A genetic screen identifies mutations in the yeast WAR1
gene, linking transcription factor phosphorylation
to weak-acid stress adaptation
Christa Gregori*, Bettina Bauer*, Chantal Schwartz, Angelika Krenà, Christoph Schu
¨
ller§
and Karl Kuchler
Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, Campus Vienna Biocenter, Austria
Weak acids have a long history as additives in food
preservation. In addition to sulfites used in wine mak-
ing, acetic, sorbic, benzoic and propionic acids are
commonly used in the food and beverage industry
to prevent spoilage [1,2]. In solution, weak acids exist
in a dynamic equilibrium between undissociated,
uncharged molecules and their anionic form. These
acids display increased antimicrobial action at low pH,
which favors the undissociated state. The uncharged
molecules can readily diffuse through the plasma
Keywords
ABC transporter; stress response; weak
organic acids; yeast; zinc finger
Correspondence
K. Kuchler, Medical University Vienna, Max
F. Perutz Laboratories, Department of
Medical Biochemistry, Campus Vienna
Biocenter, Dr Bohr-Gasse 9 ⁄ 2, A-1030,
Vienna, Austria
Fax: +43 1 4277 9618
Tel: +43 1 4277 61807
E-mail: [email protected]
Present address

properly localized in the nucleus, the War1-42p variant fails to bind the
weak-acid-response elements in the PDR12 promoter, as shown by in vivo
footprinting. Importantly, in contrast with wild-type War1p, War1-42pis
also no longer phosphorylated upon weak-acid challenge, demonstrating
that phosphorylation of War1p, its activation and DNA binding are tightly
linked processes that are essential for adaptation to weak-acid stress.
Abbreviations
GST, glutathione S-transferase; MHR, middle homology region; NLS, nuclear localization signal; PDR, pleiotropic drug resistance; WARE,
weak-acid-response element; YPD, yeast peptone ⁄ dextrose.
3094 FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS
membrane. In the cytoplasm, weak acids encounter a
more neutral pH, causing their dissociation into acid
anions and protons. The protons lead to cytoplasmic
acidification, thereby inhibiting important metabolic
processes such as glycolysis [3], possibly interfering
with active transport and signal transduction [1]. Fur-
thermore, sorbate and benzoate may also act as
membrane-damaging substances [4] and, at least under
aerobic conditions, cause severe oxidative stress [5,6].
The antimicrobial action of weak-acid preservatives
is usually characterized by extended lag phases and cell
stasis, although microbial killing does not occur. How-
ever, cells can adapt to the presence of weak acid and
resume growth. In Saccharomyces cerevisiae, this adap-
tation requires induction of the Pdr12p plasma mem-
brane ATP-binding cassette (ABC) transporter [7].
Together with the plasma membrane H
+
-ATPase,
Pma1p, the activity of which is also regulated by weak

S. cerevisiae. Hence, we pursued two different strategies.
First, we used a functional genomics approach and
screened all putative nonessential transcription factor
deletions of the EUROSCARF collection [14] (http://
www.uni-frankfurt.de/fb15/mikro/eroscarf/) for sorbate
hypersensitivity. This approach identified the regulator
War1p (weak acid resistance) as the main inducer of
PDR12 [15]. War1p is a nuclear transcription factor,
which decorates at least one weak-acid-response element
(WARE) in the PDR12 promoter. War1p is rapidly
phosphorylated upon stress challenge, and phosphory-
lation is somehow coupled to War1p activation [15].
Interestingly, War1p is required for PDR12 up-regu-
lation in response to exogenous weak-acid stress, but it
appears also to be involved in the metabolism-derived
endogenous fusel acid stress response [12].
The War1p protein belongs to the fungal-specific
Zn(II)
2
Cys
6
zinc finger family of transcriptional regula-
tors with some 54 other putative members in S. cere-
visiae [16]. These are implicated in various important
cellular processes, including amino-acid [17] and galac-
tose [18] metabolism, nitrogen source utilization [16],
peroxisomal proliferation [19,20], respiration [21,22]
and even pleiotropic drug resistance (PDR) [11]. For
example, Pdr1p and Pdr3p are key players in yeast
PDR development, because they control ABC drug

and the Pdr12p efflux pump. The defective War1-42 p
mutant is no longer phosphorylated upon stress and
C. Gregori et al. Yeast weak organic acid stress adaptation
FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS 3095
unable to bind to cis-acting WARE motifs, suggesting
that activation of War1p or its binding to WARE is
tightly linked to its post-translational modification.
Results
Isolation of sorbate-sensitive mutant strains
To identify components of the stress response pathway
that mediates induction of the Pdr12p efflux pump, we
set up a classical mutagenesis screen. For the isolation
of mutant cells that fail to induce PDR12 upon weak
acid challenge, we constructed a reporter strain carrying
the lacZ gene driven by the PDR12 promoter integrated
into the ura3 loci of two different genetic backgrounds,
creating the strains YCS12ZI and YAK3. These strains
were grown to the exponential growth phase, plated
and irradiated with UV light to randomly introduce
mutations. After a 2-day incubation, colonies were
replica-plated on plates containing 5 mm sorbate and
the dye X-Gal to induce the PDR12 promoter and to
visualize LacZ expression. In a first round of screening,
we obtained 111 white colonies (62 for YAK3 and 49
for YCS12Z-I). To determine if the white color resulted
from a lack of PDR12 promoter induction, and thus no
expression of lacZ, these colonies were re-screened for
their Pdr12p protein concentrations by immuno-
blotting. Although several mutants showed reduced
Pdr12p concentrations under stress conditions (data not

hence weak-acid resistance, War1p [15]. To determine
if the mutants isolated in the classical genetic screen
are allelic to WAR1, appropriate selection markers
were integrated and the strains subjected to com-
plementation analysis. Figure 2 shows the growth
phenotypes of the resulting diploid strains on yeast pep-
tone ⁄ dextrose (YPD), pH 4.5, with different sorbate
A
B
WT war1-28 war1-42
pdr12Δ
WT
war1-28
war1-42
pdr12Δ
YPD pH 4.5
control
0.25 m
M
0.5 m
M
1 m
M
+ Sorbate
Pdr12p
control
sorbate
-+-+-+-+
Fig. 1. war1 mutants are sorbate-sensitive and fail to induce
Pdr12p upon sorbate stress. (A) The strains W303-1A (WT), YAK4

42 were crossed with the war1D strain, the diploid
strains remained hypersensitive to sorbate, and dis-
played the same growth behavior as the pdr12D
control strain. These data suggest that the mutants
isolated in the UV mutagenesis screen were allelic to
WAR1. Thus, the mutant alleles were named War1-28
and War1-42, respectively. Interestingly, diploid War1-
28 ⁄ War1-42 cells were more resistant than war1D ⁄
mutant diploids, suggesting a possible cross-comple-
mentation of mutant alleles, and implying that War1p
acts as a dimer [15].
Identification of the mutations in war1
To identify the actual mutations leading to the loss-of-
function phenotypes, the defective war1 alleles were
amplified by PCR from genomic DNA obtained from
the mutant strains and subjected to DNA sequencing.
Sequencing of both DNA strands of War1-28 identi-
fied an A to T mutation at position 1286, and a
change of C to T at position 1288, the latter introdu-
cing a translational stop codon (Fig. 3). At the amino-
acid level, these mutations resulted in a N429I residue
exchange, and the nonsense mutation leads to trun-
cated War1-28p protein (Fig. 3A). For the mutant
War1-42 allele, four clustered mutations were found:
deletion of A2286, T2287 and T2288, and the G2291T
transversion. These four mutations caused three amino
acid changes, namely K762N, F763M and the R764D
deletion. The rest of the protein remained unchanged.
As depicted in the cartoon (Fig. 3A), War1p is repre-
sentative of the binuclear Zn(II)

protein is affected by the different mutations.
To address this point, we performed cycloheximide
chase experiments. The strains, YAK111, YBB30 and
YBB31, were grown in YPD to an A
600
of  1; then
cycloheximide was added to block protein synthesis,
and samples were collected at the indicated time points
for immunoblotting (Fig. 3C). The results show that
the wild-type protein was quite stable, with a half-life
of 100–120 min. Likewise, War1-28p-3HA was detect-
able throughout the whole chase period (Fig. 3C). In
contrast, War1-42p-3HA displayed a much faster pro-
teolytic turnover, as it was already below the detection
limit 40 min after cycloheximide addition (Fig. 3C).
Thus, the low steady-state concentrations of War1-
42p-3HA may be explained by its reduced stability.
Notably, sorbate failed to influence War1p stability, as
the half-life was unchanged under stress (data not
shown).
WT/WT
WT/war1Δ
WT/28
WT/42
war1Δ/28
war1Δ/42
28/42
pdr12Δ
YPD pH 4.5
control

of  1, split, and one half was treated with 8 mm
sorbate. After 30 min, cells were harvested, and protein
extracts prepared and subjected to immunoblotting. As
reported previously [15], wild-type War1p migrated as
a double band in unstressed cells and shifted to slower
mobility forms upon sorbate addition (Fig. 3D). In
contrast, no mobility shift was detectable for War1-
42p-3HA, as it migrated as a single band under both
stressed and nonstressed conditions (Fig. 3D). There-
fore, the post-translational modification pattern of
War1p, which is intimately linked to PDR12 stress
induction, is absent in the War1-42p-3HA mutant, indi-
cating that phosphorylation may be an essential step
in War1p activation. Remarkably, War1-42p-3HA
from unstressed cells exhibited a faster mobility on
SDS ⁄ polyacrylamide gels than authentic War1p
(Fig. 3D), suggesting that the basal modification in the
absence of stress was also affected in War1-42p-3HA.
Functional analysis of single-residue changes,
K762N, F763M and R764D
The War1-42 allele contains a cluster of four muta-
tions, leading to three residue changes. To address
which mutation alone or in combination with another
one causes the phenotype, we constructed the CEN-
based plasmids pCGWAR1-K762N, pCGWAR1-
F763M and pCGWAR1-R764D carrying the single
mutations, respectively. Each of the three plasmids
expressed a mutated version War1p with only one of
three residue changes of War1-42p. To determine the
phosphorylation status of War1p-K762N, War1p-

WT
WT
D
unstressed + 8 mM sorbate
30 min
War1p-3HA
Fig. 3. Organization, expression, stability and modification of War1p
variants. (A) The cartoon depicts the localization of mutations in the
WAR1 gene abrogating its function as a specific Pdr12p regulator.
Zn, zinc finger; AD, activation domain. Cartoon not drawn to scale.
(B) Expression and stability of the wild-type and mutant War1p vari-
ants. Cultures of the strains YAK111 (WT-3HA), YBB31 (28-3HA),
YBB30 (42-3HA) and YPH499 (WT) were grown in YPD to an A
600
of  1 and harvested. Yeast crude protein extracts equivalent to 1
A
600
were separated by PAGE (10% gel) and analyzed by immuno-
blotting using the 12CA5 HA antibody. Cross-reactions to the HA
antibody served as a loading control. (C) The strains YAK111
(War1p-3HA), YBB31 (War1-28p-3HA) and YBB30 (War1-42p-3HA)
were grown in YPD to an A
600
of  1, then cycloheximide (CHX)
was added to a final concentration of 0.1 mgÆmL
)1
, and samples
were taken at the indicated time points. Extracts (0.5 A
600
for

cultures were split in half; one half was treated with
8mm sorbate for 30 min, and the other left unstressed.
Cells were then harvested, and protein extracts were
prepared and subjected to immunoblotting. Whereas
War1p-F763M behaved as the wild-type War1p under
unstressed conditions, War1p-K762N showed a slightly
different modification pattern in unstressed cells. In
contrast with wild-type War1p, which migrated as a
double band under nonstressed conditions, the lower-
migrating band was hardly detectable in War1p-K762N
(Fig. 4A). However, sorbate shifted both War1p-
K762N and War1p-F763M to a slower mobility, as
was also observed for the wild-type control. In con-
trast, War1p-R764D remained unmodified in response
to sorbate stress. The mobility shift in response to
stress is tightly linked to PDR12 induction, which was
absent in the strain expressing War1p-R764D (Fig. 4A).
In contrast, strains expressing the mutants War1p-
K762N and War1p-F763M showed greatly increased
Pdr12p concentrations in both the absence and pres-
ence of weak-acid stress. Addition of sorbate did not
further increase Pdr12p expression levels (Fig. 4A),
demonstrating that the War1p-K762N and War1p-
F763M single mutants are gain-of-function variants.
However, their hyperactivity is suppressed by the pres-
ence of the additional R764D deletion in War1-42p.
Furthermore, we tested the strains expressing the
mutant War1p variants for their ability to grow on
YPD, pH 4.5, in the presence or absence of different
sorbate concentrations (Fig. 4B). Consistent with the

Pdr5p
WT
war1Δ
K762N
F763M
R764
Δ
YPD pH 4.5
+ 0.5 m
M sorbate + 1 mM sorbate + 2 mM sorbate
0 20 40 60 80 100 120 min CHX
War1p
War1p-R764Δ
War1-42p
B
A
C
Fig. 4. War1p-764D causes sorbate hypersensitivity and fails to
induce Pdr12p. (A) Cultures of YAK120 cells were transformed with
pCGWAR1, pCGWAR1-K762N, pCGWAR1-F763M or pCGWAR1-
R764D and grown in YPD to an A
600
of  1; cultures were split in
half, and one half was treated with 8 m
M sorbate for 30 min, while
the other remained untreated. Crude cell extracts (0.5 A
600
) were
separated by SDS ⁄ PAGE (7% gel), and immunodetected using
polyclonal antibodies against War1p, Pdr12p and Pdr5p. (B) YAK120

tion, change War1p folding, thereby destabilizing
War1p more than the loss of a single amino acid as in
War1-R764Dp.
War1-42p localizes to the nucleus but is unable
to bind to the WARE in vivo
We have previously demonstrated that War1p is a nuc-
lear protein [15]. Although the mutational changes in
War1-42p left the DNA-binding domain and both NLS
unaffected, we wanted to test whether War1-42p is also
properly localized to the nucleus. Hence, we carried out
fractionation experiments using purified nuclear frac-
tions from various strains. Subcellular fractions were
isolated by following a gentle cell lysis procedure to
preserve nuclear integrity, and subjected to immuno-
blotting using polyclonal antibodies specific for War1p,
the nuclear marker protein Swi6p and the cytoplasmic
hexokinase Hxk1p (Fig. 5A). As shown in Fig. 5A,
War1-42p, like the wild-type control War1p, localized
to the nucleus in the steady state. As expected, Hxk1p
was predominantly found in the soluble cytoplasmic
fraction. The signal for Hxk1p in the nuclear fraction is
due to normal unavoidable contamination of the nuc-
lear fraction with cytosolic proteins. However, the nuc-
lear marker, Swi6p, entirely cofractionated with both
War1-42p and wild-type War1p (Fig. 5A), demonstra-
ting that the normal nuclear localization of War1-42p
is unaffected by the mutations. No immunoreactive
material was detectable in war1D cells, confirming the
specificity of the polyclonal anti-War1p serum. Nota-
bly, the polyclonal antibodies also detected a War1p

war1Δ
CNSCSCNSN
War1p
Swi6p
Hxk1p
B
A
Fig. 5. Nuclear War1-42p is unable to decorate the PDR12 promoter
in vivo. (A) Subcellular fractions were prepared from wild-type cells
(WT), War1-42 mutants (42) and cells lacking War1p (war1D) as des-
cribed in Experimental procedures. About 2 A
600
equivalents were
subjected to immunoblotting using polyclonal antibodies against
War1p, Swi6p and Hxk1p. S, Total input; N, nuclear pellet; C, cyto-
plasmic fraction. (B) YPH499 wild-type (WT), YAK110 (war1D) and
YCS42-D4 (War1-42) cells were grown to the early exponential
growth phase and treated with dimethyl sulfoxide to methylate
DNA. For in vivo footprinting, chromosomal DNA was prepared and
used as a template for primer extension with a labeled oligonucleo-
tide primer corresponding to )497 to )472 of the PDR12 promoter.
The reaction mixture was resolved through a sequencing gel,
exposed to a phosphoimaging screen, and signals were quantified.
Intensities of traces are compared in the indicated combinations. An
asterisk marks a protected G ()631) in the War1-42 allele. Differ-
ences are indicated by bars for deprotected G residues ()643,
)642, )617, )618) and aligned with the sequence of the region.
Yeast weak organic acid stress adaptation C. Gregori et al.
3100 FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS
would be a consequence of the impaired growth of

Discussion
We are interested in dissecting the response pathway
necessary for cellular adaptation to stress from weak
organic acids in the yeast, S. cerevisiae. Using a func-
tional genomic approach, we have identified the
War1p regulator as the dedicated transcription factor
required for Pdr12p induction following weak-acid
stress exposure [15]. In this study, we report the iso-
lation and characterization of two loss-of-function
war1 alleles that give rise to War1p variants that are
unable to mediate Pdr12p induction in response to
sorbate stress. We exploited a classical genetic screen,
taking advantage of a lacZ reporter driven by the
PDR12 promoter, which otherwise controls expression
of the Pdr12p weak-acid anion-efflux pump [7]. After
UV mutagenesis, we screened more than 10 genome
equivalents of mutant colonies for their capacity in
PDR12 induction. We expected to isolate mutants in
membrane sensors, signaling components such as
kinases, phosphatases and perhaps transcriptional
regulators. However, most remarkably, only two yeast
mutants were isolated in which sorbate-mediated
induction of Pdr12p was completely abolished
(Fig. 1A). The weak-acid hypersensitivity of both
mutant strains was attributed to mutations in the
WAR1 gene (Fig. 3). DNA sequencing identified the
mutations in the nonfunctional war1 alleles. Whereas
War1-28 encoded a truncated regulator which was due
to a stop codon, War1-42 carried three residue chan-
ges close to the C-terminus. Hence, the genetic

Cys
6
transcription factors [33]. Notably, the truncated
War1-28p protein, although expressed at higher levels
than the wild-type, does not interfere with the function
of authentic War1p in diploid cells (Fig. 2), which
might be a direct consequence of impaired dimer for-
mation of War1p or a lack of DNA binding. Indeed,
in the case of War1-42p, in vivo footprinting data
(Fig. 5) indicate an inability to bind to the WARE,
which is normally decorated by wild-type War1p in the
presence or absence of the stress agent [15]. Thus, the
lack of PDR12 stress induction by War1-42p is per-
haps due to its inability to bind to the promoter
WARE of its target gene. Alternatively, the mutations
may also reduce the binding affinity of the War1-42p,
C. Gregori et al. Yeast weak organic acid stress adaptation
FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS 3101
thereby causing impaired assembly of the active tran-
scription complex.
A lack of DNA binding by War1-42p may be
explained in several ways. First, immunoblotting
cycloheximide chase experiments indicated that War1-
42p shows lower steady-state protein concentrations
and decreased stability compared with the wild-type
War1p (Fig. 4). Hence, the amount of protein might
simply be too low to allow binding to the target DNA.
Secondly, the mutations may reduce affinity for
WARE binding or prevent the formation of War1p
dimers, which appears necessary for War1p function

(Fig. 3D). Hence, the R764D mutation can be consid-
ered dominant for War1p loss-of-function when pre-
sent in combination with the hyper-activating
mutations, K762N and F763M.
The basal modification status of War1-42p is differ-
ent from wild-type War1p, as they display distinct
mobilities on immunoblotting. DNA binding in vivo
may well require basal post-translational modifications,
as present in wild-type War1p but absent in the
mutant variant (Fig. 3D). These modifications either
directly influence the DNA-binding capability or are
necessary for the interaction with another as yet
unknown cofactor that would facilitate binding to the
PDR12 promoter.
The fact that loss-of- function mutations reside out-
side the zinc finger or the NLS suggests an altered
conformation or structure. This is consistent with the
apparent absence of stress-induced phosphorylation in
War1p-R764D and its reduced protein stability. Thus,
only massive folding changes can explain the inactiv-
ity of War1p-R764D, which may hinder phosphoryla-
tion. Mutations may also affect the structure of
confined domains such as the MHR rather than the
whole tertiary structure. Hence, the lack of phos-
phorylation is most likely a consequence of massively
altered War1p conformation rather than altered struc-
ture of the kinase targets themselves. Further, the
residue changes in War1-42p do not involve serine,
threonine, tyrosine or histidine (Fig. 3A). In any case,
the nonphosphorylated war1 alleles are nonfunctional,

identify intragenic suppressor mutations. Furthermore,
high-copy or second-site suppressors may lead to the
identification of unknown War1p-interacting partners.
This seems to be a feasible and promising approach
Yeast weak organic acid stress adaptation C. Gregori et al.
3102 FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS
considering the suppression of the constitutive activity
of K762N and F763M by an additional R764D dele-
tion. Finally, the War1-42 mutant allele should be use-
ful as a tool for learning more about the molecular
structure, as well as the protein–protein and protein–
DNA interactions, of this binuclear zinc transcriptional
regulator. The polyclonal antibodies to War1p should
be useful in identifying the upstream components of the
response pathway, including War1p-specific kinases
and phosphatases implicated in the modulation of
War1p activity during adaptation to stress induced by
weak organic acids.
Experimental procedures
Yeast strains, growth conditions, and growth
inhibition assays
Rich medium (YPD) and synthetic medium were prepared
essentially as described elsewhere [35]. Unless otherwise
indicated, all yeast strains were grown routinely at 30 °C.
S. cerevisiae strains used in this study are listed in Table 1.
To determine weak-acid susceptibility, exponentially grow-
ing cultures were adjusted to A
600
of 0.2 and diluted 1 : 10,
1 : 100 and 1 : 1000. Equal volumes of these serial dilutions

YAK2 MATa ura3-1::pCS12ZI URA3 (isogenic to W303-1B) [15]
YAK3 MATa ura3-1::pCS12ZI URA3 LEU2 (isogenic to W303-1B) [15]
YAK4 MATa ura3-1::pCS12ZI URA3 LEU2 War1-28 (isogenic to W303-1B) [15]
YAK110 MATa war1D::HIS3MX6 (isogenic to YPH499) This study
YAK111 MATa WAR1-3HA KANMX6 (isogenic to YPH499) [15]
YAK120 MATa war1D::HIS3MX6 (isogenic to W303-1A) [15]
YCS42-D4 MATa ura3-52::pCS12ZI URA3 War1-42 (isogenic to YPH499) This study
YBB14 MATa pdr12::hisG-URA3-hisG (isogenic to W303-1A) This study
YBB20 MATa ⁄ a LEU2 war1D::HIS3MX6 (isogenic to W303-D) This study
YBB22 MATa ⁄ a ura3-1 leu2-3112 trp1-1 ade2-1 can1-100 ura3-52::pCS12ZI
URA3 leu2-D1 his3-D200 trp1-
D1 ade2-10
oc
lys2-801
a
War1-42
This study
YBB23 MATa ⁄ a ura3-1::pCS12ZI URA3 LEU2 his3-11,15 trp1-1 ade2-1 can1-100
War1-28 ura3-52::pCS12ZI URA3 leu2-D HIS3 trp1-D1 ade2-10
oc
lys2-801
a
War1-42
This study
YBB24 MATa ⁄ a ura3-1::pCS12ZI URA3 LEU2 HIS3 trp1-1 ade2-1 can1-100 War1-28
(isogenic to W303-D)
This study
YBB25 MATa ⁄ a ura3-1::pCS12ZI URA3 LEU2 his3-11,15 trp1-1 ade2-1 can1-100 War1-28
war1D::HIS3MX6 (isogenic to W303-D)
This study

side) plates containing 5 mm sorbate and grown for another
2 days. For YAK3, about 80 000 independent yeast colon-
ies, and for YCS12Z-I about 50 000 colonies, were screened
for the loss of sorbate-mediated lacZ induction. White col-
onies were re-streaked to single colonies on X-Gal plates
containing 5 mm sorbate for easier inspection of color devel-
opment. All colonies that remained white were re-screened
for Pdr12p concentrations by immunoblotting using poly-
clonal antibodies to Pdr12p [7]. Isolated mutant cells were
back-crossed at least three times to clean up the genetic
background and to verify 2 : 2 segregation of sorbate sensi-
tivity.
Site-directed mutagenesis and plasmid
construction
To generate the single-residue changes K762N, F763M and
R764D, a PCR fragment containing 170 bp of the WAR1
promoter and 2568 bp of the WAR1 ORF was ligated to
the vector pGEMT-easy (Promega, Mannheim, Germany)
resulting in the plasmid pIF1. This plasmid served as a tem-
plate for site-directed mutagenesis reactions using the
QuickChange Site-Directed Mutagenesis kit (Stratagene).
Site-directed mutagenesis was carried out exactly as recom-
mended by the manufacturer using the customized oligonu-
cleotide primers listed in Table 2. Mutations in the WAR1
sequence are indicated in bold italic letters in the primer
sequence. The plasmids obtained were named pIF1-762,
pIF1-763 and pIF-764, and successful mutagenesis was veri-
fied by DNA sequencing.
Fragments containing the indicated mutations were
cloned into a yeast vector as follows. A 4.32-kb PCR frag-

600
of 0.5.
Expression of the GST-War1p fusion protein was induced
at 30 °C for 2 h by adding isopropyl b-d-thiogalacto-
pyranoside to a final concentration of 0.2 mm. The
Table 2. Oligonucleotides for site-directed mutagenesis of WAR1. Bases leading to residue changes in mutagenic oligonucleotides are given
in bold italic letters.
Name Oligonucleotide sequence Source
K762Ns 5¢-CCCTTCAACAACTCTCTTTAC
AAC TTTAGGTATGTTATTGCG-3¢ This study
K762Nas 5¢-CGCAATAACATACCTAAA
GTT GTAAAGAGAGTTGTTGAAGGG-3¢ This study
F763Ms 5¢-CTTCAACAACTCTCTTTACAAA
ATG AGGTATGTTATTGCGTTATTTTG-3¢ This study
F763Mas 5¢-CAAAATAACGCAATAACATACCT
CAT TTTGTAAAGAGAGTTGTTGAAG-3¢ This study
R764Ds5¢-CCCTTCAACAACTCTCTTTACAAATTTTATGTTATTGCGTTATTTTGTC-3¢ This study
R764Das 5¢-GACAAAATAACGCAATAACATAAAATTTGTAAAGAGAGTTGTTGAAGGG-3¢ This study
Yeast weak organic acid stress adaptation C. Gregori et al.
3104 FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS
GST-War1p fusion protein was purified as described else-
where [15], except that the binding to the glutathione–Seph-
arose beads (Amersham) was performed at 4 °C for 16 h
and elution was carried out with 0.05% SDS. Removal of
SDS and concentration of the GST-War1p fusion protein
was carried out in a Centricon YM-10 centrifugal filter
device (Millipore, Billerica, MA). Antiserum to the purified
GST-War1p fusion protein was raised in rabbits using a
standard immunization regimen as described elsewhere [41].
Preparation of yeast cell extracts and immunoblotting

of 1.0 were harvested, washed and
pretreated with 2-mercaptoethanol [43]. For spheroblasting,
cells were resuspended in 2 mL S-buffer (1.0 m sorbitol,
25 mm KH
2
PO
4
pH 6.5, 0.4 mm CaCl
2
), Zymolyase
100.000 was added (25 UÆmL
)1
), and cells were incubated
with gentle shaking at 30 ° C for 1 h. Spheroblasts were cen-
trifuged at 3600 g for 10 min and washed once with S-buf-
fer. An aliquot was mixed with sample buffer for direct
lysis (total input). Spheroblasts were lysed by adding 5 vol-
umes of N-buffer (18% Ficoll, 20 mm KH
2
PO
4
pH 6.5,
0.5 mm CaCl
2
,1mm phenylmethanesulfonyl fluoride) and
vigorous vortex-mixing. After centrifugation at 2.500 g, the
supernatant was re-centrifuged at 20 000 g for 30 min. The
supernatant representing the cytoplasmic fraction was care-
fully removed; aliquots of the cytoplasmic fraction and the
pellet with the nuclear fraction were mixed with sample

and treated with 5 lL dimethyl sulfoxide. The reaction was
stopped after 5 min, and chromosomal DNA prepared. Pri-
mer extension was carried out with a
32
P-labeled oligonu-
cleotide corresponding to the residues )497 to )472 of the
PDR12 promoter, resolved through a 8% sequencing gel,
exposed to a phosphoimager screen, and quantified. Traces
were captured using ImageQuant software, converted into
vector graphs, and aligned. For identification of the muta-
tions in war1 alleles, the WAR1 gene was amplified from
genomic DNA. To eliminate the danger of PCR-derived
mutations, the pool of PCR fragments was subjected
directly to DNA sequencing using the BigDye Terminator
Cycle Sequencing Kit version 3.0 according to the instruc-
tions of the manufacturer and the ABI PRISM Sequencing
System 310 (Applied Biosystems, Foster City, CA).
Acknowledgements
We thank Manuela Schu
¨
tzer-Mu
¨
hlbauer and all labor-
atory members for critical reading of the manuscript
and helpful discussions. Peter Piper and Mehdi Molla-
pour are acknowledged for sharing unpublished infor-
mation and their long-standing collaboration. We
appreciate the gifts of polyclonal anti-Swi6p and anti-
Hxk1p sera from Kim Nasmyth and Rudolf Schweyen,
respectively. This work was supported by a grant

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