Báo cáo khoa học: Sensitivity to Hsp90-targeting drugs can arise with mutation to the Hsp90 chaperone, cochaperones and plasma membrane ATP binding cassette transporters of yeast docx

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.
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