INTRODUCTION

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Multiple drug resistance refers to a limited number of genetic alterations giving rise to a complex spectrum of tolerance to cytotoxic compounds having different intracellular targets (32). In human tumor cells, multiple drug resistance is often associated with overproduction of ATP binding cassette (ABC) transporter-encoding genes, such as MDR1 (22) or MRP (11). The overproduction of these gene products leads to enhanced efflux of toxic drugs across the plasma membrane and permits tolerance of otherwise lethal dosages.

In the yeast Saccharomyces cerevisiae, a similar multidrug tolerance phenotype can be observed and is referred to as pleiotropic drug resistance. The first PDR gene identified and cloned was designated PDR1 (2). DNA sequence analysis of this locus indicated that Pdr1p was a zinc finger-containing protein that showed strong sequence similarity to other fungal transcriptional regulatory proteins (2). Strains lacking PDR1 were hypersensitive to a broad range of drugs, including cycloheximide and oligomycin. Sequence analysis of S. cerevisiae chromosome II indicated that a homolog of PDR1, PDR3 (16), was present at this location and encoded a protein showing 36% amino acid identity with Pdr1p (15). Genetic and biochemical experiments demonstrated that Pdr1p and Pdr3p act to influence pleiotropic drug resistance (15, 33). However, mutants lacking PDR3 were not observed to have a pronounced defect in drug resistance, unlike pdr1 strains, suggesting that Pdr1p was the major contributor of drug tolerance (15, 33).

PDR1 was originally identified on the basis of semidominant mutant alleles that produced high-level resistance to cycloheximide and oligomycin, among other compounds (reviewed in reference 3). Epistasis and Northern blot experiments demonstrated that PDR1 conferred cycloheximide resistance through the transcriptional activation of the PDR5 gene (43). PDR5 was shown to encode an ABC transporter protein (5, 6, 28) that is located in the plasma membrane (5, 17) and that can act as a drug efflux pump (36, 38). Deletion mapping and DNase I footprinting analysis indicated that both Pdr1p and Pdr3p bound to several sites upstream of the PDR5 transcription start site and activated the expression of this gene (33, 34). These binding sites were named Pdr1p/Pdr3p response elements (PDREs).

While semidominant PDR1 mutants required PDR5 to mediate cycloheximide resistance, the loss of PDR5 did not affect PDR1-mediated oligomycin resistance. We screened a high-copy-number plasmid library for sequences that would elevate oligomycin resistance and recovered several different genes (35). One of these loci was found to encode an ABC transporter protein resembling the MRP gene product and was designated YOR1 (35). The loss of YOR1 leads to a large decrease in Pdr1p-mediated oligomycin resistance but has no effect on cycloheximide resistance. A second gene identified in this screen elevated both oligomycin and cycloheximide tolerance. We refer to this gene as PDR13.

In this study, we demonstrate that PDR13 encodes an Hsp70 homolog that acts to elevate the function of Pdr1p, leading to increased expression of PDRE-containing genes and drug resistance. Strains lacking PDR13 are compromised for growth and induce the expression of stress-responsive genes. A gain-of-function mutant form of Pdr13p is able to complement the growth defect of a pdr13 strain and elevates both PDRE-containing gene expression and drug resistance. Taken together, these data implicate Pdr13p as an upstream modulator of the expression of PDR genes through control of the transcription factor Pdr1p.

	MATERIALS AND METHODS

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Yeast strains and media. The genotypes of the yeast strains used in this study are listed in Table 1. Yeast transformations were performed with the lithium acetate procedure of Ito et al. (29) or a high-efficiency technique (21). Standard YPD medium and minimal medium were used for the growth of cells and drug resistance assays (49). Drug resistance assays were performed by spot tests (53). -Galactosidase activity was measured as previously described (24).

Plasmids. An integrating PDR5-lacZ fusion gene was constructed by transferring an EcoRI/SalI fragment from pKV2 (33) into pRS303 (50) to form pTH120. Strain TCH4 was generated by transforming TCH1 with StuI-cut pTH120, which directs recombination to PDR5. A SalI/NotI fragment carrying the wild-type PDR13 gene was cloned into the 2µm-containing vector pRS424 (10) to form pTH87. The same PDR13 fragment was also inserted into pRS316 (50) to generate pTH86 and into pRS314 (50) to produce pTH143. The original YEp24-based recombinant carrying PDR13 was designated pDOC10-2. The GAL1-PDR1 fusion gene contained a PDR1 fragment (extending from the ATG to a SalI site located 700 bp downstream of the translation stop codon) cloned downstream of the GAL1 promoter carried in the low-copy-number plasmid pSEYC68-GAL (44). The resulting construct was named pTH7. The YOR1-lacZ fusion plasmid was previously described (26), and a mutated variant of this clone that lacked the PDREs was constructed.

Construction of a PDR13 mutant library and selection of hyperactive PDR13 alleles. pTH86 DNA (20 µg) was mutagenized with 100 µl of 45% formic acid for 1 min. The DNA was recovered by ethanol precipitation, and the PDR13 gene was amplified by PCR from this mutagenized template by use of the flanking T3 and T7 universal primers. The PCR product was cleaved with SalI/NotI and cloned into similarly digested pRS314. The library was amplified in bacteria, and plasmid DNA was prepared.

Immunological methods. Rabbit polyclonal antisera were generated by standard immunization techniques (27) against Pdr1p and Pdr13p expressed in bacteria. Both S. cerevisiae proteins were produced as fusion proteins with glutathione S-transferase (GST) carried in plasmid pGEX-KG (23). The GST-Pdr13p fusion protein was constructed by cloning into SacI/NotI-cleaved pGEX-KG full-length Pdr13p downstream of the GST cassette carried in pGEX-KG as a SacI/NotI fragment. The GST-Pdr1p fusion protein was generated by cloning an EcoRI fragment encoding Pdr1p residues 768 to 1063 into the EcoRI site of pGEX4. Both fusion proteins were purified through the use of glutathione-agarose columns as described previously (19).

Protein extraction and analysis. Cells were grown in minimal medium to an A₆₀₀ of 0.5 to 0.7, harvested at 4°C by centrifugation, and resuspended in sorbitol breaking buffer (0.3 M sorbitol, 0.1 M NaCl, 5 mM MgCl₂, 10 mM Tris [pH 7.4]), with protease inhibitors. Cells were broken by agitation in the presence of glass beads for 25 min at 4°C and centrifuged (Eppendorf 5415C) at 12,000 rpm for 5 min. Protein concentrations were determined by the method of Lowry et al. (41). Equal amounts of protein were resuspended in Laemmli sodium dodecyl sulfate (SDS) loading buffer (37) and analyzed by Western blotting with the anti-Pdr1p or anti-Pdr13p antisera. Antigen-antibody complexes were visualized with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin secondary antibody and enhanced chemiluminescence reagents (Pierce Supersignal).

Gene disruption. A PDR13 disruption allele was produced by deletion of a BglII fragment from the wild-type PDR13 gene carried as a SalI/NotI fragment in pRT5. The deleted BglII fragment was replaced with the hisG-URA3-hisG fragment from pNKY51 (1). The resulting plasmid replaced PDR13 DNA from 41 bp upstream of the putative ATG to residue 203 with hisG-URA3-hisG. This plasmid was designated pRT6 and was cleaved with SalI/NotI prior to transformation into wild-type cells (SEY6210). URA3 transformants were selected, and correct integration was confirmed by Southern blotting (47). The URA3 gene was removed from a selected disruptant by treatment with 5-fluoro-orotic acid (7), and the resulting pdr13-1::hisG strain was designated YRT9.

	RESULTS

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Identification of PDR13 as a pleiotropic drug resistance gene. We previously reported a high-copy-number plasmid library screen in which colonies that exhibited elevated oligomycin resistance were identified by replica plating (35). Two different loci that were both capable of consistently elevating oligomycin tolerance were recovered in this screen. Characterization of the YOR1 structural gene, encoding an ABC transporter protein, has already been reported (35). The second locus found was designated PDR13.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Drug resistance conferred by the Hsp70 homolog Pdr13p requires the presence of PDR genes. (A) A phylogenetic tree showing the relationship of the Pdr13p sequence to sequences of other Hsp70 homologs was generated by use of the Megalign routine of Lasergene software (DNAstar). Sequences were aligned by use of the Jotun-Hein algorithm. The Hsp70 proteins are from S. cerevisiae, with the following four exceptions: Hsc70 (bovine), DnaK (E. coli), Sp Pdr13p (S. pombe Pdr13p homolog), and CHO Hsp110 (Cricetulus griseus). The numbers on the bottom of the panel indicate the evolutionary distances between the sequences. (B) The functional dependence of Pdr13p on the presence of PDR5 was tested by placing 1,000 cells of the indicated genotype on a YPD (49) plate containing a gradient of cycloheximide (indicated by the bar of increasing width). Isogenic wild-type (wt) or pdr5 cells were transformed with a high-copy-number vector plasmid (pRS426) or the same plasmid carrying the PDR13 structural gene. (C) The indicated transformants were streaked on YPGE medium (49) or YPGE medium plus oligomycin at 0.3 µg/ml and incubated at 30°C. Wild-type (wt) cells are SEY6210, while the yor1 (DKY7) mutant is an isogenic derivative. Plasmids were as in panel B.

High-copy-number PDR13 elevates the expression of Pdr1p target genes. To determine if high-copy-number PDR13 influenced the level of expression of PDR loci, we used gene fusions between the PDR5 and YOR1 structural genes and lacZ. Each of these plasmid-borne fusion genes has been demonstrated to respond to the same transcriptional control signals as its chromosomal counterpart (33, 35). A TRP5-lacZ fusion gene was used as a control for a locus unrelated to the PDR network. Each fusion gene was introduced into a wild-type cell along with either a high-copy-number plasmid containing PDR13 or the empty vector alone. -Galactosidase activities were then determined for transformants carrying these plasmids.

PDR13 affects the function of Pdr1p but not Pdr3p. To test the requirement of Pdr1p and/or Pdr3p for normal Pdr13p activity, we used a series of isogenic strains lacking PDR1, PDR3, or both genes (33). Each strain was transformed with a 2µm vector plasmid or the same plasmid carrying PDR13. Transformants were then tested for their cycloheximide resistance phenotypes by spot test assays (Fig. 2).

View larger version (73K): [in this window] [in a new window]   FIG. 2.   Elevation of cycloheximide resistance by high-copy-number PDR13 requires the presence of PDR1. A strain lacking both the PDR1 and the PDR3 structural genes (PB4) was transformed with the indicated plasmids, resulting in low- or high-copy-number forms of these genes. Along with the plasmids expressing PDR1 and/or PDR3, a 2µm clone of PDR13 or a high-copy-number vector was introduced into the cells. Transformants were grown in media to select for the presence of each plasmid and then assayed for growth on rich medium (YPD) or this same medium containing the concentrations of cycloheximide shown.

Growth defects of cells lacking PDR13. A gene disruption allele of PDR13 was prepared by replacing genomic DNA from 41 bp upstream of the potential PDR13 ATG to amino acid 203 of the coding sequence with hisG. The resulting allele, pdr13-1::hisG, has a deletion of 203 amino acids from the conserved Hsp70 ATPase domain of Pdr13p. The PDR13 locus was disrupted in both haploid and diploid cells. As we found that PDR13 is not an essential gene (see below), we focused our analysis on the isogenic wild-type and pdr13-1::hisG strains.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Cells lacking PDR13 are defective in growth. (A) One thousand cells each of isogenic wild-type and pdr13 strains were placed on YPD medium and incubated at the indicated temperatures (Celsius) for 2 days. (B) Isogenic wild-type and pdr13 cells were tested as in panel A for growth on a YPD plate containing a gradient of cycloheximide increasing from 0 to 0.25 µg/ml (indicated by the width of the bar on the left). Plates were incubated at 21, 30, or 37°C for 4 days. (C) Cells of the wild type (wt) and the isogenic pdr13 derivative were transformed with the indicated lacZ fusion genes carried on low-copy-number plasmids. Transformants were grown in minimal media at 30°C, and -galactosidase activities were determined. Values are the averages of at least two determinations.

Isolation of a hyper-drug-resistant form of Pdr13p. To dissect the action of Pdr13p on the pleiotropic drug resistance and growth phenotypes, we selected mutant forms of this protein that were able to confer elevated drug resistance but still provided normal growth. This was accomplished by constructing a mutant library of low-copy-number plasmids carrying the PDR13 gene. This mutant library was then transformed into pdr13 pdr3 cells containing an integrated PDR5-lacZ fusion gene. We selected transformants that grew normally, were hyperresistant to cycloheximide, and exhibited elevated levels of -galactosidase activity. Plasmids were recovered from these transformants and retested to confirm that the gene responsible for the mutant phenotypes was linked to the plasmid. The DNA sequence alteration of the PDR13 gene was then determined for several plasmids that exhibited linkage to the mutant phenotypes.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Enhanced drug resistance of cells expressing S295F Pdr13p. A strain lacking the PDR13 gene (TCH1) was transformed with low-copy-number plasmids expressing the wild type (PDR13) or the S295F (S295F PDR13) form of PDR13 or a high-copy-number plasmid carrying PDR13 (2µm PDR13). Appropriate transformants were tested for their relative resistance to the presence of the indicated concentrations of cycloheximide or oligomycin by spot test assays.

View larger version (48K): [in this window] [in a new window]   FIG. 5.   Equivalent expression of wild-type and S295F forms of Pdr13p. Whole-cell protein extracts were prepared from a pdr13 mutant strain carrying a vector plasmid (pRS314), a low-copy-number plasmid containing either wild-type PDR13 or the S295F allele of PDR13, and a high-copy-number plasmid (2µm PDR13) bearing the wild-type PDR13 gene. Protein (25 µg) was resolved by SDS-PAGE and analyzed by Western blotting with anti-Pdr13p antiserum. Numbers on the left are in kilodaltons.

Pdr13p acts posttranslationally to regulate Pdr1p. A central issue that emerges from these studies concerns the details of the activation of Pdr1p by Pdr13p. Pdr13p may change the level of expression of Pdr1p or, alternatively, may regulate the activity of Pdr1p. To examine which of these explanations was likely to be correct, we carried out two different experiments. First, the PDR1 coding sequence was placed under the control of the GAL1 promoter, and the resulting GAL1-PDR1 chimera was examined for its response to different PDR13 gene dosages. Second, an antiserum was produced against Pdr1p and used to directly examine the steady-state levels of Pdr1p in strains containing different PDR13 gene doses.

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Pdr13p does not act on the PDR1 promoter region. A wild-type strain was transformed with a low-copy-number plasmid containing a GAL1-PDR1 fusion gene. Along with this fusion gene, a high-copy-number plasmid containing the PDR13 gene (2µm PDR13) or the plasmid vector alone (vector) was introduced. Transformants containing each pair of plasmids were tested for their ability to grow on YPGE-galactose medium (YPGE-Gal) (49) lacking or containing cycloxheximide.

View larger version (21K): [in this window] [in a new window]   FIG. 7.   Pdr13p regulates Pdr1p function at a posttranslational step. Whole-cell protein extracts (100 µg) were electrophoresed by SDS-PAGE. Protein extracts were made from pdr1 pdr3 cells containing (+) or lacking () the indicated high-copy-number plasmids. After electrophoresis, the polypeptides were transferred to nitrocellulose and analyzed by blotting with anti-Pdr1p antiserum.

	DISCUSSION

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While the role of Hsp70 proteins in polypeptide metabolism has long been appreciated, recent work has indicated that Hsp70 proteins can act as regulatory factors that modulate the activity of transcription factors (20). One of the earliest examples of Hsp70 proteins influencing the activity of a transcription factor came from analysis of expression of the SSA4 gene in S. cerevisiae (8). S. cerevisiae strains lacking the Hsp70-encoding loci SSA1 and SSA2 overproduce the SSA4 transcript and protein through activation of the heat shock transcription factor (25). Detailed analysis of the maturation of the progesterone receptor has shown that Hsp70 proteins must act on this factor so that it can achieve its active conformation (9). Escherichia coli Hsp70 DnaK acts to negatively regulate its own transcription (reviewed in reference 13).

The data presented here argue that Pdr13p is a positive regulator of Pdr1p. Increases in the level of Pdr13p lead to an enhancement in the activity of Pdr1p. While the action of Pdr13p is clearly not restricted to its effect on Pdr1p (see below), Pdr13p does not affect the function of Pdr3p. Pdr3p shows 36% sequence identity to Pdr1p, binds to the same DNA element as Pdr1p, and shows extensive functional overlap with Pdr1p (15, 33). pdr1 cells containing a 2µm clone of PDR3 exhibit higher cycloheximide resistance than wild-type cells (Fig. 2). However, the function of even this presumably elevated level of Pdr3p cannot be regulated by Pdr13p, further supporting the notion that this Hsp70 protein controls Pdr1p but not Pdr3p activity.

This observation is important in light of what is known about the phenotype of cells lacking either the PDR1 or the PDR3 gene. Loss of PDR1 from the cell has a pronounced phenotypic effect on cycloheximide resistance, with a pdr1 cell exhibiting hypersensitivity to a broad range of compounds (2, 15, 33). In opposition to the effect of PDR1 removal, a pdr3 cell is not markedly different from a wild-type cell in terms of cycloheximide tolerance (15, 33). Surprisingly, expression studies with PDR5, the principal Pdr1p-Pdr3p target gene for cycloheximide resistance, indicate that both mRNA and PDR5-lacZ expression levels are similar in pdr1 and pdr3 strains (33). A key difference in these two experiments is that one is done in the presence of cycloheximide (phenotype testing), while one is performed in its absence (expression measurements). A hypothesis to explain these data is that drug exposure acts through Pdr13p to stimulate Pdr1p activity, and this activation of Pdr1p is required for normal drug resistance. The fact that Pdr1p but not Pdr3p can respond to Pdr13p would explain the observed phenotypes of strains individually lacking either of these transcription factors. This hypothesis is currently being evaluated.

While Pdr13p does not affect the function of Pdr3p, there are clearly targets of Pdr13p action other than Pdr1p. The reduction in growth rate exhibited by pdr13 cells indicates that Pdr1p cannot be the only downstream target of Pdr13p. Strains lacking Pdr1p, Pdr1p and Pdr3p, and downstream Pdr1p-Pdr3p target genes have not been found to exhibit growth phenotypes in the absence of drugs (reviewed in reference 4). In contrast, a pdr13 strain displays a growth phenotype that is partially suppressed at 37°C (Fig. 3). This finding suggests the possibility that some other Hsp70 protein expressed at 37°C is able to provide some degree of Pdr13p function. There is precedence for Hsp70 gene disruptions leading to cold-sensitive phenotypes, as mutant strains lacking both SSB1 and SSB2 exhibit a cold-sensitive phenotype that can be suppressed by elevating the growth temperature (14).

An intriguing possibility suggested by the appearance of a growth defect in pdr13 cells is that Pdr13p may act as a link between essential cellular functions and the PDR pathway. Perhaps Pdr13p coordinates the activity of the PDR genes with fundamental cell metabolism and activates Pdr1p function in response to an as-yet-unknown signal. It is important to bear in mind that drugs such as cycloheximide and oligomycin are unlikely to represent the natural substrates of the PDR genes, making the true function of these genes a still-unresolved question.

The identification of the S295F form of Pdr13p as a hyperactive regulator of Pdr1p function is interesting for three different reasons. First, this particular mutation provides a new class of Hsp70 mutants with altered function. Mutant Hsp70 derivatives with increased function have not been reported before. Isolation of this class of mutant Hsp70 proteins likely has been slowed by the complex, pleiotropic nature of the roles of these proteins. Our ability to mutationally separate the essential function(s) of Pdr13p from its role in the control of Pdr1p activity will allow genetics to be used to analyze the molecular details underlying this regulatory interaction. Second, the S295F mutation is located in the most highly conserved segment of Hsp70 proteins, the ATPase domain (18). This idea is consistent with the work of James et al. (30), who showed that at least part of the specificity of action of S. cerevisiae Hsp70 proteins was determined by this N-terminal ATPase domain. dnaK mutants with a partial loss of function also mapped to this domain (52). Finally, the change from serine to phenylalanine suggests the possibility that phosphorylation is involved. Alternatively, the large change in amino acid identity between serine and phenylalanine may lead to the observed phenotype. Discrimination between these models requires direct biochemical experiments aimed at addressing the regulation and activity of Pdr13p.