PDR16 and PDR17, Two Homologous Genes ofSaccharomyces cerevisiae, Affect Lipid Biosynthesis and Resistance to Multiple Drugs
1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês
10.1074/jbc.274.4.1934
ISSN1083-351X
AutoresH. Bart van den Hazel, Harald Pichler, Maria Adelaide do Valle Matta, Erich Leitner, André Goffeau, Günther Daum,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe Saccharomyces cerevisiae open reading frame YNL231C was recently found to be controlled by the multiple drug resistance regulator Pdr1p. Here we characterizeYNL231C (PDR16) and its homologueYNL264C (PDR17). Deletion of PDR16resulted in hypersensitivity of yeast to azole inhibitors of ergosterol biosynthesis. While no increase in drug sensitivity was found upon deletion of PDR17 alone, a Δpdr16,Δpdr17double mutant was hypersensitive to a broad range of drugs. Both mutations caused significant changes of the lipid composition of plasma membrane and total cell extracts. Deletion of PDR16 had pronounced effects on the sterol composition, whereas PDR17deletion mainly affected the phospholipid composition. Thus, Pdr16p and Pdr17p may regulate yeast lipid synthesis like their distant homologue, Sec14p. The azole sensitivity of the PDR16-deleted strain may be the result of imbalanced ergosterol synthesis. Impaired plasma membrane barrier function resulting from a change in the lipid composition appears to cause the increased drug sensitivity of the double mutant strain Δpdr16,Δpdr17. The uptake rate of rhodamine-6-G into de-energized cells was shown to be almost 2-fold increased in a Δpdr16,Δpdr17 strain as compared with wild-type and Δpdr5 strains. Collectively, our results indicate that PDR16 and PDR17 control levels of various lipids in various compartments of the cell and thereby provide a mechanism for multidrug resistance unrecognized so far. The Saccharomyces cerevisiae open reading frame YNL231C was recently found to be controlled by the multiple drug resistance regulator Pdr1p. Here we characterizeYNL231C (PDR16) and its homologueYNL264C (PDR17). Deletion of PDR16resulted in hypersensitivity of yeast to azole inhibitors of ergosterol biosynthesis. While no increase in drug sensitivity was found upon deletion of PDR17 alone, a Δpdr16,Δpdr17double mutant was hypersensitive to a broad range of drugs. Both mutations caused significant changes of the lipid composition of plasma membrane and total cell extracts. Deletion of PDR16 had pronounced effects on the sterol composition, whereas PDR17deletion mainly affected the phospholipid composition. Thus, Pdr16p and Pdr17p may regulate yeast lipid synthesis like their distant homologue, Sec14p. The azole sensitivity of the PDR16-deleted strain may be the result of imbalanced ergosterol synthesis. Impaired plasma membrane barrier function resulting from a change in the lipid composition appears to cause the increased drug sensitivity of the double mutant strain Δpdr16,Δpdr17. The uptake rate of rhodamine-6-G into de-energized cells was shown to be almost 2-fold increased in a Δpdr16,Δpdr17 strain as compared with wild-type and Δpdr5 strains. Collectively, our results indicate that PDR16 and PDR17 control levels of various lipids in various compartments of the cell and thereby provide a mechanism for multidrug resistance unrecognized so far. phosphatidylinositol phosphatidylcholine phosphatidylethanolamine phosphatidylserine yeast extract/peptone/dextrose medium yeast/peptone/glycerol medium gas-liquid chromatography/mass spectroscopy trimethylammonium diphenylhexatriene 4-morpholineethanesulfonic acid. The yeast Saccharomyces cerevisiae has, like many other organisms, the ability to acquire multiple drug resistance,i.e. become less sensitive to a broad range of chemically and functionally unrelated cytotoxic compounds (1Balzi E. Goffeau A. Biochim. Biophys. Acta. 1994; 1187: 152-162Crossref PubMed Scopus (139) Google Scholar, 2Balzi E. Goffeau A. J. Bioenerg. Biomembr. 1995; 27: 71-76Crossref PubMed Scopus (229) Google Scholar). In yeast this phenomenon can be provoked by a regulatory disorder, namely a mutation in the transcription factors Pdr1p or Pdr3p. Pdr1p and Pdr3p are homologous Zn2Cys6 DNA-binding proteins which control the expression of drug efflux pump-encoding genes (3Balzi E. Chen W. Ulaszewski S. Capieaux E. Goffeau A. J. Biol. Chem. 1987; 262: 16871-16879Abstract Full Text PDF PubMed Google Scholar, 4Delaveau T. Delahodde A. Carvajal E. Subik J. Jacq C. Mol. Gen. Genet. 1994; 244: 501-512Crossref PubMed Scopus (177) Google Scholar). Gain-of-function mutations in the PDR1 or PDR3genes may result in increased production of these efflux pumps, leading to drug resistance (5Carvajal E. van den Hazel H.B. Cybularz-Kolaczkowska A. Balzi E. Goffeau A. Mol. Gen. Genet. 1997; 256: 406-415Crossref PubMed Scopus (148) Google Scholar, 6Nourani A. Papajova D. Delahodde A. Jacq C. Subik J. Mol. Gen. Genet. 1997; 256: 397-405Crossref PubMed Scopus (97) Google Scholar). Neither Pdr1p or Pdr3p nor the drug efflux pumps which they regulate are required for growth of yeast in the absence of drugs. It is not known whether the true physiological function of these drug resistance determinants is to protect the cell from external toxic compounds or whether they may play other roles. In order to get more insight into the physiological role of Pdr1p, we recently screened for target genes regulated by this transcription factor. This screening resulted in the identification of a broad range of novel Pdr1p target genes, one of which was the open reading frame with the systematic name YNL231C (PDR16). Expression of the PDR16 gene is five times higher in strains carrying pdr1–3, a strong constitutive allele ofPDR1, as compared with isogenic wild-type strains or strains deleted forPDR1. 1H. B. van den Hazel and A. Goffeau, unpublished observation. 1H. B. van den Hazel and A. Goffeau, unpublished observation. The PDR16 gene encodes for a protein of 351 amino acids. This protein is 49% identical and 75% similar to the product of theYNL264C (PDR17) gene of S. cerevisiae.Neither PDR16 nor PDR17 has been functionally characterized. The Pdr16p is also 23% identical and 54% similar to the product of the S. cerevisiae SEC14 gene. This homology is spread throughout the protein sequence. However, three sequence blocks which are strongly conserved among the SEC14 proteins from different yeasts (around amino acid positions 55–60, 205–210, and 235–240) (7Monteoliva L. Sanchez M. Pla J. Gil C. Nombela C. Yeast. 1996; 12: 1097-1105Crossref PubMed Scopus (37) Google Scholar) are also the most conserved areas between these proteins and PDR16 and PDR17. Sec14p was initially identified as a PtdIns2 transfer protein (PITP) which can perform transfer of phospholipids between membranesin vitro (8Daum G. Paltauf F. Biochim. Biophys. Acta. 1984; 794: 385-391Crossref Scopus (75) Google Scholar, 9Szolderits G. Hermetter A. Paltauf F. Daum G. Biochim. Biophys. Acta. 1989; 986: 301-309Crossref PubMed Scopus (51) Google Scholar). Subsequently, it was shown that Sec14p/PITP is required for transport of proteins through the Golgi complex (10Bankaitis V.A. Aitken J.R. Cleves A.E. Dowhan W. Nature. 1990; 347: 561-562Crossref PubMed Scopus (435) Google Scholar). It has been proposed that in vivo Sec14p senses the levels of PtdIns and PtdCho in the Golgi complex and exerts negative feedback on PtdCho synthesis through the Kennedy pathway (11Skinner H.B. McGee T.P. McMaster C.R. Fry M.R. Bell R.M. Bankaitis V.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 112-116Crossref PubMed Scopus (136) Google Scholar). More recently, it was suggested that Sec14p/PITP may also regulate formation of secretory vesicles from the Golgi by stimulating the turnover of phospholipids (12Kearns B.G. McGee T.P. Mayinger P. Gedvilaite A. Phillips S.E. Kagiwada S. Bankaitis V.A. Nature. 1997; 387: 101-105Crossref PubMed Scopus (222) Google Scholar, 13Patton-Vogt J.L. Griac P. Sreenivas A. Bruno V. Dowd S. Swede M.J. Henry S.A. J. Biol. Chem. 1997; 272: 20873-20883Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The involvement of PITP in signal transduction of higher eukaryotes has been discussed (14Cunningham E. Tan S.K. Swigart P. Hsuan J. Bankaitis V. Cockcroft S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6589-6593Crossref PubMed Scopus (99) Google Scholar, 15Cockcroft S. Ball A. Fensome A. Hara S. Jones D. Prosser S. Swigart P. Biochem. Soc. Trans. 1997; 25: 1125-1131Crossref PubMed Scopus (11) Google Scholar, 16Cockcroft S. FEBS Lett. 1997; 410: 44-48Crossref PubMed Scopus (18) Google Scholar). In the present work, we analyze the role of the distant yeastSEC14 homologues, PDR16 and PDR17, in drug resistance and lipid biosynthesis/sorting in a wild-type and apdr1–3 background. We find that deletion ofPDR16 leads to a strongly increased sensitivity to azole antifungals. Deletion of both PDR16 and PDR17leads to reduced resistance to a broad range of drugs. We, furthermore, show that the mutations affect the phospholipid and sterol composition of the plasma membrane, and that they change the total yeast lipid composition. We propose that the azole sensitivity of theΔpdr16 single mutant is mainly due to impaired sterol synthesis and that the broad increase in drug sensitivity of the double mutant is a result of a more general change in plasma membrane composition. The possible roles of Pdr16p and Pdr17p in lipid biosynthesis and sorting are discussed. Escherichia colistrain DH5α (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) was used for plasmid propagation. S. cerevisiae strain US50–18c (MATα pdr1–3 ura3 his1) (3Balzi E. Chen W. Ulaszewski S. Capieaux E. Goffeau A. J. Biol. Chem. 1987; 262: 16871-16879Abstract Full Text PDF PubMed Google Scholar), its derivative AD3 (MATα pdr1–3 ura3 his1 pdr5::hisG) (18Decottignies A. Grant A.M. Nichols J.W. de Wet H. McIntosh D. Goffeau A. J. Biol. Chem. 1998; 273: 12612-12622Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), and strain FY1679–28C (MAT a ura3–52 leu2Δ1 his3Δ200 trpΔ63 GAL2+) (derivative of FY1679) (19Thierry A. Fairhead C. Dujon B. Yeast. 1990; 6: 521-534Crossref PubMed Scopus (86) Google Scholar) were used for the construction of strains deleted for the PDR16, PDR17, andYOR1 genes, and as reference strains in drug sensitivity assays. The US50–18c-derivatives AD1 (MATα pdr1–3 ura3 his1 yor1::hisG) (18Decottignies A. Grant A.M. Nichols J.W. de Wet H. McIntosh D. Goffeau A. J. Biol. Chem. 1998; 273: 12612-12622Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar) and AD13 (MATα pdr1–3 ura3 his1 pdr5::hisG yor1::hisG) (19Thierry A. Fairhead C. Dujon B. Yeast. 1990; 6: 521-534Crossref PubMed Scopus (86) Google Scholar) were used as reference strains in drug sensitivity assays. PDR5-deleted strain FYMK-1/1 (20Kolaczkowski M. van der Rest M. Cyburlarz-Kolaczkowska A. Soumillion J.P. Konings W.N. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) was used for drug uptake experiments. A construct for the disruption of the YOR1 gene (pDK30) (21Katzmann D.J. Hallstrom T.C. Voet M. Wysock W. Golin J. Volckaert G. Goffeau A. Mol. Cell. Biol. 1995; 15: 6875-6883Crossref PubMed Scopus (199) Google Scholar) was kindly supplied by Dr. W. Scott Moye-Rowley. All deletions of thePDR16 gene and the deletions of the PDR17 gene in US50–18c and its derivatives were constructed using the procedure described by Alani et al. (22Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (747) Google Scholar). A DNA fragment corresponding to the 5′-flanking region upstream of the gene was generated by PCR and cloned into pSK+ (Stratagene). For the PDR16 gene, the primers for the PCR were 5′-GCACGAATTCTCAAAGACGGCGGATTCA-3′ and 5′-AACCGGATCCCCTGGGTCTTCTGGAGCCC-3′, while for the PDR17gene, the primers were 5′-ACCGAATTCTGATTGAAGAGATCAAAGA-3′ and 5′-TAAGGATCCGGCAGGAGGGTCCAA-3′. Subsequently, a 3′-flanking fragment, encompassing the 3′ end of the gene, was generated by PCR. For the PDR16 gene, the PCR primers were 5′-TTTGGATCCTTGGTTAGCATGG-3′ and 5′-TTGGAGCTCAGTGCATATAGACGCG-3′, while for PDR17, they were: 5′-ATGGGATCCTTGGAGGCATTGTCGGA-3′ and 5′-CGGGAGCTCGATTGATTAGCTGGAAC-3′. This 3′-flanking PCR product was subcloned into the plasmid that already contained the 5′-upstream fragment. The resulting plasmids were termed pBVH742 forPDR16 and pBVH1068 for PDR17. In a final subcloning step, a 3.8-kilobase BamHI-BglII fragment containing a hisG-URA3-hisG cassette (22Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (747) Google Scholar) was inserted into the BamHI site between the 5′- and 3′-flanking regions of the gene, resulting in the plasmids pBVH778 (PDR16) and pBVH1127 (PDR17). Each of the resulting plasmids was treated with EcoRI andSacI to generate a linear fragment consisting of the 5′- and 3′-flanking regions of the gene interrupted byhisG-URA3-hisG. The fragment was transformed into yeast and cells that had become Ura+ due to chromosomal replacement of wild-type gene by the linear fragment were selected on SC plates lacking uracil. Subsequently, the URA3 gene was "looped-out" by growth on non-selective media, allowing recombination between the hisG sequences. Cells in which a loop-out event had occurred were selected by plating on media containing 5-fluoroorotic acid (22Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (747) Google Scholar). Deletions of PDR17 in strain FY1679–28C and its derivatives were generated by one-step gene replacement as described by Winstonet al. (23Winston F. Chumley F. Fink G.R. Methods Enzymol. 1983; 101: 211-228Crossref PubMed Scopus (159) Google Scholar). A 1.15-kilobase BamHI fragment from plasmid YDp-H, containing the HIS3 gene, was subcloned into pBVH1068, resulting in plasmid pBVH1451. The pBVH1451 plasmid was subsequently treated with EcoRI and SacI to generate a linear fragment consisting of the 5′- and 3′-flanking regions of the PDR17 gene interrupted by HIS3. This fragment was transformed into yeast, and cells that had become His+ due to chromosomal replacement of the wild-typePDR17 gene by the linear fragment were selected on SC plates lacking histidine. All deletions were verified by PCR amplification of a chromosomal region encompassing the deletion, followed by analysis of the PCR product by agarose gel electrophoresis. Furthermore, the absence of an mRNA signal from PDR16 in the US50–18c derivative deleted for this gene was verified by Northern blotting, using a fragment of the gene as a probe. Centromeric plasmids containing the PDR16 and PDR17 genes were obtained by "gap repair." An EcoRI-SacI fragment consisting of the 5′- and 3′-flanking regions of the PDR16 gene was subcloned from pBVH742 into the URA3 centromere plasmid pRS316 (24Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), resulting in plasmid pBVH1222. Similarly, aClaI-SacI fragment consisting of the 5′- and 3′-flanking regions of the PDR17 gene was subcloned from pBVH1068 into pRS316, giving pBVH1378. The pBVH1222 and pBVH1378 plasmids were linearized using BamHI and transformed into yeast strain US50–18c. Cells in which recombination between the linearized plasmid and the chromosomal locus had generated a circular centromeric plasmid containing the entire gene were selected on media lacking uracil. Plasmids were isolated from yeast, transformed intoE. coli, and restriction analysis of plasmid preparations was performed in order to verify the presence of the gene. A multicopy plasmid containing PDR16 was generated by insertion of aSalI-SacI fragment from the single-copy plasmid containing the gene into pRS426 (25Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1429) Google Scholar). E. coliwas grown in standard Luria broth medium (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Yeast was grown on standard rich glucose (YPD) or glycerol (YPG) media, or on SC medium lacking appropriate amino acids for plasmid maintenance (26Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2537) Google Scholar). For drug resistance assays on solid media, drugs were added to the media immediately prior to pouring. The drug concentrations tested were the following: 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 4, and 8 μg of cycloheximide (Sigma, stock in ethanol) per ml of YPD; 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 200 μg of rhodamine-6-G (Merck, stock in ethanol) per ml of YPG; 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2, and 5 μg of oligomycin (Sigma, stock in ethanol) per ml of YPG; 0.1, 0.25, 0.5, 1, 2, 3.5, 5, and 10 μg of 4-nitroquinoline oxide (Sigma, stock in dimethyl sulfoxide) per ml of YPD; 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, 10, and 20 μg of miconazole (kindly supplied by Janssen Pharmaceutica, stock in dimethyl sulfoxide) per ml of YPD; 0.1, 0.25, 0.5, 1, 2, 5, and 10 μg of ethidium bromide (Boehringer Mannheim, stock in water) per ml of YPG; 2, 5, 10, 15, 20, 25, 30, 40, 50, and 80 μg of nystatin per ml of YPD. 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, and 50 μg of ketoconazole (kindly supplied by Janssen Pharmaceutica, stock in dimethyl sulfoxide) per ml of YPD; 0.5, 1, 2, 5, 10, 25, 50, and 100 μg of itraconazole (kindly supplied by Janssen Pharmaceutica, stock in dimethyl sulfoxide) per ml of YPD; 0.25, 0.5, 1, 2, 5, 10, and 20 μg of crystal violet per ml of YPD; 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1 μg of antimycin A (Sigma, stock in dimethyl sulfoxide) per ml of YPG. Growth tests on non-fermentable carbon sources, at high pH, osmolarity and temperature were performed as described by Rank et al. (27Rank G.H. Gerlach J.H. Robertson A.J. Mol. Gen. Genet. 1976; 144: 281-288Crossref PubMed Scopus (19) Google Scholar), i.e. on solid media containing 0.1% yeast extract, 0.5% bacto-peptone, and 100 mm potassium phosphate, pH 7.1, supplemented with 1% glucose (with or without 0.5 mpotassium chloride), glycerol or ethanol. Plates were incubated at 30 or 37 °C. Approximately 107cells from an overnight culture were inoculated in 20 ml of YPD and grown for 3 h at 30 °C. About 5.6 × 108 cells were pelleted and washed three times with buffer A (50 mmHEPES/NaOH, pH 7.0). The cells were subsequently resuspended in 4 ml of de-energization buffer (1 μm antimycin A, 5 mm 2-deoxy-d-glucose in buffer A), incubated for 2 h 30 min at 30 °C, and transferred to a water bath at 20 °C. A 200-μl aliquot of the de-energized cell suspension was pelleted, washed once with 200 μl of cold buffer A, and resuspended in 2 ml of the same cold buffer. Cell fluorescence background was measured using an SLM Aminco 48000 S spectrofluorimeter. The excitation wavelength was 529 nm (4 nm slit), and the emission wavelength was 553 nm (4 nm slit). Rhodamine-6-G was added to the remaining cell suspension to a final concentration of 5 mm, and 200-μl aliquots were taken every 5 min up to 1 h. The cells of each aliquot were pelleted, washed, and resuspended as described above and immediately subjected to fluorescence measurements. The cell fluorescence background values after 0- and 1-h incubation were averaged, and this average was subtracted from each fluorescence value. Cells were grown on YPD medium containing 2% glucose, 2% peptone (Oxoid), and 1% yeast extract (Oxoid) under aerobic conditions at 30 °C. Growth of yeast cells was monitored by measuring the OD (600 nm). Highly purified plasma membrane was isolated as follows. The pellet of crude plasma membrane (28Serrano R. Methods Enzymol. 1988; 157: 533-544Crossref PubMed Scopus (233) Google Scholar) was suspended in 5 mm Mes, 0.2 mm EDTA, pH 6.0, with 10 strokes in a loose-fitting Dounce homogenizer and layered on top of a sucrose density gradient made of 10 ml of 38% (w/w), 10 ml of 43% (w/w), and 10 ml of 53% (w/w) sucrose in 5 mm Mes, 0.2 mm EDTA, pH 6.0. Centrifugation was carried out at 100,000 × g for 2.5 h in an SW-28 rotor (Beckman). The highly purified plasma membrane was withdrawn from the 43/53% sucrose interface, the suspension was diluted 3-fold with 10 mm Tris-HCl, pH 7.4, and the plasma membrane was sedimented at 48,000 × gfor 20 min in an SS-34 rotor (Sorvall). The plasma membrane pellet was suspended in 10 mm Tris-HCl, pH 7.4, using a loose-fitting Dounce homogenizer and stored at −70 °C. Yeast spheroplasts and mitochondria were isolated by published procedures (29Daum G. 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Data were collected by scanning from 150 to 600 atomic mass units at 1.6 scans/s. Alternatively, GLC was performed on an HP 5890 equipped with a flame ionization detector (FID) operated at 320 °C using a capillary column (HP5, 30 m × 0.32 mm × 0.25-μm film thickness). After a 1-min hold at 50 °C the temperature was increased to 310 °C at 10 °C/min. The final temperature was held for 10 min. Nitrogen was used as carrier gas and 1-μl aliquots of samples were injected cool on column. Relative retention times of sterols were similar as described previously (38Nes W.D. Xu S. Haddon W.F. Steroids. 1989; 53: 533-558Crossref PubMed Scopus (55) Google Scholar, 39Xu S. Norton R.A. Crumley F.G. Nes W.D. J. Chromatogr. 1988; 452: 377-398Crossref PubMed Scopus (88) Google Scholar, 40Patterson G.W. Anal. Chem. 1971; 43: 1165-1170Crossref Scopus (201) Google Scholar). Lipid transfer activity of cytosolic fractions and peripheral organelle membrane proteins was measured according to Ceolotto et al. 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We recently identified PDR16 (YNL231C) as one of several novel genes controlled by the yeast multiple drug resistance regulator Pdr1p.1 To investigate whether PDR16like other Pdr1p targets is involved in multiple drug resistance, we deleted the gene and studied the effects on drug sensitivity. In order to maximize the possible effects, the deletion was made in apdr1–3 genetic background (strain US50–18c) which led to overexpression of Pdr16p. Deletion of PDR16 had no effect on yeast growth in the absence of drugs. However, the PDR16-deleted strain (Δpdr16) exhibited a strongly increased sensitivity to miconazole and ketoconazole as compared with the parental strain US50–18c (Table I). For miconazole, the sensitivity of Δpdr16 was increased approximately 20-fold over the control: while the minimal inhibitory concentration was 2 μg/ml for the parental strain, it was only 0.1 μg/ml for theΔpdr16 strain. Sensitivity to ketoconazole increased about 10 times. Similar results were obtained with itraconazole (data not shown). The Δpdr16 strain was also slightly more sensitive to nystatin than the parental strain. No significant changes in sensitivity to any of the other drugs tested were observed (TableI).Table IDrug sensitivity of yeast strain US50–18c and various derivatives after 3 days of growth at 30 °CMinimal inhibitory drug concentrationUS50–18cUS50–18c Δpdr16US50–18c Δpdr17US50–18c Δpdr16, Δpdr17μg/ml of mediaMiconazole20.120.025Ketoconazole101100.25Nystatin25202520Cycloheximide1.51.51.50.5Rhodamine-6-G>200>200>200200Oligomycin1110.54-NQOa4-NQO, 4-nitroquinoline-N-oxide.553.52Antimycin A0.10.10.10.05Ethidium bromide2222Crystal violet>20>20>2020a 4-NQO, 4-nitroquinoline-N-oxide. Open table in a new tab To verify whether the drug sensitivity phenotype was indeed due to loss of PDR16 gene function, we introduced a single-copy plasmid carrying the intact PDR16 gene in the Δpdr16mutant. The resulting transformant had a level of miconazole resistance identical to that of the US50–18c parental strain, indicating that the mutant phenotype was indeed due to loss of PDR16 function (data not shown). The PDR16 gene has a close homologue in S. cerevisiae termed PDR17 (YNL264C). To test whether there is a functional relationship between these two genes, we generated an isogenic strain deleted for PDR17, and a double mutant deleted for both PDR16 and PDR17. The single PDR17-deleted strain (Δpdr17) did not exhibit a growth defect as compared with US50–18c. Moreover, theΔpdr17 strain did not show increased drug sensitivity, except for a minor increase in sensitivity to 4-nitroquinoline-N-oxide (Table I). The growth rate of the double mutant strain Δpdr16,Δpdr17, on the other hand, was slightly decreased on rich media as compared with the parental and the single mutant strains (data not shown). Growth of the various strains was also tested on non-fermentable carbon sources, at high pH, osmolarity, and temperature. While most of
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