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: karl.kuchler@meduniwien.ac.at
Present address
Universite
´de Nice-Sophia Antipolis,
Inserm, U636, Centre de Biochimie, UFR
Sciences, Parc Valrose, Nice, France
àInstitute of Biochemistry and Genetics,
Department of Clinical and Biological
Research (DKBW), Basel, Switzerland
§University of Vienna, Max F. Perutz
Laboratories, Department of Biochemistry &
Molecular and Cellular Biology, Campus
Vienna Biocenter, Austria
*
These authors contributed equally to this
work
(Received 11 January 2007, revised 4 April
2007, accepted 19 April 2007)
doi:10.1111/j.1742-4658.2007.05837.x
Exposure of the yeast Saccharomyces cerevisiae to weak organic acids such
as the food preservatives sorbate, benzoate and propionate leads to the
pronounced induction of the plasma membrane ATP-binding cassette
(ABC) transporter, Pdr12p. This protein mediates efflux of weak acid ani-
ons, which is essential for stress adaptation. Recently, we identified War1p
as the dedicated transcriptional regulator required for PDR12 stress
induction. Here, we report the results from a genetic screen that led to the
isolation of two war1 alleles encoding mutant variants, War1-28p and
War1-42p, which are unable to support cell growth in the presence of sorb-
ate. DNA sequencing revealed that War1-28 encodes a truncated form of
the transcriptional regulator, and War1-42 carries three clustered mutations
near the C-terminal activation domain. Although War1-42 is expressed and
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 ª2007 The Authors Journal compilation ª2007 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
acid stress [8,9], Pdr12p becomes one of the most abun-
dant surface proteins in stressed cells [7]. Whereas
Pma1p effluxes protons, Pdr12p mediates cellular
extrusion of weak acid anions [7]. Notably, other mem-
bers of the fungal ABC transporter family transport a
wide variety of different xenobiotics across the plasma
membrane or membranes of subcellular compartments
[10,11]. Pdr12p is the essential component of this stress
response pathway, as cells are hypersensitive to sorbic,
benzoic and propionic acid [7] and fail to adapt to such
stress conditions in the absence of Pdr12p. Moreover,
recent data indicate the involvement of Pdr12p in the
export of by-products of amino-acid catabolism, as a
pdr12Dstrain displays hypersensitivity to fusel acids
derived from leucine, isoleucine, valine, phenylalanine
and tryptophan [12]. Therefore, Pdr12p is not only
required for adaptation to weak-acid stress, but might
also efflux weak-acid metabolites. Notably, PDR12 is
rapidly induced by weak-acid challenge [7], but also in
cultures grown with leucine, methionine or phenylalan-
ine as sole nitrogen source [12]. A recent study [13]
attempted to identify Pdr12p-like proteins in other food
spoilage yeasts. A sorbic-inducible protein cross-react-
ing with S. cerevisiae Pdr12p antibodies in Saccharomy-
ces bayanus was found. In contrast, proteins detectable
with the same antibodies in Zygosaccharomyces bailii
and Zygosaccharomyces lentus were not up-regulated
upon sorbate challenge.
We are interested in identifying components of the
signaling pathway required for this efficient response in
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
efflux pumps such as Pdr5p [23,24], Snq2p [25,26] and
Yor1p [27], all of which are involved in PDR [10,11].
Most regulators harbor a binuclear DNA-binding zinc
cluster at the N-terminus, whereas the acidic activation
domain is usually present at the C-terminus. The mid-
dle homology region (MHR) bridging the DNA-bind-
ing and the activation domain may control the activity
or specificity of the transcription factor, as deletions or
mutations in this region often result in constitutive
activity [22,28–30]. Notably, a WAR1 orthologue has
been identified in the human fungal pathogen Candida
albicans [31]. Consistent with its role in S. cerevisiae,
this WAR1 is also required for sorbate tolerance.
Moreover, our group recently identified the Candida
glabrata homologue of War1p (C. Gregori and
K. Kuchler, unpublished work). Preliminary experi-
ments show that it is also required for a response to
weak organic acids in the human fungal pathogen
C. glabrata, demonstrating the evolutionary conserva-
tion of this weak-acid stress in the fungal kingdom
(C. Gregori and K. Kuchler, unpublished work).
Secondly, we applied a classical genetic screen using
aPDR12prom-lacZ reporter gene to identify compo-
nents of the weak-acid response pathway. Here, we
report the results of the genetic approach, which leads
to the isolation of two war1 mutant alleles that are
unable to drive Pdr12p induction. Remarkably, the
genetic screen identified mutations in the WAR1 gene
only, indicating that weak-acid stress response requires
two major components, a dedicated stress regulator
and the Pdr12p efflux pump. The defective War1-42p
mutant is no longer phosphorylated upon stress and
C. Gregori et al.Yeast weak organic acid stress adaptation
FEBS Journal 274 (2007) 3094–3107 ª2007 The Authors Journal compilation ª2007 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 mmsorbate 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
shown), only two mutant strains, 42 and 28, lacked
detectable Pdr12p induction upon sorbate stress
(Fig. 1A). Both mutants were back-crossed with the
wild-type several times to clean up the genetic back-
ground and determine whether the phenotype was
caused by mutations in a single gene. As tetrad analysis
revealed a 2 : 2 cosegregation of sorbate sensitivity with
the inability of lacZ induction, both mutations must
reside in a single gene (data not shown). Growth-inhibi-
tion assays (Fig. 1B) showed that mutants 28 and 42
grew at a sorbate concentration of up to 0.25 mm. The
pdr12Dcontrol strain was viable, but exhibited reduced
growth on 0.5 mmsorbate plates, and failed to grow on
1mmsorbate, whereas the wild-type control even grew
at concentrations above 1 mm. Therefore, we isolated
two yeast mutants with defects in a single gene repre-
senting at least one component of the weak-acid
response machinery that acts through Pdr12p induction
to trigger adaptation.
Identification of mutated genes
In addition to the classical genetic approach, we
recently pursued a functional genomics approach to
identify regulators of weak organic acid resistance.
Making use of the EUROSCARF haploid deletion
strain collection of S. cerevisiae [14] (EUROSCARF,
Germany; http//http://www.uni-frankfurt.de/fb15/
mikro/euroscarf/), we tested all viable transcription
factor deletion strains for their ability to grow in the
presence of sorbic acid. This approach identified the
transcription factor essential for Pdr12p induction and
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 pdr12∆
WT
war1-28
war1-42
pdr12∆
YPD pH 4.5
control
0.25 mM0.5 mM1 mM
+ 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
(War1-28), YCS42-D4 (War1-42) and control strain YBB14 (pdr12D)
were grown in YPD to an A
600
of 1. The cultures were split and
one half was stressed with 8 mMsorbate for 1 h. Cell extracts
equivalent to 0.5 A
600
were separated by SDS ⁄PAGE (7% gel), and
the immunoblots were decorated with polyclonal anti-Pdr12p
serum. A cross-reaction to the antibodies served as loading control.
(B) The strains W303-1A (WT), YAK4 (War1-28), YCS42-D4 (War1-
42) and YBB14 (pdr12D) were grown in YPD to an A
600
of 1.
Then the A
600
was adjusted to 0.2, and the cells were spotted
along with three 1 : 10 serial dilutions on YPD, pH 4.5, containing
the indicated sorbate concentrations. Growth was monitored after
a 48-h incubation at 30 C.
Yeast weak organic acid stress adaptation C. Gregori et al.
3096 FEBS Journal 274 (2007) 3094–3107 ª2007 The Authors Journal compilation ª2007 FEBS
concentrations. Mutants 28 and 42, as well as the
war1Ddeletion, when in combination with a wild-type
gene, displayed similar growth to the diploid wild-type
strain. Thus, the mutant alleles, 28 and 42, are reces-
sive for sorbate growth, and a heterozygous wild-
type ⁄war1Dstrain did not show any haplo-insufficiency
phenotypes (Fig. 2). However, when mutants 28 and
42 were crossed with the war1Dstrain, 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)
2
Cys
6
transcription
factor family, all members of which contain a DNA-
binding zinc finger at their N-terminus (amino acids
75–111), followed by two predicted nuclear localization
signals (NLS amino acids 106–123 and 286–303), and
a coiled-coil domain mediating protein–protein interac-
tions. The putative transcriptional activation domain is
located near the C-terminus, residues 911–937. Hence,
a loss-of-function phenotype of War1-28 may easily be
explained by the absence of the activation domain,
whereas the effect of the mutations in War1-42 on
War1p function is not immediately obvious.
Characterization of War1-42p and its
post-translational modification
To determine if the mutant proteins are properly
expressed, we epitope-tagged both War1-28p and
War1-42p at the C-terminus by genomic integration of
a triple 3HA epitope, creating the strains YBB30
and YBB31, respectively. Immunoblotting of protein
extracts from exponentially growing cultures revealed
that both mutant proteins displayed a mobility corres-
ponding to their predicted molecular mass (Fig. 3B).
However, when compared with the wild-type, the
steady-state concentrations of mutant War1-42p-3HA
appeared to be markedly reduced. Notably, the con-
centrations of the truncated War1-28p-3HA appeared
slightly increased, implying that the stability of the
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/war1∆
WT/28
WT/42
war1∆/28
war1∆/42
28/42
pdr12∆
YPD pH 4.5
control
0.5 mM1 mM
+ Sorbate
Fig. 2. The sorbate-sensitive mutants carry loss-of-function alleles
of WAR1. The strains W303-D (WT ⁄WT), YBB21 (WT ⁄war1D),
YBB24 (WT ⁄28), YBB22 (WT ⁄42), YBB25 (war1D⁄28), YBB26
(war1D⁄42), YBB23 (28 ⁄42) and YBB14 (pdr12D) were grown to an
A
600
of 1, diluted to A
600
of 0.2 and spotted on to YPD, pH 4.5,
agar plates containing the indicated sorbate concentrations along
with three 1 : 10 serial dilutions. Colony growth was inspected
after 48 h at 30 C.
C. Gregori et al.Yeast weak organic acid stress adaptation
FEBS Journal 274 (2007) 3094–3107 ª2007 The Authors Journal compilation ª2007 FEBS 3097
Stress-induced phosphorylation is absent
in War1-42p
Whereas for War1-28p the inability to induce PDR12
transcription was attributable to the lack of the activa-
tion domain, the explanation was not obvious for
War1-42p. Notably, we have previously shown that
PDR12 induction coincides with War1p phosphoryla-
tion [15]. Therefore, we determined the post-transla-
tional modification status of War1-42p-3HA under
both stressed and nonstressed conditions using immu-
noblotting (Fig. 3D). The cultures were grown to an
A
600
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-R764Dcarrying 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-
war1-28
N429I
STOP
war1-42
K762N
F763M
R764∆
Zn
NLS
AD
NLS
A
WT-3HA
28-3HA
42-3HA
WT
loading control
cross reaction
B
War1p-3HA
War1-28p-3HA
War1p-3HA
War1-28p-3HA
War1-42p-3HA
0 20 40 60 80 100 120 min CHX
C
WT-3HA
WT-3HA
42-3HA
42-3HA
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
War1p-3HA and War1-28p-3HA, 1.5 A
600
for War1-42p-3HA) were
fractionated by SDS ⁄PAGE (10% gel), followed by immunodetec-
tion of the War1p-3HA and variants by monoclonal 12CA5 HA anti-
body. (D) The strains YAK111 (WT-3HA), YBB30 (42-3HA) and
YPH499 (WT) were grown in YPD to an A
600
of 1, then the cul-
tures were split, and one half was treated with 8 mMsorbate for
30 min. Crude cell extracts (equivalent to 1.5 A
600
for 42-HA and
0.5 A
600
for WT-HA and WT) were separated by SDS ⁄PAGE (7%
gel) and immunodetected using the monoclonal 12CA5 HA anti-
body.
Yeast weak organic acid stress adaptation C. Gregori et al.
3098 FEBS Journal 274 (2007) 3094–3107 ª2007 The Authors Journal compilation ª2007 FEBS