The EMBO Journal Vol.17 No.15 pp.4257–4265, 1998
The Pdr12 ABC transporter is required for the
development of weak organic acid resistance in
Peter Piper1, Yannick Mahe ´2,3,
Suzanne Thompson1, Rudy Pandjaitan2,
Caroline Holyoak4, Ralf Egner2,
Manuela Mu ¨hlbauer2, Peter Coote4and
2Department of Molecular Genetics, University and Biocenter of
Vienna, A-1030 Vienna, Austria,1Department of Biochemistry and
Molecular Biology, University College London, London WC1E 6BT
and4Microbiology Department, Unilever Research,
Colworth Laboratory, Sharnbrook, Bedford MK44 1LQ, UK
3Present address: Institut Curie, INSERM U-248, Section de
Recherche, 26, rue d’Ulm, 75248 Paris, Cedex 05, France
P.Piper and Y.Mahe ´ contributed equally to this work
Exposure of Saccharomyces cerevisiae to sorbic acid
strongly induces two plasma membrane proteins, one
of which is identified in this study as the ATP-binding
cassette (ABC) transporter Pdr12. In the absence of
weak acid stress, yeast cells grown at pH 7.0 express
causes a dramatic induction of Pdr12 in the plasma
membrane. Pdr12 is essential for the adaptation of
yeast to growth under weak acid stress, since ∆pdr12
mutants are hypersensitive at low pH to the food
preservatives sorbic, benzoic and propionic acids, as
well as high acetate levels. Moreover, active benzoate
effluxisseverely impairedin∆pdr12cells. Hence,Pdr12
confers weak acid resistance by mediating energy-
dependent extrusion of water-soluble carboxylate
anions. The normal physiological function of Pdr12 is
perhaps to protect against the potential toxicity of
weak organic acids secreted by competitor organisms,
acids that will accumulate to inhibitory levels in cells
at low pH. This is the first demonstration that regulated
expression of a eukaryotic ABC transporter mediates
weak organic acid resistance development, the cause
of widespread food spoilage by yeasts. The data also
have important biotechnological implications, as they
suggest that the inhibition of this transporter could be
a strategy for preventing food spoilage.
Keywords: ABC protein/adaptation/Saccharomyces
cerevisiae/stress response/weak organic acid tolerance
Weak acid preservatives are generally considered safe
antimicrobials, consistent with the long history and now
widespread use of these compounds for the preservation
of foods and beverages. For instance, the use of sulfite
for the sterilization of wine vessels is centuries old and
© Oxford University Press
still used in wine making. In solution, these acids are
in a dynamic, pH-dependent equilibrium between their
undissociated molecules and anionic states. An acidic pH
favours the undissociated, uncharged state, a state in which
weak acid preservatives exert much stronger antimicrobial
action. This is probably because such action largely
involves the uncharged acid diffusing through the plasma
membrane into the cytoplasm, where it encounters a more
neutral pH and consequently dissociates. This dissociation
releases protons, the resulting intracellular acidification
inhibiting several metabolic processes (Krebs et al., 1983).
an extended lag phase and cell stasis, rather than cell
death. The ability of certain yeast species to grow at low
pH in the presence of weak organic acid food preservatives
enables them to act as important agents of food spoilage
which can cause considerable economic losses (Deak,
1991; Fleet, 1992). Certain strains of Saccharomyces
cerevisiae will grow in the presence of up to 3 mM sorbic
acid at pH 4.5, although the presence of the preservative
causes both a drastic lag phase extension and a reduction
of final biomass yield (Stratford and Anslow, 1996; Piper
et al., 1997). Although S.cerevisiae is sometimes identified
as a food spoilage organism, other even more weak acid-
tolerant and osmotolerant yeasts such as Zygosaccharo-
myces bailii are more frequently found causing food
spoilage. These yeast species are sometimes capable of
adapting to growth in the presence of the highest levels
of weak organic acids allowed in commercial food preserv-
ation, at pH values less than the pKas of these acids (Deak,
1991; Fleet, 1992).
We have been investigating whether weak acid adapta-
tion by S.cerevisiae involves a novel stress response or is
the manifestation of an already identified stress response
pathway. In this yeast, we found that weak organic acid
treatment at low pH rapidly renders cells refractory to the
well-studied heat shock response, inhibiting both heat
shock protein (Hsp) and thermotolerance induction by
sublethal heat stress (Cheng et al., 1994). Instead, sorbic
acid treatment at pH 4.5 stimulates a hitherto unknown
stress response pathway, leading to a strong induction of
two plasma membrane proteins, one of which was identi-
fied earlier as Hsp30, a protein that is also induced by
heat shock and ethanol (Piper et al., 1997). Hsp30 assists
weak acid adaptation, since cultures lacking Hsp30 show
reduced biomass yields and take longer to adapt to growth
in the presence of sorbate (Piper et al., 1997).
In this study, we identify the larger sorbate-induced
protein as the ATP-binding cassette (ABC) transporter
Pdr12, a homologue of the Snq2 (Servos et al., 1993) and
Pdr5 (Balzi et al., 1994; Bissinger and Kuchler, 1994)
of Pdr12 plays a pivotal role in the acquisition of tolerance
to weak organic acid preservatives such as sorbate and
P.Piper et al.
Fig. 1. Purified plasma membrane fractions from sorbate-treated yeast
cells show a highly induced (S) membrane protein. (A) Wild-type cells
were cultured overnight (ON) in pH 4.5 YPD in the absence (1) and
presence (2) of 1 mM sorbate. About 40 µg total plasma membrane
protein per lane were separated through a 9% SDS–polyacrylamide gel
and stained with Coomassie blue. (B) About 8 µg of total plasma
membrane proteins from wild-type (3) and ∆pdr12 (4) cells grown for
6 h in pH 4.5 YPD in the presence of 1 mM sorbate were analysed by
SDS–PAGE and silver-staining. The main 100 kDa band represents the
Pma1 plasma membrane H?-ATPase.
benzoate. Weak acid-mediated Pdr12 induction and con-
comitant development of tolerance is independent of the
Yap1 (Kuge and Jones, 1994) and Msn2/Msn4 (Martinez-
Pastor et al., 1996) transcription factors, all of which are
important stress response regulators. Surprisingly, sorbate
resistance was enhanced in ∆pdr1 and ∆pdr1 ∆pdr3
deletion mutants, implying a functional cross-talk between
a yet unknown sorbate response pathway and the pleio-
tropic drug resistance (PDR) network (Decottignies and
Goffeau, 1997; Kuchler and Egner, 1997).
We have been investigating plasma membrane proteins
induced in S.cerevisiae during adaptation to growth at
pH 4.5 in the presence of sorbic acid, a non-metabolized
weak acid food preservative. Microsequencing of a highly
induced 170 kDa protein (Figure 1B, lane 3) in purified
plasma membrane fractions from sorbate-treated cells
yielded four peptide sequences that were perfect matches
to the regions 287–300, 366–383, 838–859 and 1062–
1078 of a large open reading frame, YPL058c, present
in the yeast Proteome Database. YPL058c residing on
chromosome XVI encodes the 1511-residue protein Pdr12,
a typical member of the ABC protein superfamily
(Decottignies and Goffeau, 1997; Kuchler and Egner,
1997). The predicted topology of the Pdr12 transporter
includes 12 putative transmembrane-spanning α-helices
and two highly conserved nucleotide binding domains,
the hallmark domains of all ABC proteins (data not
shown). Pdr12 is highly homologous to two previously
identified yeast ABC drug efflux pumps, Snq2 (Servos
et al., 1993; Decottignies et al., 1995; Mahe ´ et al., 1996b)
and Pdr5 (Balzi et al., 1994; Bissinger and Kuchler, 1994),
sharing 46% and 37% primary sequence identity with
these latter parameters, respectively.
Next, a ∆pdr12 deletion strain was constructed and the
protein patterns of plasma membrane fractions of both
wild-type and isogenic ∆pdr12 cells were analysed. It was
apparent from silver-stained gels (Figure 1B, lane 4) that
a sorbate-induced protein of 170 kDa (S) was completely
band in sorbate-treated wild-type cells (Figure 1A, lane
2). Based on these results, we investigated the effects of
different stress conditions on the mRNA levels of three
ABC transporter genes, namely PDR5, SNQ2 and PDR12.
Weak acid stress strongly induces PDR12 mRNA
Northern analysis of total yeast RNA showed that the
PDR12 mRNA was increased in response to ethanol
treatment or severe osmostress (2 M sorbitol, 1 M NaCl
or 1 M KCl) at pH 7.0 (Figure 2A, lanes 4, 6, 8 and 9).
Consistent with earlier studies (Miyahara et al., 1996),
PDR5 and SNQ2 mRNAs were also slightly induced in
response to various stresses (Figure 2A). PDR12 mRNA
was also detectable in pH 4.5 cultures in the absence of
weak acid, but became much more strongly induced by
addition of either 1 mM or 9 mM sorbate (Figure 2B).
The quantification of Northern blots by laser-scanning
densitometry indicated that PDR12 mRNA was induced
at least 15-fold in wild-type cells following 9 mM sorbate
treatment. Surprisingly, PDR5 mRNA levels were severely
reduced in response to sorbate stress, while SNQ2 mRNA
levels remained essentially unchanged (Figure 2B). HSP30
encoding the second known sorbate-induced plasma mem-
brane protein (Piper et al., 1997) required higher sorbate
levels for a strong induction than PDR12 (Figure 2B).
However, HSP30 was more strongly heat shock-inducible
and less osmostress-inducible than PDR12 (Figure 2A).
These results show that PDR12 mRNA is stress-
inducible, with a particularly strong induction in response
to sorbic acid treatment. To test whether the transcription
factors implicated in cellular stress response or PDR
development contribute to PDR12 regulation, we analysed
stress induction of PDR12 in appropriate yeast strains
deleted for the YAP1 (Moye-Rowley et al., 1989; Wemmie
et al., 1994; Li et al., 1996), PDR1 (Balzi et al., 1987)
and PDR3 (Delaveau et al., 1994) genes. The levels of
PDR5, SNQ2 and PDR12 mRNAs in response to sorbate
exposure were investigated by Northern analysis of RNAs
isolated from wild-type and isogenic ∆yap1 and ∆pdr1
∆pdr3 cells. In agreement with our earlier work (Mahe ´
et al., 1996b), PDR5 expression was almost abolished and
SNQ2 mRNA levels were reduced in the ∆pdr1 ∆pdr3
mutant (Figure 2B). Notably, normalizing for RNA
amounts indicated slightly elevated PDR12 mRNA levels
in ∆pdr1 ∆pdr3 cells treated with sorbate (Figure 2B).
However, PDR12 mRNA was essentially unchanged in
response to low pH and sorbate in ∆yap1 cells when
compared with wild-type YAP1 cells. These results suggest
that the induction of Pdr12 by weak organic acids such
as sorbate does not require the transcriptional regulators
Pdr1, Pdr3 or Yap1.
Finally, a strong Pdr12 induction was also found in
Pdr12 ABC transporter in yeast
Fig. 2. Northern analysis of total RNA from yeast cells subjected to
different stresses. Hybridization to radiolabelled probes specific for the
genes indicated to the left of the figure panels was carried out by
routine methods. An actin-specific probe (ACT1) served as a control
for equal RNA loading. (A) About 20 µg total RNA each from
unstressed control cells (lane 1); or cells heat-shocked at 40°C for 1 h
(lane 2); cold-shocked at 15°C for 3 h (lane 3); or osmostressed at
30°C for 1 h with either 2 M sorbitol (lane 4), 0.5 M or 1 M NaCl
(lanes 5, 6), 0.5 M or 1.0 M KCl (lanes 7, 8), and 6% (w/v) ethanol at
30°C for 1 h (lane 9) were fractionated through agarose gels as
described in Materials and methods. (B) Northern analysis of total
RNA from wild-type (FY1679-28C) and isogenic ∆yap1 and ∆pdr1
∆pdr3 strains grown in pH 4.5 YPD medium and treated with 0, 1 or
9 mM sorbate for 1 h. RNA samples of 10 µg per lane were separated
through a 1% agarose formaldehyde gel. Both short (10 min) and long
(1 h) exposures of the blot hybridized to the PDR12-specific probe are
cells lacking Msn2 and Msn4 (data not shown), both of
which are transcriptional regulators of a stress response
pathway acting through a promoter motif known as STRE
(for ‘stress response element’; Ruis and Schu ¨ller, 1995;
Martinez-Pastor et al., 1996). Taken together, these data
show that the induction of PDR12 by weak organic acid
stress does not require the transcriptional regulators Pdr1,
Fig. 3. Immunological detection of Pdr12 in wild-type and sorbate-
treated cells. (A) Total cell extracts of wild-type and ∆pdr12 cells
grown at pH4.5 were immunoblotted using a polyclonal antiserum
raised against a GST–Pdr12 fusion protein. (B) Cell extracts from
untreated (–) and 9 mM sorbate-treated (?) pH 4.5 and pH 7.0
FY1769-28C cultures were analysed for Pdr12 expression by
immunoblotting. The non-specific cross-reaction at higher molecular
mass serves as an internal standard for equal protein loading in each
that as yet unidentified stress-responsive transcription
factors are required for the response to weak organic acid
stress in S.cerevisiae.
Low pH and sorbate-mediated induction of Pdr12
The PDR12 open reading frame of 4533 bp potentially
encodes a 1511-residue protein with a predicted molecular
mass of 171 kDa. To demonstrate that PDR12 is over-
expressed at the protein level following weak acid treat-
ment, a polyclonal anti-Pdr12 antiserum was raised in
rabbits using a bacterially expressed GST–Pdr12 fusion
protein as the antigen. Total cellular extracts were prepared
from both wild-type and isogenic ∆pdr12 cells and sub-
jected to immunoblotting (Figure 3A). A polypeptide band
with an expected molecular mass of ~175 kDa was
specifically recognized by the antiserum in wild-type cell
extracts, whereas no protein in this molecular mass range
was detectable in extracts from ∆pdr12 cells (Figure 3A).
A possible sorbate-mediated induction of Pdr12 was also
tested by immunoblotting. Cells from an overnight culture
of wild-type FY1679-28C were inoculated into fresh
pH 4.5 and pH 7.0 YPD medium. Both cultures were then
grown to an OD600of 0.7–1.0, whereupon sorbate was
added to a final concentration of 9 mM to half of
each culture. After another 2 h incubation, extracts were
analysed for Pdr12 expression by immunoblotting (Figure
3B). Pdr12 expression was extremely low at pH 7.0 in
the absence of sorbate. However, sorbate addition to such
pH 7.0 cultures resulted in 50-fold higher levels of Pdr12
(Figure 3B). Notably, pH 4.5 cultures, when compared
with pH 7.0 cultures, also displayed a 10-fold elevated
Pdr12 expression even in the absence of sorbate (Figure
ally induce Pdr12 protein levels. No signal in the Pdr12
P.Piper et al.
Fig. 4. Subcellular localization of Pdr12. (A) Wild-type and ∆pdr12
cells were grown in pH 7.0 YPD in the absence (pH 7.0) and presence
of sorbate (pH 4.5 ? 0.5 mM sorbate). After fixation of cells, Pdr12
localization was analysed by indirect immunofluorescence using the
FITC filter set. Nuclear DNA was stained and visualized with DAPI.
(B) The fluorescence of a Pdr12–GFP fusion was visualized
microscopically in living cells of strain YYMMI-2 grown in pH 7.0
YPD to mid-logarithmic growth phase.
size range was observed in extracts from the ∆pdr12
strain, even after sorbate treatment (data not shown),
demonstrating that the induced protein is Pdr12.
Subcellular localization of Pdr12
To determine the subcellular localization of Pdr12 in wild-
type and in sorbate-treated cells, we performed subcellular
fractionation and indirect immunofluorescence experi-
ments. Wild-type cells were grown in complete YPD
medium in the absence and presence of sorbic acid. A
ring-like fluorescence staining in sorbate-induced cells
was apparent, revealing a cell surface localization of Pdr12
(Figure 4A). The antibodies failed to detect Pdr12 in
non-treated wild-type cells, presumably because Pdr12
expression under these conditions is too low to allow for
a detection by this method. As expected, no fluorescence
was observed in non-induced or induced control ∆pdr12
cells (Figure 4A). However, sucrose gradient fractionation
experiments of cell-free extracts did confirm a plasma
membrane localization of Pdr12 in uninduced wild-type
cells (data not shown).
Finally, we used the ∆pdr5 ∆snq2 strain YYMMI-2 to
genomically tag PDR12 at the C-terminus with green
fluorescent protein (GFP), yielding a Pdr12–GFP fusion
that is fully functional in vivo (data not shown). Again, a
ring-like fluorescence showed that the Pdr12–GFP fusion
protein was localized in the plasma membrane of living
cells, while no fluorescence was observed in control cells
expressing Pdr12 without the GFP tag (Figure 4B). In
summary, these results show unequivocally a plasma
membrane localization of Pdr12, and demonstrate that
increased Pdr12 levels are due to a sorbate-induced PDR12
overexpression, rather than regulated cell surface targeting
of pre-existing intracellular Pdr12 pools.
A ∆pdr12 deletion strain is hypersensitive to weak
The sorbate induction of Pdr12 is remarkably strong,
raising the possibility that Pdr12 may be required for
adaptation to growth in the presence of weak acid stress.
Thus, both wild-type and isogenic ∆pdr12 strains were
containing various commonly used food preservatives.
Furthermore, we have also tested the sorbate resistance
phenotypes of isogenic strains carrying ∆yap1, ∆pdr1,
∆pdr3 and ∆pdr1 ∆pdr3 deletions (Figure 5). This analysis
revealed a striking hypersensitivity of ∆pdr12 cells to
sorbate at pH 4.5 when compared with the wild-type
strain, as ∆pdr12 mutants failed to grow in the presence
of 0.5 mM sorbate (Figure 5). In separate experiments,
we have also determined the IC50values for sorbate,
benzoate and acetate. The results showed that ∆pdr12
cells gave IC50values of ~0.20 mM for sorbate and
benzoate, and 20 mM for acetate, while isogenic wild-
acid inhibition, respectively (data not shown). Similar
experiments also revealed a hypersensitivity of ∆pdr12
cells to propionate at pH 4.5, though not to sulfite (data
not shown). Surprisingly though, loss of Pdr1, but not
Pdr3 or Yap1, led to an increased sorbate resistance,
implying that under these conditions perhaps other so far
unknown Pdr1 target genes can also contribute to weak
acid resistance development (Figure 5).
Next, we investigated in more detail the effects of
different sorbate (pKa4.76), benzoate (pKa4.19) and
acetate (pKa4.75) concentrations on the growth behaviour
of wild-type and ∆pdr12 cells at pH 4.5 (Figure 6), pH 3.8
and pH 5.7 (data not shown). At all three pH values,
∆pdr12 cells grew slightly slower in the absence of weak
acid when compared with the wild-type. This subtle slow-
growth phenotype was manifested as a longer lag-phase
period (Figure 6). At pH 5.7, a pH at which all tested weak
acids are almost completely dissociated and relatively non-
toxic to cells, the presence of 0.8 mM benzoate or
0.9 mM sorbate produced little extension to the lag phase;
moreover, they caused practically no difference to the
growth of ∆pdr12 and wild-type cells (data not shown).
In contrast, at pH 4.5, where an appreciable fraction of
each acid is undissociated, the same amounts of benzoate
and sorbate severely reduced both growth rate and biomass
yield of wild-type cells (Figure 6A and C). Furthermore,
Pdr12 ABC transporter in yeast
Fig. 5. PDR12 is essential for adaptation of yeast cells to growth in the presence of weak acids. Growth of wild-type and ∆pdr12 cells and isogenic
strains carrying ∆yap1, ∆pdr1, ∆pdr3 and ∆pdr1 ∆pdr3 deletions was monitored on sorbate plates. Cell suspensions of OD600? 0.025 as well as
1:10 serial dilutions were spotted onto pH 4.5 YPD plates with the indicated concentrations of sorbate. The plates were photographed after 2.5 days
incubation at 30°C.
∆pdr12 cells displayed a marked hypersensitivity to weak
acids at this pH, since they were unable to grow at
benzoate levels ?0.2 mM (Figure 6B). While ∆pdr12
cells could still adapt to 0.45 mM sorbate at pH 4.5, they
failed completely to grow in the presence of 0.9 mM
sorbate (Figure 6D).
Although acetic acid can be used as a carbon source
by non-glucose-repressed yeast, high acetate levels are
inhibitory in glucose-grown cultures (Figure 6E and F).
Wild-type cells, although totally inhibited by 90 mM
acetate, grew in the presence of 45 mM acetate at pH 4.5
(Figure 6E) and at pH 3.8 and pH 5.7 (data not shown).
However, no growth was observed when ∆pdr12 cells
were grown for 65 h in the presence of 45 mM acetate at
pH 4.5 (Figure 6F), as well as at pH 3.8 and pH 5.7 (data
not shown). Thus, ∆pdr12 cells are defective in glucose
growth in the presence of high levels of acetate. Under
these conditions the monocarboxylate uptake systems of
S.cerevisiae are repressed (Casal et al., 1996), so that
acetate will enter the cells primarily by diffusion of the
undissociated acid. Hence, the data (Figure 6E and F)
indicate that Pdr12 is capable of catalysing an active
extrusion of acetate. Finally, we also tested strongly
membrane-disruptive compounds, including ethanol, the
ate, the latter a highly lipophilic weak acid with a long
aliphatic chain (Stratford and Anslow, 1996). However,
loss of Pdr12 had no effect on growth inhibition caused
by these compounds (data not shown). This suggests that
Pdr12 confers no protection against compounds that are
highly liposoluble and primarily membrane-disruptive in
their cytotoxic effects.
∆pdr12 mutants show impaired benzoate
We used [14C]benzoate in efflux experiments to test
whether ∆pdr12 cells display any defects in benzoic acid
extrusion. Both wild-type and ∆pdr12 cells were cultured
at pH 4.5 to the mid-exponential growth phase. Half of
each culture was treated with 1 mM sorbic acid for 2 h.
Cells were then harvested and resuspended in glucose-
free pH 4.5 buffer. Next, [14C]benzoate was added, fol-
lowed 5 min later by the addition of glucose. Both the
intracellular accumulation of radiolabelled benzoate and
its rapid efflux after glucose addition were followed
(Henriques et al., 1997). The benzoate initially taken up
by the cells represented one-quarter to one-third of the
added radiolabel for the non-adapted cells (Figure 7A),
and half of the added benzoate for the sorbate-pretreated
cells (Figure 7B). Although sorbate-pretreated wild-type
Fig. 6. Bioscreen monitoring of the growth of wild-type (A, C, E) and
∆pdr12 (B, D, F) cells in liquid pH 4.5 YPD medium containing
increasing concentrations of benzoate (A, B), sorbate (C, D) or acetate
Fig. 7. Intracellular accumulation of [14C]benzoate by wild-type (j)
and ∆pdr12 cells (e) before and after glucose addition marked by an
vertical arrow (at 5 min). Cells were grown at either pH 4.5 (A) or at
pH 4.5, then pre-treated with 1 mM sorbate for 2 h (B) as described in
Materials and methods. Each point represents the SEM of three
separate measurements made on the same batch of cells.
cells accumulated less [14C]benzoate, presumably because
their intracellular pH was lower, they still displayed a
rapid extrusion of much of this benzoate after glucose
addition (Figure 7B). However, although the initial
[14C]benzoate accumulation of ∆pdr12 cells was similar
to that of wild-type, energy-dependent benzoate efflux by
P.Piper et al.
the mutant was severely impaired (Figure 7). For both the
non-adapted and the sorbate-pretreated wild-type cells,
70–80% of the accumulated [14C]benzoate was rapidly
extruded after glucose addition. In contrast, non-adapted
andsorbate-pretreated ∆pdr12cells extrudedonly ~50%of
their intracellular [14C]benzoate under the same conditions
(Figure 7B). These differences between the ∆pdr12 mutant
and its isogenic parent were maintained for at least 1 h,
zoate over this period (Figure 7). In summary, these
results demonstrate that benzoate is a substrate for Pdr12-
mediated extrusion, and that Pdr12 is a major catalyst of
energy-dependent benzoate efflux in yeast.
This study provides, for the first time, genetic and bio-
chemical evidence that adaptation of yeast cells to growth
in the presence of toxic weak acids involves the induction
of a system for energy-dependent weak organic acid
extrusion. Our studies identify Pdr12 as the ABC protein
of S.cerevisiae strongly induced in response to sorbate
exposure (Figures 1 and 3). Pdr12 is a major determinant
conferring resistance to sorbate, benzoate and acetate
(Figures 5 and 6) and it provides much of the cellular
capacity for active benzoate extrusion (Figure 7). Pdr12
is strongly stress-inducible, its induction being essential
for the development of weak acid resistance. Furthermore,
our results strongly support the notion that certain yeast
ABC transporters, through their actions in cellular detoxi-
fication and defence against toxic compounds in the
environment, have important physiological roles in adapta-
tion to adverse conditions (Decottignies and Goffeau,
1997; Kuchler and Egner, 1997).
Ycf1 is another example of an ABC transporter that is
both stress-inducible and which assists stress survival.
Ycf1 is induced by oxidative stress in a Yap1-dependent
manner (Wemmie et al., 1994) and it confers resistance
to high cadmium levels (Szczypka et al., 1994) and
glutathione-conjugated molecules (Li et al., 1996). Our
data indicate that Pdr12 induction, at least under the
experimental conditions used, is independent of the Pdr3,
Yap1 and Msn2/Msn4 transcription factors, all of which
are important mediators of PDR development and the
general stress response pathway, respectively. Indeed,
consensus 5?-TCCGCGGA-3? PDRE (for PDR responsive
element) motifs found in PDR-responsive genes such as
PDR5 (Delahodde et al., 1995; Katzmann et al., 1996),
YOR1 (Katzmann et al., 1995), PDR10 and PDR15
Likewise, both the STRE consensus motif 5?-AGGGG-3?
mediating the general stress response (Martinez-Pastor
et al., 1996) and the Yap1 consensus 5?-TGACTCA-3?
(Kuge and Jones, 1994) are absent from the PDR12
promoter. Interestingly, a recently identified novel Yap1-
binding motif, 5?-TTACTAA-3? (Fernandes et al., 1997),
is found at position –64 from the initiating methionine,
implying a possible role for Yap-homologues in Pdr12
regulation under weak acid stress (Fernandes et al., 1997).
Unexpectedly, ∆pdr1 and ∆pdr1 ∆pdr3 cells exhibited
increased sorbate resistance (Figure 5), indicating that
certain as yet unknown Pdr1 target genes may exert a
negative influence on weak acid adaptation. Such a nega-
Fig. 8. Schematic representation of the effects of substantial amounts
of undissociated weak organic acid (XCCOH) on unadapted yeast cells
(A). As mentioned in the Discussion, the induction of a weak acid
efflux pump (Pdr12) poses potential problems for homeostasis
maintenance in cells adapted to these acids (B), unless there is also
simultaneous induction of a system restricting free diffusional entry of
the undissociated acid. Pma1 is the proton-translocating plasma
tive effect could perhaps operate through a degenerate
PDRE-like motif (5?-TCGCCGGA-3?) at position –486
relative to the PDR12 translational start site. The Pdr12
pump shares ?37% primary sequence identity with Pdr5.
Nevertheless, their expression, regulation and functions
seem quite different. The reason for the drastic reduction
of PDR5 mRNA under weak acid stress (Figure 2) is
unclear at the moment, but one could argue that Pdr5
could somehow interfere with Pdr12 function in stressed
cells. Thus, it will be interesting to determine whether or
induction under weak acid stress is also responsible for
Pdr5 repression in weak acid-treated cells.
The mechanism of Pdr12 in weak acid resistance
A schematic model of how Pdr12 function might aid
acidified yeast cultures in counteracting the inhibitory
effects of water-soluble weak organic acids is depicted in
Figure 8. In both unadapted (A) and acid-adapted cells
(B), the protonated uncharged form of the acid (XCOOH)
is shown as freely permeable to the cell membrane and
readily entering the cell by passive diffusion. In unadapted
cells (Figure 8A), the XCOOH concentration inside and
outside should be about the same. However, the higher
pH environment of the cytoplasm will cause a substantial
fraction of the intracellular acid to dissociate to the anion
(XCOO–) which, being charged, is relatively membrane-
impermeable and therefore accumulates inside the cell.
Moreover, this dissociation also releases protons, resulting
in a cytoplasmic acidification that inhibits many metabolic
processes. The electrochemical potential (Z∆pH) across
the plasma membrane, largely maintained through the
plasma membrane ATPase (Pma1)-catalysed proton extru-
sion, is essential for many aspects of cellular metabolism.
Thus, weak acid influx in (A) will act to dissipate the
∆pH, though not the charge (Z) component of this gradient.
The extent to which the weak acid-induced cytoplasmic
acidification in (A) can be counteracted by increased Pma1
activity may be severely limited, since the high levels of
additional proton extrusion needed will also require greater
increases to the electrostatic charge across the plasma
membrane (Z) than can be generated by the Pma1 ATPase.
Pdr12 ABC transporter in yeast
In weak acid-adapted cells (Figure 8B), the proposed
intracellular organic acid levels and, by moving a charge
compensating for the charge on a Pma1-extruded proton,
enable greater levels of catalysed proton extrusion than
would otherwise be possible. The latter process, though
energetically expensive, could assist weak acid-stressed
cells to elevate their intracellular pH to the point where
substantial metabolic activity and cell growth can resume.
However, induction of Pdr12-catalysed acid anion extru-
sion alone would seem to be pointless without simul-
taneous limitation to the diffusional uptake of the
undissociated acid (XCOOH). Without such a limitation,
acid could potentially diffuse in as fast as Pdr12 pumps
it out in a futile cycle that, besides consuming large
quantities of ATP, will also cause substantial influx of
protons (Figure 8B). How weak acid diffusion across the
cell envelope is restricted in adapted cells, whether by
cell wall or membrane alteration, is at present unknown.
However, it is noteworthy that there exists an inverse
correlation between the rates with which different yeast
species take up benzoic acid and the resistances of these
yeasts to benzoate (Warth, 1989). Thus, although our data
indicate that Pdr12-mediated anion efflux is essential for
weak acid adaptation, it appears likely that Pdr12 is not
the only component of the adaptation system.
Whether or not the induction of a weak acid efflux
pump is important for weak acid resistance by yeasts has
been a contentious issue for several years (Warth, 1977;
Cole and Keenan, 1987). It has now been resolved by this
study. Earlier work had established that adaptation of
S.cerevisiae (Henriques et al., 1997) and Z.bailii (Warth,
1977) to growth in the presence of 1 mM benzoic
acid caused cells to maintain an intracellular versus
extracellular distribution of benzoate that is not in equilib-
rium. Since benzoate (Henriques et al., 1997) is not
metabolized by S.cerevisiae, these data were fully con-
sistent withthe induction ofan energy-dependent extrusion
system for the anion in response to benzoate exposure.
The substrate specificity and normal physiological
roles of Pdr12
Because PDR12 encodes a close homologue of the Snq2
ABC drug efflux pump (Servos et al., 1993), we also
tested the sensitivity phenotypes of ∆snq2 and ∆pdr12
strains. However, despite a high primary identity of Pdr12
and Snq2, their substrate specificity does not overlap. A
∆pdr12 mutant is not hypersensitive to 4-nitroquinoline-
N-oxide (4-NQO), a typical Snq2 substrate, while a ∆snq2
acids (data not shown). Instead, ∆snq2 ∆pdr5 double
mutants even exhibited increased resistance to sorbic acid
(Y.Mahe ´ and K.Kuchler, unpublished results). Although
we cannot formally exclude the possibility that Pdr12
might transport other cytotoxic drugs or toxic metabolites,
it appears as if its main function is in mediating cellular
efflux of weak organic acids (Figures 5–7). The substrates
that we have identified to date are all water-soluble
carboxylic acids, suggesting that Pdr12 primarily pumps
molecules that partition preferentially into the membrane
It follows that the normal physiological function of the
Pdr12 ABC transporter may be to minimize the effects of
water-soluble organic acids. These may accumulate to
toxic levels within yeast cells growing in environments
of slightly acidic pH (Figure 8A). Weak organic acids will
often be present in plant materials, such as ripe fruits and
cacti, where yeasts grow as saprophytes. These environ-
ments, with their plentiful supply of water and carbohyd-
rates, provide niches where growth does not need a
high degree of evolutionary specialization and where
competition among different microbes will be extreme.
Acetic acid, for example, will often be present at quite
high concentrations in such situations as it is both a
product of bacterial fermentation and a compound secreted
in high levels by certain non-Saccharomyces yeasts such
as Brettanomyces and Dekkera. The S.cerevisiae in wine
mush is frequently inhibited, especially at the early stage
of fermentation, by the high acetic acid levels caused by
such microbes. It therefore seems plausible that the strong
Pdr12 induction by weak acid stress protects S.cerevisiae
against the toxicity of high organic acid levels under
these conditions. Still further protection, on fermentative
substrates, will come from the high ethanol yield of
S.cerevisiae and the fact that this is one of the most
ethanol-tolerant organisms known.
Materials and methods
Yeast strains and media
Rich medium (YPD) and synthetic medium (SD), supplemented with
(Kaiser et al., 1994). Unless otherwise indicated, all yeast strains listed
in Table I were grown routinely at 30°C. The ∆pdr12::hisG disruption
strain YYM19 was constructed through a one-step gene replacement
procedure (Rothstein, 1983) by transforming FY1679-28C with the
BglII–XhoI ∆pdr12::hisG-URA3-hisG fragment isolated from plasmid
pYM63. Transformants were grown on plates containing 5-fluoro-orotic
acid (Boeke et al., 1987) to select for the pop-out of the URA3 marker.
Correct genomic integration ofdeletion constructs and proper looping-out
was confirmed by PCR analysis of genomic DNA (Mahe ´ et al., 1996a).
A glutathione-S-transferase (GST)–Pdr12 gene fusion was constructed
as follows. A 500 bp PCR fragment of PDR12 was generated from a
genomic DNA template using the custom primers PDR12-8: 5?-CGA-
CTG-ACG-AAT-TCA-TTG-AGA-AAG-3? and PDR12-528: 5?-CAT-
TTC-ACC-GAA-TTC-AAC-GAC-ACC-3?. The PCR product was
digested with EcoRI and cloned into the EcoRI site of plasmid pGEX-
expression of the N-terminal 164 Pdr12 residues (aa 8–172) fused to the
C-terminus of GST.
The ∆pdr12::hisG-URA3-hisG deletion plasmid was constructed in
two steps. First, the above-mentioned 500 bp EcoRI fragment obtained
by PCR with primers PDR12-8 and PDR12-528 was inserted in the
EcoRI site of plasmid pYM28, which contains the hisG-URA3-hisG
element (Mahe ´ et al., 1996a), resulting in plasmid pYMI14. In the
second step, the 3? end of the PDR12 gene was cloned as a 840 bp
BamHI–XhoI fragment, generated by PCR using the primers PDR12-
31: 5?-CGT-GCA-TCT-CAT-GCA-GG-3? and PDR12-32: 5?-GCC-ATT-
pYMI14 to yield plasmid pYM63.
Drug resistance and weak acid susceptibility assays
Drug resistance and weak acid susceptibility of yeast strains was initially
tested by spotting serial dilutions of exponentially growing cultures onto
YPD plates supplemented with the indicated compounds (Bissinger and
Kuchler, 1994; Mahe ´ et al., 1996a). For studies of the effects of pH and
weak acids on glucose batch fermentation cultures, the strains FY1679-
28C and YYM19 were grown to late exponential phase at 30°C on
YEPD medium containing no stress agent. Cultures were diluted to an
OD600of 0.8, followed by another 100-fold dilution with YPD of
P.Piper et al.
Table I. Genotypes of S.cerevisiae strains used in this study
MATa ura3-52 his3-∆200 leu2-∆1 trp1-∆63
MATa ∆pdr12::hisG (otherwise isogenic to FY1679-28c)
MATa ∆pdr1::TRP1 (otherwise isogenic to FY1679-28c)
MATa ∆pdr3::HIS3 (otherwise isogenic to FY1679-28c)
MATa ∆pdr1::TRP1 ∆pdr3::HIS3 (otherwise isogenic to FY1679-28c)
MATa ∆yap1::hisG (otherwise isogenic to FY1679-28c)
MATa can1-100 ade2-1ochis3-11,-15 leu2-3,-112 trp1-1 ura3-1
MATa msn2-∆3::HIS3 msn4-1::TRP1 (otherwise isogenic to W303-1A)
MATa ura3-52 his3-∆200 leu2-∆1 trp1-∆63 lys2-801ambade2-101oc
MATa ∆pdr5::TRP1 ∆snq2::hisG (otherwise isogenic to YPH499)
MATα ∆pdr5::TRP1 ∆snq2::hisG:URA3:hisG (otherwise isogenic to YPH499)
Delaveau et al. (1994)
Delaveau et al. (1994)
Delaveau et al. (1994)
Delaveau et al. (1994)
Wendler et al. (1997)
Martinez-Pastor et al. (1996)
Sikorski and Hieter (1989)
Egner et al. (1998)
pH 5.74, pH 4.5 or pH 3.8, with or without the indicated concentrations
of weak acid, giving ~5?103cells/ml and placed into the wells of a
Bioscreen plate. The Bioscreen plate was then placed into a Bioscreen
turbidometric analyser (Labsystems OY, Helsinki, Finland) that was
programmed to provide both continuous shaking at 30°C and to monitor
RNA isolation, radiolabelling and Northern analysis
Total yeast RNA was isolated, fractionated through agarose gels and
hybridized to radiolabelled probes using standard methods (Piper, 1994).
DNA fragments were radiolabelled using a Megaprime Labelling Kit
under conditions recommended by the manufacturer (Amersham). The
PDR12-specific probe (?8 to ?4787 region of PDR12) was amplified
by PCR from total yeast genomic DNA using the primers PDR12-8 and
PDR12-32 under standard PCR conditions (Mahe ´ et al., 1996b).
Preparation of a polyclonal anti-Pdr12 antiserum
The Escherichia coli strain DH5α carrying plasmid pYM53 was grown
at 30°C to an OD600of 0.7. Expression of the GST–Pdr12 fusion protein
was induced by adding 0.1 mM isopropyl β-D-thiogalactopyranoside for
4 h. Purification of the GST–Pdr12 fusion protein was done exactly as
described previously (Mahe ´ et al., 1996b). Removal of glutathione and
concentration of the eluted GST fusion protein was carried out in a
Centricon 10 microconcentrator (Amicon Division). The purified GST–
Pdr12 fusion protein was used to immunize rabbits according to routine
injection regimes (Harlow and Lane, 1988).
Plasma membrane isolation, microsequencing and
Yeast plasma membrane fractions were partially purified and fractionated
by one-dimensional SDS–PAGE exactly as described previously (Piper
et al., 1997). Peptide microsequencing was performed on protein samples
blotted onto PVDF membranes by routine laboratory methods (Harlow
and Lane, 1988). Protein extracts from whole yeast cells were isolated
essentially as described elsewhere (Egner et al., 1995). Proteins on
immunoblots were visualized with the ECL system (Vieira et al., 1994)
under conditions recommended by the manufacturer (Amersham).
Indirect immunofluorescence and subcellular fractionation
Immunofluorescence of yeast cells was carried out as previously pub-
lished (Kuchler et al., 1993) using the following modifications. Wild-
type and ∆pdr12 cells were grown in complete YPD medium to an
OD600of ~0.5. After addition of 0.5 mM sorbate to one-half of each
culture, cells were cultivated for another 3 h. Further treatment of cells
was exactly as previously published (Egner et al., 1995). Fluorescence
staining of Pdr12 was visualized with a Zeiss Axiovert 10 fluorescence
microscope equipped with an appropriate FITC filter set. Photomicro-
graphs were taken with a Kodak TMY400 black and white film.
Genomic tagging of the PDR12 C-terminus with GFP (Cubitt et al.,
1995) was carried out by a PCR-based method (Wach et al., 1997) using
the primer pair PDR12-GFPn 5?-ATT-TTC-CAA-ACA-GTT-CCA-GGT-
CCG-GG-3? and PDR12-GFPc 5?-GTA-AAA-TCA-AAT-GTA-AAA-
TTC-GAG-CTC-G-3?. PCR fragments were transformed into yeast strain
YYMMI-2 by electroporation as previously published (Mahe ´ et al.,
1996b). GFP-fluorescence in living cells was observed microscopically
using a FITC filter set.
Subcellular fractionation of yeast cells was performed following a
previously published protocol (Egner et al., 1998). Yeast strain YYM4
containing the plasmid pCKSF1 (Bissinger and Kuchler, 1994) was
grown in synthetic medium at pH 4.5 without sorbate to logarithmic
phase (OD600? 2). Cell-free extracts were fractionated in a sucrose
gradient and fractions were analysed by SDS–PAGE. Immunoblotting
with polyclonal antisera against Pdr12, Pdr5, Pdr12 and Pma1 was
performed by standard laboratory procedures (Egner et al., 1995).
Measurement of benzoic acid efflux
Overnight FY1679-28c and ∆pdr12 cultures were diluted 100-fold in
water, then inoculated into two flasks with 100 ml pH 4.5 YPD and
grown to an OD600of 0.7–1.0. Each culture was then divided into two
50 ml portions, with the addition of 1 mM sorbic acid to one of these.
After a further 2 h incubation at 30°C, the cells were harvested, washed
in ice-cold water and resuspended in 5.4 ml 20 mM sodium citrate
pH 4.5 at room temperature. After a 10 min incubation in this buffer,
5 µCi [7-14C]benzoic acid (740 MBq/mmol; NEN) was added, followed
5 min later by the addition of 0.6 ml 20% (w/v) glucose. After several
time intervals, 0.5 ml samples of the cell suspension were filtered on
Whatman GF/C filters, the filters briefly washed in pH 4.5 citrate buffer.
Filter-bound radioactivity of air-dried filters was determined by liquid
We would like to thank Helmut Ruis, Achim Wach and Ramon Serrano
for providing yeast strains and reagents. Many critical and helpful
comments by Helga Edelmann, Hubert Wolfger, Ian Booth and Graeme
Walker are also thankfully acknowledged. The help of Alexandra Pacher
with immunofluorescence analysis is highly appreciated. This work was
supported by the grant P12661-BIO from the ‘Fonds zur Fo ¨rderung
der wissenschaftlichen Forschung’ to K.K. and by the BBSRC grant
FQS02267 to P.W.P. M.M. was a recipient of a Emil Boral Fellowship,
and S.T. a BBSRC CASE studentship supported by Unilever.
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Received February 19, 1998; revised May 19, 1998;
accepted June 2, 1998