Eur. J. Biochem. 271, 1145–1152 (2004) Ó FEBS 2004
doi:10.1111/j.1432-1033.2004.04018.x
The transporters Pdr5p and Snq2p mediate diazaborine resistance
and are under the control of the gain-of-function allele PDR1-12
Eva Wehrschütz-Sigl*, Helmut Jungwirth*, Helmut Bergler and Gregor Högenauer
Institut für Molekularbiologie, Biochemie und Mikrobiologie, Karl-Franzens-Universität Graz, Austria
The spontaneous acquisition of resistance to a variety of
unrelated cytotoxic compounds has important implications
in medical treatment of infectious diseases and anticancer
therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux
pumps and is called pleiotropic drug resistance. We have
found that allelic forms of the genes for the transcription
activators Pdr1p and Pdr3p, designated PDR1-12 and
PDR3-33, respectively, mediate resistance to diazaborine.
Here we demonstrate that the transporters Pdr5p and Snq2p
are involved in diazaborine detoxification. We report that
in the PDR3-33 mutant diazaborine resistance is exerted
mainly via overexpression of the PDR5 and SNQ2 genes,
while in the PDR1-12 mutant, additional genes, i.e. the
Yap1p target genes FLR1 and YCF1, are also involved in
diazaborine detoxification. In addition, we show that in
the presence of cycloheximide or diazaborine PDR5 can be
activated by additional transcription factors beside Pdr1p
and Pdr3p.
Yeast Saccharomyces cerevisiae is equipped with a detoxification mechanism called the pleiotropic drug resistance
(PDR) network, which protects the cell from a number of
structurally and functionally unrelated toxic compounds
[1,2]. The PDR-system involves complex interactions
between a set of regulatory proteins and membranelocated efflux pumps from the ATP-binding-cassette
(ABC) or the major facilitator superfamily (MFS) type,
which eliminate toxic compounds from the cell [1,3]. The
master regulators of the PDR-network are the zinccontaining transcription activators, Pdr1p and Pdr3p [4,5].
The plasma membrane located efflux pumps Pdr5p and
Snq2p are by now the best characterized yeast ABC
transporters involved in pleiotropic drug resistance. Both
proteins, Pdr5p and Snq2p, are under the genetic control
of the transcription factors Pdr1p and Pdr3p (reviewed
in [3]). Various mutations in both Pdr1p and Pdr3p or
multicopy overproduction of the wild-type genes are
known to cause overexpression of the efflux pumps Pdr5p
and Snq2p thereby mediating drug resistance (reviewed in
[1]).
The second group of transcription factors regulating
efflux protein expression is the Yap-family, which contains a
bZIP structural motif [3,6]. We recently reported that the
ABC transporter, Ycf1p and the MFS transporter, Flr1p
play a crucial role in YAP1-mediated diazaborine resistance
[7]. Diazaborines are heterocyclic boron containing compounds which exhibit strong antibacterial activity and also
inhibit growth of yeast cells [8–10]. Furthermore, our studies
on the effect of diazaborine on yeast cells showed that gainof-function alleles of both PDR1 and PDR3, designated
PDR1-12 and PDR3-33, also mediate high level diazaborine
resistance phenotypes. Interestingly, the PDR1-12 allele
was much more resistant to the drug than the PDR3-33
allele [10].
In this paper we describe the relevance of the transporters
Pdr5p and Snq2p in the Pdr1p- or the Pdr3p- mediated
diazaborine resistance. Furthermore, we found that Flr1p
and Ycf1p, which are two efflux-pumps under the control of
Yap1p are necessary for full development of the resistant
phenotype in the PDR1-12 mutant. We also found that
PDR5-mRNA is increased in a strain lacking the PDR1
and the PDR3 genes in the presence of cycloheximide or
diazaborine, indicating that yet another transcription factor
must regulate this gene. We also demonstrate an increase
of the PDR3 mRNA level due to the gain-of-function
allele PDR1-12.
Correspondence to G. Högenauer, Institut für Molekularbiologie,
Biochemie und Mikrobiologie, Karl-Franzens-Universität Graz,
Universitätsplatz 2, A-8010 Graz, Austria.
Fax: + 43 316 380 9898, Tel.: + 43 316 380 5683,
E-mail: [email protected]
Abbreviations: ABC transporter, ATP-binding cassette transporter;
MFS transporter, major facilitator superfamily transporter; PDR,
pleiotropic drug resistance; PDRE, Pdr1p/Pdr3p response element.
*Note: These authors contributed equally to this work.
Note: A website is available at http://www.kfunigraz.ac.at/imbmwww/
(Received 2 September 2003, revised 8 January 2004,
accepted 30 January 2004)
Keywords: diazaborine; ABC transporters; transcriptional
regulation; Saccharomyces cerevisiae.
Material and methods
Yeast strains and media
S. cerevisiae strains used in this study are listed in Table 1.
Cells were grown in rich medium (YPD) or synthetic
medium (SD), supplemented with appropriate nutrients for
maintenance of plasmids, as described by Sherman et al.
[11]. Yeast was grown routinely at 30 °C.
Ó FEBS 2004
1146 E. Wehrschütz-Sigl et al. (Eur. J. Biochem. 271)
Table 1. Genotypes of strains used in this study.
Strain
Genotype
Source/reference
FY1679–28C
FY1679–28C Dpdr1Dpdr3
FY1679–28C Dpdr1Dpdr3Dyap1
A2
KPHJ1
KPHJ2
W303a
W303Dflr1Dycf1
YPH500
YKKB-13
YYM3
YYM5
MATa ura3 leu2 his3 trp1
MATa ura3 leu2 his3 trp1 pdr1D::TRP1, pdr3D::HIS3
MATa ura3 leu2 his3 trp1 pdr1D::TRP1, pdr3D::HIS3 yap1D::hisG
MATa leu2 his3 can1
MATa leu2 his3 can1 PDR1-12::HIS3
MATa leu2 his3 can1 PDR3-33::HIS3
MATa ura3 leu2 his3 trp1 ade2
MATa ura3 leu2 his3 trp1 ade2 can1 flr1D::URA3 ycf1D::kanMX6
MATa ura3 his3 leu2 trp1 lys2 ade2
MATa ura3 his3 leu2 trp1 lys2 ade2 pdr5D::TRP1
MATa ura3 his3 leu2 trp1 lys2 ade2, pdr5D::TRP1 snq2D::hisG
MATa ura3 his3 leu2 trp1 lys2 ade2 snq2D::hisG
K. Kuchler
K. Kuchler
K. Kuchler
V. L. MacKay
[7]
[7]
S. D. Kohlwein
[7]
K. Kuchler
K. Kuchler
K. Kuchler
K. Kuchler
Plasmids
The multicopy plasmids, YEp13, pYSTS1, containing gene
PDR5 in YEp13 and pSYS1 (SNQ2) were a gift from
K. Kuchler. The multicopy plasmids, YEp351, pKP100
(PDR1-12) and pKP300 (PDR3-33), containing allelic
forms of genes PDR1 and PDR3 in YEp351, were described
before [10]. Plasmids were transformed into S. cerevisiae
using the lithium acetate method of Gietz et al. [12].
Measurement of growth inhibition by diazaborine
The effects of diazaborine on different yeast strains were
quantitated by growing them overnight in YPD medium
or, when plasmid carrying strains were used, in minimal
medium lacking leucine. The cell suspension was diluted
to an A600 of 0.1. This suspension was then spotted on
YPD plates containing a gradient of diazaborine (maximal
concentration of 200 lgÆmL)1; Novartis Research Institute, Vienna, Austria) or a gradient of cycloheximide
(maximal concentration of 0.5 lgÆmL)1). Alternatively, the
diluted overnight cultures were further diluted in tenfold
increments. Five microlitres of each of these dilutions were
spotted on YPD plates with or without inhibitor. Each
experiment was repeated at least three times.
Results
Overexpression of PDR5 and SNQ2 causes diazaborine
resistance
We have shown that a pdr5 deletion strain was more
sensitive to diazaborine than the isogenic wild-type strain
[10]. This experiment reveals a possible link between PDR5
and diazaborine detoxification. To investigate whether
overexpression of PDR5 causes diazaborine resistance,
yeast strain W303 was transformed with the multicopy
plasmid pYSTS1, carrying gene PDR5, or the empty vector
alone, which served as a control. These strains were tested
for growth in the presence of various diazaborine concentrations. As shown in Fig. 1, the strain overexpressing
PDR5 was more resistant to diazaborine than the strain
with the empty vector. In order to asses a possible role of the
efflux pump Snq2p in diazaborine resistance, the multicopy
plasmid pYSNQ2 with the gene SNQ2 was transformed
Northern blot analysis
Yeast strains were grown overnight in YPD medium. The
cells were diluted to a D600 of 0.05 and cultured to a D600 of
0.3–0.4. Samples of total RNA were prepared from each
aliquot as described in the Qiagen RNeasy Handbook.
RNA samples (20 lg) were denatured and separated on a
1.2% (w/v) agarose gel containing 1.2 M formaldehyde and
transferred to Hybond N (Amersham) filters. Hybridization was performed in 0.5 M Na2HPO4, pH 7.2, 1 mM
EDTA, 7% (w/v) SDS, 1% (w/v) BSA and 100 lgÆmL)1
salmon-sperm DNA with 32P-labeled DNA probes at 65 °C
for 12–16 h. The filters were washed four times in 40 mM
Na2HPO4, pH 7.2, 1% (w/v) SDS, at 65 °C for 25 min,
and the radioactivity was made visible by autoradiography
at )70 °C and quantitated on a densitometer. As an
internal control, the yeast gene ACT1 (Harata et al. [13])
was used.
Fig. 1. Overexpression of PDR5 and SNQ2 genes confers diazaborine
resistance. The strain W303 carrying either the empty vector pYEp13
(2 lm), PDR5 on the multicopy plasmid pYSTS1 (2 lm PDR5), or
SNQ2 on the multicopy plasmid pYSNQ2 (2 lm SNQ2), were spotted
on YPD-plates containing no or 80 lgÆmL)1 diazaborine.
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Pdr5p and Snq2p mediate diazaborine resistance (Eur. J. Biochem. 271) 1147
into S. cerevisiae W303. The SNQ2 overexpressing strain
was again more resistant to diazaborine relative to a strain
carrying the empty vector (Fig. 1). These results show
that both efflux pumps, Pdr5p and Snq2p, contribute to
diazaborine detoxification.
shown). However, when both, PDR5 and SNQ2, were
deleted, PDR3-33 was unable to confer resistance. Hence,
we conclude that PDR3-33 mediated diazaborine resistance
is exclusively dependent on the ABC transporters PDR5
and SNQ2.
PDR3-33 mediated diazaborine resistance depends
on functional PDR5 and SNQ2 genes
Additional transporters are involved in PDR1-12
mediated diazaborine resistance
To define the contribution of Pdr5p and Snq2p on PDR3
mediated diazaborine resistance, yeast strains YKKB13 and
YYM5, which are disrupted in genes SNQ2 and PDR5,
respectively, were transformed with the multicopy plasmid
pKP300, carrying the PDR3-33 allele. Similarly, the double
mutant Dpdr5Dsnq2 and the wild-type strain YPH500 were
also transformed with the same plasmid. Additionally, a
control, containing the empty vector, was carried along.
Diazaborine resistance was determined on a plate containing a drug gradient. As observed before, the Dpdr5 strain
containing the empty vector grew poorly in the presence of
the drug, while the Dsnq2 strain grew like the wild-type [10].
Thus, the deletion of SNQ2 does not influence the resistance
of the respective strain. Although Pdr5p seems to contribute
mostly to the drug tolerance of the wild-type, the allelic form
of the relevant transcription factor, PDR3-33, could still
confer diazaborine resistance in the Dpdr5 strain (Fig. 2).
Similarly, deletion of SNQ2 alone did not influence the level
of PDR3-33 mediated diazaborine resistance (data not
In an earlier publication, we have shown that strains
carrying the PDR1-12 allele are much more resistant to
the drug diazaborine than those carrying the PDR3-33
allele [10]. This effect is partially due to elevated transcript
levels of PDR5 and SNQ2 in the PDR1-12 mutant relative
to the PDR3-33 mutant strain as shown by the Northern
blot in Fig. 3. However, involvement of additional transporters could not be excluded. Therefore, the double
disruptant Dpdr5Dsnq2 and the wild-type strain were
transformed with the plasmids pKP100 (PDR1-12), or the
empty vector. These strains were again tested on a plate
containing a drug gradient. As shown in Fig. 4, the
Dpdr5Dsnq2 strain carrying the plasmid pKP100 was less
resistant than the wild-type strain with the plasmid.
However, the Dpdr5Dsnq2 disruptant strain with pKP100
was still more resistant than the strain with the empty
vector. These results support the idea that PDR1-12
activates at least one additional transporter which contributes to diazaborine resistance.
Fig. 2. Overexpression of the allelic form PDR3-33 does not confer
diazaborine resistance in the Dpdr5Dsnq2 strain. The strains YPH500
(wt), YKKB-13 (Dpdr5), and YYM3 (Dpdr5Dsnq2) carrying either the
empty vector pYEp351 (2 lm), or the allelic form PDR3-33 on the
multicopy plasmid pKP300 (2 lm PDR3-33), were spotted onto YPD
plates containing a gradient of diazaborine (maximal concentration
150 lgÆmL)1) and tested for growth. The direction and relative
strength of the gradient is indicated below.
Fig. 3. Northern blots showing expression of PDR5 and SNQ2 mRNA.
Total RNA was extracted from S. cerevisiae A2 (wild-type), KPHJ1
(PDR1-12) and KPHJ2 (PDR3-33). Radioactive probes of PDR5 (A)
and SNQ2 (B) were used in this experiment and the positions of the
mRNAs are indicated on the right. The amount of 26S and 18S rRNA
was used with each sample as a control for monitoring RNA loading.
1148 E. Wehrschütz-Sigl et al. (Eur. J. Biochem. 271)
Fig. 4. Overexpression of the allelic form PDR1-12 does still confer
diazaborine resistance in the Dpdr5Dsnq2 strain. The strains, YPH500
(wt), YKKB-13 (Dpdr5) and YYM3 (Dpdr5Dsnq2) carrying either the
empty vector, pYEp351 (2 lm), or the allelic form, PDR1-12, on the
multicopy plasmid pKP100 (2 lm PDR1-12), were spotted onto YPD
plates containing a gradient of diazaborine (maximal concentration
150 lgÆmL)1) and tested for growth. The direction and relative
strength of the gradient is indicated below.
FLR1 and YCF1 contribute to PDR1-12 mediated
diazaborine resistance
As we had earlier observed a crosstalk between the Yap1p
controlled detoxification system and the PDR network [10],
we decided to examine a possible regulation of the YAP1
controlled efflux pumps Ycf1p and Flr1p by the transcription factor Pdr1p. Thus, the wild-type strain W303 and
the strain Dflr1Dycf1 were transformed with the plasmid
pKP100 (PDR1-12) or the empty vector. Diazaborine
resistance of the transformants was determined. As shown
in Fig. 5, PDR1-12 could not confer resistance to high
concentrations of diazaborine in the double disruptant. This
result indicates that, besides Pdr5p and Snq2p, the YAP1
controlled efflux pumps Ycf1p and Flr1p play a crucial role
in PDR1-12 mediated diazaborine resistance. In addition,
this finding, together with our previous finding that YAP1
mediated diazaborine resistance is strongly dependent on
functional PDR1 and PDR3 genes, could indicate that
PDR1 and/or PDR3 are important for full expression of
YCF1 and FLR1. For FLR1 regulation, the involvement of
Pdr3p was shown previously [14]. A plausible explanation
for this effect would be that genes YCF1 and FLR1 were
directly under the control of transcription factor Pdr1. We
tested this possibility by measuring the mRNA levels of
FLR1 and YCF1 in the PDR1-12 strain. No significant
differences as compared to the wild-type levels were
observed (data not shown). Therefore a different mechanism than an enhanced transcription by Pdr1–12p has to
Ó FEBS 2004
Fig. 5. Overexpression of PDR1-12 in a Dycf1Dflr1 strain does not
mediate resistance. The strains, W303 and the isogenic Dycf1Dflr1
disruptant carrying either the empty vector, pYEp351 (2 lm), or the
allelic form, PDR1-12, on the multicopy plasmid, pKP100 (2 lm
PDR1-12), were spotted on YPD-plates containing no or 50 lgÆmL)1
diazaborine.
account for the lack of resistance in the Dflr1Dycf1 double
mutant.
Pdr1p and Pdr3p are not the only transcriptional
activators regulating PDR5 expression
To examine a possible involvement of additional transcriptional transactivators on the PDR5 expression, its gene on
a multicopy plasmid was transformed into the Dpdr1Dpdr3
strain. When we tested the double mutant strain for
resistance against diazaborine and cycloheximide, we found
that the strain overexpressing PDR5 was clearly more
resistant than the strain with the empty vector (Fig. 6). This
result shows that PDR1 and PDR3 cannot be the only
transcription activators involved in PDR5 expression. As
YAP1 was connected with heat shock-induced PDR5
expression [15], we performed the same experiment with
a Dpdr1Dpdr3Dyap1 triple mutant. As shown in Fig. 6,
deletion of YAP1 had no effect on PDR5-mediated
resistance to diazaborine and cycloheximide. A similar
behaviour was observed for the gene SNQ2, which still
mediated diazaborine resistance in the absence of YAP1,
PDR1 and PDR3 (Fig. 7). To investigate the expression of
PDR5 in the presence of the inhibitors, RNA from the drug
treated Dpdr1Dpdr3 and Dpdr1Dpdr3Dyap1 strains was
extracted and analyzed by Northern blotting using a
PDR5 specific probe. Treatment of the wild-type strain
with cycloheximide resulted in increased expression of the
PDR5 gene. Figure 8 shows the result after cycloheximide
and diazaborine treatment. In the untreated Dpdr1Dpdr3
strain, no PDR5 mRNA could be detected. However, when
this strain was treated with cycloheximide, a clear signal was
found. This signal was much stronger when PDR5 was on
a multicopy plasmid. The Dpdr1Dpdr3Dyap1 triple mutant
behaved similarly to the double mutant. We infer from these
experiments that YAP1 has no influence on drug induced
Ó FEBS 2004
Pdr5p and Snq2p mediate diazaborine resistance (Eur. J. Biochem. 271) 1149
Fig. 7. Deletion of YAP1 has no effect on SNQ2-mediated diazaborine
resistance. The wild-type strain, FY1679–28C (wt) and the deletion
strains, Dpdr1Dpdr3 and Dpdr1Dpdr3Dyap1, carrying either SNQ2 on a
multicopy plasmid (2 lm SNQ2), or the empty vector (2 lm), were
spotted on YPD plates containing a gradient of diazaborine (maximal
concentration 50 lgÆmL)1) and tested for growth. The direction and
relative strength of the gradient is indicated below.
Fig. 6. Overexpression of PDR5 in a Dpdr1Dpdr3 strain still confers
resistance. The deletion strains, Dpdr1Dpdr3 and Dpdr1Dpdr3Dyap1
and the appropriate wild-type strain, FY1679–28C (wt) containing
either PDR5 on a multicopy plasmid (2 lm PDR5), or the empty
vector (2-lm), were tested for growth on plates containing a gradient
of diazaborine (maximal concentration 40 lgÆmL)1) or cycloheximide
(maximal concentration 0.5 lgÆmL)1). The direction and relative
strength of the gradient is indicated below.
expression of PDR5 and SNQ2. However, the important
finding from these experiments is that there must be an
additional transcription factor for PDR5 expression that
becomes active only in the presence of the inhibitors.
The mutation in PDR1-12 leads to an increase
of PDR1 and PDR3 mRNA
Pdr1p belongs to the large family of Zn(II)2Cys6 transcription factors and binds to a DNA-element, called Pdr1p/
Pdr3p response element (PDRE) [3–5,16,17]. The fact that
gene PDR3 contains two PDREs and is regulated by
Pdr1p, prompted us to test whether PDR1-12 has any effect
on PDR3 mRNA expression. Northern blot experiments
showed higher levels of PDR3 mRNA in the PDR1-12
mutant strain relative to the isogenic wild-type (Fig. 9). This
result shows that the gain-of-function allele PDR1-12 results
in an increased production of PDR3 mRNA.
Delahodde et al. [18] have shown that gene PDR3 is
autoregulated and that this transcriptional autoregulation
process is relevant for the response to cycloheximide. Due to
the functional homologies of Pdr3p and Pdr1p, we investigated the PDR1-12 transcript level in the PDR1-12 mutant
strain. We measured the abundance of PDR1-12 mRNA by
Northern blot analysis. Our studies demonstrated that in
the PDR1-12 mutant, the amount of PDR1-12 transcript
was increased when compared to the isogenic wild-type
strain (Fig. 10). This result might be an indication for a
positive autoregulatory feedback loop in the case of Pdr1p.
However, a reporter construct with the PDR1 promoter
followed by a lacZ gene showed no increased b-galactosidase levels. The higher PDR1-mRNA level needs another
explanation, e.g. an enhanced mRNA stability in the
PDR1-12 transformant.
Discussion
We have shown previously that allelic forms of PDR1 and
PDR, designated PDR1-12 and PDR3-33, cause resistance
to the drug diazaborine in yeast. Like other multiple drug
resistant PDR1 and PDR3 mutants, the strains with the
diazaborine resistant alleles showed increased levels of
Pdr5p and Snq2p [10]. Interestingly, a strain deleted for
PDR5 and SNQ2 proved to be more sensitive to the drug
than the wild-type strain. This finding indicated that these
two efflux pumps could be responsible for PDR1-12 or
PDR3-33 mediated diazaborine resistances. In this paper,
we report that overexpression of both genes, PDR5 and
SNQ2, confers diazaborine resistance. Northern blot analyses revealed that the gain-of-function alleles, PDR1-12
1150 E. Wehrschütz-Sigl et al. (Eur. J. Biochem. 271)
Ó FEBS 2004
Fig. 9. Northern blot showing expression of PDR3 mRNA in a PDR112 mutant strain. The RNA samples were derived from S. cerevisiae A2
(wild-type) and KPHJ1 (PDR1-12). The radioactive probe of PDR3
used in this experiment and the mRNA band is indicated at the right
margin. The UV-illuminated gel with the 26S and 18S rRNA bands,
which served as a control for monitoring RNA loading, is shown
below the blot.
Fig. 8. Northern blots showing expression of PDR5 mRNA in the
presence of cycloheximide (top) and diazaborine (bottom). Total RNA
from the drug treated wild-type strain, FY1679–28C (wt) and the
deletion strains, Dpdr1Dpdr3 and Dpdr1Dpdr3Dyap1, carrying either
the empty vector, pYEp351 (2 lm), or PDR5 on the multicopy plasmid (2 lm PDR5), was extracted and analyzed by Northern blotting
using a PDR5 specific probe. ACT1 was used as an internal standard.
and PDR3-33 lead to elevated transcript levels of PDR5 and
SNQ2, as compared to the wild-type strain. This behavior is
consistent with various reports in the literature that other
mutations in the genes encoding the transcription factors
Pdr1p and Pdr3p were associated with overexpression of
transcripts of PDR5 and SNQ2 [1,19–21]. The increased
transcript levels result in increased amounts of the proteins
Pdr5p and Snq2p, which in turn causes diazaborine
resistance. The fact that the allelic forms of PDR1 and
PDR3 mediate diazaborine resistance by activating the
genes for the efflux pumps Pdr5p and Snq2p can also be
inferred from our experiments where the genes of the
transporters have been deleted either alone or both together.
Single deletions gave strains which still showed resistance
phenotypes. However, the strain with the double-deletion
was no longer resistant when expressing the gain-offunction allele PDR3-33. We explain the behaviour of the
singly defective mutants by a compensatory mechanism
in each of the remaining pump proteins. Apparently, either
efflux pump, Pdr5p and Snq2p that are highly homologous,
can compensate the loss of the other of these proteins. In the
doubly defective mutant lacking both efflux pumps, PDR333 is no longer able to cause resistance because this
compensatory potential is missing. In contrast to the PDR333 mutant, we still observed diazaborine resistance when the
gain-of-function allele PDR1-12 was introduced into the
Fig. 10. Northern blot showing expression of PDR1-12 mRNA in a
PDR1-12 mutant strain. Total RNA was extracted from the strains A2
(wild-type) and KPHJ1 (PDR1-12) and was probed with radiolabeled
PDR1. The amount of 26S and 18S rRNA was used with each sample
as a control for monitoring RNA loading.
double mutant. We explain this result by the presence of
additional efflux pumps that only Pdr1–12p but not Pdr3–
33p could activate. We have shown recently that the ABC
transporter, Ycf1p and the MFS transporter, Flr1p, which
are transactivated by Yap1p, are able to remove diazaborine
from yeast cells and thus effectively lead to detoxification
[7]. The allele, PDR1-12 that mediated high level resistance
in a wild-type strain, was lacking this phenotype in the
Dflr1Dycf1 mutant. One of the two pump proteins encoded
by these genes must contribute to the overall diazaborine
resistance. In a Dycf1 mutant expressing the PDR1-12 allele
on a plasmid, we saw less diazaborine resistance when
compared to the wild-type with the plasmid, suggesting that
the vacuolar pump, Ycf1p is being activated by the gain-offunction protein, Pdr1–12p. However, Northern blotting
did not support this assumption. A different mechanism has
to be invoked which could involve Yap1p or another
transcription factor yet to be identified.
We have shown that Pdr5p is a major efflux pump in
diazaborine detoxification. It was therefore important to
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Pdr5p and Snq2p mediate diazaborine resistance (Eur. J. Biochem. 271) 1151
follow the expression pattern of the PDR5 gene in a
Dpdr1Dpdr3 background. Surprisingly the PDR5 gene
proved to be under the control of an as yet unidentified
transcription factor that became active only in cells treated
with cycloheximide or diazaborine. We interpret this result
to mean that treatment of the yeast cells with the inhibitors
activates a stress response mechanism that activates PDR5
transcription. Cycloheximide is known to arrest polysome
structures. Therefore, cycloheximide could cause PDR5
mRNA stabilization. However, as diazaborine treatment
resulted in a similar outcome as in Fig. 8, we believe that the
enhanced PDR5 mRNA concentration can be explained by
an induction. As it was shown that in the case of heat shock
Yap1p is involved in PDR5 expression [15], we tested a
triple disruptant Dpdr1Dpdr3Dyap1 overexpressing PDR5
on a multicopy plasmid for a resistant phenotype and for
the expression of PDR5 mRNA. The triple disruptant
behaved like the Dpdr1Dpdr3 double disruptant, demonstrating that YAP1 is not involved in increased PDR5
expression in the presence of diazaborine or cycloheximide.
We observed higher steady state levels of PDR1 mRNA
in the PDR1-12 strain. The elevated PDR1 mRNA level
in the presence of PDR1-12 is probably not due to a
stimulation of the PDR1-promoter by PDR1-12 because a
reporter construct with the PDR1-promoter showed no
increased b-galactosidase levels as measured by immunoblotting.
We also found that the gain-of-function allele PDR1-12
leads to an enhanced level of PDR3 mRNA when compared
to the isogenic wild-type strain. Pdr3p is also known to be
responsible for the transactivation of PDR5 and SNQ2
[22,23]. Overexpression of PDR3 is known to increase levels
of Pdr5p and Snq2p and causes increased resistance to
cycloheximide, oligomycin and benomyl [14,22]. According
to our experiments, the mutation in the allele, PDR1-12,
results in an increase of the PDR1-12 mRNA level and
leads to a large increase of the PDR3 mRNA content. The
enhanced levels of transcripts of both genes might lead to
elevated levels of both, Pdr1–12p and Pdr3p. The increased
amount of both transcriptional activators in the PDR1-12
strain might have a synergistic effect on the expression of
PDR5 and SNQ2. This might explain why the PDR1-12
strain shows much more resistance to diazaborine than the
PDR3-33 strain [10].
Acknowledgements
We are greatly indebted to V. L. MacKay (University of Washington,
Seattle, WA, USA), S. W. Moye-Rowley (University of Iowa, Iowa
City, IO, USA), K. Kuchler (Vienna Biocenter, University of Vienna,
Vienna, Austria), S. D. Kohlwein (IMBM, University of Graz, Graz,
Austria) and U. Wintersberger (Institut für Krebsforschung, University
of Vienna, Vienna, Austria) for providing strains and plasmids. This
work was supported by the Fonds zur Förderung der wissenschaftlichen
Forschung, grants no. P9260, P13000, P15458 and S007.
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