Multiple mechanisms contribute to the development of clinically significant azole resistance in Aspergillus fumigatus.

Infections caused by the filamentous fungus Aspergillus fumigatus are a significant clinical issue and represent the second most-common form of fungal infection. Azole drugs are effective against this pathogen but resistant isolates are being found more frequently. Infections associated with azole resistant A. fumigatus have a significantly increased mortality making understanding drug resistance in this organism a priority. The target of azole drugs is the lanosterol α-14 demethylase enzyme encoded by the cyp51A gene in A. fumigatus. Mutations in cyp51A have been described that give rise to azole resistance and been argued to be the primary, if not sole, contributor to azole resistance. Here, I discuss recent developments that indicate multiple mechanisms, including increased expression of ATP-binding cassette (ABC) transporter proteins, contribute to azole resistance. ABC transporters are well-established determinants of drug resistance in other fungal pathogens and seem likely to play a similar role in A. fumigatus.

Retrograde regulation of multidrug resistance in Saccharomyces cerevisiae.

Communication between the mitochondria and the nucleus is essential to ensure correct metabolic coordination of the cell. Signaling pathways leading from the mitochondria to the nucleus are referred to as retrograde signaling and were first discovered in the yeast Saccharomyces cerevisiae. Cells that lack their mitochondrial genome (rho0 cells) trigger expression of the nuclear CIT2 gene in order to ensure adequate amino acid biosynthesis. More recently, it has been found that a different set of genes involved in multidrug resistance in S. cerevisiae is strongly induced in rho0 cells. During a search for negative regulators of the ATP-binding cassette (ABC) transporter-encoding gene PDR5, it was observed that rho0 mutants exhibited dramatic up-regulation of the transcript of this gene. This induction was due to the post-translational activation of a direct upstream regulator of PDR5 that was designated Pdr3p. Loss of the LGE1 gene led to a block in rho0-mediated induction of PDR5 expression. Lge1p has been observed by others to be involved in histone H2B ubiquitination along with the ubiquitin-conjugating enzyme Rad6p and the ubiquitin ligase Bre1p. Our studies provide evidence that Lge1p has another function unique from H2B ubiquitination that is required for retrograde regulation of PDR5 transcription. We have also found that the Pdr pathway regulates expression of several genes involved in sphingolipid biosynthesis. These findings suggest that the physiological role of the PDR genes might be to regulate membrane homeostasis and rho0-triggered changes in this parameter may be the signal controlling PDR gene expression.

Saccharomyces cerevisiae multidrug resistance gene expression inversely correlates with the status of the F(0) component of the mitochondrial ATPase.

Loss of the mitochondrial genome (rho(0) cell) or elimination of the mitochondrial inner membrane protein Oxa1p causes a dramatic increase in expression of the ATP binding cassette transporter-encoding gene PDR5 in the yeast Saccharomyces cerevisiae. This increase in gene expression occurs via activation of the function of the Cys(6)-Zn(II)(2) cluster transcription factor Pdr3p, which in turn autoregulates expression of its structural gene. Surprisingly, the acquisition of PDR5-dependent multidrug resistance occurs at a very high frequency, consistent with the appearance of rho(-) cells in a fermentatively growing culture (approximately 2%). The degree of activation of Pdr3p target genes was found to vary considerably and to be influenced by the presence of the homologous protein, Pdr1p. Mutagenesis and overexpression studies provided evidence that the control of Pdr3p expression was the major control point of this transcription factor by mitochondrial retrograde signaling. Because both rho(0) and oxa1 mutant cells have multiple defects including loss of normal respiratory chain function and oxidative phosphorylation, a series of mutant strains with more selective defects in mitochondrial function was employed to identify the molecular signal that triggers PDR5 transcriptional activation. Only mutations that influenced the functional status of the F(0) subunit of the mitochondrial ATPase were found to lead to activation of PDR5 expression.

Coordinate control of sphingolipid biosynthesis and multidrug resistance in Saccharomyces cerevisiae.

Multiple or pleiotropic drug resistance often occurs in the yeast Saccharomyces cerevisiae through genetic activation of the Cys(6)-Zn(II) transcription factors Pdr1p and Pdr3p. Hyperactive alleles of these proteins cause overproduction of target genes that include drug efflux pumps, which in turn confer high level drug resistance. Here we provide evidence that both Pdr1p and Pdr3p act to regulate production of an enzyme involved in sphingolipid biosynthesis in S. cerevisiae. The last step in formation of the major sphingolipid in the yeast plasma membrane, mannosyldiinositol phosphorylceramide, is catalyzed by the product of the IPT1 gene, inositol phosphotransferase (Ipt1p). Transcription of the IPT1 gene is responsive to changes in activity of Pdr1p and Pdr3p. A single Pdr1p/Pdr3p response element is present in the IPT1 promoter and is required for regulation by these factors. Loss of IPT1 has complex effects on drug resistance of the resulting strain, consistent with an important role for mannosyldiinositol phosphorylceramide in normal plasma membrane function. Direct assay for lipid contents of cells demonstrates that changes in sphingolipid composition correlate with changes in the activity of Pdr3p. These data suggest that Pdr1p and Pdr3p may act to modulate the lipid composition of membranes in S. cerevisiae through activation of sphingolipid biosynthesis along with other target genes.

Cross-talk between transcriptional regulators of multidrug resistance in Saccharomyces cerevisiae.

Multiple or pleiotropic drug resistance often arises in the yeast Saccharomyces cerevisiae due to genetic alterations of the functional state of the Cys(6)-Zn(II)(2) transcription factors Pdr1p and Pdr3p. Single amino acid substitutions give rise to hyperactive forms of these regulatory proteins, which in turn cause overproduction of downstream target genes that directly mediate multidrug resistance. Previous work has identified a novel Cys(6)-Zn(II)(2) transcription factor designated Yrr1p as mutant forms of this protein confer high level resistance to the cell cycle inhibitor reveromycin A and DNA damaging agent 4-nitroquinoline-N-oxide. In the present study, we demonstrate that Yrr1p also mediates oligomycin resistance through activation of the ATP-binding cassette transporter-encoding gene YOR1. Additionally, insertion of triplicated copies of the hemagglutinin epitope in the C-terminal region of Yrr1p causes the protein to behave as a hyperactive regulator of transcription. We have found that YRR1 expression is both controlled in a Pdr1p/Pdr3p-dependent manner and autoregulated. Chromatin immunoprecipitation experiments also show that Yrr1p associates with target promoters in vivo. Together these data argue that the signal generated by activation of Pdr1p and/or Pdr3p can be amplified through the action of these transcriptional regulatory proteins on downstream target genes, like YRR1, that also encode transcription factors.

Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae.

The action of gamma-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H(2)O(2) and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the cross-reaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H(2)O(2) exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.

Multiple signals from dysfunctional mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces cerevisiae.

Multiple or pleiotropic drug resistance most often occurs in Saccharomyces cerevisiae due to substitution mutations within the Cys(6)-Zn(II) transcription factors Pdr1p and Pdr3p. These dominant transcriptional regulatory proteins cause elevated drug resistance and overexpression of the ATP-binding cassette transporter-encoding gene, PDR5. We have carried out a genetic screen to identify negative regulators of PDR5 expression and found that loss of the mitochondrial genome (rho(o) cells) causes up-regulation of Pdr3p but not Pdr1p function. Additionally, loss of the mitochondrial inner membrane protein Oxa1p generates a signal that results in increased Pdr3p activity. Both of these mitochondrial defects lead to increased expression of the PDR3 structural gene. Importantly, the signaling pathway used to enhance Pdr3p function in rho(o) cells is not the same as in oxa1 cells. Loss of previously described nuclear-mitochondrial signaling genes like RTG1 reduce the level of PDR5 expression and drug resistance seen in rho(o) cells but has no effect on oxa1-induced phenotypes. These data uncover a new regulatory pathway connecting expression of multidrug resistance genes with mitochondrial function.

Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p.

CAP1 encodes a basic region-leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant-regulated localization of Yap1p to the nucleus requires the presence of a carboxy-terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Deltayap1 S. cerevisiae strain. Whereas a Yap1p-responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p-expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post-translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function.

Hyperactive forms of the Pdr1p transcription factor fail to respond to positive regulation by the hsp70 protein Pdr13p.

Multidrug resistance in Saccharomyces cerevisiae is commonly associated with the overproduction of ATP-binding cassette transporter proteins such as Pdr5p or Yor1p. The Cys6-Zn(II)2 cluster-containing transcription factors Pdr1p and Pdr3p are key regulators of expression of these pleiotropic drug resistance (PDR) loci. Previous experiments have demonstrated that the Hsp70 protein encoded by the PDR13 gene is a positive regulator of Pdr1p function. We have examined the mechanism underlying the control of Pdr1p by Pdr13p. Expression of deletion, insertion and amino acid substitution mutant variants of Pdr1p suggest that the centre region of the transcription factor is the target for Pdr13p-mediated positive regulation. Immunological and fusion protein analyses demonstrate that Pdr13p is located in the cytoplasm, while Pdr1p is found in the nucleus. Biochemical fractionation experiments indicate that Pdr13p is associated with a high-molecular-weight complex and suggest the association of some fraction of Pdr13p with ribosomes.

Yap1p activates gene transcription in an oxidant-specific fashion.

Positive regulation of gene expression by the yeast Saccharomyces cerevisiae transcription factor Yap1p is required for normal tolerance of oxidative stress elicited by the redox-active agents diamide and H(2)O(2). Several groups have provided evidence that a cluster of cysteine residues in the extreme C terminus of the factor are required for normal modulation of Yap1p by oxidant challenge. Deletion of this C-terminal cysteine-rich domain (c-CRD) produces a protein that is highly active under both stressed and nonstressed conditions and is constitutively located in the nucleus. We have found that a variety of different c-CRD mutant proteins are hyperactive in terms of their ability to confer diamide tolerance to cells but fail to provide even normal levels of H(2)O(2) resistance. Although the c-CRD mutant forms of Yap1p activate an artificial Yap1p-responsive gene to the same high level in the presence of either diamide or H(2)O(2), these mutant factors confer hyperresistance to diamide but hypersensitivity to H(2)O(2). To address this discrepancy, we have examined the ability of c-CRD mutant forms of Yap1p to activate expression of an authentic target gene required for H(2)O(2) tolerance, TRX2. When assayed in the presence of c-CRD mutant forms of Yap1p, a TRX2-lacZ fusion gene fails to induce in response to H(2)O(2). We have also identified a second cysteine-rich domain, in the N terminus (n-CRD), that is required for H(2)O(2) but not diamide resistance and influences the localization of the protein. These data are consistent with the idea that the function of Yap1p is different at promoters of loci involved in H(2)O(2) tolerance from promoters of genes involved in diamide resistance.

Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein.

The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overproduced from a high-copy-number plasmid. Analysis of the predicted amino acid sequence of Yor1p indicated that this protein was a new member of a subfamily of ABC transporter proteins defined by the multidrug resistance protein (MRP). In this work, Yor1p is demonstrated to localize to the Saccharomyces cerevisiae plasma membrane by both indirect immunofluorescence and biochemical fractionation studies. Several mutations were generated in the amino-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high degree of sequence conservation in this region of the protein was important for function. Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticulum (ER) and that was unstable. As shown by others, deletion of the analogous residue from a second mammalian MRP family member, the cystic fibrosis transmembrane conductance regulator (CFTR), also led to retention of this normally plasma membrane-localized protein in the ER. Changes in the spacing between or the sequences flanking functional motifs of Yor1p NBD1 led to defective trafficking or decreased activity of the mutant proteins. Analyses of the degradation of wild-type and DeltaF670 Yor1p indicated that the half-life of DeltaF670 Yor1p was dramatically shortened. While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of DeltaF670 Yor1p was found to be more complex with both proteasomal and vacuolar contributions.

Regulation of transcription factor Pdr1p function by an Hsp70 protein in Saccharomyces cerevisiae.

Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae requires the expression of several ATP binding cassette transporter-encoding genes under the control of the zinc finger-containing transcription factor Pdrlp. The ATP binding cassette transporter-encoding genes regulated by Pdrlp include PDR5 and YOR1, which are required for normal cycloheximide and oligomycin tolerances, respectively. We have isolated a new member of the PDR gene family that encodes a member of the Hsp70 family of proteins found in this organism. This gene has been designated PDR13 and is required for normal growth. Overexpression of Pdr13p leads to an increase in both the expression of PDR5 and YOR1 and a corresponding enhancement in drug resistance. Pdr13p requires the presence of both the PDR1 structural gene and the Pdr1p binding sites in target promoters to mediate its effect on drug resistance and gene expression. A dominant, gain-of-function mutant allele of PDR13 was isolated and shown to have the same phenotypic effects as when the gene is present on a 2microm plasmid. Genetic and Western blotting experiments indicated that Pdr13p exerts its effect on Pdr1p at a posttranslational step. These data support the view that Pdr13p influences pleiotropic drug resistance by enhancing the function of the transcriptional regulatory protein Pdr1p.

Divergent transcriptional control of multidrug resistance genes in Saccharomyces cerevisiae.

Improper control of expression of ATP binding cassette transporter-encoding genes is an important contributor to acquisition of multidrug resistance in human tumor cells. In this study, we have analyzed the function of the promoter region of the Saccharomyces cerevisiae YOR1 gene, which encodes an ATP binding cassette transporter protein that is required for multidrug tolerance in S. cerevisiae. Deletion analysis of a YOR1-lacZ fusion gene defines three important transcriptional regulatory elements. Two of these elements serve to positively regulate expression of YOR1, and the third element is a negative regulatory site. One positive element corresponds to a Pdr1p/Pdr3p response element, a site required for transcriptional control by the homologous zinc finger transcription factors Pdr1p and Pdr3p in other promoters. The second positive element is located between nucleotides -535 and -299 and is referred to as UASYOR1 (where UAS is upstream activation sequence). Interestingly, function of UASYOR1 is inhibited by the downstream negative regulatory site. Promoter fusions constructed between UASYOR1 and the PDR5 promoter, another gene under Pdr1p/Pdr3p control, are active, whereas analogous promoter fusions constructed with the CYC1 promoter are not. This suggests the possibility that UASYOR1 has promoter-specific sequence requirements that are satisfied by another Pdr1p/Pdr3p-regulated gene but not by a heterologous promoter.

Saccharomyces cerevisiae basic region-leucine zipper protein regulatory networks converge at the ATR1 structural gene.

Saccharomyces cerevisiae cells express a family of transcription factors belonging to the basic region-leucine zipper family. Two of these proteins, yAP-1 and Gcn4p, are known to be involved in oxidative stress tolerance and general control of amino acid biosynthesis, respectively. Strains lacking the YAP1 or GCN4 structural gene have very different phenotypes, which have been taken as evidence that these transcriptional regulatory proteins control separate batteries of target genes. In this study, we provide evidence that both yAP-1 and Gcn4p control the expression of a putative integral membrane protein, Atr1p. Both yAP-1 and Gcn4p can elevate resistance to 3-amino-1,2,4-triazole and 4-nitroquinoline-N-oxide but only if the ATR1 gene is intact. Expression of ATR1 is enhanced in the presence of constitutively active alleles of YAP1 and GCN4. Regulation of ATR1 transcription by yAP-1 and Gcn4p occurs through a common DNA element related to the yAP-1 recognition element found upstream of other yAP-1-regulated genes. These data provide the first indication of overlap between the regulatory networks defined by yAP-1 and Gcn4p.

Mutational analysis of the Saccharomyces cerevisiae ATP-binding cassette transporter protein Ycf1p.

Ycf1p is a member of the ATP-binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein. These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.

The Saccharomyces cerevisiae AP-1 protein discriminates between oxidative stress elicited by the oxidants H2O2 and diamide.

The Saccharomyces cerevisiae AP-1 protein (yAP-1) is a key mediator of oxidative stress tolerance. Transcriptional activation by yAP-1 has been shown to be inducible by exposure of cells to H2O2 and diamide, among other oxidative stress eliciting compounds. Here we define the segments of the yAP-1 protein that are required to respond to this environmental challenge. Western blotting analyses indicated that levels of yAP-1 do not change during oxidative stress. Deletion mutagenesis and gene fusion experiments indicate that two different segments of yAP-1 are required for oxidative stress inducibility. These two domains function differentially depending on the type of oxidant used to generate oxidative stress. Three repeated cysteine-serine-glutamate sequences located in the carboxyl terminus are required for normal regulation of yAP-1 function during oxidative stress. Replacement of these cysteine-serine-glutamate repeats by alanine residues does not similarly affect H2O2 and diamide regulation of yAP-1 function. While yAP-1 transactivation is enhanced by exposure to either H2O2 or diamide, the protein responds to the oxidative stress produced by these compounds in nonidentical ways.

Multiple Pdr1p/Pdr3p binding sites are essential for normal expression of the ATP binding cassette transporter protein-encoding gene PDR5.

Saccharomyces cerevisiae has large number of genes that can be genetically altered to produce a multiple or pleiotropic drug resistance phenotype. The homologous zinc finger transcription factors Pdr1p and Pdr3p both elevate resistance to many drugs, including cycloheximide. This elevation in cycloheximide tolerance only occurs in the presence of an intact copy of the PDR5 gene that encodes a plasma membrane-localized ATP binding cassette transporter protein. Previously, we have found that a single binding site for Pdr3p present in the PDR5 promoter is sufficient to provide Pdr3p-responsive gene expression. In this study, we have found that there are three sites in the PDR5 5'-noncoding region that are closely related to one another and are bound by both Pdr1p and Pdr3p. These elements have been designated Pdr1p/Pdr3p response elements (PDREs), and their role in the maintenance of normal PDR5 expression has been analyzed. Mutations have been constructed in each PDRE and shown to eliminate Pdr1p/Pdr3p binding in vitro. Analysis of the effect of these mutant PDREs on normal PDR5 promoter function indicates that each element is required for wild-type expression and drug resistance. A single PDRE placed upstream of a yeast gene lacking its normal upstream activation sequence is sufficient to confer Pdr1p responsiveness to this heterologous promoter.

ROD1, a novel gene conferring multiple resistance phenotypes in Saccharomyces cerevisiae.

Glutathione-dependent detoxification reactions are catalyzed by the enzyme glutathione S-transferase and are important in drug resistance in organisms ranging from bacteria to humans. The yeast Issatchenkia orientalis expresses a glutathione S-transferase (GST) protein that is induced when the GST substrate o-dinitrobenzene (o-DNB) is added to the culture. In this study, we show that overproduction of the I. orientalis GST in Saccharomyces cerevisiae leads to an increase in o-dinitrobenzene resistance in S. cerevisiae cells. To recover genes that influence o-DNB resistance in S. cerevisiae, a high copy plasmid library was screened for loci that elevate o-DNB tolerance. One gene was recovered and designated ROD1 (resistance to o-dinitrobenzene). This locus was found to encode a novel protein with no significant sequence similarity with proteins of known function in the data base. An epitope-tagged version of Rod1p was produced in S. cerevisiae and shown to function properly. Subcellular fractionation experiments indicated that this factor was found in the particulate fraction by differential centrifugation. Overproduction of Rod1p leads to resistance to not only o-DNB but also zinc and calcium. Strains that lack the ROD1 gene are hypersensitive to these same compounds. Rod1p represents a new type of molecule influencing drug tolerance in eukaryotes.

Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae.

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

Analysis of second-site mutations that suppress the multiple drug resistance phenotype of the yeast PDR1-7 allele.

The yeast PDR1 locus encodes a member of the C6 zinc cluster family of transcriptional regulatory proteins. Among the targets of PDR1 is the yeast PDR5 locus. The product of this gene is a member of the ATP-binding cassette (ABC) transmembrane protein family and plays a major role in inhibitor efflux. Mutations in PDR1 affect the relative level of PDR5 transcript and can therefore result in increased or decreased drug resistance. We isolated three second-site suppressors of a PDR1-7 semidominant hyper-resistant mutation. These mutants were drug hypersensitive, as compared with isogenic controls. Two of the three mutations contained alterations in a putative DNA-binding domain. Significantly, the mutant proteins exhibited reduced DNA-binding capacity.