INTRODUCTION

One of several possibilities by which cells exert resistance to inhibitory substances is the extrusion of these toxic compounds by membrane-located efflux pumps. In eukaryotic cells, certain pumps may be able to transport unspecifically a variety of unrelated drugs, resulting in resistance to more than one inhibitor, a phenomenon called pleiotropic drug resistance (1). The genes involved in this process are referred to as pleiotropic drug resistance (PDR)¹ genes. In general, pleiotropic drug resistance involves the overexpression of membrane pumps from the ABC (ATP-binding cassette) or MFS (major facilitators superfamily) type, all of which transport toxic compounds out of the cell or into the vacuole, thus preventing inactivation of the drug target (1-3). Well known examples for such pumps in Saccharomyces cerevisiae are Pdr5p, Snq2p, and Yor1p (4-7). Each of these transporters manages the export of several unrelated compounds. To date, the best characterized ABC protein involved in pleiotropic drug resistance is Pdr5p. This 175-kDa protein of the ABC transporter family contains a duplicated six-membrane span domain with a repeated ATP-binding motif (4, 5). Pdr5p displays ATPase activity and confers upon overexpression resistance to cycloheximide, sulfomethuron methyl, chloramphenicol, erythromycin, sporidesmin, and cerulenin, to mention a few examples. Disruption of gene PDR5 is not lethal but results in marked hypersensitivity to the same drugs to which it confers resistance upon overexpression (4, 5).

Multicopy overproduction of transcriptional regulatory proteins or gain-of-function mutations in their genes also result in resistance to a variety of toxic compounds. For example, the wild type gene of the yeast AP1 homologue, YAP1, causes, when overexpressed on multicopy plasmids, resistance to cycloheximide, sulfomethuron methyl, 4-NQO, N-methyl-N-nitro-N-nitrosoguanidine, 1,10-phenanthroline, heavy metal ions like cadmium or zinc, and reactive oxygen species (1). Yap1p-mediated cycloheximide resistance is independent of PDR5 overexpression and is thought to involve an as yet unidentified transporter. Yap1p is a transcription activator belonging to the bZIP type family (8).

The PDR1 and PDR3 genes have also been found to mediate resistance to a wide variety of compounds, since mutations in the respective genes lead to resistance to many structurally unrelated drugs (1). PDR1 and PDR3 code for homologous transcriptional regulators of the Zn(₂)Cys(₆) family, and both gene products were demonstrated to interact with the PDR5, SNQ2, and YOR1 promoters where they exert transcription activator functions (5, 7, 9, 10, 13). Gain-of-function alleles of both PDR1 and PDR3 or overexpression of the wild type genes was reported to cause overexpression of the Pdr5p and Snq2p proteins, explaining the correlation of these regulatory proteins and resistance (11-13). PDR1-dependent resistance to cycloheximide and chloramphenicol requires a functional PDR5 gene (14). In addition to PDR5, SNQ2, and YOR1, Pdr1p appears to regulate several other genes, some of which are involved in pleiotropic drug resistance development (1).

In this paper, we report the results from our studies on diazaborine resistance in S. cerevisiae. Diazaborines are heterocyclic boron-containing compounds that exhibit strong antibacterial activity (15, 16). These drugs cause inhibition of fatty acid and phospholipid biosyntheses in Gram-negative bacteria, like Escherichia coli or Salmonella typhimurium (17). The target for diazaborine in E. coli and S. typhimurium was identified as the enoyl-(acyl carrier protein) reductase (FabI), catalyzing a key regulatory step in the elongation cycle of fatty acid biosynthesis (18). Diazaborine was found to bind to the purified FabI protein only in the presence of NAD(H), thereby inhibiting its essential enzymatic function. Diazaborine-resistant mutants were isolated and characterized from E. coli and S. typhimurium and found to contain allelic forms of the fabI gene (19).

We demonstrate here that a eukaryotic organism, the yeast S. cerevisiae, is also sensitive to diazaborine. To study the mechanism of action of diazaborine in yeast, we used UV mutagenesis to generate drug-resistant mutants. Three classes of resistant mutants were obtained. We show that allelic forms of the genes AFG2, PDR1, and PDR3 cause resistance and that overexpression of YAP1 also leads to diazaborine resistance in yeast. YAP1-mediated resistance is dependent on functional PDR1 and PDR3 genes, demonstrating for the first time a functional interaction between YAP1-mediated gene regulation and the yeast PDR network.

EXPERIMENTAL PROCEDURES

All S. cerevisiae strains used in this study are listed in Table I. An isogenic set of wild type and PDR3-2 strains was obtained by back-crossing a PDR3-2 strain (kindly provided by J. Subik) several times into the W303-1A background to generate the strain YYMI4-A4 (MATa PDR3-2 ura3 leu2 his3 trp1 ade2) and its isogenic PDR3 segregant YYMI4-O3 (MATa PDR3 ura3 leu2 his3 trp1 ade2). Synthetic medium (SD), supplemented with appropriate nutrients for maintenance of plasmids, or rich medium (YPD), were prepared exactly as described by Sherman et al. (20). Yeast were grown routinely at 30 °C. Yeast transformations were carried out by the method of Ito et al. (21).

Table I. Genotypes of strains used in this study Strain Genotype                     Source A2 MAT leu2-2,112 his3-11,15 can1 Vivian L. MacKay L1544 MATa lys9 Ref. 44 ADR6 MAT leu2-2,112 his3-11,15 can1 DRG1-1 This study AL1 MATa lys9 DRG1-1 This study DM6 MAT leu2-2,112 his3-11,15 can1 DRG2-1 pet This study DM12 MAT leu2-2,112 his3-11,15 can1 DRG3-1 pet This study FW52 MAT leu2-2,112 his3-11,15 can1 YAP1::HIS3 This study DM6 (yap1) MAT leu2-2,112 his3-11,15 can1 DRG2-1 pet yap1::HIS3 This study DM12 (yap1) MAT leu2-2,112 his3-11,15 can1 DRG3-1 pet yap1::HIS3 This study KPHJ1 MAT leu2-2,112 his3-11,15 can1 PDR1-12::HIS3 pet This study KPHJ2 MAT leu2-2,112 his3-11,15 can1 PDR3-33::HIS3 pet This study YYMI4-03 MATa PDR3 trp1 his3 ura3 leu2 ade2 This study YYMIA-A4 (PDR3-2) MATa PDR3-2 trp1 his3 ura3 leu2 ade2 This study YALA-B1 MATa ura3-52 leu2-3,112 his3-11, 115 trp1-1 PDR1 Ref. 13 YALA-G4 (PDR1-3) MATa ura3-52 leu2-3,112 his3-11, 115 trp1-1 PDR1-3 Ref. 13 FY1679-28C MATa ura3-52 leu21 his3200 trp163 Refs. 13 and 28 FY1679-28C (pdr1) MATa ura3-52 leu21 his3200 trp163 pdr3::TRP1 Ref. 28 FY1679-28C (pdr3) MATa ura3-52 leu21 his3200 trp163 pdr3::HIS3 Ref. 28 FY1679-28C (pdr1 pdr3) MATa ura3-52 leu21 his3200 trp163 pdr1::TRP1, pdr3::HIS3 Refs. 13 and 28 W303-1A MATa ura3-1 leu2-3 his3-11,15 trp1-1 ade2-1 can1-100 Ref. 44 YPH500 MAT ura3-52 leu21 his3200 trp163 lys2-801amb ade2-101oc Ref. 29 YKKB-13 (pdr5) MAT ura3-52 leu21 his3200 trp163 lys2-801amb ade2-101oc pdr5::TRP1 Ref. 4 YYM4 (pdr5 snq2) MATa ura3-52 leu21 his3200 trp163 lys2-801amb ade2-101oc pdr5::TRP1 snq2::hisG Ref. 29

UV mutagenesis of wild type strain A2 was performed according to the protocol of Jandrositz et al. (22). It allowed the isolation of 17 stable mutants with diazaborine-resistant phenotypes. To test if resistance is dominant or recessive, we crossed the mutants DM1 to DM17, all of them MAT, with the diazaborine-sensitive strain L1544 (MATa, lys9). All diploids from these crosses were diazaborine-resistant, indicating that the mutations are dominant. The diploid strains were sporulated and subjected to tetrad analysis. From every cross, at least eight complete tetrads were examined. All of them were found to segregate 2:2 for diazaborine resistance and diazaborine sensitivity, demonstrating that the mutations reside at a single genetic locus.

From one of the mutants we cloned an allelic form of AFG2 (DRG1-1).² DRG1-1 was introduced into the wild type strain A2, resulting in strain ADR6. To determine the number of complementation groups, strain ADR6 was crossed with the diazaborine-sensitive strain L1544 (MATa lys9). After sporulation and tetrad analysis, we isolated a haploid strain, AL1 (MATa lys9, DRG1-1). With this strain, we back-crossed all of the remaining original 16 mutants. After sporulation and tetrad analysis, the segregation pattern of the diazaborine-resistant phenotype was studied. At least six complete tetrads from each cross were checked. Only resistant spores were obtained in 10 cases, indicating that all 10 mutants fall into the same complementation group as ADR6, i.e. drg1. To examine if the remaining six mutants form a homogeneous complementation group, we crossed one of these mutants (DM6) with strain L1544 (MATa lys9), sporulated the diploid, and isolated resistant haploid MATa cells. One was selected to be used for a further cross. Each of the remaining five mutants of the second recombination group was crossed with this strain and subjected to tetrad dissection. At least eight complete tetrads were examined. In three cases, only resistant spores could be found, indicating that the cells harbor mutations in the same locus. A similar analysis revealed that the remaining two mutants belong to a third recombination group. These recombination groups were designated drg2 and drg3, respectively.

The genomic libraries of yeast strains DM6, DM12, DM6 (yap1), and DM12 (yap1) were constructed in BamHI-digested plasmid YEp351 by using Sau3A-digested genomic DNA with a size of fragments in the range of 5-10 kb. After transformation into E. coli in all cases, at least 4.5 × 10⁵ transformants were obtained. Transformants from each library were pooled, and a plasmid preparation was performed.

Yeast strain A2 was transformed with a 2-µm YEp351-based genomic library from DM6 or DM12, respectively, to leucin prototrophy and diazaborine resistance (50 µg/ml) under double selection conditions. In each case about 100,000 transformants were screened. Eleven transformants from the DM6 and 17 from the DM12-derived library were obtained. Plasmid loss and retransformation of plasmids into the sensitive strain A2 confirmed plasmid-dependent resistance. For further subcloning procedures, plasmid pFW100 derived from the DM6 library was selected. By fragment exclusion experiments, a minimal 2.8-kb PstI/SphI subfragment was identified as the cause of diazaborine resistance. The plasmid harboring this fragment was designated pFW300. The presence of a common consensus fragment in all of the isolated plasmids from either the DM6 or the DM12 library was verified by Southern blot analysis with a digoxigenin-labeled 1.8-kb PstI/SacI fragment. Furthermore, a 4.3-kb PstI fragment from plasmid pFW100 was cloned into pBluescript, resulting in plasmid pBS300. From this plasmid, a 1-kb KpnI and a 0.6-kb KpnI/BamHI fragment were again cloned into pBluescript, and both ends of the inserts were sequenced using T3 and T7 primers by the Sanger dideoxy chain termination method (23). Searches for homology, using the FASTA algorithm (24), were performed on this sequence and showed the presence of gene YAP1.

Genomic libraries of DM6 (yap1) and DM12 (yap1) were used to transform the diazaborine-sensitive strain S. cerevisiae A2. Growth of the transformants on synthetic media lacking leucine yielded at least 4.5 × 10⁵ transformants for each library. After double selection on plates lacking leucine but containing 15 µg/ml diazaborine for the DM6 (yap1) library, or 10 µg/ml diazaborine for the DM12 (yap1) library, 13 diazaborine-resistant transformants were obtained in the first instance, and 11 in the second. Upon retransformation into sensitive yeast A2, all plasmids from these strains were able again to create resistant phenotypes. The flanking regions of the inserts of two plasmids obtained from the DM6 (yap1) library were sequenced with forward and reverse primers for pBluescript. Sequence comparison revealed that both plasmids contained the PDR1 gene. A digoxigenin-labeled 1-kb PstI fragment of PDR1 was used to perform Southern blot analysis of the remaining 11 plasmids, which showed a common sequence in all 13 plasmids. The insert of the smallest plasmid, pKP100, was sequenced entirely. Similarly, sequencing of the flanking regions of the inserts of three plasmids derived from the DM12 (yap1) library showed that all plasmids contained the PDR3 gene. A common sequence in all 11 plasmids was confirmed by Southern blot analysis using a digoxigenin-labeled 1.4-kb NciI fragment of PDR3. The smallest plasmid obtained from the DM12 (yap1) library, pKP300, was further reduced in size by using the singular SalI site. The insert of the resulting plasmid pKP310 was completely sequenced.

The starting material for construction of the YAP1 gene, marked with a HIS3 cassette, was plasmid pBS300. A 1.75-kb HIS3 BamHI cassette was cloned into the singular SphI site, and a linear EcoRI/XbaI fragment was used to transform strain A2, resulting in strain FW52. Appropriate recombinants were identified by Southern blotting (25). Furthermore, this fragment was cloned into YEp351 and transformed into A2 to exclude any side effects due to the insertion of the HIS3 cassette.

Gene disruptions were constructed using the one-step gene disruption method of Rothstein (26). Disruption was performed as above except that the HIS3 cassette was inserted into a singular HpaI site. The resulting strains were designated DM6 (yap1) and DM12 (yap1).

To allow selection after the allele exchange experiment of gene PDR1-12, a 1.75-kb HIS3 cassette was introduced into the downstream ApaI site on plasmid pKP100, giving plasmid pKP120, which still mediated resistance. Strain KPHJ1 was obtained by transformation of the linear 6.1-kb SacI/SalI fragment from plasmid pKP120 into the diazaborine-sensitive strain A2. Transformants were tested for correct chromosomal integration by polymerase chain reaction.

The allele exchange of PDR3-33 was performed after introducing a 1.75-kb HIS3 cassette into the downstream BssHII site of plasmid pKP300, resulting in plasmid pKP320, which still conferred resistance. The linear 6.4-kb SacI/SalI fragment of pKP320 was transformed into the diazaborine-sensitive strain A2. The strain obtained was histidine-prototrophic and diazaborine-resistant and was designated KPHJ2. Correct integration of the construct was confirmed by polymerase chain reaction and Southern hybridization.

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. From this cell suspension, 100 µl were used to inoculate 2 ml of fresh YPD medium containing different concentrations of diazaborine. After 24 h, the A₆₀₀ was measured spectrophotometrically to quantitate the effects of diazaborine on each strain. Each experiment was repeated at least three times.

Exponentially growing cells were harvested and washed once with water to prepare cell extracts as described before (27). Briefly, after repelleting the cells, one A₆₀₀ equivalent of cells was treated for 10 min on ice with 150 µl of 1.85 M NaOH containing 7.5% mercaptoethanol. 150 µl of 50% trichloroacetic acid were added, and the samples were incubated for an additional 10 min on ice. Precipitates were collected by centrifugation at 5000 rpm for 5 min and washed once with ice-cold 2% trichloroacetic acid. The samples were resuspended in SDS-PAGE sample buffer (40 mM Tris-HCl, pH 6.8, 8 M urea, 5% SDS, 0.1 mM EDTA, 1% mercaptoethanol, 0.01% bromphenol blue) and neutralized by the addition of volume of 1 M Tris base. After heating at 37 °C for 15 min, insoluble material was removed by a 10-min high speed centrifugation in an Eppendorf microcentrifuge. Approximately 0.5 A₆₀₀ equivalent or 10 µl of each sample was fractionated by SDS-polyacrylamide gel electrophoresis on a 7.5% polyacrylamide gel and analyzed by immunoblotting. The polyclonal anti-Pdr5p and anti-Snq2p antibodies used for immunoblotting have been described elsewhere (13, 27).

RESULTS

The yeast S. cerevisiae is sensitive to the antibacterially active compound diazaborine. We selected the strain A2 whose minimum inhibitory concentration value is 12.5 µg/ml diazaborine in YPD medium. As a first step toward identifying the cellular target of the inhibitor, we generated drug-resistant mutants by UV mutagenesis. These mutants were studied by classical yeast genetics and cloning procedures to identify the genes involved in the resistance mechanisms.

After UV treatment of strain A2, we obtained a large number of resistant colonies. Seventeen mutants proved to be stable and could be divided into three complementation groups, i.e. drg1, drg2, and drg3. Eleven mutants were in group drg1, four in drg2, and two in drg3. Mating and subsequent spore analysis showed that the resistance phenotype of each mutant was dominant and resided at a single genetic locus. One of the mutants of the first complementation group was further analyzed by cloning the DNA fragment conferring resistance. It was found to carry an allelic form of gene AFG2. This gene codes for a protein with two "Walker motifs" specifying ATP binding sites. Protein Afg2p is a member of a novel protein family that has been designated AAA (ATPases associated to a variety of cellular activities). We are exploring the function of protein Afg2p in detail and will report about this study in a separate work. For further genetic analysis, the isolated AFG2 allele DRG1-1 was reintroduced by linear transformation into the parental wild type strain A2, and the resulting recombinant strain designated ADR6.

For the mutants of the second and third groups, but not for the one of the first complementation group, collateral phenotypes were associated with the diazaborine-resistant phenotype. Mutations in the drg2 and the drg3 locus also showed a cycloheximide resistance phenotype. (Fig. 1). In addition, a pet phenotype cosegregated with diazaborine resistance in the second recombination group, as we showed by back-crossing and tetrad analysis of the DM6 mutant of this group. However, the significance of this observation is unknown. No increased sensitivity to osmotic stress, heat shock, or sensitivity to pH changes was detected for any of the 17 mutants.

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To identify genes causing resistance in the second and third complementation groups, we prepared DNA libraries from two mutants from each the drg2 and the drg3 group, i.e. strain DM6 and strain DM12, respectively. From a first screen of both libraries, we identified the open reading frame of the YAP1 gene on a minimal consensus fragment, which could confer diazaborine resistance. From the DM12-derived library we also isolated a C-terminally truncated version of the YAP1 gene, which caused resistance to both diazaborine and cycloheximide (Fig. 2). This truncated Yap1p version lacks the last 212 amino acids of the C-terminal portion of Yap1p.

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To test whether a mutation in the YAP1 gene or overexpression of the wild type gene causes drug resistance, we linked the isolated gene with a HIS3 cassette to allow selection and introduced it into the isogenic background of the wild type strain A2 via homologous recombination. The resulting strain FW52 showed no drug resistance. We conclude from this experiment that resistance due to YAP1 is the result of overexpression of the wild type gene and not of a mutation.

Since we were able to clone only the YAP1 gene from the libraries prepared from strains DM6 and DM12, we decided to inactivate YAP1 in these two mutants. We were particularly interested to see whether elimination of YAP1 would influence the resistance phenotype of these two strains. YAP1 gene disruptions were made in both strains, DM6 and DM12. The resulting yap1 mutant strains were subsequently tested for their ability to grow in the presence of diazaborine. We could not detect any changes in drug resistance in yap1-disrupted strains of either DM6 or DM12 background. However, they showed hypersensitivity to hydrogen peroxide, a characteristic property of yap1 disruptants (30).

Since gene YAP1 was obliterating the search for genes causing resistance in strains DM6 and DM12, we prepared genomic libraries from the disrupted strains DM6 (yap1) and DM12 (yap1), respectively. These genomic libraries were screened for plasmids that conferred diazaborine resistance to the sensitive strain A2. A total of 13 diazaborine-resistant transformants were obtained from the DM6 (yap1)-derived and 11 from the DM12 (yap1)-derived library. Upon retransformation into sensitive yeast, all plasmids were able to mediate drug resistance. For still unknown reasons, these strains showed a somewhat retarded growth on complete medium (Fig. 1).

Southern blotting and restriction analysis demonstrated that all 13 plasmids derived from the DM6 (yap1) library contained fragments sharing a common sequence (data not shown). The recombinant plasmid with the smallest insert of 4.3 kb from the DM6 (yap1) library was designated pKP100 (Fig. 2) and subjected to DNA sequencing. Comparison with the Saccharomyces Genome Database revealed that this fragment was identical to a sequence of the S. cerevisiae chromosome VII containing gene PDR1, except for a single nucleotide exchange. This T to A transversion changes codon 1044 (CTA  CAA), resulting in a L1044Q exchange in the amino acid sequence of Pdr1p. In addition, we sequenced the wild type PDR1 gene from our parent strain A2, verifying the L1044Q mutation in the new PDR1 gain-of-function allele, which we designated PDR1-12. This result suggested that PDR1-12 might be responsible for diazaborine resistance. To provide conclusive proof, an allelic exchange experiment was performed, replacing the wild type PDR1 gene in strain A2 with the mutated PDR1-12 allele to generate strain KPHJ1. Strain KPHJ1 was diazaborine-resistant, showing that the mutation was indeed responsible for the resistance phenotype.

All 11 plasmids isolated from the DM12 (yap1) library contained fragments with a common DNA sequence different from the PDR1-specific fragments. The plasmid from this library carrying the smallest insert of 5.1 kb was designated pKP300. Shortening this fragment to 4.6 kb using the single SalI site in the insert yielded plasmid pKP310, which still conferred diazaborine resistance. The insert of pKP310 contained only one complete open reading frame, which was identified as the PDR3 gene (Fig. 2). A deletion affecting the PDR3 gene up to the SphI site resulted in complete loss of diazaborine resistance. Hence, we conclude that resistance is due to PDR3 expression. The nucleotide sequence of the pKP310 insert was determined, revealing three differences to the published PDR3 sequence in the Saccharomyces Genome Database. One mutation was a T to C transition (TAT  CAT) changing amino acid Tyr²⁷⁶ to His in the Pdr3p polypeptide chain. The second and third transitions were found to lie in the 3-untranslated region outside the open reading frame. They involve nucleotides 10 and 11 after the stop codon and change AG to GA. DNA sequencing of the wild type PDR3 gene in A2 showed that the latter two-base exchanges were also present in the wild type background. Thus, only the mutation in the coding region, which is specific for the mutant, can account for the resistance phenotype. To examine whether the newly cloned PDR3 allele, which was named PDR3-33, can cause diazaborine resistance upon chromosomal integration, we again performed an allelic replacement of wild type PDR3. The wild type sequence in the sensitive strain A2 was replaced for the mutant allele, resulting in strain KPHJ2. It was diazaborine-resistant, showing that its associated resistance phenotype is the consequence of the Y276H amino acid exchange in PDR3-33.

Strains carrying the isolated PDR1-12 and PDR3-33 alleles were subsequently tested for overexpression of Pdr5p and Snq2p. As shown in Fig. 3, the PDR1-12 allele leads to similar levels of Pdr5p and Snq2p as compared with PDR1-3. The PDR3-33 allele leads to even higher levels of these two pumps than the previously described multiple drug-resistant PDR3-2 allele.

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To investigate if other known PDR gain-of-function alleles can also confer diazaborine resistance and to test if strains deleted in the PDR1 and PDR3 genes are supersensitive to the drug, strains YALA-G4 (PDR1-3), YYMIA-A4 (PDR3-2), and FY1679-28C (pdr1 pdr3) were tested for their ability to grow in the presence of diazaborine. As shown in Fig. 4, the strains YALA-G4 and YYMIA-A4 are resistant to diazaborine but exhibited differences in the level of resistance i.e. the PDR1-3 strain tolerates the presence of higher concentrations of diazaborine than the PDR3-2 strain. This closely resembles the situation found with our PDR1-12 allele, which was more resistant than the PDR3-33 allele (data not shown). Strain FY1679-28C (pdr1 pdr3) proved to be hypersensitive to diazaborine. The minimum inhibitory concentration of this strain was 15 µg/ml, while that of the wild type strain FY1679-28C was 30 µg/ml.

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We also studied the effects of the drug on mutants carrying deletions in genes coding for ABC transporters in an attempt to directly identify the pumps involved in diazaborine resistance. Deletion strains such as snq2 and pdr5 were tested for their sensitivity to diazaborine. Only the pdr5 deletion strain was more sensitive to diazaborine than the isogenic wild type strain. Notably, no increase in sensitivity was observed when, in addition to PDR5, SNQ2 was deleted. However, when compared with the pdr1 pdr3 double disruptant, the sensitivity of the pdr5 strain was much less pronounced (data not shown). This suggests that PDR5 contributed somewhat to diazaborine resistance but that there are likely to exist additional genes regulated by PDR1 and PDR3 that can lead to diazaborine resistance.

The diazaborine supersensitivity of the pdr1 pdr3 strain prompted us to investigate whether resistance caused by YAP1 overexpression depends on functional PDR1/PDR3 genes. While the wild type strain carrying pFW300 was diazaborine-resistant, the double disruptant with the same plasmid was sensitive to the drug (Fig. 5). In contrast, when we tested the pdr1 pdr3 deletion strain with a plasmid carrying a diazaborine-resistant allele DRG1-1 of the AFG2 gene, no differences were observed as compared with the wild type strain. This result indicates that the YAP1-mediated diazaborine resistance is dependent on functional PDR1 and PDR3 genes, while the mechanism of DRG1-1 mediated resistance is independent of the components of multiple drug resistance. To investigate whether PDR1, PDR3, or both genes are responsible for diazaborine resistance mediated by YAP1, we also determined diazaborine resistance in the single mutants carrying pFW300. As shown in Fig. 6, either PDR1 or PDR3 is required for YAP1-mediated resistance, although deletion of PDR3 resulted in a more pronounced reduction of resistance. Deletion of PDR1 had only a minor effect on diazaborine resistance caused by YAP1. This result indicates that Yap1-mediated diazaborine resistance is exerted mainly via PDR3. Dependence on PDR3 was also found for Yap1p-mediated resistance to 4-NQO. In contrast, YAP1 overexpression in the pdr1 pdr3 background still caused cycloheximide and 1,10-phenanthroline resistance (data not shown). Thus, different resistance mechanisms are operative on the two groups of inhibitors, diazaborine and 4-NQO on one hand and cycloheximide and 1,10-phenanthroline on the other.

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DISCUSSION

Within the last few years, the antibacterial drug diazaborine has gained considerable attention due to its unique mode of action (31) and because it inhibits the Gram-negative homologue of protein InhA, which is involved in isoniazide resistance of clinical isolates of Mycobacterium tuberculosis (32). Although serious side effects are known, diazaborine could serve as a model compound for novel drug design once the cause for toxicity on eukaryotic cells is understood. To investigate the mechanism of action of diazaborine in eukaryotic cells, we used the yeast S. cerevisiae as a model system. We have shown that diazaborine inhibits the growth of yeast cells and have isolated several drug-resistant yeast mutants. They belong to three different complementation groups. The genes responsible for resistance in each of these groups were allelic forms of the genes AFG2, PDR1, and PDR3. In addition, YAP1 overexpression was identified as another cause of diazaborine resistance. PDR1 and PDR3 are involved in multiple drug resistance via overproduction of drug efflux pumps (5, 7, 9, 10). In contrast, Afg2p belongs to the family of AAA proteins (33, 34). Up to now, no AAA protein has been associated with resistance to growth inhibitors. The mechanism of resistance specified by AFG2 is unknown, but it appears to be different from that of gene YAP1 and the known genetic mechanisms leading to multiple or pleiotropic drug resistance in yeast. As expected, we did not isolate the yeast enoyl-(acyl carrier protein) reductase, because this enzymatic activity is part of the multifunctional fatty acid synthase and therefore structurally very different from that in E. coli (35).

Here we describe our results on PDR1 and PDR3 and, for the first time, show a link to the transcription factor Yap1p, which also causes diazaborine resistance when overexpressed. Pdr1p and Pdr3p are highly related regulatory proteins of the Zn(2)Cys(6) family. They act on the same upstream sequences of certain PDR-responsive genes (1) and functionally overlap, at least for the genes of the efflux pumps PDR5, SNQ2, and YOR1 (5, 7, 9, 10, 13). The gain-of-function alleles of PDR1 and PDR3 that we have isolated here contain mutations that lead to a single amino acid change in each of the encoded proteins. The exchange in Pdr1p is located in the C-terminal part of the protein and results in an L1044Q exchange. Interestingly, this region of Pdr1p shows significant homology to the C-terminal portion of Pdr3p and Gal4p (Fig. 7). In Gal4p, this segment is involved in transactivation and interaction with the negative regulator Gal80p (36). Moreover, the transactivation domain of Pdr1p resides within the last 251 amino acids of the protein (37). Martens et al. (37) also demonstrated that the C-terminal region of Pdr1p and Pdr3p interact with the ADA coactivator/repressor complex. This association inhibits the transactivation activity of Pdr1p. The gain-of-function mutation in our PDR1-12 allele could therefore affect either the transactivation itself or the interaction with a negative regulator. In contrast to Pdr1p, the exchange in PDR3-33 leading to diazaborine resistance is located in the middle portion of the protein, a region without any significant homology to Gal4p. This region, however, is conserved in the amino acid sequence of Pdr1p and the putative regulatory protein YER184C, which belongs to the same family of regulatory proteins. This motif may therefore be of functional importance (Fig. 7).

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Pdr1p and Pdr3p could regulate another yet unidentified target protein that might be the major resistance determinant. This is confirmed by the fact that a double deletion mutant exhibits very high sensitivity, surpassing each single deletion mutant. Since transcriptional regulation of PDR3 is known to involve both control by Pdr1p, and by Pdr3p itself via an autoregulatory loop (38), it is still an open question whether Pdr1 and Pdr3 act simultaneously on the same or neighboring promoter elements (pleiotropic drug resistance elements) of their target genes or whether there is a sequential gene activation.

Moreover, we have found that overexpression of the wild type YAP1 gene also generates diazaborine resistance. We have also isolated a truncated version of this gene, which was sufficient to confer resistance when overexpressed. This deletion lacks the 212 C-terminal codons of YAP1. Very recently, it was demonstrated that yeast cells relocalize Yap1p from the cytoplasm to the nucleus upon exposure to reactive oxygen (39). The information for this regulated process is located within a C-terminal, cysteine-rich, domain of the Yap1p protein. Mutations exchanging these cysteines or a deletion of the whole domain result in nuclear localization of Yap1p. Such truncated Yap1p proteins were shown to have retained their potential to transactivate and to constitutively express target genes (39). Since Yap1p is involved in multiple drug resistance and is also required in response to oxidative stress (1), we tested a variety of mutants that had disruptions in genes known to be activated by Yap1p, such as GSH1, GLR1, and TRX2 (40-42). However, we found no change in the level of resistance to diazaborine. No resistance developed when we overexpressed GSH1, TRX2, and YCF1 (43) (data not shown). Notably, disruption of YAP1 did not result in hypersensitivity to diazaborine in a wild type strain or in a drop in resistance in the gain-of-function mutants DM6 and DM12. These data show that the transcription factor Yap1p is not alone responsible for activation of a target gene mediating diazaborine resistance.

Our finding that YAP1 overexpression gives rise to diazaborine resistance only in a wild type but not in a pdr1 pdr3 background indicates that Yap1p acts either in concert with these two transcription factors on a common gene or participates in the sequential activation of an unknown target gene. A different possibility could be that Pdr1p and Pdr3p are under the influence of a common regulatory protein, e.g. Gal4p is repressed by Gal80p. The gain-of-function mutations would weaken or impair the association between the transcription factor and the inhibiting protein, resulting in a constitutive activity of the transactivator. An intracellular increase of Yap1p in wild type cells could influence the abundance or activity of the putative inhibitory protein, thereby partially removing it from Pdr1p and Pdr3p. This way, Pdr1p and Pdr3p would be liberated, resulting in an increased production of detoxifying pumps. This idea also seems to explain why the yap1 disruptant shows increased sensitivity to hydrogen peroxide but not to diazaborine. Thus, Yap1p may be only indirectly influencing diazaborine but directly influencing hydrogen peroxide resistance. In addition to diazaborine resistance, PDR3 is required for Yap1p-mediated resistance to 4-NQO but not for the inhibitors cycloheximide and 1,10-phenanthroline. This shows that resistance to some drugs caused by YAP1 overexpression is mediated via the PDR3 gene, while other detoxifying genes might be directly activated by Yap1p (Fig. 8). Taken together, we describe here for the first time a functional connection of the Yap1p activity with the master regulators of the pleiotropic drug resistance network, namely Pdr1p and Pdr3p.

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