©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of SNQ2, a New Multidrug ATP Binding Cassette Transporter of the Yeast Plasma Membrane (*)

(Received for publication, April 17, 1995)

Anabelle Decottignies (1) Laurence Lambert (1) Patrice Catty (1) Herv Degand (1) Eric A. Epping (2) W. Scott Moye-Rowley (2) Elisabetta Balzi (1) Andr Goffeau (1)(§)

From the  (1)Unit de Biochimie Physiologique, Universit Catholique de Louvain, Place Croix du Sud 2/20, B-1348 Louvain-la-Neuve, Belgium and the (2)Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The SNQ2 gene of Saccharomyces cerevisiae, which encodes an ATP binding cassette protein responsible for resistance to the mutagen 4-nitroquinoline oxide, is regulated by the DNA-binding proteins PDR1 and PDR3. In a plasma membrane-enriched fraction from a pdr1 mutant, the SNQ2 protein is found in the 160-kDa overexpressed band, together with PDR5. The SNQ2 protein was solubilized with n-dodecyl -D-maltoside from the plasma membranes of a PDR5-deleted strain and separated from the PMA1 H-ATPase by sucrose gradient centrifugation. The enzyme shows a nucleoside triphosphatase activity that differs biochemically from that of PDR5 (Decottignies, A., Kolaczkowski, M., Balzi, E., and Goffeau, A.(1994) J. Biol. Chem. 269, 12797-12803) and is sensitive to vanadate, erythrosine B, and Triton X-100 but not to oligomycin, which inhibits the PDR5 activity only. Disruption of both PDR5 and SNQ2 in a pdr1 mutant decreases the cell growth rate and reveals the presence of at least two other ATP binding cassette proteins in the 160-kDa overexpressed band that have been identified by amino-terminal microsequencing.


INTRODUCTION

Several genes sharing homology with the MDR genes, which confer multidrug resistance in man and mouse (recently reviewed by Gottesman and Pastan(1993)), have been identified in Saccharomyces cerevisiae. The predicted topography of the products of these genes is typical of the ABC()superfamily, consisting of two hydrophobic domains of six transmembrane segments each and two cytoplasmic nucleotide-binding folds (reviewed by Balzi and Goffeau (1994)). Yeast ABC proteins include the a mating factor transporter STE6 (McGrath and Varshavsky, 1989; Kuchler et al., 1989), the multidrug transporters PDR5 (Balzi et al., 1994; Bissinger and Kuchler, 1994; Hirata et al., 1994) and SNQ2 (Servos et al., 1993), the yeast cadmium factor YCF1 (Szczypka et al., 1994), the oligomycin resistance factor YOR1,()and several newly discovered genes of totally unknown function like PDR10()and PDR11 (accession no. Z38113, NCBI gi:558389``) (reviewed by Balzi and Goffeau(1994)). When overexpressed, the PDR5 gene confers resistance to several unrelated drugs, such as the protein synthesis inhibitor cycloheximide and the acetolactate synthase inhibitor sulfomethuron methyl. Overexpression of SNQ2 confers resistance to the mutagen 4-NQO and to sulfomethuron methyl (reviewed by Balzi and Goffeau (1994)). With the exception of STE6, the physiological substrates of the yeast ABC transporters remain unknown.

The MDR gene product, called P-glycoprotein, has been shown to hydrolyze ATP in vitro and to mediate ATP-dependent vanadate-sensitive drug efflux when reconstituted in plasma membrane vesicles (Horio et al., 1988). PDR5 was the first yeast ABC transporter shown to hydrolyze ATP in vitro (Decottignies et al., 1994), but no biochemical information is presently available for SNQ2 or any other yeast multidrug resistance protein.

In S. cerevisiae, a complex genetic network is responsible for the regulation of the PDR phenotype. At least two transcription factors, called PDR1 (Balzi et al., 1987) and PDR3 (Delaveau et al., 1992, 1994), have been shown to interact with the PDR5 promoter (Katzmann et al., 1994; Delahodde et al., 1995). Quite remarkably, point mutations in PDR1 lead to pleiotropic drug resistance correlated with overexpression of PDR5 mRNA (Meyers et al., 1992). Impressive amounts of PDR5 protein were found in the plasma membrane-enriched fraction from the pdr1-3 mutant (Balzi et al., 1994; Decottignies et al., 1994). On the other hand, disruption of PDR1 and/or PDR3 leads to hypersensitivity to drugs and decreases the level of PDR5 mRNA (Delaveau et al., 1994; Katzmann et al., 1994). Mutations in the PDR7 and PDR9 genes also influence the level of PDR5 transcript (Dexter et al., 1994).

In this study, we show that the level of SNQ2 transcript is controlled by PDR1 and PDR3 and that disruption of SNQ2 affects cell growth. We have located the SNQ2 protein in the plasma membrane-enriched fraction together with PDR5. By selectively deleting or disrupting either the PDR5 or the SNQ2 gene in a pdr1-3 mutant, we were able to distinguish, at the plasma membrane level, the nucleotide triphosphatase activities of the two corresponding proteins independently overexpressed. In a double PDR5,SNQ2 disruptant, the two new ABC transporters PDR11 and YOR1 were detected in the overexpressed 160-kDa protein band of the pdr1-3 mutant. Our set of genetically purified strains was used to solubilize the SNQ2, PDR11, and YOR1 proteins and to separate them from the plasma membrane H-ATPase PMA1 by centrifugation on sucrose gradients.


EXPERIMENTAL PROCEDURES

Chemicals

n-Dodecyl -D-maltoside and Triton X-100 were purchased from Boehringer; ATP, UTP, GTP, CTP, ITP, bovine serum albumin, lysolecithin, o-nitrophenyl -D-galactopyranoside, cycloheximide, and 4-NQO were from Sigma; erythrosine B and oligomycin were from Janssen Chemica; molecular weight markers (range, 53,000-212,000 Da) were from Pharmacia Biotech Inc.; nylon membranes (Hybond N) were purchased from Amersham Corp. Yeast extract was either from KAT or from DIFCO. Asolectin was from Associated Concentrates (Woodside, Long Island, NY). All other reagents were of analytical grade.

Yeast Strains and Growth Media

The S. cerevisiae strains used in this study are listed in Table 1. To disrupt PDR5, a 4.7-kb PvuII fragment of plasmid pDR3.3 containing the PDR5 gene (Leppert et al., 1990) was cut by double digestion with SmaI/SalI to eliminate 1,243 bp of the promoter and 1,390 bp of the PDR5 open reading frame. The remainder was blunt ended by Klenow polymerase treatment, ligated overnight to the URA3 gene isolated by BamHI digestion of the YDp-U vector (Berben et al., 1991), and blunt ended by Klenow treatment. Escherichia coli strain C600 was transformed as in Hanahan(1983). The extracted plasmid, designated as ppdr5::URA3, was cleaved with BstEII, BglI, and EcoRI to generate the 2,852-bp fragment containing the first 368 bp of the PDR5 promoter, the complete 1,108-bp URA3 gene, and the 1,373-bp SalI-BstEII fragment of the PDR5 open reading frame. The haploid yeast strain US50-18C was then transformed as in Kuo and Campbell(1983). Transformants (US50-D5) appeared after 3 days at 30 °C on a selective uracil-free dropout medium. The chromosomal structure of the disrupted pdr5 allele was checked by Southern blotting analysis. The PDR5 null allele obtained was designated as pdr5-3::URA3 (two other PDR5 null alleles were previously constructed by Katzmann et al.(1994) and Hirata et al.(1994). The ura3 auxotrophic marker was recovered by selection on 5-fluoroorotic acid, and disruption of the SNQ2 gene was carried out as follows. The linearized snq2::Tn10-LUK fragment obtained by XbaI-SalI cleavage of pBS2218 (Servos et al., 1993), kindly provided by M. Brendel and J. Servos (Goethe Universitat, Frankfurt, Germany), was used to transform the yeast strains US50-18C and US50-D5. Disruption of the SNQ2 gene in the strains US50-D2 (SNQ2 disruption in US50-18C strain) and US50-D25 (SNQ2 disruption in US50-D5 strain) was checked by Southern blotting analysis. The strains, grown on standard medium as previously described (Ulawzewski et al., 1987), were tested for drug resistance on solid complex medium containing 1% yeast extract (Difco), 2% glucose, and either cycloheximide dissolved in ethanol or 4-NQO dissolved in acetone. A 32-well replicator was used for replica plating, and drug resistance was scored after 3-4 days at 30 °C as described by Balzi et al.(1987).



For plasma membrane preparations and growth curves, the cells were grown in 5.8% glucose and 2% yeast extract (KAT) adjusted to pH 4.5 with HCl.

Northern Blot Analysis

Total RNA was extracted by the ``hot phenol'' procedure of Schmitt et al.(1990). Standard techniques were used for agarose-formaldehyde electrophoresis (Sambrook et al., 1989) and for transfer to nylon membranes. A 4.7-kb PvuII fragment of plasmid pDR3.3, a 1.0-kb EcoRI fragment of plasmid pBS2218, and a 1.0-kb BamHI-HindIII fragment of plasmid pACT (kind gift of J. Verdi re, Gif-sur-Yvette, France) were used as probes, specific for PDR5, SNQ2, and actin, respectively, after labeling by nick translation with [-P]dCTP. Hybridizations were carried out for 15 h at 42 °C in 40% formamide, 5 Denhardt's, 0.5% SDS, 5 SSPE, and 20 µg/ml denatured salmon sperm DNA. The membranes were washed at 55 °C in 1 SSC, 0.2% SDS and autoradiographed (KODAK x-ray film). For rehybridization, the membranes were immersed once or twice in boiling 0.5% SDS and allowed to cool at room temperature.

Isolation of Plasma Membranes

Plasma membranes were isolated from the particulate fraction pelleted at 15,000 g for 40 min by selective precipitation at pH 5.2 as described by Goffeau and Dufour(1988).

Protein Microsequencing

The broad 160-kDa band obtained from the plasma membranes of the PDR5-deleted strain US50-D5:pdr1-3,pdr5-3::URA3 was excised from an SDS-PAGE gel and electroeluted overnight in 25 mM Tris, 192 mM glycine, 0.2% (w/v) SDS. Electroeluted proteins were precipitated with 10% (w/v) trichloroacetic acid and centrifuged for 15 min at 15,000 g at 4 °C. SDS was removed by acetone precipitation as described by le Maire et al.(1993). After lyophilization, the sample was dissolved in 0.1 M NHHCO, 10% (v/v) acetonitrile, pH 8.0, and incubated overnight at 37 °C in the presence of 1 µg of trypsin. The digestion product was lyophilized, dissolved in 100 µl of 0.1% (w/v) trifluoroacetic acid, and fractionated by reverse phase high pressure liquid chromatography (C; buffer A, 0.06% (w/v) trifluoroacetic acid; buffer B, 0.052% (w/v) trifluoroacetic acid, 80% acetonitrile; gradient: 0-10 min, 5% B, flow rate 0.3 ml/min; 10-10.01 min, 5% B, flow rate 0.3 ml/min; 10.01-90 min, 75% B, flow rate 0.15 ml/min). The peaks, collected manually, were monitored for their OD at 210 mm. A peptide selected from the fractionation was sequenced with an Applied Biosystems sequenator (model 477A) equipped with an on-line amino acid derivative analyzer (model 120A).

The amino-terminal sequences of YOR1 and PDR11 were determined as follows. After pre-electrophoresis for 5 h at 50 mA/gel in separating gel buffer containing 0.1 mM thioglycolate, 150 µg of plasma membrane proteins from the US50-D25 strain with deletions in both the PDR5 and SNQ2 genes were separated on an SDS-7% polyacrylamide gel as previously described (Laemmli, 1970), except that polyacrylamide was replaced with 1% agarose in the stacking gel as described by Moos et al. (1988). After electrophoresis at 50 mA, the proteins were blotted onto a polyvinylidene difluoride membrane (Problott, Applied Biosystems) overnight at 40 V. The 160-kDa Serva blue-stained band was excised with a razor blade and sequenced.

Solubilization of PDR5 and SNQ2

US50-D5, US50-D2, and US50-D25 plasma membrane proteins (5 mg/ml) were solubilized with 0.2% (w/v) n-dodecyl -D-maltoside in the presence of 8 mM Tris-HCl, pH 7.5, as described by Decottignies et al.(1994).

Centrifugation on a Continuous Sucrose Gradient

The solubilized proteins (7 mg) were separated on a 33.3-ml linear sucrose gradient containing 6-30% (w/v) sucrose, 10 mM Tris, 1 mM EDTA, 1 mM ATP, pH 7.5 (AcOH). After centrifugation for 16 h at 76,000 g in a Beckman type SW28 rotor, the sucrose gradient was divided into 30 1-ml fractions.

Nucleoside Triphosphatase Assays

Nucleotide hydrolysis was measured by incubation of the sample at 30 °C in a final volume of 100 µl containing either 6 mM NTP, 7.6 mM MgSO, 10 mM NaN (to inhibit mitochondrial ATPase), and 59 mM MES-NaOH (from pH 5.0 to 7.0) or 6 mM NTP, 7 mM MgSO, 10 mM NaN, and 59 mM Tris-HCl (from pH 7.3 to 9.0). Assays were carried out as described by Decottignies et al.(1994).

Other Methods

The protein content was measured as described by Lowry et al.(1951), with bovine serum albumin as the standard. Protein samples were electrophoresed on SDS-7% polyacrylamide gels according to Laemmli(1970) and stained with Coomassie Blue. -Galactosidase activity was measured as described by Sambrook et al.(1989) after transformation of the yeast with a plasmid bearing an SNQ2 promoter-lacZ gene fusion constructed as follows. 726 bp upstream of the SNQ2 ATG were amplified by polymerase chain reaction, and an in-frame BamHI site was placed immediately downstream of this ATG. Primer sequences were as follows: 5`-CGGGATATCCACGGCAAGGAAGTGGCGCG and 3`-GCGGGATCCATTGAATTCTCTTTACGTA. After sequencing, the SNQ2 promoter fragment was inserted as an EcoRV-BamHI fragment into SmaI-BamHI-cleaved lacZ fusion vector pSEYC102. The yeast cells were transformed as in Kuo and Campbell (1983). When necessary, the ura3 auxotrophic marker was obtained by selection on 5-fluoroorotic acid. Each value given is the mean ± S.D. of at least 10 trials.


RESULTS

Transcriptional Control of the PDR5 and SNQ2 Genes by the PDR1 and PDR3 Gene Products

The transcriptional control of the PDR5 gene by the PDR1 gene product has been previously demonstrated (Meyers et al., 1992; Balzi et al., 1994; Dexter et al., 1994; Katzmann et al., 1994; Delahodde et al., 1995). The PDR3 gene encodes a transcription factor highly homologous but not identical to PDR1 and also controls the expression of the PDR5 gene by binding to the same nucleotide sequence as PDR1 (Delaveau et al., 1994; Katzmann et al., 1994). SNQ2 is highly homologous to PDR5; the two protein sequences display 40% identity over their entire length. Several PDR1/PDR3 consensus binding sites are present in the SNQ2 promoter. To test whether transcription of SNQ2 is affected by PDR1 and/or PDR3, Northern blot hybridizations were carried out on total RNA from strains bearing either mutant PDR1 alleles or null PDR1 and/or PDR3 alleles and from the corresponding parental strains. The PDR5 transcript level was also analyzed with a specific probe (Fig. 1).


Figure 1: Northern blot analysis of PDR5 and SNQ2 transcripts in different strains. 30 µg of total RNA was run in each well of a 1% agarose-formaldehyde gel, transferred to a Hybond-N membrane, and separately hybridized with probes specific for PDR5 (4.7-kb PvuII fragment from plasmid pDR3.3), SNQ2 (1.0-kb EcoRI fragment from plasmid pBS22-LB18), and actin (1.0-kb BamHI-HindIII fragment from pACT plasmid). After autoradiography, the membrane was stripped of one probe and rehybridized with another probe. The actin gene was used as a control for the mRNA levels. Lane2, D1 (pdr1-1::URA3) strain obtained by deletion of PDR1 in the wild-type strain 2229-5C (lane1); lane4, D1-3/3 (pdr1-1::URA3) strain obtained by deletion of pdr1 in the pdr1-3 strain US50-18C (lane3); lane6, D1-2 (pdr1-1::URA3) strain obtained by deletion of pdr1 in the pdr1-2 strain US54-17B (lane5); lane7, wild-type strain FY1679-28C; lane8, strain FY1679-28C/EC (pdr1-2::TRP1); lane9, strain FY1679-28C/TD (pdr3-1::HIS3); and lane10, strain FY1679-28C/TDEC (pdr1-2::TRP1, pdr3-1::HIS3).



Transcription of both PDR5 and SNQ2 is greatly enhanced in the pdr1-3 (Fig. 1, lane3) and pdr1-2 (Fig. 1, lane5) mutant strains, as in the pdr1-6 and pdr1-7 mutants (data not shown). When the gene bearing the point mutation is replaced with a partially deleted PDR1 allele (pdr1-1), both PDR5 and SNQ2 transcript levels decrease drastically (Fig. 1, lane4versuslane3 and lane6versuslane5). As compared to a parental wild type, total deletion of both PDR1 (pdr1-2) and PDR3 (pdr3-1) reduces the levels of both the PDR5 and SNQ2 transcripts, the effect being more marked for PDR5 than for SNQ2 (Fig. 1, lanes10versus7). When only PDR1 or PDR3 is deleted, the PDR5 and SNQ2 transcript levels are but slightly affected (lane2versus1 and lanes7-9).

The regulation of SNQ2 transcription by PDR1 and PDR3 was confirmed by measuring the -galactosidase activity expressed from an SNQ2 promoter-lacZ fusion (Table 2). The results clearly show the induction of lacZ expression in the pdr1-3 mutant compared to its parental strains as well as the decreased -galactosidase activity when the mutated allele (pdr1-3 or pdr1-2) is deleted. As compared to a parental wild type, a deletion mutant lacking either PDR1 or PDR3 expresses only slightly less -galactosidase from the SNQ2 promoter-lacZ fusion gene, while deletion of both genes reduces the -galactosidase activity by 50%.



Taken together, these results clearly show not only that the pdr1 mutations increase transcription of SNQ2 (and PDR5) but also that the wild-type PDR1 and PDR3 proteins jointly control transcription level of both SNQ2 and PDR5.

Analysis of PDR5 and SNQ2 in the Plasma Membrane-enriched Fraction

We used SDS-PAGE to analyze the proteins of the plasma-membrane-enriched fractions isolated from a set of strains, all derived from the drug-resistant pdr1-3 mutant and isogenic but for the deletions/disruptions mentioned (Fig. 2, lanes4-8). The results are also given for the drug-sensitive parental strains (Fig. 2, lanes2 and 3). Several components are present in the overexpressed 160-kDa band from the drug-resistant pdr1-3 mutant US50-18C (Fig. 2, lane4). This band almost completely disappears after deletion of pdr1 in the drug-hypersensitive strain D1-3/3 (Balzi et al., 1987; Decottignies et al., 1994). Densitometric quantitation of the pdr1-3 plasma membrane proteins separated by SDS-PAGE revealed that PDR5 and SNQ2 amount, respectively, to 20 and 10% of the total membrane protein, while PMA1 H-ATPase totals 23%. As previously reported (Balzi et al., 1994; Decottignies et al., 1994), PDR5 is the major component of the overexpressed 160-kDa band. The intensity of this band is reduced by 66% after deletion of the PDR5 gene in the US50-D5 strain (Fig. 2, lane6). We detected no SNQ2 amino-terminal sequence in the remaining band, but microsequencing of a tryptic product revealed the(1243)AIASR(1247) sequence belonging to SNQ2. Analysis of the 160-kDa band from the SNQ2 disruptant showed that PDR5 occupies the lower part of the band (Fig. 2, lane7) and that SNQ2 amounts to 27% of the total protein content of the band from the parental strain US50-18C. When both PDR5 and SNQ2 were disrupted in strain US50-D25, two additional proteins were detected in the band (lane8). These were shown by microsequencing to contain the amino-terminal sequences SLSKYFNPIP and SITVGDAVXE, respectively, matching those deduced from the PDR11 and YOR1 gene sequences without the initiating methionine. A comparison of the pdr1-3 mutant (lane8) and the null mutant (lane5) suggests that levels of both proteins are controlled by PDR1.


Figure 2: Protein levels of PDR5, SNQ2, YOR1, and PDR11 in the plasma membrane. Coomassie Blue-stained SDS-PAGE of plasma membrane-enriched fractions is shown. Lanes1 and 10, molecular mass markers; lane2, IL125-2B (PDR1, PDR5, SNQ2); lane3, 2229-5C (PDR1, PDR5, SNQ2); lane 4, US50-18C (pdr1-3, PDR5, SNQ2); lane 5, D1-3/3 (pdr1-1::URA3, PDR5, SNQ2); lane 6, US50-D5 (pdr1-3, pdr5-3::URA3, SNQ2); lane 7, US50-D2 (pdr1-3, PDR5, snq2::Tn10-LUK); lane 8, US50-D25 (pdr1-3, pdr5-3::URA3, snq2::Tn10-LUK). Each lane contained 150 µg of protein. PDR5, SNQ2, YOR1, and PDR11 were found in the 160-kDa overexpressed band.



Disruption of SNQ2 Alters Cell Growth

The growth curves obtained with four of the above-mentioned strains in rich medium (Fig. 3) show that a strain lacking the PDR5 gene reaches a higher cell density than a strain overexpressing PDR5 due to a pdr1-3 mutation (the two strains being isogenic apart from the features just mentioned). The PDR5 protein would thus appear to become toxic when overexpressed, since wild-type PDR1 strains grow better than the pdr1-3 mutant (data not shown). SNQ2 disruption, on the other hand, enhances the lag phase without changing the exponential growth rate. This suggests that the transition from the stationary phase to exponential growth may require the SNQ2 transporter. Double PDR5-SNQ2 disruption alters both the lag and the log phase. Since simultaneous loss of both genes results in slower growth and although neither PDR5 nor SNQ2 is an essential gene, the products of these genes do seem to perform, albeit redundantly, a function or functions that affect growth.


Figure 3: Effect of PDR5 and/or SNQ2 disruption on growth in rich medium. The culture medium was inoculated at 0.5 10 cells/ml from stationary phase precultures of US50-18C (pdr1-3), US50-D5 (pdr1-3, pdr5-3), US50-D2 (pdr1-3, snq2::Tn), and US50-D25 (pdr1-3, pdr5-3, snq2::Tn).



PDR5 and SNQ2 Confer Drug Resistance in Vivo

PDR5 and SNQ2 are both involved in multidrug resistance (Leppert et al., 1990; Meyers et al., 1992; Servos et al., 1993; Balzi et al., 1994; Hirata et al., 1994; Bissinger and Kuchler, 1994), but the resistance spectra associated with these genes are different. Resistance to cycloheximide is PDR5-specific; resistance to 4-NQO is SNQ2-specific; resistance to staurosporine and fluphenazine is attributable to both genes (Hirata et al., 1994). We therefore compared the sensitivities to cycloheximide and 4-NQO of mutant and parental strains (Fig. 4). The SNQ2 disruptants (Fig. 4C, 6 and 7) could not grow on plates containing 4-NQO at 0.5 µg/ml, a concentration which failed to inhibit growth of the PDR1 wild-type strain, the pdr1 mutant, or the deletion mutants lacking PDR1, PDR3, or both (Fig. 4C, 1-5 and 8-23). In contrast, deletion of PDR5 did not affect growth on 4-NQO (Fig. 4C, 5). In the presence of 1.0 µg of 4-NQO/ml, the pdr1 mutant strains could still grow while the corresponding (and otherwise isogenic) strains carrying the wild-type allele or lacking the PDR1 gene grew more slowly or not at all (Fig. 4D, 3/4, 9/8, 10/11, 13/12, 15/14, 18/19). Deletion of PDR1 or PDR3 in a wild-type strain enhanced the sensitivity of the strain to 4-NQO (Fig. 4D, 20-22). Deletion of both PDR1 and PDR3 was even more drastic in this respect (Fig. 4D, 23). These results show that SNQ2-mediated resistance to 4-NQO is under the control of PDR1 and PDR3, thus confirming the results obtained by Northern blot analysis. When PDR5 was disrupted, the disruptant cells failed to grow on plates containing 0.1 µg/ml cycloheximide (Fig. 4E, 5 and 7) while SNQ2 disruption had no effect (Fig. 4E, 5 and 7). The pdr1 deleted strains were more sensitive to cycloheximide than the otherwise isogenic pdr1 mutant strains (Fig. 4E, 4/3, 11/10). Disruption of both PDR1 and PDR3 in a wild-type strain caused cell death on 0.1 µg of cycloheximide/ml (Fig. 4E, 23). These results fully confirm previous data showing that PDR1 and PDR3 jointly control PDR5-mediated resistance to cycloheximide. They also show that SNQ2 has no influence on cycloheximide resistance but mediates resistance to 4-NQO through transcriptional regulation involving both PDR1 and PDR3.


Figure 4: In vivo resistance to cycloheximide and 4-nitroquinoline oxide. Cells were spotted onto YPD agar plates containing either 4-NQO (0.5 and 1.0 µg/ml) or cycloheximide (0.1 µg/ml) and incubated for 3 days at 30 °C. A: 1, IL125-2B (wild type); 2, 2229-5C (wild type); 3, US50-18C (pdr1-3); 4, D1-3/3 (pdr1-1); 5, US50-D5 (pdr1-3, pdr5-3); 6, US50-D2 (pdr1-3, snq2::Tn); 7, US50-D25 (pdr1-3, pdr5-3, snq2::Tn); 8, FM11 (wild type); 9, 2-20 (pdr1-2); 10, US54-17B (pdr1-2); 11, D1-2 (pdr1-1); 12, IL125-2B (wild type); 13, DRI9-T8 (pdr1-3); 14, D286-2A (wild type); 15, BOR2-XI (pdr1-6); 16, JG200 (wild type); 17, JG204 (pdr1-7); 18, 2229-5C (wild type); 19, D1 (pdr1-1); 20, FY1679-28C (wild type); 21, FY1679-28C/EC (pdr1-2); 22, FY1679-28C/TD (pdr3-1); 23, FY1679-28C/TDEC (pdr1-2, pdr3-1). Strains with isogenic backgrounds are grouped.



SNQ2 Hydrolyzes Nucleoside Triphosphate in Vitro

To investigate the ATPase activity of SNQ2, we measured this activity at different pH values in plasma membrane preparations from four strains (isogenic but for the features mentioned): the parental pdr1-3 mutant (strain US50-18C), the PDR5-deleted pdr1-3 strain US50-D5 (pdr5-3::URA3), the SNQ2-disruptant pdr1-3 strain US50-D2 (snq2::Tn10-LUK), and the double PDR5-SNQ2 knockout pdr1-3 strain US50-D25 (pdr5-3::URA3, snq2::Tn10-LUK). The ATPase activities of PDR5 and SNQ2 were estimated by subtracting the ATPase activity of the doubly deleted strain US50-D25 from the activities measured for the single disruptants US50-D5 or US50-D2 or by calculating the loss of activity observed in strains US50-D5 and US50-D2 as compared to their parental strain US50-18C. This enabled us to eliminate the contribution of the plasma membrane H-ATPase PMA1 and possibly of other ATPases (Fig. 5). The SNQ2 ATPase activity plotted against the pH for strain US50-D5 or US50-18C shows a pH optimum at 6.0-6.5; the activity profile is shifted toward slightly more alkaline pH values than that of the PMA1 H-ATPase (Borst-Pauwels and Peters, 1977; Dufour and Goffeau, 1980). The peak is rather sharp, contrasting with the much broader profile obtained for PDR5, in strain US50-D2 or US50-18C. The ATP-hydrolyzing ability of PDR5 indeed remains almost constant from pH 6.0 to 9.0, as already shown by Decottignies et al.(1994). Specific ATPase activities in the plasma membrane-enriched fraction amount to 2.5 µmol Pminmg for PDR5 at pH 6.3 and 1.7 µmol Pminmg for SNQ2 at pH 6.0 (Fig. 5).


Figure 5: Effect of pH on plasma membrane-bound PDR5- and SNQ2-related ATPase activities. ATPase activity was measured at different pH values, in the presence of 6 mM Mg-ATP and 59 mM MES (pH values from 5.2 to 7.0) or Tris (pH values from 7.3 to 9.0), in plasma membrane-enriched fractions from strains US50-18C, US50-D5, US50-D2, and US50-D25. The pH was adjusted with NaOH or HCl. PDR5- and SNQ2-related activities were calculated as described in the text for three independent sets of four strains derived from the same wild type. Data are expressed as means with the S.D. (, PDR5; , SNQ2).



Further NTPase activity measurements were carried out at pH 6.3 for both SNQ2 and PDR5. Fig. 6shows that both PDR5 and SNQ2 hydrolyze magnesium nucleotides at pH 6.3 in the plasma membrane but that the specificity of PDR5 is broader. SNQ2 exhibits a marked ``preference'' for ATP.


Figure 6: Nucleoside phosphatase activities of plasma membrane-bound PDR5 and SNQ2. Plasma membrane-enriched fractions from strains US50-18C, US50-D5, US50-D2, and US50-D25 were assayed at 35 °C in buffers containing 6 mM Mg-NTP and 59 mM MES-NaOH, pH 6.3. PDR5 and SNQ2 NTPase activities were estimated as described in Fig. 5. The data are expressed as means with the S.D.



Taken together, these results show that despite their high sequence homology, the PDR5 and SNQ2 enzymes have different activity versus pH profiles and different substrate specificities.

Inhibition of PDR5 and SNQ2 UTPase Activity

We have previously shown that oligomycin and erythrosine B inhibit only partially the UTPase activity exhibited by pdr1-3 plasma membranes. In contrast, inhibition by vanadate or Triton X-100 is almost total (Decottignies et al., 1994). We show here that oligomycin strongly (I = 0.07 µg/ml) and almost totally inhibits the PDR5 UTPase activity at pH 6.3, as reported for P-glycoprotein (Sarkadi et al., 1992; Al-Shawi and Senior, 1993); it has very little effect, on the other hand, on SNQ2 UTPase activity (Fig. 7A). As for erythrosine B, it reduces the PDR5 UTPase activity by only 20% (Fig. 7B), whereas erythrosine B causes 65% inhibition of SNQ2 UTPase activity at 100 µM and 50% inhibition at 35 µM. Vanadate strongly inhibits, to approximately the same extent, both PDR5- and SNQ2-related UTPase activity, with I values around 3-5 µM. Triton X-100 at 1 mM concentration totally inhibits both the SNQ2 and the PDR5 activities, as shown in Fig. 7D. Both enzymes are half-inhibited at 40 µM Triton X-100. This result shows that the H-ATPase activity does not interfere with the UTPase measurements, since at these concentrations Triton X-100 considerably enhances the H-ATPase activity instead of inhibiting it (Goffeau and Dufour, 1988).


Figure 7: Effect of inhibitors on plasma membrane-bound UTPase activities. The effects of oligomycin (A), erythrosine B (B), vanadate (C), and Triton X-100 (D) on UTPase activity at pH 6.3 were tested on samples of the plasma membrane-enriched fractions from strains US50-D5, US50-D2, and US50-D25. The UTPase activities from the strain US50-D25 measured as described in Fig. 6were subtracted from those measured in strains US50-D5 and US50-D2 to yield estimates of SNQ2 and PDR5 inhibition, respectively.



Solubilization and Partial Purification of PDR5 and SNQ2

At a detergent-to-protein ratio of 0.4 (w/v), n-dodecyl -D-maltoside solubilized the components of the overexpressed 160-kDa protein band obtained from plasma membranes of the pdr1-3 mutant. Solubilization by this detergent led to the loss of 62% of the PDR5 UTPase activity measured at pH 6.3 and 54% of the SNQ2 UTPase activity. The remaining activity was about 20% stimulated by addition of 0.25 mg/ml asolectin to the assay medium.

After solubilization with n-dodecyl -D-maltoside, the plasma membrane proteins from the pdr1-3 strains US50-D5, US50-D2, and US50-D25 were separated on continuous 6-30% sucrose gradients. Fractions 20-23 of each sucrose gradient were found to be enriched in the overexpressed proteins of the 160-kDa band and devoid of the 100-kDa major subunit of PMA1, detected in fractions where the sucrose density exceeded that of fraction 19. Fig. 8shows the protein content of fraction 22 from each gradient. In the US50-D5 strain gradient fractions, at least two protein bands can be distinguished in the 150-160 kDa range (Fig. 8, lane 2). One of them is SNQ2, since it disappears in the corresponding fractions of the US50-D25 strain (Fig. 8, lane 3). Of the fractions ``enriched'' in solubilized SNQ2, fraction 21 showed the highest ATPase activity: 0.4 µmol of Pminmg. Much higher ATPase activity was found in the PDR5-enriched fractions 20-24: 1.7 µmol of Pminmg. ATPase activities of about 4 µmol of Pminmg were found at pH 6.3 in the PMA1-enriched fractions from all strains.


Figure 8: Partial purification of PDR5 and SNQ2. Solubilized plasma membrane proteins (7 mg) from strains US50-D2 (lane1), US50-D5 (lane 2), and US50-D25 (lane 3) were separated by centrifugation through a continuous sucrose gradient as described under ``Experimental Procedures.'' 22 fractions (50 µl), corresponding to a sucrose concentration of 12% (w/v), were analyzed by Coomassie Blue staining of SDS-polyacrylamide gels.




DISCUSSION

SNQ2 has been isolated as a gene conferring hyper-resistance to the mutagens 4-nitroquinoline-N-oxide and triaziquone when introduced on a multi-copy plasmid (Haase et al., 1992). It also confers resistance to sulfomethuron methyl and phenanthroline (Servos et al., 1993) as well as to staurosporine and fluphenazine (Hirata et al., 1994). Apart from the observations that it encodes a putative ABC protein (Servos et al., 1993) whose amino acid sequence is 40% identical to that of PDR5 (Balzi et al., 1994; Hirata et al., 1994) and that its transcription is enhanced by heat shock or treatment with a drug such as cycloheximide, 4-nitroquinoline-N-oxide or fluphenazine (Hirata et al., 1994), nothing is known about the SNQ2 gene nor has the SNQ2 protein been biochemically characterized.

Our study establishes three important new facts about SNQ2. First, transcription of the SNQ2 gene is controlled by PDR1 and PDR3 as shown by Northern blot analysis and by expression of an SNQ2 promoter-lacZ fusion gene in a wild-type strain compared to otherwise isogenic pdr1 or pdr3 point or null mutants. This tallies with our observation that mutations in PDR1 enable the mutated strain to grow in the presence of higher concentrations of 4-NQO, while deletion of PDR1 and/or PDR3 renders the strain more sensitive.

Second, disruption of the SNQ2 gene in a PDR5/SNQ2-overexpressing pdr1-3 strain markedly lengthens the adaptation phase, during which stationary cells undergo metabolic and physiological changes before starting exponential growth. Yet, the exponential growth rate itself is not affected. In contrast, deletion of PDR5 slightly enhances cell growth as compared to strains overexpressing PDR5. This is in agreement with the suggestion made by Romanos et al.(1992) that overexpressed membrane proteins generally disturb yeast functions. It may also explain why wild-type PDR1 strains grow slightly better than the overexpressing pdr1-3 mutant (data not shown). Disruption of both PDR5 and SNQ2 genes reduces the exponential cell growth rate, showing that the presence of either PDR5 or SNQ2 is important for normal growth. As suggested by Hirata et al. (1994), these transporters may extrude intracellular cytotoxic metabolites accumulated during growth.

Third, the SNQ2 gene product exhibits nucleoside triphosphatase activity. This activity, like that of PDR5, is located in the plasma membrane. The study of the SNQ2 activity was made possible by the development of three key procedures: 1) the use of mutants affected in the transcription factor PDR1, which overexpress SNQ2; 2) the use of mutants where PDR5 and/or SNQ2 is/are disrupted, making it possible to compare enzyme activities in plasma membranes lacking either PDR5, SNQ2, or both; and 3) the use of magnesium nucleotides other than Mg-ATP to measure activities not supported by the H-ATPase PMA1, previously reported to be very specific for Mg-ATP (Borst-Pauwels and Peters, 1977).

We were also able to physically separate SNQ2 or PDR5 from PMA1 by solubilizing the plasma membranes with n-dodecyl -D-maltoside and centrifuging overnight on sucrose gradients. This procedure, however, reduced the SNQ2 ATPase activity from 1.7 µmol of Pminmg in the plasma membrane to 0.4 and the PDR5 ATPase activity from 2.4 to 1.7 µmol of Pminmg. The purification enabled us, nevertheless, to show that SNQ2 is much more ATP-specific than PDR5; moreover, erythrosine B specifically inhibits the SNQ2-related nucleoside triphosphatase activity and oligomycin the PDR5-related activity. Vanadate and Triton X-100 inhibit both enzymes. Also, the SNQ2 ATPase activity profile peaks sharply at pH 6.0, while PDR5 can hydrolyze ATP from pH 6.0 to 9.0 with approximately constant efficiency.

If, as predicted, PDR5 and SNQ2 are ``drug efflux pumps,'' one might expect a certain substrate specificity for each pump. Hirata et al.(1994) have indeed shown that multiple copies of the SNQ2 gene confer resistance to 4-nitroquinoline-N-oxide, for instance, but not to cycloheximide, while the opposite is true for the PDR5 gene under the same conditions. Direct proof of this prediction and the study of the mechanism involved require the development of new procedures for reconstituting transport activities either in sealed plasma membrane vesicles or in proteoliposomes containing the purified proteins. It is noteworthy, in this respect, that Pawagi et al.(1994) suggest that the transmembrane distribution of aromatic amino acids in P-glycoprotein may play a functional role in drug specificity. Amino acid sequence analysis of PDR5, SNQ2, and CDR1 (the homolog of PDR5 in Candida albicans, which also confers resistance to cycloheximide (Prasad et al., 1995)) reveals that aromatic residues in transmembrane segments are more highly conserved between PDR5 and CDR1 (75%) than between PDR5 and SNQ2 (50%). Whether this difference in aromatic residue distribution is related to the difference in substrate specificity is a matter of speculation. The putative ATP binding sites of PDR5 and SNQ2 are also interesting; although the GK(S/T) motif is found in the Walker A motifs of all ABC transporters (reviewed by Thomas and Pedersen(1993)) and although the middle lysine is said to be strictly required for ATPase activity (Sung et al., 1988; Reinstein et al., 1990; Tian et al., 1990), this lysine is replaced by a cysteine in the amino-terminal Walker A motif of both PDR5 and SNQ2, both of which nevertheless actively hydrolyze ATP.

After disruption of both PDR5 and SNQ2 in the pdr1-3 mutant, at least two additional proteins, identified as PDR11 and YOR1, were found in the 160-kDa overexpressed band. Both are ABC transporters; PDR11 is homologous to SNQ2()and yor1 mutants are sensitive to oligomycin. These proteins seem to be in relatively low amount in the plasma membrane, as judged by the intensity of Coomassie Blue staining and by the low residual nucleoside triphosphatase activity in plasma membranes isolated from the double deletion mutant lacking both PDR5 and SNQ2. Nevertheless, a further reconstitution study should use preparations genetically purified of PDR11 and YOR1.


FOOTNOTES

*
This work was supported in part by grants from the Service de la Politique Scientifique, Action Sciences de la vie, and by the Fonds National de la Recherche Scientifique (Belgium), by National Institutes of Health Grant GM49825, and by NATO Collaborative Research Grant CRG940493. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests and correspondence should be addressed. Tel.: 32-10-47-36-14; Fax: 32-10-47-38-72.

The abbreviations used are: ABC, ATP binding cassette; 4-NQO, 4-nitroquinoline oxide; PDR, pleiotropic drug resistance; YOR, yeast oligomycin resistance; PMA, plasma membrane ATPase; PAGE, polyacrylamide gel electrophoresis; MES, 2-(N-morpholino)ethanesulfonic acid; kb, kilobase(s); bp, base pair(s).

G. Volckaert and W. S. Moye-Rowley, personal communication.

K. Wolfe, personal communication.

B. Barrell and M. A. Rajandream, personal communication.


ACKNOWLEDGEMENTS

We gratefully thank M. Brendel and J. Servos for the gift of the snq2::Tn10-LUK plasmid and G. R. Fink for the gift of the FM11 strain. We also thank J. Nader for helpful technical assistance.


REFERENCES

  1. Al-Shawi, M. K., and Senior, A. E.(1993)J. Biol. Chem. 268, 4197-4206 [Abstract/Free Full Text]
  2. Balzi, E., and Goffeau, A.(1994)Biochim. Biophys. Acta 1187, 151-162
  3. Balzi, E., Chen, W., Ulawzewski, S., Capieaux, E., and Goffeau, A.(1987) J. Biol. Chem. 262, 16871-16879 [Abstract/Free Full Text]
  4. Balzi, E., Wang, M., Leterme, S., Van Dyck, L., and Goffeau, A.(1994)J. Biol. Chem. 269, 2206-2214 [Abstract/Free Full Text]
  5. Berben, G., Dumont, J., Gilliquet, V., Bolle, P.-A., and Hilger, F.(1991) Yeast 7, 475-477 [Medline] [Order article via Infotrieve]
  6. Bissinger, P. H., and Kuchler, K.(1994)J. Biol. Chem. 269, 4180-4186 [Abstract/Free Full Text]
  7. Borst-Pauwels, G. W. F. H., and Peters, P. H. J.(1977)Biochim. Biophys. Acta 466, 488-495 [Medline] [Order article via Infotrieve]
  8. Decottignies, A., Kolaczkowski, M., Balzi, E., and Goffeau, A.(1994)J. Biol. Chem. 269, 12797-12803 [Abstract/Free Full Text]
  9. Delahodde, A., Delaveau, Th., and Jacq, C. (1995) Mol. Cell. Biol., in press
  10. Delaveau, Th., Jacq, C., and Perea, J.(1992)Yeast 8, 761-768 [Medline] [Order article via Infotrieve]
  11. Delaveau, Th., Delahodde, A., Carvajal, E., Subik, J., and Jacq, C.(1994) Mol. & Gen. Genet. 244, 501-511
  12. Dexter, D., Moye-Rowley, W. S, Wu, A., and Golin, J.(1994)Genetics 136, 505-515 [Abstract/Free Full Text]
  13. Dufour, J.-P., and Goffeau, A.(1980)Eur. J. Biochem. 105, 145-154 [Medline] [Order article via Infotrieve]
  14. Goffeau, A., and Dufour, J.-P.(1988)Methods Enzymol. 157, 528-533 [Medline] [Order article via Infotrieve]
  15. Gottesman, M. M., and Pastan, I.(1993)Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  16. Gurineau, P., Slomninski, P. P., and Avner, P. R.(1974) Biochem. Biophys. Res. Commun. 61, 462-469 [Medline] [Order article via Infotrieve]
  17. Haase, E., Servos, J., and Brendel, M.(1992)Curr. Genet. 21, 319-324 [Medline] [Order article via Infotrieve]
  18. Hanahan, D.(1983) J. Mol. Biol.166,557-580 [Medline] [Order article via Infotrieve]
  19. Hirata, D., Yano, K., Miyahara, K., and Miyakawa, T.(1994)Curr. Genet. 26, 285-294 [Medline] [Order article via Infotrieve]
  20. Horio, M., Gottesman, M. M., and Pastan, I.(1988)Proc. Natl. Acad. Sci. U. S. A. 85, 3580-3584 [Abstract]
  21. Katzmann, D. J., Burnett, P. E., Golin, J., Mah, Y., and Moye-Rowley, W. S.(1994)Mol. Cell. Biol. 14, 4653-4661 [Abstract]
  22. Kuchler, K., Sterne, R. E., and Thorner, J.(1989)EMBO J. 8, 3973-3984 [Abstract]
  23. Kuo, C.-L., and Campbell, J. L.(1983)Mol. Cell. Biol. 3, 1730-1737 [Medline] [Order article via Infotrieve]
  24. Laemmli, U. K. (1970)Nature227,680-685 [Medline] [Order article via Infotrieve]
  25. le Maire, M., Deschamps, S, Moller, J. V., Le Caer, J.-P., and Rossier, J.(1993) Anal. Biochem. 214, 50-57 [CrossRef][Medline] [Order article via Infotrieve]
  26. Leppert, G., McDevitt, R., Falco, S. C., Van Dyk, T., Ficke, M., and Golin, J.(1990) Genetics 125, 13-20 [Abstract/Free Full Text]
  27. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.(1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  28. McGrath, J. P., and Varshavsky, A.(1989)Nature 340, 400-404 [CrossRef][Medline] [Order article via Infotrieve]
  29. Meyers, S., Schauer, W., Balzi, E., Wagner, M., Goffeau, A., and Golin, J.(1992) Curr. Genet. 21, 431-436 [Medline] [Order article via Infotrieve]
  30. Miller, J. H. (ed) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  31. Moos, M., Jr., Nguyen, N. Y., and Liu, T.-Y.(1988)J. Biol. Chem. 263, 6005-6008 [Abstract/Free Full Text]
  32. Nass, G., and Poralla, K.(1976)Mol. & Gen. Genet. 147, 39-43
  33. Pawagi, A. B., Wang, J., Silverman, M., Reithmeier, R. A. F., and Deber, C. M.(1994) J. Mol. Biol. 235, 554-564 [CrossRef][Medline] [Order article via Infotrieve]
  34. Prasad, R., De Wergifosse, P., Goffeau, A., and Balzi, E.(1995) Curr. Genet. 27, 320-329 [Medline] [Order article via Infotrieve]
  35. Rank, G. H., Gerlach, J. H., and Robertson, A. J.(1976)Mol. & Gen. Genet. 144, 281-288
  36. Reinstein, J., Schlichting, I., and Wittinghofer, A.(1990) Biochemistry 29, 7451-7459 [Medline] [Order article via Infotrieve]
  37. Romanos, M. A., Scorer, C. A., and Clare, J. J.(1992)Yeast8,423-488 [Medline] [Order article via Infotrieve]
  38. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  39. Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992)J. Biol. Chem. 267, 4854-4858 [Abstract/Free Full Text]
  40. Schmitt, M. E., Brown, T. A., and Trumpower, B. L.(1990)Nucleic Acids Res. 18, 3091-3092 [Medline] [Order article via Infotrieve]
  41. Servos, J., Haase, E., and Martin, B.(1993)Mol. & Gen. Genet. 236, 214-218
  42. Sung, P., Higgins, D., Prakash, L., and Prakash, S.(1988)EMBO J. 7, 3263-3269 [Abstract]
  43. Szczypka, M. S., Wemmie, J. A., Moye-Rowley, W. S., and Thiele, D. J.(1994)J. Biol. Chem. 269, 22853-22857 [Abstract/Free Full Text]
  44. Thomas, P. J., and Pedersen, P.(1993)J. Bioenerg. Biomembr. 25, 11-19 [Medline] [Order article via Infotrieve]
  45. Tian, G., Yan, H., Jiang, R. T., Kishi, F., Nakazawa, A., and Tsai, M. D.(1990) Biochemistry 29, 4296-4304 [Medline] [Order article via Infotrieve]
  46. Ulawzewski, S., Balzi, E., and Goffeau, A.(1987)Mol. & Gen. Genet. 207,38-46

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.