 plasmid containing a lacZ reporter under heat shock element control (HSE-lacZ [25]). A Beckmann BioMek robot was used to add 20 lLof minus uracil dropout medium (SD-ura), either with or without RD, to 16 replicate 25 °C 100 lL [26] cultures of each transformant. Immediately thereafter, eight of these cultures were maintained for 1 h at 25 °C while the remaining eight were heat shocked to 39 °Cfor1hafter which, 50 lLof1 M sodium carbonate was added. The cells were then collected by centrifugation and their lacZ activity measured by a permeabilized cell assay [25]. Results Certain point mutations in the native Hsp90 of yeast render cells much more sensitive to Hsp90 inhibitor drugs To test whether mutations in the native Hsp90 chaperone of yeast influence sensitivity to Hsp90 inhibitors, we tested eight S. cerevisiae hsp82 mutants bearing point mutations in their single functional Hsp90 gene (HSP82) (Table 1) for their sensitivities to GA and RD (Fig. 1). This set of mutants had originally been isolated by Nathan and Lindquist as corres- ponding to mutant forms of Hsp90 that cause temperature- sensitive (ts) yeast growth at 37 °C [27]. They therefore represent mutations in the Hsp82 isoform of yeast Hsp90 that cause partial, rather than total, loss of the essential Table 1. The yeast strains employed for this study and their sensitization to Hsp90 drugs. Strain Increase in GA/RD sensitivity Genotype Strain origin P82a – W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-HSP82 a [27] T22I Slight (GA) b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(T22I) a [27] A41V No b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(A41V) a [27] G81S No b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(G81S) a [27] T101I Yes b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(T101I) a [27] G170D Slight (RD) b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(G170D) a [27] G313S Slight (RD) b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(G313S) a [27] E381K Slight (GA) b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(E381K) a [27] A587T Yes b W303–1a hsc82::LEU2 hsp82::LEU2 HIS3-GPD-hsp82(A587T) a [27] W303–1a – MATa ura3–1 trp1–1 leu2–3112 his3–11 ade2–1 can1–100 ssd1-d2 Euroscarf Dsba1 No c W303–1a sba1DkanMX4 This study Dsti1 Yes c W303–1a sti1DHIS3MX6 This study Dcpr6 No c W303–1a cpr6::URA3 [49] Dcpr7 Moderate c W303–1a cpr7::TRP1 [49] Dcpr6,Dcpr7 Moderate c W303–1a cpr6::URA3 cpr7::TRP1 [49] Dsti1,Dcpr6 Yes c W303–1a sti1DHIS3MX6 cpr6::URA3 This study Dsti1,Dsba1 Yes c W303–1a sti1DHIS3MX6 sba1DkanMX4 This study YPH500 – MATa, ura3–52, lys2–801 am , ade2–101 oc , trp1-D63,his3-D200, leu2-D1 [50] Dpdr5 (YKKB-13) Yes d YPH500 pdr5::TRP1 [51] Dsnq2 (YYM5) Slight (RD) d YPH500 snq2::hisG [51] Dpdr5,Dsnq2 (YYM4) Yes d YPH500 pdr5::TRP1 snq2::hisG [51] a This integrated wild-type (HSP82) or mutant (hsp82) gene for Hsp90 is the only functional Hsp90 gene in these strains and is expressed under the control of the constitutively active glyceraldehyde-3-phosphate (GPD1) gene promoter [27]. b Relative to P82a (Fig. 1A). c relative to W303–1a parent (Fig. 4A). d Relative to YPH500 parent (see Fig. 4B). 4690 P. W. Piper et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Hsp90 function, or that prevent the higher levels of Hsp90 activity needed for yeast growth at high temperatures [28,29]. Growth of these mutants relative to the strain expressing the wild-type Hsp82 (p82a) on GA- or RD-containing plates (Fig. 1A) revealed that a number of these hsp82 alleles render the cells hypersensitive to GA (A587T, T101I; also to lesser extent, T22I, G313S and E381K). Only two (T101I and A587T) were associated with pronounced sensitivity to RD (Fig. 1A). It was these latter two mutations, alleles causing extreme sensitivity to both GA and RD, that we chose to investigate further. This is because the in vivo effects of GA may not be limited to its capacity to inhibit Hsp90. GA possesses a benzoquinone ring, readily reduced in vivo by NAD(P)H-dependent oxidoreductases. It therefore has the potential to cause oxidative stress. RD, in contrast, is not a redox-cycling compound [30]. We were surprised initially by the strong inhibitory action of RD on certain yeast mutants (Fig. 1A) as mammalian studies had indicated that RD derivatives may not be very stable in vivo [16,30,31]. These increases in drug sensitivity could arise through certain of the mutations causing lowered intracellular levels of the drug target, Hsp90, itself. This possibility could be discounted as these hsp82 mutants all expressed similar levels of Hsp82 (their sole Hsp90 isoform). Hsp82 levels in the GA and RD-sensitive T101I and A587T hsp82 mutants were essentially unaltered with respect to the p82a cells expressing the wild-type Hsp82 (Fig. 1B). The IC 50 for GA inhibition of the intrinsic ATPase of purified Hsp82 is unaffected by the A587T mutation We recently presented temperature/activity profiles for the in vitro ATPase activity of purified mutant forms of Hsp82, the same mutant forms that are expressed in the hsp82 mutants in Fig. 1 [32]. There is no apparent correlation between this in vitro ATPase activity and the in vivo sensitivity to Hsp90 drugs (Fig. 1A), despite the fact that GA and RD are both potent inhibitors of this ATPase [7,8]. Of the two Hsp82 mutations associated with high in vivo sensitivity to Hsp90 drugs, T101I dramatically reduces the in vitro ATPase activity of purified Hsp82, whereas, A587T exerts little effect [32]. Yet another inhibitor of the in vitro ATPase of Hsp90 is Sti1p, a cochaperone protein that also affects drug resistance (see below). When Sti1p, the functional equivalent of mammalian Hop, binds to Hsp90 in the GA/Hsp90 complex it displaces the bound GA [33]. If the increased Hsp90 drug sensitivity of the T101I and A587T hsp82 mutants (Fig. 1A) was due to these mutations causing tighter drug binding to the chaperone, these mutations should render the in vitro ATPase of Hsp82 more sensitive to drug inhibition. We determined if A587T renders the ATPase of the purified chaperone more susceptible to inhibition by either GA or Sti1p in assays using wild-type and A587T mutant forms of Hsp82 (inhibition of the T101I mutant protein was not determined, its extremely low activity [32] making it much more difficult to obtain definitive data). The A587T mutation did not affect the GA or Sti1p inhibitions of in vitro chaperone ATPase (Fig. 2). Adenosine 5¢-(b,c-imino)triphosphate (AMP-PCP) binding to purified Hsp82 was also essentially unaffected by the A587T and T101I mutations (K d values for AMP-PCP Fig. 1. GA and RD sensitivities of a collection of yeast strains expres- sing either wild-type (p82a; w + ) or mutant forms of Hsp82. (A) Strains were streaked onto YPDA agar containing the indicated concentra- tions of GA or RD. The plates were then photographed after 5 days of growth at 20–22 °C. (B) Western blot measurement of the levels of Hsp82 and Sba1p (loading control) in these cells cultured at 22 °C. Fig. 2. GA and Sti1p inhibition of the intrinsic ATPase of purified wild- type and A587T mutant Hsp82. Assays were conducted at 37 °Cas described previously [33], using 2 l M Hsp82 protein and either the indicated level of GA (A) or zero, 2 l M and 8 l M Sti(B).Activityat 100%  5000 pmol ATPÆmin )1 Æmg )1 for both protein samples; the IC 50 forGAinthisassay 3 l M [8,23]. Ó FEBS 2003 Mutations sensitizing yeast to Hsp90 inhibitor antibiotics (Eur. J. Biochem. 270) 4691 binding to the wild-type, T101I and A587T forms of Hsp82 measured by CD spectroscopy being 33, 37 and 37 l M , respectively; G. Siligardi, unpublished observation). The T101I and A587T mutations allow RD to potentiate the yeast heat shock response Figure 2 reveals that the in vivo manifestation of increased GA sensitivity in the A587T hsp82 mutant (Fig. 1A) is not due to increased drug affinity for the chaperone, suggesting that it may require additional components of the Hsp90 chaperone machinery and possibly an assembled Hsp90/ cochaperone/client complex. To seek evidence for whether this is the case, we determined if, through the expression of the T101I and A598T mutant forms of Hsp82, an Hsp90- dependent process becomes more sensitive to Hsp90 drug inhibition in vivo. In a variety of cell systems, Hsp90 inhibitor administra- tion acts almost immediately to activate the heat shock response [25,34,35]. This reflects the requirement for Hsp90 in deactivation of the transcriptional activator of heat shock genes, heat shock factor (HSF). When heat shocked to 37– 39 °C, the hsp82 mutants in Fig. 1 all display considerably higher levels of HSF activation relative to the wild-type [25,35]. They are therefore defective in this down-regulation of HSF activity at these temperatures. One of these mutants (E381K hsp82) even displays a high HSF activity at low temperatures of growth [25], indicating that Hsp90 is also required in order to maintain HSF in its basal activity state, the form present in unstressed cells. As mentioned above, GA is potentially a source of oxidative stress in vivo through its capacity to act as a redox-cycling drug. Oxidative stress is known to activate HSF [36–38]. We therefore used RD, a non redox-active compound, in investigating whether the expression of T101I and A587T mutant forms of Hsp82 influences the capacity of an Hsp90 inhibitor to activate the heat shock response. In the absence of heat stress, a 1 h, 1 l M RD administration caused moderate increases in HSF activity, both in wild- type, T101I hsp82 and A587T hsp82 cells (Fig. 3). Heat shock induced this activity still further, yet it was only in the cells expressing the T101I and A587T mutant Hsp82s, not cells expressing wild-type Hsp82, that such low amounts of RD could potentiate this heat-induced increase in HSF activity (Fig. 3). Heat shocked cells of these two hsp82 mutants are therefore more responsive to RD administra- tion (ÔresponsivenessÕ measured as HSF activity). Losses of Hsp90 system cochaperones and plasma membrane ATP binding cassette (ABC) transporters can sensitize cells to Hsp90 drugs Hsp90 works in association with a number of cochaperone proteins. These may, in many cases, stabilize discrete multiprotein complex intermediates of the Hsp90 chaperone cycle, thereby improving the overall efficiency of client protein activation by Hsp90. At least nine such Hsp90 system cochaperones have now been identified in yeast {Sti1p(Hop), Cdc37p, Cns1p, Sba1p(p23), Cpr6p, Cpr7p, Sse1p, Hch1p, Aha1p [39–42]}. We tested whether Hsp90 drug resistance is affected by the loss of several of the cochaperones that are nonessential for viability of yeast (Sti1p, Sba1p, Cpr6p, Cpr7p, Hch1p and Aha1p). Sse1p and essential cochaperones such as Cdc37p and Cns1p were not included in this screen. At 22 °C there were no appreciable effects of the loss of Sba1p, Cpr6p (Fig. 4A), Hch1p or Aha1p (not shown) on drug sensitivity, whereas the loss of Sti1p increased sensitivity to both GA and RD (Fig. 4A). With loss of Cpr7p, the cyclophilin whose loss causes the most marked phenotype in yeast [43], drug sensitivities were slightly increased (an increased sensitivity of cpr7 cells to GA had been reported previously [44]). However, in these cells with a W303-1a genetic background, these effects on drug sensitivity due to the loss of Cpr7p were appreciably smaller than those due to the loss of Sti1p (Fig. 4A). An increased GA sensitivity of sti1D cells was noted in an earlier study, work that also identified increased GA sensitivity with the loss of the Sse1p cochaperone [41]. We have since found these effects of the sti1D mutation on drug sensitivity to be influenced strongly by the isoform of Hsp90 that is expressed (at a similar level) in the cells. Increased GA and RD sensitivity with the loss of Sti1p was most marked in S. cerevisiae expressing the Candida albicans Hsp90, less in cells expressing solely the native S. cerevisiae Hsp82 and negligible in cells expressing solely the S. cere- visiae Hsc82 [22]. These differences are quite remarkable as the two isoforms of S. cerevisiae Hsp90 (Hsc82 and Hsp82) share no less than 97% sequence identity [28]. It is probable that yeast cells use plasma membrane pumps to catalyse a cellular efflux of Hsp90-targeting drugs, just as they actively efflux very many other xenobiotics and antitumour agents [45,46]. We therefore investigated whe- ther the pleiotropic drug resistance (PDR) system contri- butes to Hsp90 inhibitor resistance. Strains lacking two of the major plasma membrane ATP-binding cassette (ABC) transporter determinants of drug resistance (Pdr5p and Snq2p [46]) were streaked onto plates containing GA and RD. This revealed the Dpdr5 mutant to be hypersensitive to both drugs and the Dsnq2 mutant to be slightly sensitive to RD (Fig. 4B). Pdr5p is a broad-specificity ABC transporter that provides resistance to a wide range of hydrophobic and Fig. 3. Expression of a HSE-LacZ reporter of HSF activity in cells expressing the wild-type (p82a), or T101I and A587T mutant forms of Hsp82. Basal and heat-induced (1 h 39 °C) HSE-LacZ activity (open and solid bars, respectively) was determined both in the absence (–) and presence (+) of 1 l M RD. Data represents the mean and SD of eight assays. 4692 P. W. Piper et al. (Eur. J. Biochem. 270) Ó FEBS 2003 cationic compounds in yeast. Its substrate specificity is remarkably similar to that of the human ABC transporter (Mdr1) overexpressed in a number of multiple drug- resistant tumours [45]. Discussion This study is the first to reveal that an increased sensitivity to Hsp90 drugs can arise with mutations to Hsp90 itself (Fig. 1) and with specific ABC transporter defects (Fig. 4B). Previously, an increased GA sensitivity had been shown to result from the loss of certain cochaperones [41,44]; results that have been partly confirmed and extended in this work (Fig. 4A). The IC 50 for inhibition of the in vitro ATPase of purified Hsp90 is around 3 l M for GA and 1 l M for RD [8,23] (Fig. 2). The effects of short-term exposure of yeast cells to the latter low RD concentration also are quite readily monitored (Fig. 3). In contrast, on Petri dishes where cells are growing for extended periods, levels of these drugs in excess of 100 l M can still permit the growth of wild- type cells, though certain mutants are clearly inhibited [22](Figs 1 and 4). This resistance to long-term Hsp90 drug exposure is attributable partly to the actions of the membrane drug pumps (Fig. 4B). These drug efflux activ- ities cause yeast to be remarkably resistant to a wide range of inhibitory compounds and can limit the effectiveness of yeast-based drug screens [45]. Instability of the Hsp90 drug compounds themselves may be another factor in this resistance to long-term Hsp90 drug exposure (GA is readily oxidized; while RD possesses dienone and epoxide groups that are potentially reactive and a lactone ring that presents possibilities for esterase action [30]). The sensitization of yeast to Hsp90 drugs, whether through expression of the T101I or A587T mutant forms of the native Hsp82 (Fig. 1) or through heterologous expres- sion of the human Hsp90b [22], is not a reflection of a higher binding affinity of the chaperone for the drug. The IC 50 for GA inhibition of the in vitro ATPase of purified yeast Hsp82 is unaffected by the A587T mutation (Fig. 2) and similar for both yeast and human Hsp90s [47]. Are the T101I and A587T hsp82 mutations acting selectively to sensitize the assembled Hsp90/cochaperone/client protein complex to drug inhibition of progression through the chaperone cycle, or is it simply that these mutations are reducing Hsp90 activity in vivo, which in turn leads to increased sensitivity to Hsp90 drugs in a general way? A number of indicators suggest the former. Yeast needs higher levels of Hsp90 for high temperature growth [28,29], so that mutations causing a substantially reduced Hsp90 activity should all present ts phenotypes. Nevertheless there appears to be no correlation between the degrees of drug sensitivity and temperature sensitivity displayed by these hsp82 strains (compare Fig. 1A of this report with Fig. 2B of [27]). The mutant with the most severe ts phenotype (T22I hsp82 [27]) is not the most drug-sensitive (Fig. 1A). There is also no corre- lation between the in vivo drug sensitivity of each hsp82 mutant and the in vitro ATPase of the corresponding purified chaperone [32]. Glucocorticoid receptor activity Fig. 4. Analysis of cochaperone and ABC transporter mutants for Hsp90 drug sensitivity. (A) RD and GA sensitivities of strains bearing deletions in genes for Hsp90 system cochaperones. Wild-type cells (w + ), Dsti1, Dsba1, Dcpr6 or Dcpr7 single mutants, also Dsba1,Dsti1 and Dcpr6,Dsti1 double mutants, all from a W303–1a genetic background, were photographed after 2 days of growth at 30 °ConYPDAintheabsenceorpresenceofthe indicated concentrations of GA or RD. (B) RD and GA sensitivities of strains bearing deletions of the Pdr5 and Snq2 plasma membrane ABC transporters. Wild-type cells (wt), Dpdr5 or Dsnq2 single mutants, and a Dpdr5,Dsnq2 double mutant, all of YPH500 genetic background, were photographed after 2 days of growth at 30 °C on YPDA in the presence of the indicated concentrations of GA or RD. Ó FEBS 2003 Mutations sensitizing yeast to Hsp90 inhibitor antibiotics (Eur. J. Biochem. 270) 4693 measurements in these strains indicate that the different hsp82 alleles, rather than all simply lowering Hsp90 activity, exert diverse in vivo pleiotropic effects on Hsp90 client protein activation/deactivation processes [27]. Furthermore the deactivation of heat-induced HSF activity is more sensitive to drug inhibition in cells expressing the T101I or A587T mutant forms of Hsp82 (Fig. 3). This though is only an indication, not formal proof, that these two specific mutant Hsp82s may allow the drug to exert stronger inhibitory effects on the Hsp90 chaperone cycle. In yeast expressing wild-type Hsp90s, increased drug sensitivity is generally apparent with the loss of the Sti1p (Hop) cochaperone (Fig. 4A). Sti1p binding to Hsp90 may help stabilize the ATP/ADP-free state of Hsp90 [33], ready for its loading with a fresh substrate client protein [the latter probably as a complex with Hsc70 and Ydj1(Hsp40)]. ATP binding to the Hsp90 N-terminal domains in the Hsp90 dimer then causes these N-domains to associate [32]. This ATP-induced conformational change may also be the signal for Hsc70, Ydj1 and Sti(Hop) to be displaced from the complex and for other cochaperone proteins, including Sba1(p23), to bind so as to produce the later multiprotein complexes of the Hsp90 chaperone cycle. Hsp90 drugs inhibit ATP binding [6–8], therefore progression to these later stages of the chaperone cycle. It may be the progression to these later complexes that is more sensitive to Hsp90 drugs in yeast expressing the T101I or A587T mutant forms of Hsp82 (Fig. 1) or the human Hsp90b [22]. Although Sti1p contacts the C-terminus of Hsp90 [48], its binding also displaces bound GA, indicating that there is also an interaction of Sti1p with the ADP/ATP binding site of the chaperone [33]. The increased drug sensitivity of sti1D mutant cells (Fig. 4A) might therefore be attributable to the absence of a protein that limits access of the drug to its binding site on Hsp90. Such a model is probably oversim- plistic as we have found the increased drug sensitivity with the loss of Sti1p to be strongly dependent on the form of Hsp90 being expressed in the yeast [22]. Though this study has focussed on mutations that cause an increased sensitivity to Hsp90 drugs, it is probable that increased resistance can also arise (e.g. through gain-of- function PDR mutations leading to the overproduction or overactivation of membrane pumps catalysing drug efflux from the cell). Mdr1p, the human ABC transporter overexpressed in a number of multiple drug-resistant tumours, has a spectrum of diverse substrates that overlap quite remarkably with those of Pdr5p (a major yeast ABC transporter determinant of Hsp90 drug resistance; Fig. 4B) [45]. It remains to be established whether increased resist- ance to Hsp90 drugs can arise with mutational alteration to the Hsp90 chaperone machine. Acknowledgements We are indebted to Susan Lindquist, Didier Picard, Richard Gaber, and Karl Kuchler for gifts of strains. Part of this work was supported by the Wellcome Trust. References 1. Csermely,P.,Schnaider,T.,Soti,C.,Prohaszka,Z.&Nardai,G. (1998) The 90kDa molecular chaperone family: structure, function and clinical applications. A comprensive review. Pharmacol. Ther. 79, 1–39. 2. Pearl, L.H. & Prodromou, C. (2000) Structure and in vivo function of Hsp90. Curr. Opin. Struct. Biol. 10, 46–51. 3. Picard, D. (2002) Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol Life Sci. 59, 1640–1648. 4. DeBoer,C.,Meulman,P.A.,Wnuk,R.J.&Petersen,D.H.(1970) Geldanamycin, a new antibiotic. J. Antibiot. (Tokyo). 23, 442– 447. 5. Wicklow, D.T., Joshi, B.K., Gamble, W.R., Gloer, J.B. & Dowd, P.F. (1998) Antifungal metabolites (monorden, monocillin IV, and cerebrosides) from Humicola fuscoatra traaen NRRL 22980, a mycoparasite of Aspergillus flavus sclerotia. Appl. Environ. Microbiol. 64, 4482–4484. 6. Stebbins,C.E.,Russo,A.A.,Schneider,C.,Rosen,N.,Hartl,F.U. & Pavletich, N.P. (1997) Crystal structure of an Hsp90-geldana- mycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89, 239–250. 7. Prodromou, C., Roe, S.M., O’Brien, R., Ladbury, J.E., Piper, P.W. & Pearl, L.H. (1997) Identification and structural char- acterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90, 65–75. 8. Roe, S.M., Prodromou, C., O’Brien, R., Ladbury, J.E., Piper, P.W. & Pearl, L.H. (1999) Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J. Med. Chem. 42, 260–266. 9. Schulte, T.W., Akinaga, S., Murakata, T., Agatsuma, T., Sugi- moto, S., Nakano, H., Lee, Y.S., Simen, B.B., Argon, Y., Felts, S., Toft, D.O., Neckers, L.M. & Sharma, S.V. (1999) Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones. Mol. Endocrinol. 13, 1435–1448. 10. Uehara,Y.,Hori,M.,Takeuchi,T.&Umezawa,H.(1986)Phe- notypic change from transformed to normal induced by benzo- quinoid ansamycins acccompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus. Mol. Cell. Biol. 6, 2198–2206. 11. Kwon, H.J., Yoshida, M., Fukui, Y., Horinouchi, S. & Beppu, T. (1992) Potent and specific inhibition of p60v-src protein kinase both in vivo and in vitro by radicicol. Cancer Res. 52, 6926–6930. 12. Schneider, C., Sepp-Lorenzino, L., Nimmesgern, E., Ouerfelli, O., Danishefsky, S., Rosen, N. & Hartl, F.U. (1996) Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc. Natl Acad. Sci. USA. 93, 14536–14541. 13. Schulte, T.W., Blagosklonny, M.V., Ingui, C. & Neckers, L. (1995) Disruption of the raf-1-Hsp90 molecular complex results in destabilisation of raf-1 and the loss of raf)1–ras association. J. Biol. Chem. 270, 24585–24588. 14. Whitesell, L., Sutphin, P., An, W.G., Schulte, T., Blagosklonny, M.V. & Neckers, L. (1997) Geldanamycin-stimulated destabili- sation of mutated p53 is mediated by the proteasome in vivo. Oncogene 14, 2809–2816. 15. Bagatell, R., Khan, O., Paine-Murrieta, G., Taylor, C.W., Akinaga, S. & Whitesell, L. (2001) Destabilisation of steroid receptors by heat shock protein 90-binding drugs: a ligand- independent approach to hormonal therapy of breast cancer. Clin. Cancer Res. 7, 2076–2084. 16. Workman, P. & Maloney, A. (2002) HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin. Biol. Ther. 2, 3–24. 17. Srethapakdi, M., Liu, F., Tavorath, R. & Rosen, N. (2000) Inhibition of Hsp90 function by ansamycins causes retino- blastoma gene product-dependent G1 arrest. Cancer Res. 60, 3940–3946. 18. Supko,J.G.,Hickman,R.L.,Grever,M.R.&Malspeis,L.(1995) Prelinical pharmacological evaluation of geldanamycin as an antitumour agent. Cancer Chemother. Pharmacol. 36, 305–315. 4694 P. W. Piper et al. (Eur. J. Biochem. 270) Ó FEBS 2003 19. Aherne, W., Maloney, A., Prodromou, C., Rowlands, M.G., Hardcastle, A., Boxall, K., Clarke, P., Walton, M.I., Pearl, L. & Workman, P. (2003) Assays for HSP90 and inhibitors. Methods Mol. Med. 85, 149–161. 20. Brown, A.J.P. & Tuite, M.F. (1998) Yeast Gene Analysis.Aca- demic Press, San Diego. 21. Ling, M., Merante, F. & Robinson, B.H. (1995) A rapid and reliable DNA preparation method for screening a large number of yeast clones by polymerase chain reaction. Nuceic. Acids Res. 23, 4924–4925. 22. Piper, P.W., Panaretou, B., Millson, S.H., Truman, A., Molla- pour, M., Pearl, L.H. & Prodromou, C. (2003) Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90b, a property that can be exploited in screens for new Hsp90 chaperone inhibi- tors. Gene 302, 165–170. 23. Panaretou, B., Prodromou, C., Roe, S.M., O’Brien, R., Ladbury, J.E., Piper, P.W. & Pearl, L.H. (1998) ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo. EMBO J. 17, 4829–4836. 24. Ali, J.A., Jackson, A.P., Howells, A.J. & Maxwell, A. (1993) The 43-kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzes ATP and binds coumarin drugs. Biochemistry 32, 2717–2724. 25. Harris, N., MacLean, M., Hatzianthis, K., Panaretou, B. & Piper, P.W. (2001) Increasing the stress resistance of Saccharomyces cerevisiae, by the overactivation of the heat shock response that results from Hsp90 defects, does not extend replicative life span but can be associated with a slower chronological ageing of non- dividing cells. Mol. General Genomics. 265, 258–263. 26. Adams, A., Gottschling, D.E., Kaiser, C.A. & Stearns, T. (1997) Methods in Yeast Genetics., Cold Spring. Harbor Laboratory Press, Cold Spring Harbor, New York. 27. Nathan, D.F. & Lindquist, S. (1995) Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase. Mol. Cell Biol. 15, 3917–3925. 28. Borkovich, K.A., Farrelly, F.W., Finkelstein, D.B., Taulien, J. & Lindquist, S. (1989) hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell Biol. 9, 3919–3930. 29. Morano, K.A., Santoro, N., Koch, K.A. & Thiele, D.J. (1999) A trans-activation domain in yeast heat shock transcription factor is essential for cell cycle progression during stress. Mol. Cell Biol. 19, 402–411. 30. Piper, P.W. (2001) The Hsp90 chaperone as a promising drug target. Curr. Opin. Invest Drugs. 2, 1606–1610. 31. Smith, D.F., Whitesell, L. & Katsanis, K. (1998) Molecular cha- perones: biology and prospects for pharmacological intervention. Pharmacol. Revs. 50, 493–514. 32. Prodromou, C., Panaretou, B., Chohan, S., Siligardi, G., O’Brien, R., Ladbury, J.E., Roe, S.M., Piper, P.W. & Pearl, L.H. (2000) The ATPase cycle of Hsp90 drives a molecular ÔclampÕ via tran- sient dimerization of the N-terminal domains. EMBO J. 19, 4383– 4392. 33. Prodromou, C., Siligardi, G., O’Brien, R., Woolfson, D.N., Regan, L., Panaretou, B., Ladbury, J.E., Piper, P.W. & Pearl, L.H. (1999) Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones. EMBO J. 18, 754–762. 34. Zou, J., Guo, Y., Guettouche, T., Smith, D.F. & Voellmy, R. (1998) Repression of heat shock transcription factor HSF1 acti- vation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 94, 471–480. 35. Duina, A.A., Kalton, H.M. & Gaber, R.F. (1998) Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response. J. Biol. Chem. 273, 18974–18978. 36. Bonner, J.J., Carlson, T., Fackenthal, D.L., Paddock, D., Storey, K. & Lea, K. (2000) Complex regulation of the yeast heat shock transcription factor. Mol Biol. Cell. 11, 1739–1751. 37. Lee, S., Carlson, T., Christian, N., Lea, K., Kedzie, J., Reilly, J.P. & Bonner, J.J. (2000) The yeast heat shock transcription factor changes conformation in response to superoxide and temperature. Mol. Biol. Cell. 11, 1753–1764. 38. Ahn, S.G. & Thiele, D.J. (2003) Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and pro- tection from stress. Genes Dev. 17, 516–528. 39. Chang, H.C., Nathan, D.F. & Lindquist, S. (1997) In vivo analysis of the Hsp90 cochaperone Sti1 (p60). Mol. Cell Biol. 17, 318–325. 40. Dolinski, K., Muir, S., Cardenas, M. & Heitman, J. (1997) All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae. Proc.NatlAcad.Sci.USA.94, 13093–13098. 41. Liu, X.D., Morano, K.A. & Thiele, D.J. (1999) The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone. J. Biol. Chem. 274, 26654–26660. 42. Siligardi, G., Panaretou, B., Meyer, P., Singh, S., Woolfson, D.N., Piper, P.W., Pearl, L.H. & Prodromou, C. (2002) Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37. J. Biol. Chem. 26, 26. 43. Duina, A.A., Marsh, J.A. & Gaber, R.F. (1996) Identification of two CyP-40-like cyclophilins in Saccharomyces cerevisiae, one of which is required for normal growth. Yeast 12, 943–952. 44. Dolinski, K.J., Cardenas, M.E. & Heitman, J. (1998) CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations and interacts with Hsp90. Mol. Cell Biol. 18, 7344–7352. 45. Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., Soumillion, J.P., Konings, W.N. & Goffeau, A. (1996) Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p. J. Biol. Chem. 271, 31543–31548. 46. Bauer, B.E., Wolfger, H. & Kuchler, K. (1999) Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance. Biochim. Biophys. Acta. 1461, 217–236. 47. McLaughlin, S.H., Smith, H.W. & Jackson, S.E. (2002) Stimula- tion of the Weak ATPase Activity of Human Hsp90 by a Client Protein. J. Mol. Biol. 315, 787–798. 48. Scheufler, C., Brinker, A., Bourenkov, G., Pegoraro, S., Moroder, L., Bartunik, H., Hartl, F.U. & Moarefi, I. (2000) Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101, 199–210. 49. Duina, A.A., Chang, H.C., Marsh, J.A., Lindquist, S. & Gaber, R.F. (1996) A cyclophilin function in Hsp90-dependent signal transduction. Science 274, 1713–1715. 50. Sikorski, R.S. & Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27. 51. Mahe ´ , Y., Parle-McDermott, A.G., Nourani, A., Delahodde, A., Lamprecht, A. & Kuchler, K. (1996) The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae:anovel target for the transcription factors Pdr1 and Pdr3. Mol. Microbiol. 20, 109–117. Ó FEBS 2003 Mutations sensitizing yeast to Hsp90 inhibitor antibiotics (Eur. J. Biochem. 270) 4695 . Sensitivity to Hsp90- targeting drugs can arise with mutation to the Hsp90 chaperone, cochaperones and plasma membrane ATP binding cassette transporters. cells can be rendered hypersensitive to Hsp90 inhibitors by mutation to Hsp90 itself (within the Hsp82 isoform of yeast Hsp90, the point mutations T101I and A587T);