Candida glabrata ATP-binding Cassette Transporters Cdr1p and Pdh1p Expressed in aSaccharomyces cerevisiae Strain Deficient in Membrane Transporters Show Phosphorylation-dependent Pumping Properties
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m207817200
ISSN1083-351X
AutoresShun‐ichi Wada, Masakazu Niimi, Kyoko Niimi, Ann R. Holmes, Brian C. Monk, Richard D. Cannon, Yoshimasa Uehara,
Tópico(s)Antifungal resistance and susceptibility
ResumoThe expression and drug efflux activity of the ATP binding cassette transporters Cdr1p and Pdh1p are thought to have contributed to the recent increase in the number of fungal infections caused by Candida glabrata. The function of these transporters and their pumping characteristics, however, remain ill defined. We have evaluated the function of Cdr1p and Pdh1p through their heterologous hyperexpression in a Saccharomyces cerevisiae strain deleted in seven major drug efflux transporters to minimize the background drug efflux activity. Although both Cdr1p- and Pdh1p-expressing strains CDR1-AD and PDH1-AD acquired multiple resistances to structurally unrelated compounds, CDR1-AD showed, in most cases, higher levels of resistance than PDH1-AD. CDR1-AD also showed greater rhodamine 6G efflux and resistance to pump inhibitors, although plasma membrane fractions had comparable NTPase activities. These results indicate that Cdr1p makes a larger contribution than Phd1p to the reduced susceptibility of C. glabrata to xenobiotics. Both pump proteins were phosphorylated in a glucose-dependent manner. Whereas the phosphorylation of Cdr1p affected its NTPase activity, the protein kinase A-mediated phosphorylation of Pdh1p, which was necessary for drug efflux, did not. This suggests that phosphorylation of Pdh1p may be required for efficient coupling of NTPase activity with drug efflux. The expression and drug efflux activity of the ATP binding cassette transporters Cdr1p and Pdh1p are thought to have contributed to the recent increase in the number of fungal infections caused by Candida glabrata. The function of these transporters and their pumping characteristics, however, remain ill defined. We have evaluated the function of Cdr1p and Pdh1p through their heterologous hyperexpression in a Saccharomyces cerevisiae strain deleted in seven major drug efflux transporters to minimize the background drug efflux activity. Although both Cdr1p- and Pdh1p-expressing strains CDR1-AD and PDH1-AD acquired multiple resistances to structurally unrelated compounds, CDR1-AD showed, in most cases, higher levels of resistance than PDH1-AD. CDR1-AD also showed greater rhodamine 6G efflux and resistance to pump inhibitors, although plasma membrane fractions had comparable NTPase activities. These results indicate that Cdr1p makes a larger contribution than Phd1p to the reduced susceptibility of C. glabrata to xenobiotics. Both pump proteins were phosphorylated in a glucose-dependent manner. Whereas the phosphorylation of Cdr1p affected its NTPase activity, the protein kinase A-mediated phosphorylation of Pdh1p, which was necessary for drug efflux, did not. This suggests that phosphorylation of Pdh1p may be required for efficient coupling of NTPase activity with drug efflux. Drug efflux mediated by membrane pump proteins is a major resistance mechanism in both cancer cells and pathogenic microorganisms. Whereas many of the drug efflux pumps in bacteria are antiporters that harness the pH gradient across the plasma membrane to efflux molecules (1Nikaido H. Curr. Opin. Microbiol. 1998; 1: 516-523Crossref PubMed Scopus (256) Google Scholar, 2Saier Jr., M.H. Paulsen I.T. Sliwinski M.K. Pao S.S. Skurray R.A. Nikaido H. FASEB J. 1998; 12: 265-274PubMed Google Scholar), eukaryotic organisms often use ATP-binding cassette (ABC) 1The abbreviations used for: ABC, ATP-binding cassette; MDR, multidrug resistance; MIC, minimum growth-inhibitory concentration; CSM, complete synthetic medium; MES, 4-morpholineethanesulfonic acid; nt, nucleootide(s); ORF, open reading frame; HBS, HEPES-buffered saline; PKA, protein kinase A. transporters to pump compounds out of the cell at the expense of ATP hydrolysis (3Borges-Walmsley M.I. Walmsley A.R. Trends Microbiol. 2001; 9: 71-79Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 4Higgins C.F. Res. Microbiol. 2001; 152: 205-210Crossref PubMed Scopus (491) Google Scholar, 5Wolfger H. Mamnun Y.M. Kuchler K. Res. Microbiol. 2001; 152: 375-389Crossref PubMed Scopus (122) Google Scholar, 6Litman T. Druley T.E. Stein W.D. Bates S.E. Cell. Mol. Life Sci. 2001; 58: 931-959Crossref PubMed Scopus (644) Google Scholar). A central role for drug efflux ABC transporters in multidrug resistance (MDR) has been reported for pathogenic fungi (7Sanglard D. Kuchler K. Ischer F. Pagani J.-L. Mond M. Bille J. Antimicrob. Agents Chemother. 1995; 39: 2378-2386Crossref PubMed Scopus (720) Google Scholar, 8Albertson G.D. Niimi M. Cannon R.D. Jenkinson H.F. Antimicrob. Agents Chemother. 1996; 40: 2835-2841Crossref PubMed Google Scholar, 9Sanglard D. Ischer F. Monod M. Bille J. Microbiology. 1997; 143: 405-416Crossref PubMed Scopus (509) Google Scholar, 10Del Sorbo G. Andrade A.C. van Nistelrooy J.G.M. van Kan J.A.L. Balzi E. De Waard M.A. Mol. Gen. Genet. 1997; 254: 417-426Crossref PubMed Scopus (114) Google Scholar, 11Thornewell S.J. Peery R.B. Skatrud P.L. Gene (Amst.). 1997; 201: 21-29Crossref PubMed Scopus (41) Google Scholar, 12Miyazaki H. Miyazaki Y. Geber A. Parkinson T. Hitchcock C. Falconer D.J. Ward D.J. Marsden K. Bennett J.E. Antimicrob. Agents Chemother. 1998; 42: 1695-1701Crossref PubMed Google Scholar, 13Sanglard D. Ischer F. Calabrese D. Majcherczyk P.A. Bille J. Antimicrob. Agents Chemother. 1999; 43: 2753-2765Crossref PubMed Google Scholar, 14Katiyar S.K. Edlind T.D. Med. Mycol. 2001; 39: 109-116Crossref PubMed Scopus (85) Google Scholar). Infections of immunocompromised and debilitated individuals caused byCandida sp. are the most frequent and problematic of fungal diseases. Triazole drugs, such as fluconazole and itraconazole, inhibit lanosterol 14α-demethylase and block the synthesis of ergosterol. These drugs have been used widely for the treatment of patients withCandida infections because of their limited toxicity to humans and their favorable pharmacokinetics. Fungal infections recalcitrant to triazole therapy occur frequently, however, due to the drug resistance of fungal strains. Candida albicans acquires resistance to azole drugs by overexpressing ABC transporters, antiporter proteins, or 14α-demethylase, by acquiring mutations in 14α-demethylase, or by changing its membrane composition (15Vanden Bossche H. Dromer F. Improvisi I. Lozano-Chiu M. Rex J.H. Sanglard D. Med. Mycol. 1998; 36: 119-128PubMed Google Scholar, 16Lupetti A. Danesi R. Campa M. Del Tacca M. Kelly S. Trends Mol. Med. 2002; 8: 76-81Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Although C. albicans is normally susceptible to azoles, the incidence of acquired resistance increased significantly in the 1990s before the advent of highly active antiretro-viral treatment therapy for AIDS patients. Throughout the 1990s, there was also an increase in the incidence of candidosis caused by non-C. albicans Candida species (17Coleman D.C. Rinaldi M.G. Haynes K.A. Rex J.H. Summerbell R.C. Anaissie E.J. Li A. Sullivan D.J. Med. Mycol. 1998; 36 Suppl. 1: 156-165PubMed Google Scholar) due to selection of strains with lower azole susceptibility. Candida glabratawas one the most common species responsible for these infections, which were difficult to treat (18Fidel Jr., P.L. Vazquez J.A. Sobel J.D. Clin. Microbiol. Rev. 1999; 12: 80-96Crossref PubMed Google Scholar). The susceptibilities of C. glabrata clinical isolates to azole drugs, measured as minimum growth-inhibitory concentrations (MICs), are 16–64-fold higher than those for C. albicans(19Diekema D.J. Messer S.A. Brueggemann A.B. Coffman S.L. Doern G.V. Herwaldt L.A. Pfaller M.A. J. Clin. Microbiol. 2002; 40: 1298-1302Crossref PubMed Scopus (357) Google Scholar), implying that C. glabrata is naturally more resistant to azoles. ABC transporters Cdr1p (13Sanglard D. Ischer F. Calabrese D. Majcherczyk P.A. Bille J. Antimicrob. Agents Chemother. 1999; 43: 2753-2765Crossref PubMed Google Scholar) and Pdh1p (also referred to as Cdr2p; see "Discussion") are thought to be the main contributors to the azole drug resistance of C. glabrata (12Miyazaki H. Miyazaki Y. Geber A. Parkinson T. Hitchcock C. Falconer D.J. Ward D.J. Marsden K. Bennett J.E. Antimicrob. Agents Chemother. 1998; 42: 1695-1701Crossref PubMed Google Scholar, 20Sanglard D. Ischer F. Bille J. Antimicrob. Agents Chemother. 2001; 45: 1174-1183Crossref PubMed Scopus (209) Google Scholar). Cdr1p is highly expressed in azole-resistant clinical isolates, and it has been shown to be involved in fluconazole efflux (13Sanglard D. Ischer F. Calabrese D. Majcherczyk P.A. Bille J. Antimicrob. Agents Chemother. 1999; 43: 2753-2765Crossref PubMed Google Scholar). At present there is no direct evidence that Pdh1p is involved in azole efflux, although a gradual increase in Pdh1p expression in C. glabrata strains has been reported during exposure to fluconazole in vivo(12Miyazaki H. Miyazaki Y. Geber A. Parkinson T. Hitchcock C. Falconer D.J. Ward D.J. Marsden K. Bennett J.E. Antimicrob. Agents Chemother. 1998; 42: 1695-1701Crossref PubMed Google Scholar) and in vitro (20Sanglard D. Ischer F. Bille J. Antimicrob. Agents Chemother. 2001; 45: 1174-1183Crossref PubMed Scopus (209) Google Scholar). These pumps were inferred to efflux divergent xenobiotics, because both pumps share about 70% amino acid sequence identity with Saccharomyces cerevisiae Pdr5p, an ABC efflux pump that is responsible for pleiotropic drug resistance (12Miyazaki H. Miyazaki Y. Geber A. Parkinson T. Hitchcock C. Falconer D.J. Ward D.J. Marsden K. Bennett J.E. Antimicrob. Agents Chemother. 1998; 42: 1695-1701Crossref PubMed Google Scholar, 13Sanglard D. Ischer F. Calabrese D. Majcherczyk P.A. Bille J. Antimicrob. Agents Chemother. 1999; 43: 2753-2765Crossref PubMed Google Scholar, 21Balzi E. Wang M. Leterme S. Van Dyck L. Goffeau A. J. Biol. Chem. 1994; 269: 2206-2214Abstract Full Text PDF PubMed Google Scholar). However, the precise function of the transporters and their pumping characteristics are ill defined. More detailed knowledge of their drug efflux mechanisms may enable the development of improved antifungal drugs that are not pumped out from the cells or chemosensitizers that inhibit the pumping activity of these transporters in C. glabrata. There are about 30 genes in Saccharomyces cerevisiaeencoding ABC transporters, some of which are responsible for the efflux of xenobiotics (5Wolfger H. Mamnun Y.M. Kuchler K. Res. Microbiol. 2001; 152: 375-389Crossref PubMed Scopus (122) Google Scholar, 22Decottignies A. Goffeau A. Nat. Genet. 1997; 15: 137-145Crossref PubMed Scopus (405) Google Scholar, 23Daniel T. Michaelis S. Methods Enzymol. 1998; 292: 130-162Crossref PubMed Scopus (85) Google Scholar, 24Bauer B.E. Wolfger H. Kuchler K. Biochim. Biophys. Acta. 1999; 1461: 217-236Crossref PubMed Scopus (237) Google Scholar). C. glabrata is a close relative of S. cerevisiae (18Fidel Jr., P.L. Vazquez J.A. Sobel J.D. Clin. Microbiol. Rev. 1999; 12: 80-96Crossref PubMed Google Scholar, 25Barns S.M. Lane D.J. Sogin M.L. Bibeau G. Weisburg W.G. J. Bacteriol. 1991; 173: 2250-2255Crossref PubMed Google Scholar) and probably has a similar number of transporters. Thus, the background activities of other endogenous pumps are likely to be problematic for the precise analysis of the efflux mechanism of individual drug efflux pumps in intact C. glabrata cells. We recently reported the functional expression ofC. albicans drug efflux pump CaCdr1p in S. cerevisiae strain AD1–8u−, which was used to resolve the problem of endogenous background drug efflux (26Nakamura K. Niimi M. Niimi K. Holmes A.R. Yates J.E. Decottignies A. Monk B.C. Goffeau A. Cannon R.D. Antimicrob. Agents Chemother. 2001; 45: 3366-3374Crossref PubMed Scopus (171) Google Scholar). Strain AD1–8u− was deleted in seven major drug efflux transporters and has a PDR1 gain-of-function mutation that highly activates the PDR5 promoter (26Nakamura K. Niimi M. Niimi K. Holmes A.R. Yates J.E. Decottignies A. Monk B.C. Goffeau A. Cannon R.D. Antimicrob. Agents Chemother. 2001; 45: 3366-3374Crossref PubMed Scopus (171) Google Scholar, 27Decottignies A. Grant A.M. Nichols J.W. de Wet H. McIntoch D.B. Goffeau A. J. Biol. Chem. 1998; 273: 12612-12622Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Drug efflux pump genes inserted at the PDR5 locus of this strain are highly expressed, and the pumping activities of the transporters can be measured at both the cellular level and in the plasma membrane fraction against a diminished background of endogenous drug efflux activity. In this study, Cdr1p and Pdh1p were hyperexpressed in S. cerevisiae, and the chemical specificities, drug efflux activities, and NTPase activities of plasma membrane fractions from the resultant yeast strains were analyzed. The post-translational modification of the fungal drug efflux transporters by phosphorylation and its differential effects on the regulation of drug efflux function were also investigated. Plasmids were maintained in Escherichia coli XL1-Blue. E. coliwas cultured in Luria-Bertani medium (Difco), to which ampicillin was added (50 μg/ml) as required. The genes described in this study were obtained from C. glabrata CBS138 and C. albicansATCC 10261. The S. cerevisiae strains used were AD1–8u− (MATa, pdr1–3,his1, ura3,Δyor1::hisG,Δsnq2::hisG,Δpdr5::hisG,Δpdr10::hisG,Δpdr11::hisG,Δycf1::hisG,Δpdr3::hisG Δpdr15::hisG) (27Decottignies A. Grant A.M. Nichols J.W. de Wet H. McIntoch D.B. Goffeau A. J. Biol. Chem. 1998; 273: 12612-12622Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar) (provided by Dr. A. Decottignies and Prof. A. Goffeau, Université Catholique de Louvain, Belgium) and its derivatives expressing C. glabrataABC transporters. The yeast strains were cultured in YEPD broth (Difco) or complete synthetic medium (CSM; 790 mg/liter of complete supplement mixture (Bio 101, Vista, CA) and 26.7 g/liter Dropout Base (Bio 101)). For agar plates, 2% (w/v) Bacto agar (Difco) was added to the medium. CSM was buffered with 10 mm2-(N-morpholino)ethanesulfonic acid (MES) and 18 mm HEPES, pH 7.0, for MIC assays and pump phosphorylation experiments. In the latter case, yeast nitrogen base (1.7 g/liter; Difco) and ammonium sulfate (5 g/liter) were added to the medium, instead of Dropout Base, to prepare CSM without glucose (CSM−Gluc). For the selection and maintenance of Ura+ strains, complete synthetic medium without uracil (CSM−URA; Bio 101) was used. Genomic DNA was prepared from C. glabrata CBS138 and C. albicans ATCC 10261 as described previously (28Scherer S. Stevens D.A. J. Clin. Microbiol. 1987; 25: 675-679Crossref PubMed Google Scholar). Genes required for the construction of expression vectors were amplified by PCR, with the combinations of templates and primers indicated in Table I using KOD (+) DNA polymerase (Toyobo, Osaka, Japan). PCR products were digested with restriction enzymes and inserted into pSK-PDR5PPUS vector plasmid (26Nakamura K. Niimi M. Niimi K. Holmes A.R. Yates J.E. Decottignies A. Monk B.C. Goffeau A. Cannon R.D. Antimicrob. Agents Chemother. 2001; 45: 3366-3374Crossref PubMed Scopus (171) Google Scholar), which had previously been digested with restriction enzymes and treated with calf intestinal alkaline phosphatase (New England Biolabs, Beverly, MA) as shown in Fig. 1. Correct vector construction was confirmed by DNA sequencing with the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences) and an ABI 373 DNA sequencer. The resultant vectors, named pSK-CDR1 and pSK-PDH1, were digested with XhoI and NotI or KpnI and NotI, respectively, to prepare their transformation cassettes for gene transfer. These cassettes and a PCR fragment ofPDH1 digested with HindIII (1–3 μg) were used to transform S. cerevisiae AD1–8u− by the lithium acetate transformation protocol (Alkali-Cation Yeast kit; Bio 101). Cdr1p- and Pdh1p-expressing strains were selected by growth on CSM−URA agar followed by growth on YEPD agar containing fluconazole (5 μg/ml). CDR1 and PDH1 genes from the resultant transformants were amplified by PCR using the primers GC-1 and GC-2 or GP-1 and GP-2 (Table I), respectively, and the DNA sequences of the PCR fragments were obtained as described above.Table IMaterials used in PCR amplifications for plasmid and yeast constructionGeneaNucleotide positions shown are from the open reading frames numbered in the 5′ to 3′ direction with the first base of the translation start codon being +1.Template DNAPrimerSequence (oligonucleotide name)bRestriction sites for the cloning are underlined.CaCDR2 terminator (4501–4871)Genomic DNA from C. albicans10261Sense5′-GACTAGTCGGATGGGGTCTTATTTTACAAT (AC2–1)Antisense5′-GACTAGTCTTTTAACTGGGACCCTGC (AC2–2)CDR1 (1–4500)Genomic DNA from C. glabrataCBS138Sense5′-CCCAAGCTTGATGTCTCTTGCAAGTGACAA (GC-1)Antisense5′-CGGAATTCCGTTATTTCTTGGCAAGTTTAC (GC-2)PDH1 -U (1–530)Genomic DNA from C. glabrataCBS138Sense5′-CCCAAGCTTATGAACACACCCGATGACTCT (GP-1)Antisense5′-GGAATTCGGCTTTAGGATGCGGAATGTATC (GP-2)PDH1 -L (4532–5486)Genomic DNA from C. glabrataCBS138Sense5′-TCCCCCCGGGCAAGAGTGCCTAAAACCAAT (GP-3)Antisense5′-GACTAGTCTGGACAAGAGAATATATCCTCG (GP-4)PDH1 (1–5486)Genomic DNA from C. glabrataCBS138Sense5′-CCCAAGCTTATGAACACACCCGATGACTCT (GP-1)Antisense5′-GACTAGTCTGGACAAGAGAATATATCCTCG (GP-4)a Nucleotide positions shown are from the open reading frames numbered in the 5′ to 3′ direction with the first base of the translation start codon being +1.b Restriction sites for the cloning are underlined. Open table in a new tab Total RNA (8 μg) extracted from S. cerevisiae cells (29Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1175) Google Scholar) was electrophoresed in an agarose gel, blotted onto Hybond-N+ nylon membrane (Amersham Biosciences), and fixed with 50 mm NaOH. The membranes were hybridized at 50 °C for 16 h with digoxygenin-labeled DNA probes that had been prepared with a BcaBESTTM DIG Labeling Kit (Takara, Kusatsu, Shiga, Japan). The probes consisted of nt 1–1331 of CDR1 ORF, nt 428–1957 of PDH1 ORF, and the complete S. cerevisiae PMA1 (plasma membrane H+-ATPase) ORF as a control. After washing and blocking the blots with the appropriate buffers (Roche Diagnostics), the membranes were incubated with alkaline phosphatase-conjugated anti-digoxygenin Fab fragments (Roche Diagnostics) and CDP-star (Amersham Biosciences). The chemiluminescence of CDR-star was detected with x-ray film. Plasma membrane fractions of yeast cells were prepared as described previously (30Monk B.C. Kurtz M.B. Marrinan J.A. Perlin D.S. J. Bacteriol. 1991; 173: 6826-6836Crossref PubMed Google Scholar) with some modifications. S. cerevisiae cells were cultured in YEPD broth at 27 °C from an initial A 600 = 0.2 for 12 h with continuous shaking. These yeasts were transferred to CSM−Gluc or labeled with 20 μCi/ml [32P]orthophosphate, depending on the experiment. The following procedures were performed on ice or at 4 °C. The culture (5 ml) was centrifuged at 3,000 × g for 5 min, and the yeast cell pellet was washed with 1 ml of ice cold 2% (w/v) glucose (or water in some cases). The cells were harvested by centrifugation at 3,000 × g for 5 min and suspended in 250 μl of homogenizing buffer (50 mm Tris-HCl (pH 7.5), 2 mm EDTA, and 2% (w/v) glucose). The following were added to the cell suspension: 1 mm phenylmethanesulfonyl fluoride, 1.2 μm antipain, 1.6 μmleupeptin, 1.1 μm pepstatin A, and 400 μl of glass beads (Sigma). The yeast cells were lysed using a MT-360 micro tube mixer (TOMY Seiko Co. Ltd., Tokyo, Japan) at maximum vibration for 10 min. The cell extract was collected, and the glass beads were washed with up to 1 ml of homogenizing buffer containing 1 mmphenylmethanesulfonyl fluoride. The cell extract was centrifuged (2,000 × g for 10 min) to remove unbroken cells and cellular debris, and the supernatant was centrifuged at 20,000 ×g for 45 min. The pellet was washed with 1 ml of GTED-20 buffer (10 mm Tris-HCl (pH 7.0), 0.5 mm EDTA, and 20% (v/v) glycerol) containing 1 mmphenylmethanesulfonyl fluoride and centrifuged at 20,000 ×g for 45 min. The pellet was resuspended in 100 μl of GTED-20 buffer and used as the crude membrane fraction for measuring pump protein expression and for Western blots. For the measurement of plasma membrane NTPase activity, a large scale crude membrane fraction was acidified with 0.1 m acetate to pH 5.0 and incubated for 5 min. The precipitated mitochondria were removed by centrifugation at either 5,000 × g for 30 s for glucose-fermenting cells or 8,000 × g for 10 min for glucose-starved cells. The supernatants were immediately neutralized (to pH 7.5) with 1 m Tris and centrifuged at 20,000 ×g for 45 min. The precipitated plasma membrane fractions were resuspended in GTED-20 buffer to a protein concentration of approximately 1 mg/ml and used in the NTPase assay. The protein concentrations of these membrane fractions were determined by a BCA assay (Pierce) with bovine serum albumin as the standard. Crude membrane samples were separated by SDS-PAGE (8% acrylamide (w/v)) (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (215638) Google Scholar) and either stained with Coomassie Brilliant Blue R-250 or electroblotted onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA) at 12 V for 1 h. The membranes were blocked with phosphate-buffered saline containing 5% (w/v) skim milk and 0.05% (v/v) Tween 20 (t-PBS) at room temperature for 2 h. After washing twice with PBS for 5 min, the membranes were incubated with anti-phosphoprotein kinase A substrate antibody (Cell Signaling Technology, Beverly, MA) diluted 1:1000 in t-PBS containing 1% (w/v) bovine serum albumin at 4 °C for 16 h. The blot was washed with PBS three times and then incubated with secondary antibody (anti-rabbit IgG conjugated with horseradish peroxidase; AmershamBiosciences) diluted 1:5000 in t-PBS containing 1% (w/v) skim milk for 1 h. The membranes were washed with t-PBS containing 5% (w/v) skim milk for 15 min and twice with PBS alone for 15 min. Signal from the horseradish peroxidase was detected with an ECL system (AmershamBiosciences). For the identification of pump proteins, membrane fractions were incubated with 80 mm iodoacetamide for 30 min before SDS-PAGE. The putative pump-containing bands were excised from the gel, and the proteins were extracted with 1% (w/v) SDS buffered with 20 mm Tris-HCl, pH 8.0. After concentration by precipitation with 50% acetone, the samples in 10 mmTris-HCl, pH 9.0, were partially digested with lysyl endopeptidase (Wako, Osaka, Japan; at an enzyme/substrate molar ratio of 1:100) at 37 °C for 1 h, electrophoresed, and blotted onto polyvinylidene fluoride membrane. N-terminal sequences of several of the digested fragments were analyzed with an Applied Biosystems model 473A Protein Sequencer through the courtesy of Prof. Watabe and Dr. Nakaya at the University of Tokyo. Drug susceptibility of yeast strains was measured by filter disk assays on YEPD plates containing 1.5% (w/v) agar. Exponentially growing yeast cells were seeded at a concentration of 6 × 104cells/ml in the agar. Sterile Whatman paper disks, on which drug solutions had been spotted and dried at room temperature for 1 h to remove excess solvent, were placed on the agar plates. Miconazole, ketoconazole, nystatin, amphotericin B, flucytosine, cerulenin, cycloheximide, nigericin, monensin, rhodamine 123, rhodamine 6G, staurosporine, cytochalasin D, bafilomycin A1, 4-nitroquinolineN-oxide, trifluoperazine, carbonyl cyanidem-chlorophenylhydrazone, verapamil, tri-n-methyltin chloride, and tri-n-butyltin chloride were purchased from Sigma. Oligomycin, cyclosporin A, and protein kinase A inhibitor 14–22 amide were purchased from Calbiochem. The sources of other drugs used in this study were as follows: Fluconazole (Pfizer Ltd., Sandwich, Kent, UK), Itraconazole (Jannssen Research Foundation, Beerse, Belgium), terbinafine HCl (Novartis Pharma K.K., Tokyo, Japan), adriamycin (Kyowa Hakko, Tokyo, Japan), latrunculin A (Wako), aureobasidin A (Takara), G418 (Invitrogen), tri-n-ethyltin chloride (Strem Chemicals, Newburyport, MA), tri-n-propyltin chloride (Merck), tri-n-pentyltin chloride (Kanto Chemicals Co., Inc., Tokyo, Japan), FK506 (Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan), H-89 and H-8 (Seikagaku Corp., Tokyo, Japan). Theonellamide F and calyculin A were kindly provided by Prof. Fusetani (University of Tokyo). In some experiments, drug susceptibility of yeasts grown in YEPD was measured using a 96-well microtiter plate assay. Yeasts cultured in YEPD until early stationary phase were diluted toA 600 = 0.016 in YEPD in microtiter plate wells (Nunc, Roskilde, Denmark), drugs were added to the indicated final concentration, and the cells were incubated at 30 °C for 48 h. The growth of the cells in individual wells was measured with a microplate reader (EL 312e; Bio-Tek Instruments, Winooski, VT) at 590 nm. The MICs of antifungal agents forS. cerevisiae cells were determined by a microdilution test based on the macrodilution reference method of the National Committee for Clinical Laboratory Standards (32National Committee for Clinical Laboratory Standards Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast, Approved standard M27-A. National Committee for Clinical Laboratory Standards, Wayne, PA1997Google Scholar). Cells (10-μl suspension of 2 × 105 cells/ml) were inoculated into 90 μl of CSM buffered with MES and HEPES in microtiter plate wells. The wells contained doubling dilutions of antifungal agents in the CSM (final concentrations were as follows: fluconazole, 0.058–1024 μg/ml; itraconazole, 0.004–128 μg/ml; ketoconazole and miconazole, 0.002–32 μg/ml; flucytosine and nystatin, 0.031–64 μg/ml; amphotericin B, 0.0078–16 μg/ml; tri-n-methyltin chloride, 0.098–50 μm; other tri-n-alkyltin chlorides, 0.0098–5 μm). The microtiter plates were incubated at 35 °C for 48 h, and then the growth of cells in individual wells was measured with a microplate reader. The MIC80 was the lowest concentration of drug that inhibited the growth yield by at least 80% compared with the growth for a no-drug control. The efflux of rhodamine 6G from S. cerevisiae cells was determined as previously reported (26Nakamura K. Niimi M. Niimi K. Holmes A.R. Yates J.E. Decottignies A. Monk B.C. Goffeau A. Cannon R.D. Antimicrob. Agents Chemother. 2001; 45: 3366-3374Crossref PubMed Scopus (171) Google Scholar, 33Kolackowski M. van der Rest M. Cybularz-Kolaczkowska A. Soumillion J.-P. Konings W.N. Goffeau A. J. Biol. Chem. 1996; 271: 31543-32548Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) with slight modification. Yeast cells (1 × 109 cells) from YEPD cultures in late exponential growth phase (A 600 = 5.0–8.0) were collected by centrifugation (3,000 × g, 5 min, 20 °C) and washed twice with HEPES-buffered saline (HBS) containing 50 mm HEPES-NaOH (pH 7.0) and 100 mm NaCl. After 2 h of incubation in HBS at 27 °C with shaking (150 rpm), the cells were centrifuged as above and suspended in 4 ml of HBS supplemented with 5 mm 2-deoxyglucose and 10 μm rhodamine 6G. The cell suspension was incubated at 27 °C with shaking for 90 min to allow rhodamine accumulation under glucose starvation conditions. The starved cells were washed twice in HBS and finally suspended in 7.6 ml of HBS. Glucose (25 μl, 40 mm) was added to a portion of the cell suspension (475 μl) to initiate rhodamine 6G efflux. At specified intervals after the addition of glucose, the cells were removed by centrifugation, and triplicate 100-μl volumes of the supernatants were transferred to the wells of 96-well flat-bottom microtiter plates. The rhodamine 6G fluorescence of the samples was measured with a CytoFluor Series 4000 spectrofluorimeter (PerSeptive Biosystems, Inc., Framingham, MA). The excitation wavelength was selected using a 530/525 filter, and emission was detected using a 580/550 filter. Nucleotide triphosphatase (NTPase) activity of plasma membrane fractions was measured by adapting a previously described method (26Nakamura K. Niimi M. Niimi K. Holmes A.R. Yates J.E. Decottignies A. Monk B.C. Goffeau A. Cannon R.D. Antimicrob. Agents Chemother. 2001; 45: 3366-3374Crossref PubMed Scopus (171) Google Scholar). Purified plasma membrane samples (2.5 μg of protein), prepared as described above, were incubated in a solution (final volume 30 μl) containing 6 mm NTP and 7 mm MgSO4 in 59 mm MES-Tris buffer (pH 4.0–8.5). To eliminate possible contributions from nonspecific phosphatases and vacuolar or mitochondrial ATPases, 0.2 mmammonium molybdate, 50 mm KNO3, and 10 mm NaN3 were included, respectively, in the assay mixtures (30Monk B.C. Kurtz M.B. Marrinan J.A. Perlin D.S. J. Bacteriol. 1991; 173: 6826-6836Crossref PubMed Google Scholar). Oligomycin (20 μm) was added to the assay for the control reactions. After a 30-min incubation at 30 °C, the reaction was stopped by the addition of 32.5 μl of a solution containing 1% (w/v) SDS, 0.6 mH2SO4, 1.2% (w/v) ammonium molybdate, and 1.6% (w/v) ascorbic acid. The amount of inorganic phosphate released from NTPs was measured at 690 nm after 10 min of incubation at room temperature. KH2PO4 solutions (0.4–2 mm) were used to obtain a standard curve. S. cerevisiae was chosen for the analysis ofC. glabrata efflux proteins for several reasons. It is a tractable yeast amenable to molecular genetic manipulation. It has been well studied, and much is known about the genes involved in pleiotropic drug resistance (21Balzi E. Wang M. Leterme S. Van Dyck L. Goffeau A. J. Biol. Chem. 1994; 269: 2206-2214Abstract Full Text PDF PubMed Google Scholar). S. cerevisiae and C. glabrata are closely related (25Barns S.M. Lane D.J. Sogin M.L. Bibeau G. Weisburg W.G. J. Bacteriol. 1991; 173: 2250-2255Crossref PubMed Google Scholar) and probably possess similar machinery for th
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