YeastYeast 2003; 20: 865–880.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1013

Research Article

The pfr1 gene from the human pathogenic fungusParacoccidioides brasiliensis encodes a half-ABCtransporter that is transcribed in response totreatment with fluconazoleChristopher H. Gray†, M. Ines Borges-Walmsley, Gareth J. Evans and Adrian R. Walmsley*Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen’s Campus, Stockton-on-Tees TS17 6BH, UK

*Correspondence to:Adrian R. Walmsley, Centre forInfectious Diseases, WolfsonResearch Institute, University ofDurham, Queen’s Campus,Stockton-on-Tees TS176BH, UK..E-mail:a.r.walmsley@durham.ac.uk

†Present address: Section ofStructural Biology, Institute ofCancer Research, Fulham Road,London SW3 6JB, UK..

Received: 12 February 2003Accepted: 5 May 2003

AbstractWe have isolated a gene that encodes a half-ABC-transporter, designated Pfr1,from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis, whichhas high identity with members of the ABC-superfamily involved in multidrugresistance. The pfr1 gene is predicted to encode a 827 amino acid protein that, incommon with mammalian Mdr1, has a TM-NBD topology. The transcription of thepfr1 gene is induced by the triazole drug fluconazole but not by amphotericin B,suggesting a role in transport-mediated azole resistance. However, Pfr1 has greatestidentity to the mitochondrial ABC transporters Mdl1 and Mdl2 from Saccharomycescerevisiae and mammalian ABC-me, with identities of 47.2%, 40.6% and 39.5%,respectively, over the length of these proteins. Furthermore, the N-terminus of Pfr1is rich in positively charged residues, a feature of mitochondrial targeting sequences.Considering these features, it seems likely that Pfr1 is a mitochondrial protein.Previous studies have revealed that the acquisition of azole resistance in S. cerevisiaeis linked to mitochondrial loss and, conversely, that mitochondrial dysfunction canlead to the upregulation of PDR transporters mediated by the transcription factorPdr3. Our studies suggest that a mitochondrial ABC transporter is induced as partof the cellular response to drug treatment. The promoter region of pfr1 containsa PDRE-like consensus sequence to which Pdr3 binds, which may be the elementresponsible for the upregulation of Pfr1 in response to fluconazole. The nucleotidebinding domain of Pfr1 was expressed and purified from Escherichia coli and shownto retain ATPase activity, consistent with Pfr1 functioning as a homodimeric transportATPase. Copyright 2003 John Wiley & Sons, Ltd.

Keywords: Paracoccidioides brasiliensis; ABC-transporter; antifungals; drug resis-tance; human pathogenic fungus; mitochondria

Introduction

The dimorphic, human pathogenic fungus Para-coccidioides brasiliensis is the aetiological agentof paracoccidioidomycosis, the most prevalent sys-temic mycosis in Latin America (McEwen et al.,1995). It is estimated that throughout the endemicregion, in which about 90 million people live,as many as 10 million of those individuals maybe infected (Restrepo et al., 2001). The fungus

undergoes a complex transformation in vivo, withmycelia in the environment producing conidia,which probably act as infectious propagules uponinhalation into the lungs, where they transform tothe pathogenic yeast form (Medoff et al., 1987;for reviews, see San-Blas et al., 2002; Borges-Walmsley et al., 2002). A change in temperatureis the only factor required to trigger the trans-formation (San-Blas et al., 2002). The pathogenic-ity of the fungus is intimately linked to this

Copyright 2003 John Wiley & Sons, Ltd.

866 C. H. Gray et al.

morphological transition, since strains that areunable to transform into the yeast form are avir-ulent (San-Blas and Nino-Veja, 2001). Infectioncan give rise to either an asymptomatic condi-tion or to active disease, and initially causes pul-monary lesions in the lungs but can subsequentlydisseminate to other organs and tissues (Brummeret al., 1993). Regardless of the organ involved,paracoccidioidomycosis usually heals by fibrosis,with the formation of fibrotic sequelae, which canpermanently interfere with the well-being of thepatient. Paracoccidioidomycosis occurs mainly inimmunocompromised individuals and there is astrong gender bias towards males, which is proba-bly attributable to the fact that mammalian oestro-gens inhibit the morphological transition (Aristiza-bal et al., 2002).

The treatment of paracoccidioidomycosis is usu-ally prolonged, with many patients receiving ther-apy for 1–2 years; in the absence of drug therapythe disease is usually fatal. Currently, azoles are thedrugs of choice over sulphonamides and ampho-tericin B, for which patient relapse rates are high(Mendes et al., 1994). Although azole drugs canarrest the progression of paracoccidioidomycosis,the fibrotic sequelae persist, probably constitut-ing a source of P. brasiliensis that can lead toa relapse in the disease following termination oftreatment. Of further concern are reports of clinicalisolates of P. brasiliensis that have elevated levelsof resistance to azole drugs (Rodero et al., 1999).Since multidrug pumps constitute a major mecha-nism of resistance, we have screened P. brasiliensisfor genes that encode ABC-transporters that mightconfer azole resistance. In the course of these stud-ies we have identified a half-ABC-transporter thatbelongs to the Mdr/Tap subfamily of ABC pro-teins that is upregulated in response to treatmentby fluconazole.

Methods

Strains

P. brasiliensis strain Pb 01 (ATCC, MYA-826)was used throughout this study. The Pb01 yeastand mycelium λZAPII cDNA libraries (Petrofezada Silva et al., 1999) and the λDASHII genomic(Pereira et al., 2000) library, which were a giftfrom Dr Maria Sueli Soares Felipe (University of

Brasilia), were maintained in Escherichia coli XL-1 Blue (Stratagene).

Fungal growth

Mycelium and yeast cultures were grown on solidFava-Neto medium (Favo-Neto, 1955) as describedpreviously (Petrofeza da Silva et al., 1999). Fordrug induction experiments, cultures were grownovernight at 37 ◦C in liquid Fava-Neto mediumin an orbital incubator at 180 rpm; drugs werethen added and growth continued for a further 3 h,before cells were collected by either filtration orcentrifugation.

RNA extraction

Total RNA was extracted from cells (1 g wetweight), which had been disrupted by macera-tion after freezing in liquid nitrogen, with Trizol(10 ml/0.5–1.0 g cells) according to the supplier’sinstruction (GibcoBRL). To remove any residualgenomic DNA, the RNA samples were treated with1 unit of RNAse-free DNAse I at room tempera-ture for 15 min, followed by standard procedures(Sambrook et al., 1989). Streptavidin MagnesphereParamagnetic Particles (Promega) were used toseparate mRNA from total RNA according to themanufacturer’s instructions.

Cloning of pfr1

A single 413 bp fragment from P. brasiliensis chro-mosomal DNA, which had high sequence similaritywith mdr2 from Aspergillus fumigatus, was ampli-fied by PCR using degenerate oligonucleotides(primers mdr1 and 2: 5′-GC(C/T)CTCGT(G/C)GG(G/C)CCCTC(G/C)GG-3′ and 5′-GAT(A/G)CG(C/T)TG CTT(C/T)TG(A/G)CC(A/G)CC-3′, resp-ectively) and 2.5 units HotStarTaq (in 100 µl1.5 mM MgCl2, 200 µM dNTPs and 1.5 µM eachprimer) according to the following program: 1 acti-vation cycle at 95 ◦C for 15 min; 25 cycles of 30 sat 94 ◦C, 40 s at 61 ◦C and 1 min at 72 ◦C; fol-lowed by 1 cycle of 10 min at 72 ◦C. Additionalfragments were only amplified under lower strin-gency conditions of annealing temperatures below55 ◦C. The mdr1 and mdr2 primers were used inan initial screen of a λDASHII genomic library andtwo λZAP cDNA libraries constructed from mRNAextracted from P. brasiliensis mycelial and yeastcells. A DNA fragment could only be amplified

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

The ABC-transporter pfr1 from P. brasiliensis 867

from the genomic library, suggesting that the genewas not expressed during construction of the cDNAlibraries. Accordingly, only the genomic librarywas used for further screening: 1.8 × 103 pfu wereplated and screened with the mdr1/mdr2 PCR frag-ment, which had been tagged by incorporation offluorescein nucleotides, using a Gene Images kitfrom Amersham according to the manufacturer’sinstructions. A single clone was identified. To aidin DNA sequencing, this genomic clone was sub-cloned by restriction enzyme digestion and ligationof the resulting fragments into the vector pZERO(Invitrogen). Digestion with XbaI, EcoRI and PstIgave fragments of 2.5, 3.9 and 4.8 kb, respec-tively, that were shown to be positive by South-ern blotting with the mdr1/mdr2 PCR fragment.Although the 2.5 kb XbaI fragment contained mostof the gene, including a well-defined stop codonand polyadenylation signal sequence, comparisonwith mdr2 from A. fumigatus suggested that the5′ end of the pfr1 gene was missing from thisfragment. Single specific primer (SSP)–PCR reac-tions, using a gene specific primer in combina-tion with pZERO primer with the larger (3.9 kb)EcoRI and (4.8 kb) PstI fragments, provided addi-tional sequence for the 5′ end of the pfr1 genebut not the apparent ATG start; nor could thesequence upstream of this ATG be determined by5′ RACE RT–PCR. In order to isolate the 5′ end ofthe gene, Genome-Walker libraries (Clontech) weregenerated for screening by SSP–PCR. P. brasilien-sis DNA was digested individually with the bluntcutting restriction enzymes DraI, EcoRV, PvuII,StuI and ScaI, and Genome-Walker adapters lig-ated to each of the five restriction digested DNApools. To PCR amplify the 5′ end of the pfr1gene within these libraries, a nested PCR approachwas employed, using sequentially two pairs ofprimers, each pair composed of an adaptor-directedprimer and a gene-specific primer. The two gene-specific primers used to amplify the DNA were 5′-GACGCGCTCGCGCAACGCATTTCTTTCGGAGTT-3′ and 5′-GGTACGCGAGGCACTGCGCTTTTCAGCAATGGC-3′, respectively. DNA frag-ments of 511 and 312 bp were generated fromthe EcoRV and PvuII libraries, respectively, butnot from the other three libraries, and cloned intopGEM-T-Easy and sequenced. Both gave addi-tional 5′ sequence overlapping the conceptual ATGof pfr1.

Sequencing of the P. brasiliensis pfr1 gene

Both strands of the pfr1 genomic clone weresequenced by dideoxynucleotide chain terminationprocedure and by primer-walking with syntheticprimers on an ABI377 or LiCor automated DNAsequencer (BaseClear). The pfr1 gene sequencewas assembled using Autoassembler (Perkin-Elmer); and analysed using the program VectorNTi(Informax).

RT–PCR and 5′ RACE RT–PCR

Standard RT–PCR was performed using theAccess RT–PCR system (Promega). The follow-ing oligonucleotide primers were used: P1, 5′-GTTCGGCCTCACACTCCCCGT-3′; P2, 5′-TTCACATTTGCTATCCA TCT-3′; P3, 5′-TATTGTTGCGCGACTAA-3′; hsp1, 5′-GGTCTAGAAAGCTAC GCC TA-3′; and hsp2, 5′-GGTCTAG AAAGC-TACGCC TA-3′. In order to determine thetranscriptional start site of the pfr1 gene,nested 5′ SMART RACE RT–PCR (Clon-tech) was performed with the gene-specificprimers 5′-GCAAGCAGAGTAACGCC-3′ and 5′-AGCCGAGCCTCTTCCCGC-3′.

Northern blotting

RNAs were separated by electrophoresis on formal-dehyde-containing 1.5% agarose gels and trans-ferred by vacuum blotting onto nylon membranes(Hybond-N, Amersham), which were hybridizedwith the radiolabelled pfr1 PCR fragment underhigh-stringency conditions of 0.1× SSC/0.1% w/vSDS for 2 h at 68 ◦C, according to standard proce-dures (Sambrook et al., 1989).

Nucleotide and protein sequence analysis

BLAST searches, using the ExPASy molecularbiology server http://us.expasy.org/tools/blast/,were used to identify proteins that share sequencesimilarity with Pfr1. Protein sequence alignmentswere performed with the Align program (Vec-torNTi, Informax). TATA-box elements were iden-tified using the HC tata program at http://125.itba.mi.cnr.it/∼webgene/wwwHC tata.html. Transc-riptional start sites were predicted using the Neu-rol Network Prediction Program at http://www.fruitfly.org/seq tools/promoter.html. The pfr1

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

868 C. H. Gray et al.

sequence was screened for potential transcrip-tion factor binding elements using the programTFSEARCH at http://www.cbrc.jp/research/db/TFSEARCH.html. The TMpred program at http://www.ch.embnet.org/software/TMPRED form.html was used to predict the number of transmem-brane helices.

Overexpression of the Pfr1 nucleotide bindingdomain

The sequence encoding the NBD of Pfr1, residues516–756, was amplified by RT–PCR. The forwardand reverse primers 5′-CGGGATCCGACCAGACCCGCAGTGACTGTATTT-3′ and 5′-GTGCTCGAGAACCACCGACTGCACAGCCCCCCC-3′were used to amplify the sequence and introduce 5′and 3′ flanking XhoI and BamHI sites, respectively.The cDNA fragment was A-T cloned into pGEM-T Easy (Promega), recovered by XhoI–BamHIrestriction digest, and ligated into the correspond-ing sites in pET21b (Novagen) to produce thepET21b–Pfr1NBD construct that was used totransform Epicurian coli BL21-CodonPlus (DE3)-RIL cells (Stratagene). The insert region of thepET-construct was sequenced in both directions toensure its sequence integrity and that it was in-frame with the T7 and His6-tags.

A single colony of the transformant was used toinoculate a starter culture of LB media (containing100 µg/ml carbenicillin, 100 µg/ml chlorampheni-col). Outgrowth was allowed at 37 ◦C, with shak-ing at 200 rpm, until A600 = 0.5 was reached. Thestarter culture was then added as 1/10 volume to theLB media intended for expression and growth con-tinued under the same incubation conditions untilA600 = 0.6 was attained. At this point 1 mM IPTGwas added to induce expression of the Pfr1 NBD,the incubation temperature was reduced to 18 ◦Cand growth continued overnight. For most prepa-rations we cultivated 6 l cells (e.g. 12 × 0.5 l),which were harvested by a 7 K centrifugal spin andwashed with 50 mM Tris–HCl (pH 8.0)/150 mM

NaCl/10% glycerol (TNG buffer). The cell pel-let from a 6 l culture was resuspended in 80 mlTNG buffer, containing 10 mM imidazole, 1.0 ml10 mg/ml lysozyme, 0.2 ml 10 mg/ml DNAaseand 1 protease inhibitor cocktail tablet (complete,EDTA — free, Roche). The cells were disruptedby passage through a Constant Systems cell dis-rupter (Model Z-plus 1.1 kW) operated at 4 ◦C.

Unbroken cells and cell debris was cleared from thesupernatant by a 20 min 18 K rpm centrifugal spinat 4 ◦C; which was then spun at 43 K rpm for 1.5 hat 4 ◦C, separating the soluble protein in the super-natant from the cell membranes in the pellet. Tothe supernatant, 6 ml suspended Ni2+-NTA agarose(Qiagen) and 1 protease inhibitor tablet was addedand incubated on a gently rotating (blood-tube,Bibby) rotator for 1 h at 4 ◦C. A 1.5 cm diame-ter Econo-column (BioRad) was packed with theNi2+-NTA agarose and washed with 20 vol 40 mM

imidazole in TNG buffer; before elution of theprotein, under gravity, with 250 mM imidazole inTNG buffer. The protein, which was generally col-lected at a concentration of 0.1–0.2 mg/ml, wasdialysed against TNG buffer to remove the imi-dazol and quickly frozen and stored at −80 ◦C.Protein concentrations were determined by theBCA assay (Pierce) with BSA as standard. Pro-teins were separated by SDS–PAGE on a 4–12%polyacrylamide gradient gel and stained with Gel-code Blue (Pierce); or transferred to nitrocellulosemembranes and immunoblotted with monoclonalanti-His6 antibodies (Sigma).

ATPase assays

The ATPase activity of the purified Pfr1 NBDwas assayed using a malachite green/ammoniummolybdate assay for inorganic phosphate (Pi)developed for use in a 96-well microtitre plateformat (Harder et al., 1994). The protein wasincubated with varying concentrations of ATP, inTN buffer, at 37 ◦C; the reaction was initiatedby the addition of 2 mM MgCl2 and 45 µl sam-ples removed at 10 min intervals and mixed with5 µl EDTA to stop the ATPase reaction. Sampleswere developed by the addition of 100 µl mala-chite green/ammonium molybdate in 6 N HCl;absorbances at 610 nm were determined usinga Packard plate reader, and the Pi concentrationin each assay sample determined by comparisonwith a Pi standard curve. In control experiments, noATPase activity was apparent when the MgCl2 wasomitted. For each ATP concentration, the reactionrate was determined by linear regression analysisof the seven time points (0, 10, 20, 30, 40, 50and 60 min). No deviation from a straight line wasapparent for any of the ATP concentrations, indicat-ing that our measurements are of true initial rates.The Km and Vmax were determined from a plot of

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

The ABC-transporter pfr1 from P. brasiliensis 869

the reaction rate vs. the [ATP] by non-linear regres-sion using SIGMA PLOT (SPSS Inc.).

Nucleotide sequence Accession No.

The Accession No. (EMBL) for P. brasiliensis pfr1gene is AJ 558104.

Results

Cloning and analysis of the nucleotide sequenceof the pfr1 gene from P. brasiliensis

In an attempt to identify mdr-like genes encod-ing antifungal drug transporters in P. brasilien-sis, degenerate oligonucleotides were designed totarget the ALVGPSG and GGQKQRI motifs thatare conserved in the nucleotide binding domainsof proteins displaying sequence similarity withhuman Mdr1. Using these primers, a fragment wasraised from P. brasiliensis chromosomal DNA thathad sequence similarity with a number of ABC-transporters, including human MDR1 and A. fumi-gatus Mdr2, and used to screen a P. brasiliensisgenomic library from which a clone was identified.When compared to AfuMdr2, the sequence of thisclone suggested that the homologous P. brasiliensisgene had been truncated at the 5′ end; and Genome-Walker SSP–PCR was used to obtain the entiresequence. We termed this P. brasiliensis gene Pfr1(for Paracoccidioides fungicide responsive gene 1)because, as reported below, the transcription of thisgene is induced by fluconazole.

The entire pfr1 gene sequence (Figure 1) indi-cates an open reading frame (ORF) of 2628nucleotides, interrupted by two putative intronsof 69 and 78 nucleotides at positions 1248–1317and 1953–2031, respectively. Consistent with thepredicted ORF, there are several CAAT (i.e. atpositions −180 and −45) and TATA elements(i.e. at positions −260, −227, −197, −93 and−64) upstream of the putative translational startcodon; two transcriptional start sites are predictedat −167 and −140; while a putative polyadeny-lation signal (AATAGA) overlaps the stop codon.The 5′ and 3′ ends of the two putative intronsobey the GT/AG rule for donor and acceptor splic-ing sites (Mount, 1982) and both contain inter-nally conserved sequences, TACTAAT and CAC-TAAT respectively, that resemble the consensus

TACTAAC that is important for branch site forma-tion (Langford et al., 1984). In order to preciselydefine the intron splice sites, the processing of pfr1mRNA extracted from fluconazole treated cells wasinvestigated by RT–PCR, using the forward primerP1 to target nucleotide position 663 and the reverseprimer P2, which targets the stop codon, to amplifya cDNA fragment spanning both putative introns.As expected for the spliced template, this reac-tion amplified a fragment of about 2 kb, which wascloned into pGEM-T-Easy and its DNA sequencewas determined, revealing an identical sequence tothe genomic clone but lacking the introns.

The putative 5′ UTR (untranslated region) con-tains sequences that resemble those for the bind-ing of the transcription factors Pdr1/Pdr3 (i.e.TCCT CGGA at position −161), GATA (i.e. GATAat positions −229, −155 and −35), StuAp (i.e.TCGCGC TA at position −138), Nit2 (i.e. TATCTat positions −133 and −116), Xbp1 (i.e. TCGAat positions −262 and −109), Hsf (i.e. AGAAat position −249) and E2F (i.e. TTCGGCGC andTATCGCGC at positions −190 and −141, respec-tively) and for bHLH transcription factors thatbind to an E-box (i.e. CAGCTG at position −77).Pdr1 and Pdr3 are transcription factors that con-trol the expression of a number of genes involvedin pleiotropic drug resistance in S. cerevisiae, e.g.they control the expression of the Pdr5 multidrugpump (Balzi and Goffeau, 1995). The GATA tran-scription factors, which bind a (T/A)GATA(A/G)sequence element, form a large family that controla number of processes in fungi, including nitrogenmetabolism, siderophore biosynthesis, and devel-opment (Scazzocchio, 2000). Nit2 is a GATA tran-scription factor that regulates the expression ofgenes involved in nitrogen metabolism in fungi(Chiang and Marzluf, 1994). However, it also bindsa TATCT sequence element that is complementaryto the AGATA element. Generally, two of theseelements, spaced 10–30 bases apart, are requiredfor strong binding (Chiang and Marzluf, 1994).Consistent with a binding site for a Nit2-like tran-scription factor, we note that the two TATCT ele-ments within the 5′ UTR of the pfr1 gene are 17bases apart. The presence of a putative bindingsite for Xbp1 is interesting because this transcrip-tion factor is also nitrogen-responsive, mediatingthe repression of the cell cycle regulator Clb in S.cerevisiae under nitrogen-limiting conditions andpreventing the formation of pseudohyphae (Miled

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

870 C. H. Gray et al.

-276

-240

-180

-120

Xbp1 Hsf atcgcctatagctctcgagatcctcctagaatcctc

GATA E2Faagatcctctagatatatattgtctatccgatctcttagctcttataagcttcggcgccg

PDRE GATA E2F StuAp Nit2caataatcgctataagcgctcctcggatatatacacacatatcgcgctatcttcgctatc

Nit2 E-boxggggtatctcttcgattctccgattcttatatgccgctaaatccagctgaatcaaatata

GATA

1 atggcacttgccgccttcactccacgagcatcttcacgtcgcattgctcagtttggatcg M A L A A F T P R A S S R R I A Q F G S 20

61 cgatcgcttcccttatttaactatacctccttccatgcccagagaccatcgcttgatgct R S L P L F N Y T S F H A Q R P S L D A 40

121 agcggtggcttaagacgtcaccagtcgaacgcccccatcgtaacccaacgaccagacatc S G G L R R H Q S N A P I V T Q R P D I 60

181 ctacggcagccagacatccgaaagtcatctgcaacaaatatacatgtcactatttcatct L R Q P D I R K S S A T N I H V T I S S 80

241 atcgtgggacctggttgtcttcagcaggtccgattcctctcctctacgacagcacggcga I V G P G C L Q Q V R F L S S T T A R R 100

301 aaagaagccaaaccggagcccgcgaaaaagctgcggaagccgcagaagccgcagacagag K E A K P E P A K K L R K P Q K P Q T E 120

361 ctagaaaagaaacaagaaaccgctctagaagagatacatagaggatttgagcgtacggag L E K K Q E T A L E E I H R G F E R T E 140

421 aaggcatcacaagcagcgaaattaaaccttagcgcaagattatcaaaggatgcgggcggg K A S Q A A K L N L S A R L S K D A G G 160

481 aagaggctcggctttcgcgagatatggcggttattgaaaattgcccgtccagaggccaaaK R L G F R E I W R L L K I A R P E A K

MTS180

541 atcctgtccatggcgttactctgcttgctcatctcgtcatctatcaccatgtcgattccg I L S M A L L C L L I S S S I T M S I P 200

601 ttttccatcggcaagattctcgatatcgcaacgcatagcagccctgaaggtggcaatgaa F S I G K I L D I A T H S S P E G G N E 220

661 ttgttcggcctcacactccccgtcttctacagtgtcctgggcggtgttcttttgttgggt L F G L T L P V F Y S V L G G V L L L G 240

721 gctgctgcaaactgtggtcgtattataattctacggattgtgggggaacgtattgttgcg A A A N C G R I I I L R I V G E R I V A 260

-60 agctcttagcccgatcaatactcctgatatcctattattctaatctccttgttccttaat

781 cgactaagatctaagctcttcagacgtacttttatgcaggatgcggagtttttcgatgcc R L R S K L F R R T F M Q D A E F F D A 280

841 aacagagtcggtgacttgatttcccggctaagctcggatacgataatcgtcggcaaaagt N R V G D L I S R L S S D T I I V G K S 300

Figure 1. The nucleotide and deduced amino acid sequence of the pfr1 gene from P. brasiliensis. The exon nucleotides areindicated by upper case letters, while the intron, 5′ and 3′ non-translated nucleotides are indicated by lower case letters.Base numbers are on the left and amino acid numbers on the right. Putative CAAT, TATA and polyadenylation signalsare in bold and underlined. Putative regulatory elements are boxed; where these overlap, one elements of an overlappingpair is indicated by italics and the other in bold. The amino acids that make up the Walker A and B sites, ABC signaturesequence and putative MTS are underlined

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

The ABC-transporter pfr1 from P. brasiliensis 871

1081 aacctgggtagcctgaccaaaatcgcggaggagcgtctgggaaatgtgaagacaagtcag N L G S L T K I A E E R L G N V K T S Q 380

1141 tcttttgcaggagagattttggaggtccatcgatataataggcaaaccaggagaattttt S F A G E I L E V H R Y N R Q T R R I F 400

1320 ggtttgatgggaaatatgacgattttgagtttactctatgttggtggtggaatggtccag G L M G N M T I L S L L Y V G G G M V Q 437

1380 tccggggccatttccattggagacttgacttcattcctcatgtatgcagcatacgctgga S G A I S I G D L T S F L M Y A A Y A G 457

1440 tcaagcatgtttggcttgtccagcttctattcagaactgatgaagggcgttggtgctgcc S S M F G L S S F Y S E L M K G V G A A 477

1500 agtcgactattcgagctccaagaccgccatcccactattccccttactgtaggagataaa S R L F E L Q D R H P T I P L T V G D K 497

1560 gtggtttcggcaaggggagcgatccggttcaagaatttggatttcagttatcccaccaga V V S A R G A I R F K N L D F S Y P T R 517

1620 cccgcagtgactgtatttaaagacctcgactttgagattccgcaaggaagcaacgttgca P A V T V F K D L D F E I P Q G S N V A 537

1680 atcgtcggcccctcggggggcggtaaatccacgattgcctccttgttactgcgcttctac I V G P S G G G K S T I A S L L L R F Y

Walker A

557

1740 aaacataccagaggccagatcctgatagatgggaaggacatctcctcaatgaatgcaaag K H T R G Q I L I D G K D I S S M N A K 577

1800 tctctacgaaggaaaatcggcgtcgtcgcgcaggagcccgtactcttctctggaactatc S L R R K I G V V A Q E P V L F S G T I 597

1201 gagctgggaaagagagagtctctcgttagcgcagcgttctttagcaccgtgagtacttc E L G K R E S L V S A A F F S T 416

1260 taatcactaattttcatcaaatctggtatatgctgacagctatttgtatctttgcagaccT 417

1860

1920

1979

2039

2099

gctgagaacatatcctacggcaagccccatgcgacaaggacggaaattatcgcagcggcg A E N I S Y G K P H A T R T E I I A A A

cgcaaggccaactgtcaattcatcagtgacttcgtaagtgacatctaccttcctctcca R K A N C Q F I S D F

agcattcccacctacccggtttactaatacaagactcgtttctcgattcagcccgatggc P D G

ctcgacacacacgtaggcgcccgcggcgctcaactctccggcggccaaaagcagcgaatc L D T H V G A R G A Q L S G G Q K Q R I Walker Bgccatcgcccgtgccctgattaaaaacccagacatcctcatcctcgacgaagccacgtcc A I A R A L I K N P D I L I L D E A T S ABC-signature

617

628

631

651

671

901 atcacgcagaatctttcagatggacttagggctgctgttagtggcgttgctgggtttggt I T Q N L S D G L R A A V S G V A G F G 320

961 ctgatggcctttgtcagtcttaagctgtcaagcattctccttttgctgatacccccagtg L M A F V S L K L S S I L L L L I P P V 340

1021 tcccttggagcgttcttatacggcagatcaatcagaaatattagtcgcaagattcagaag S L G A F L Y G R S I R N I S R K I Q K 360

Figure 1. Continued

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

872 C. H. Gray et al.

2339

2399

2459

2519

2579

2639

2699

2759

2819

2879

2939

2999

cgaccggacggcgcgtttacgaagcttatggagtggcagatgagctctgaagggggggct R P D G A F T K L M E W Q M S S E G G A

gtgcagtcggtggttaggggaccgccgtctgagaaggaggagttgcagcagatgctgcag V Q S V V R G P P S E K E E L Q Q M L Q

gaaggggaggaagattatggggagtatgatgatgatagtgatgcggagccggagaagctt E G E E D Y G E Y D D D S D A E P E K L

gttgagagggacggcgttgctgagggagcttctaaggagaaatatgctgtggctgctggg V E R D G V A E G A S K E K Y A V A A G Poly-Aatagaggccagtattgctacttctaagcaacagccgtcccaagaaaaatagatggatagc I E A S I A T S K Q Q P S Q E K -

aaatgtgaatactctttgcttgctgtgtcacttccctcctctttgttccccagctcctat

catcccagtcccaaaggtcacgatcctgtggtctcgtccttcaaggcctgaagaccatag

attcgtttctcctttatcctctgcccatcttaccccgtcctccatcgcataggactttgc

taaaccatacccttgaactttctcttttttctttccctccatcatctatggatgcatgat

atttggtattttaccgtgcttcctcatgcatttatatattatatctagatgcatgctcga

gcggccgccagtgtgatggatatctgcagaatttccagcacactggcggccggtactagt

gga

751

771

791

811

827

2159 gccctcgatgccgaatccgagacgctggtcaacagcgctcttgccgcgttactccgcggc A L D A E S E T L V N S A L A A L L R G 691

2219

2279

aacaatacgacaatcagcatcgcccaccgtctctccaccatcaagcgctccgacaccatc N N T T I S I A H R L S T I K R S D T I

atcgtactcagcggtgacggccacgttgcggagcaaggttcgtatcaggaactgagcgcg I V L S G D G H V A E Q G S Y Q E L S A

711

731

Figure 1. Continued

et al., 2001). StuA is a member of the family ofbasic helix–loop–helix (bHLH) transcription fac-tors, which regulate cell cycle progression and mor-phological changes in fungi (Dutton et al., 1997;Borneman et al., 2002). We have also noted thepresence of an E-box (i.e. CANNTG sequence ele-ments) within the 5′ UTR of the pfr1 gene, whichare bound by a number of bHLH transcription fac-tors, e.g. Efg1 that controls hyphal development inC. albicans (Leng et al., 2001). In addition to thesepotential regulatory sites, there is an E-box which ismore commonly a feature of mammalian and plantgenes that possess the E2F transcription factor, butthese have also been found and are functional infungi (Zhang et al., 1999). E2F is involved in cell-cycle progression; and genes involved in nitrogenmetabolism are activated at the G1 –S phase tran-sition by E2F (Farnham et al., 1993).

The deduced amino acid sequence

The deduced primary sequence reveals a proteinof 827 amino acids, with a predicted Mrof 89.522 kDa and a pI of 9.68 (Figure 1).

Comparative analysis of the deduced P. brasiliensisprotein revealed a close homology to sequencesto transporters belonging to the ABC-superfamilyfrom other organisms, but particularly with theB (i.e. MDR/Tap) subfamily (e.g. the N- andC-terminal halves of human Mdr1 have 29.4%and 30.4% identity to Pfr1 over their lengths),with the highest identity of 65.1% to AfuMdr2from A. fumigatus (Tobin et al., 1997) (Figure 2).We observed the presence of three conservedsequence motifs in Pfr1 that are characteristic ofthe ABC superfamily: (a) GPSGGGKS (residues540–547) of the Walker A motif; (b) LSGGQ(residues 643–647) of the Walker B motif;and (c) RALIKNPDILILDEATSALD (residues655–674) of the ABC signature sequence. Incontrast to many fungal ABC-transporters, suchas AtrA (Del Sorbo et al., 1997), Pdr5 (Balziet al., 1994; Bissinger and Kuchler, 1994) and Cdr1(Prasad et al., 1995), the sequences of the WalkerA and B motifs of Pfr1 are well conserved, withlittle or no degeneracy from that of human Mdr1.

An analysis of the hydropathy of Pfr1 indicatedthat the protein can be divided into hydrophilic

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

The ABC-transporter pfr1 from P. brasiliensis 873

1 50 Pfr1 (1) ----MALAAFTPRASSRRIAQFGSRSLPLFNYTSFHAQRPSLDASGGLRR AFuMDR2 (1) MRGIRSLPCWAPGLSTKRIPPR-ELFADLFPNACVISARHSARNGLIRQF ScMDL1 (1) -------------------------------------------------- ScMDL2 (1) ------------------MKTYVLLYGKLIMTTMILNTGRFEEWYKVCII NcMDL (1) ---MVTGVAVRRAVLLPQCGGSGAGLAALLPFQRSPTMTSSILSAKFCRPConsensus (1) I A L S 51 100 Pfr1 (47) HQSNAPIVTQRPDILRQPDIRKSSATNIHVTISSIVGPGCLQQVRFLSST AFuMDR2 (50) SGCSGSISNSCNPRPYRSAITSLLSANVCSKGVSAVQPRFLSTVRLFSTS ScMDL1 (1) ---------------------------MIVRMIRLCKGPKLLRSQFASAS ScMDL2 (33) ALKEKEIYVPSSPIAMLNGRLPLLRLGICRNMLSRPRLAKLPSIRFRSLV NcMDL (48) FSSVQRPSTTSFTIVSVPFSSSHPGSRPCVQSQRWTSPFGLLRQLSTSQTConsensus (51) I I ICV MIS P L VRF S S 101 150 Pfr1 (97) TARRKEAKPEPAKKLRKPQKPQTELEKKQETALEEIHRGFERTEKASQAA AFuMDR2 (100) QR-----SLEPKSNVKSTGGQVVRPELHQDQEHEDIEKGFELSERAAQAA ScMDL1 (24) ALYSTKSLFKPPMYQKAEINLIIPHRKHFLLRSIRLQSDIAQG-KKSTKP ScMDL2 (83) TPS---SSQLIPLSRLCLRSPAVGKSLILQSFRCNSSKTVPETSLPSASP NcMDL (98) RLR-EQPAKEAAAETAEEAAKDVEKVKEYTSEEHLKVHGFTKSERAHKAAConsensus (101) SS EP K V K S I KGF SEKAS AA 151 200 Pfr1 (147) KLNLSARLSKD---AGGKRLGFREIWRLLKIARPEAKILSMALLCLLISS AFuMDR2 (145) QVNLSAKLAKDG--AAGKKAGFKEIWRLLLIARPEAKKLALAFLFLLVSS ScMDL1 (73) TLKLSNANSKS--------SGFKDIKRLFVLSKPESKYIGLALLLILISS ScMDL2 (130) ISKGSARSAHAK--EQSKTDDYKDIIRLFMLAKRDWKLLLTAILLLTISC NcMDL (147) HINMSARLSKDGKSQSGTKPGFAEVWRLIKIARPEVKAMSVAFVLLLISSConsensus (151) LNLSARLSKD GKK GFKEIWRLLLIARPEAK LSLALLLLLISS 201 250 Pfr1 (194) SITMSIPFSIGKILDIATHSS-----PEGGNELFGLTLPVFYSVLGGVLL AFuMDR2 (193) GITMSIPFSIGKIMDTSTKAT-----TEGGNELFGLSLPMFYGALAGILT ScMDL1 (115) SVSMAVPSVIGKLLDLASESDGEDEEGSKSNKLYGFTKKQFFTALGAVFI ScMDL2 (178) SIGMSIPKVIGIVLDTLKTSSGS-DFFDLKIPIFSLPLYEFLSFFTVALL NcMDL (197) AVTMSIPFSIGRILDLSTQG------PAGEVRLFGLTLYQFFGGLAGLLTConsensus (201) SITMSIPFSIGKILDLAT SS EG N LFGLTL FFSALAGVLL 251 300 Pfr1 (239) LGAAANCGRIIILRIVGERIVARLRSKLFRRTFMQDAEFFDANRVGDLIS AFuMDR2 (238) LGAAANYGRIIILRIVGERIVARLRSKLFRQTFVQDAEFFDANRVGDLIS ScMDL1 (165) IGAVANASRIIILKVTGERLVARLRTRTMKAALDQDATFLDTNRVGDLIS ScMDL2 (227) IGCAANFGRFILLRILSERVVARLRANVIKKTLHQDAEFFDNHKVGDLIS NcMDL (241) LGATANFGRIIILRIVGERVVARLRTNLYRRTYVQDAEFFDANRVGDLISConsensus (251) LGAAANFGRIIILRIVGERIVARLRSKLFRRTFVQDAEFFDANRVGDLIS 301 350 Pfr1 (289) RLSSDTIIVGKSITQNLSDGLRAAVSGVAGFGLMAFVSLKLSSILLLLIP AFuMDR2 (288) RLSSDTIIVGKSITQNLSDGLRAAVSGAAGFGLMAYVSLKLSSILALLLP ScMDL1 (215) RLSSDASIVAKSVTQNVSDGTRAIIQGFVGFGMMSFLSWKLTCVMMILAP ScMDL2 (277) RLGSDAYVVSRSMTQKVSDGVKALICGVVGVGMMCSLSPQLSILLLFFTP NcMDL (291) RLNSDTVVVGKSITQNVSDGLRSMVSGAAGFAAMFWLSPKLTSIILIMVPConsensus (301) RLSSDTIIVGKSITQNVSDGLRAIVSG AGFGLMAFLS KLSSILLILIP 351 400 Pfr1 (339) PVSLGAFLYGRSIRNISRKIQKNLGSLTKIAEERLGNVKTSQSFAGEILE AFuMDR2 (338) PIGLGAFFYGRAIRNLSRQIQRNLGTLTKIAEERLGNVKTSQSFAGEVLE ScMDL1 (265) PLGAMALIYGRKIRNLSRQLQTSVGGLTKVAEEQLNATRTIQAYGGEKNE ScMDL2 (327) PVLFSASVFGKQIRNTSKDLQEATGQLTRVAEEQLSGIKTVQSFVAEGNE NcMDL (341) PIGLGAVLYGRNIRNLSRQIQKNVGSLMKIAEERLGNIKTSQAFAAEVQEConsensus (351) PIGLGA LYGR IRNLSRQIQKNLGSLTKIAEERLGNIKTSQSFAGEVNE 401 450 Pfr1 (389) VHRYNRQTRRIFELGKRESLVSAAFFS-TGLMGNMTILSLLYVGGGMVQS AFuMDR2 (388) VRRYNNQVRKIFELGKKESLISATFFSSTGFAGNMTILALLYVGGGMVQS ScMDL1 (315) VRRYAKEVRNVFHIGLKEAVTSGLFFGSTGLVGNTAMLSLLLVGTSMIQS ScMDL2 (377) LSRYNVAIRDIFQVGKTAAFTNAKFFTTTSLLGDLSFLTVLAYGSYLVLQ NcMDL (391) VGRYNKQVRKIFALGRKEAIVSGVFFSSTSYAGNLAILALLIVGGNLVRSConsensus (401) V RYNKQVRKIF LGKKEALVSA FFSSTGLLGNLTILALL VGG MVQS

Figure 2. Alignment of the amino acid sequences of P. brasiliensis Pfr1 (PbPfr1) with A. fumigatus AfuMdr2; S. cerevisiaeMdl1 and Mdl2; and N. crassa Mdl

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

874 C. H. Gray et al.

451 500 Pfr1 (438) GAISIGDLTSFLMYAAYAGSSMFGLSSFYSELMKGVGAASRLFELQDRHP AFuMDR2 (438) GAITIGELTSFLMYTAYAGSSMFGLSSFYSELMKGVGAASRLFELQDRQP ScMDL1 (365) GSMTVGELSSFMMYAVYTGSSLFGLSSFYSELMKGAGAAARVFELNDRKP ScMDL2 (427) SQLSIGDLTAFMLYTEYTGNAVFGLSTFYSEIMQGAGAASRLFELTDRKP NcMDL (441) GAMSLGDLTSFMMYTVFAGSSLFGVSGFYSELMKGVGAASRLFELEDRKPConsensus (451) GAISIGDLTSFMMYT YAGSSLFGLSSFYSELMKGVGAASRLFELQDRKP 501 550 Pfr1 (488) TIPLTVGDK-VVSARGAIRFKNLDFSYPTRPAVTVFKDLDFEIPQGSNVA AFuMDR2 (488) TISPTKGEK-VASARGPIRFENVTFSYPTRPAVPIFRDLNFEIPQGTNVA ScMDL1 (415) LIRPTIGKDPVSLAQKPIVFKNVSFTYPTRPKHQIFKDLNITIKPGEHVC ScMDL2 (477) SISPTVGHK-YKPDRGVIEFKDVSFSYPTRPSVQIFKNLNFKIAPGSSVC NcMDL (491) AIPQTVGVK-VESAQGPIKFSNVTFAYPTRPAVTIFNGLDFEIPSGTNVCConsensus (501) TI PTVG K V SARGPIRFKNVSFSYPTRPAV IFKDLNFEIP GSNVC 551 600 Pfr1 (537) IVGPSGGGKSTIASLLLRFYKHTRGQILIDGKDISSMNAKSLRRKIGVVA AFuMDR2 (537) IVGPSGGGKSTIASILLRFYSPTEGRVLIGGKDITHMNAKSLRRKIGIVS ScMDL1 (465) AVGPSGSGKSTIASLLLRYYDVNSGSIEFGDEDIRNFNLRKYRRLIGYVQ ScMDL2 (526) IVGPSGRGKSTIALLLLRYYNPTTGTITIDNQDISKLNCKSLRRHIGIVQ NcMDL (540) IVGPSGGGKSTVASLLLRFYNPTSGSITINGIDISKMNAKSLRRRIGMVSConsensus (551) IVGPSGGGKSTIASLLLRFY PTSGSI I G DIS MNAKSLRRKIGIVS 601 650 Pfr1 (587) QEPVLFSGTIAENISYGKP---HATRTEIIAAARKANCQ-FISDFPDGLD AFuMDR2 (587) QEPVLFSGTIAENIAYGKP---QAKRSEIVAAARKANCQ-FISDFPDGLD ScMDL1 (515) QEPLLFNGTILDNILYCIPPEIAEQDDRIRRAIGKANCTKFLANFPDGLQ ScMDL2 (576) QEPVLMSGTIRDNITYGLT--YTPTKEEIRSVAKQCFCHNFITKFPNTYD NcMDL (590) QEPVLFSGTIAENIAYGRP---RAPRTEIIAAAQKANCG-FISDFPEGLEConsensus (601) QEPVLFSGTIAENIAYGKP A RTEIIAAARKANC FISDFPDGLD 651 700 Pfr1 (633) THVGARGAQLSGGQKQRIAIARALIKNPDILILDEATSALDAESETLVNS AFuMDR2 (633) TQVGPRGAQLSGGQKQRIAIARALIKDPDILILDEATSALDAESETLVNS ScMDL1 (565) TMVGARGAQLSGGQKQRIALARAFLLDPAVLILDEATSALDSQSEEIVAK ScMDL2 (624) TVIGPHGTLLSGGQKQRIAIARALIKKPTILILDEATSALDVESEGAINY NcMDL (636) TQVGARGAQLSGGQKQRIAIARALLKDPDILILDEATSALDAESETLVNSConsensus (651) T VGARGAQLSGGQKQRIAIARALIKDPDILILDEATSALDAESETLVNS 701 750 Pfr1 (683) ALAALLRG-NNTTISIAHRLSTIKRSDTIIVLSGDGHVAEQGSYQELSAR AFuMDR2 (683) ALTALLRG-NNTTISIAHRLSTIKRSDTIIVLGPDGRVAEQGSYEELSAR ScMDL1 (615) NLQRRVER-GFTTISIAHRLSTIKHSTRVIVLGKHGSVVETGSFRDLIAI ScMDL2 (674) TFGQLMKSKSMTIVSIAHRLSTIRRSENVIVLGHDGSVVEMGKFKELYAN NcMDL (686) ALAELLKG-RSTTISIAHRLSTIKRSDKIIVLSSEGTVAEIGSYTELSANConsensus (701) ALA LLKG TTISIAHRLSTIKRSD IIVLG DGSVAE GSY ELSA 751 800 Pfr1 (732) PDGAFTKLMEWQMSSEGGAVQSVVRGPPSEKEELQQMLQEGEEDYGEYDD AFuMDR2 (732) PDGAFTKLMEWQMS--GGEVMDQLANTPANPVAQETSWDLQSDDGTEISE ScMDL1 (664) PNSELNALLAEQQDE--------------------------EGKGGVIDL ScMDL2 (724) PTSALSQLLNEKAAPGPSDQQLQIEKVIEKEDLNESKEHDDQKKDDNDDN NcMDL (735) KDSHFSKLMEWQMS--GGDVSPDHRPPSGDPHVSEVEEIEEEFAEAENDVConsensus (751) PDSAFSKLMEWQMS GGDV I E E E GE D 801 847 Pfr1 (782) DSDAEPE-KLVERDGVAEGASKEKYAVAAGIEASIATSKQQPSQEK- AFuMDR2 (780) DTNIPSEPRKID----------------------------------- ScMDL1 (688) DNSVAREV--------------------------------------- ScMDL2 (774) DNNHDNDSNNQSPETKDNNSDDIEKSVEHLLKDAAKEANPIKITPQP NcMDL (783) DDAVEKDVKSHKEPVRE------------------------------Consensus (801) D VE E K

Figure 2. Continued

N- (residues 1–165) and C-terminal (residues485–827) domains that sandwich a hydrophobicdomain (residues 165–485). In common withAfuMdr2, the central domain of Pfr1 was predictedto form five membrane-spanning α-helices (e.g.residues 181–203, 224–243, 312–336, 409–427

and 448–466). Pfr1 can be considered as a‘half-size’ ABC-transporter that more closely rese-mbles human Mdr1, which has a topology inwhich the membrane spanning domain (MSD)precedes the nucleotide-binding domain (NBD),rather than fungal transporters, such as AtrA,

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

The ABC-transporter pfr1 from P. brasiliensis 875

Pdr5 and Cdr1, in which the NBD precedesthe MSD.

Although Pfr1 has highest homology to Afu-Mdr2, this protein has not been characterized. Con-sequently, it is interesting to note that not onlydoes Pfr1 have high identity with the B-subfamilyof ABC transporters but has highest identity witha group of mitochondrial transporters (Lill andKispal, 2001), including Mdl1 and Mdl2 from S.cerevisiae (Dean et al., 1994; Young et al., 2001),the mouse (Shirihai et al., 2000) and human (Zhanget al., 2000) ABCB10 proteins, and the humanABCB8 protein (Hogue et al., 1999). Mdl1, whichhas 47.2% identity with Pfr1 over its length, hasbeen identified as a mitochondrial intracellular pep-tide transporter (Young et al., 2001); whilst mouseABCB10, which has 40.0% identity with Pfr1over its length, is induced by GATA-1 during ery-throid differentiation and may be involved in thetranslocation of intermediates that are utilized inthe biosynthesis of heme (Shirihai et al., 2000).In common with these proteins, Pfr1 has large N-and C-terminal extension that sandwich a puta-tive five-helix membrane spanning domain. Thelarge N-terminal domains of the human and mouseABCB10 proteins have been shown to encodemitochondrial targeting sequences (MTSs). Unfor-tunately there is little conservation of the sequencein these MTSs except that they generally incor-porate a preponderance of positively charged andhydrophobic residues with the potential to formamphipathic α-helices (Folsch et al., 1998). In thiscontext we note that the 180 amino acid sequenceof Pfr1 prior to the first putative membrane-spanning helix incorporates 40 positively chargedresidues, yielding a net positive charge of 25, and71 hydrophobic residues. There is a stretch of 20residues, starting at position 161 of Pfr1, whichincludes seven positively charged residues and 10hydrophobic residues, and has high potential asan amphipathic α-helical MTS. Considering thedegree of identity between Pfr1 and Mdl1, thesimilar topology of Pfr1 with other mitochondrialABC-transporters, and the presence of a putative N-terminal MTS in Pfr1, it is reasonable to concludethat Pfr1 is a mitochondrial homologue of Mdl1.Furthermore, all eukaryotic half ABC-transporterscharacterized to date are found in subcellularorganelles. A BLAST search and phylogeneticanalysis revealed homologues in Aspergillus fumi-gatus (Q43129), Neurospora crassa (Q8NIW1),

S. cerevisiae (Mdl1, P33310; Mdl2, P33311),Schizosaccharomyces pombe (Q9Y7M7) and Can-dida albicans (CaMdl1, P97998) (Figure 3).

Analysis of pfr1 transcription

A screen of mycelium and yeast cDNA librariesfailed to identify the pfr1 gene, suggesting thatthe gene was not constitutively transcribed butmight be inducible by potential substrates, suchas azole drugs. To test this hypothesis, mRNAwas prepared from yeast cells treated with growth-permissive concentrations of the azole drug flu-conazole (1 µg/ml) or amphotericin B (5 µg/ml) for3 h. While fluconazole is a substrate for many fun-gal ABC-transporters, amphotericin B, which inter-calates into the cell membrane, is not. RT–PCRwas used to assess the pfr1 transcript levels inthese mRNA pools. A pfr1 amplicon was only pro-duced from the mRNA from cells treated withfluconazole (Figure 4); indicating that expression

ScMDL1(3.A.1.212.1)CaMDL1

PbPfr1AFuMDR2NcMDL

ScMDL2SpMDL

MmABCB10HsABCB10

Tap1 (3.A.1.209.1)Tap2 (3.A.1.209.1)

HsABCB9HsPGP-alpha (3.A.1.201.1)HsPGP-beta (3.A.1.201.1)

HsABCB6 (3.A.1.212.1)ScAtm1 (3.A.1.210.1)HsABCB7 (3.A.1.210.2)SpHMT1 (3.A.1.210.2)

HsABCB8

Figure 3. Phylogenetic relationship of P. brasiliensis Pfr1 toother ABC transporters. A neighbour-joining bootstrap treeis derived from the amino acid sequences of representativeABC transporters within the same subgroup using theVectorNTi align program. The following proteins wereincluded in the analysis: P. brasiliensis pfr1; A. fumigatusAfuMdr2 (Q43129); N. crassa Mdl (Q8NIW1); C. albicans Mdl(P97998); S. cerevisiae Mdl1 (P33310) and Mdl2 (P33311);Sz. pombe (Q9Y7M7); Homo sapiens ABCB10 (Q9NRK6);H. sapiens Mdr1 (P08183); H. sapiens ABCB6 (Q9NP58);S. cerevisiae Atm1 (P40416); H. sapiens ABCB7 (O75027);Sz. pombe Hmt1 (Q02592); H. sapiens Tap1 (Q03518) andTap2 (Q03519); H. sapiens ABCB9 (Q9NP78); and H. sapiensABCB8 (Q9NUT2). The Mdr1, or PGP, protein was splitinto the N- (α) and C-terminal (β) halves in the analysis.Where the transporter substrate is known, the TC numberis also given alongside the gene name. It is note worthy thatall of the above half-transporters are located in subcellularorganelles

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

876 C. H. Gray et al.

1 2 3 4 5

3054 bp

2036 bp1636 bp

517 bp

298 bp

Figure 4. RT–PCR analysis of pfr1 transcription in P.brasiliensis. Total RNA was extracted from P. brasiliensis yeastcells that had been treated with either fluconazole (1 µg/ml)or amphotericin B (5 µg/ml) for 3 h; from which mRNAwas prepared and used as the target for RT–PCR analysis.The reactions to test for pfr1 expression were primed witholigonucleotides P3 and P2; these oligonucleotides, whichflank the two introns, would amplify a DNA fragment of1710 bp. A fragment of the expected size was generatedfrom the fluconazole-treated mRNA pool (lane 3) but notfrom the amphotericin B-treated mRNA pool (lane 1). As acontrol, the same primers were used with genomic DNA,raising a slightly larger fragment of 1857 bp (lane 5) as wouldbe expected, since this would include the introns. Thesefragments were ligated into pGEM-T-Easy and sequencingconfirmed that both arose from the pfr1 gene but that fromthe mRNA pool lacked the introns. As a positive control,two additional primers, hsp1 and hsp2, were used to targetthe hsp70 gene, which is constitutively expressed in yeastcells of P. brasiliensis. Furthermore, primers were chosento span the 3′ intron of the two introns present in hsp70,which have been shown to be spliced out in yeast cells. Theamplification of this region of the hsp70 gene would yield anamplicon of 412 bp and 340 bp before and after splicing outof the intron, respectively. A single hsp70 amplicon of 340bp was obtained from both the fluconazole treated (lane 4)and amphotericin B (lane 2) treated mRNA pools, and bothDNA fragments were of a similar intensity on an agarosegel; indicating that the mRNA samples were viable, of asimilar concentration, and were not contaminated by DNA.No amplification products were observed when controlreactions were performed on each RNA sample by omittingthe enzyme reverse transcriptase in the first strand cDNAsynthesis reaction (data not shown). The oligonucleotidesequences and the RT–PCR reaction conditions wereas described under Methods; and the RT–PCR reactionproducts were separated on a 2% agarose gel and stainedwith ethidium bromide

of the putative mitochondrial ABC-transporter pfr1is inducible by specific drugs that are commonsubstrates for fungal ABC-transporters that conferazole drug resistance.

Interestingly, 5′-RACE RT–PCR was used tomap the start site to 6 bp downstream of theconceptual ATG for pfr1 and a transcript size of2.6 kb was confirmed by Northern blotting (datanot shown), thus implying that the Pfr1 protein istruncated at the N-terminus because the next in-frame ATG is positioned 550 bases downstream,truncating the protein by 183 residues. Althoughother ATGs are present in the sequence, theywould change the ORF and only correspond toshort polypeptides with little similarity with ABC-transporters. Although this would argue against analternative ORF, we cannot exclude the possibilitythat translation is initiated from an in-frame GTGor TTG codon. There is only one of each of thesecodons in-frame, at positions 244 and 514, whichwould truncate the N-terminus by 81 and 171residues, respectively. It is interesting to note that,while truncation of Pfr1 at positions 244 or 514would still leave a arginine-rich sequence that couldact as a MTS, truncation at position 550 woulddelete the MTS and possibly retarget the protein.

Overexpression, purification and functionalcharacterization of the Pfr1 NBD

In order to establish that the pfr1 ORF encodesa transport-ATPase, we sought to overexpress theprotein for an analysis of its ATPase activity. Ourinitial attempts to overexpress the whole proteinwere unsuccessful, so we sought to overexpressthe NBD, since this might be soluble. This wasnot a trivial aim because, although the NBDs ofother eukaryotic half ABC-transporters, e.g. Tap1and Tap2, have been expressed, they have not beenshown to retain ATPase activity (Lapinski et al.,2000). However, this might be attributable to arequirement for the Tap1 and Tap2 NBDs to inter-act. Indeed, there is considerable evidence indi-cating that the nucleotide binding-sites of ABC-transporters are composed of residues from bothNBDs (Smith et al., 2002). Consequently, demon-strating that the NBD of Pfr1 has ATPase activitywould suggest that it functions as a homodimerictransport-ATPase.

The TMpred program indicated that the cytoso-lic NBD of Pfr1 was located between residues 485

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

The ABC-transporter pfr1 from P. brasiliensis 877

A B

Pfr1 NBD

Pfr1 NBD

1 2 1 2 3 4 5 6 7 8 9

Figure 5. Purification of the overexpressed Pfr1 NBD. (A) A gel showing the level of overexpression of the Pfr1 NBD inE. coli BL21-CodonPlus (DE3)-RIL cells before (lane 1) and after IPTG induction (lane 2). However, most of the protein wasfound in the insoluble fraction. (B) The Pfr1 NBD was purified as a soluble protein from E. coli BL21-CodonPlus (DE3)-RILcells transformed with pET–Pfr1NBD. The cells were disrupted and the debris removed by centrifugation to release thesoluble fraction (lane 1). This was applied to a 6 ml Ni2+-NTA agarose column; monitoring the flow through revealed thedisappearance of a band with a Mr of 28 kDa (lane 2), presumably the Pfr1 NBD that had bound to the column. The columnwas washed extensively with 3 × 40 ml 40 mM imidazole/TNG to elute any protein bound non-specifically to the column(lanes 3–5); protein was then eluted from the column with 250 mM imidazole/TNG and 0.5 ml samples collected (lanes6–9). The purified protein, with a Mr of 28 kDa, was confirmed as the Pfr1 NBD by a Western blot using antibodies tothe His6-tag (data not shown). Protein samples from each step of the purification were run on a 4–12% SDS–PAGE gel,which was stained with Gelcode Blue (Pierce). The five marker bands have molecular weights of 94, 67, 43, 30 and 20 kDa,respectively

and 827. However, a smaller fragment of the C-terminal NBD of murine MDR1 was overexpressedas a soluble protein that retained the nucleotidebinding-site (Conseil et al., 1998). The correspond-ing region of Pfr1 has 49.2% identity to thatof murine MDR1 and so we were encouragedto attempt the overexpression of the equivalentdomain from Pfr1, using pET21b in E. coli strainBL21-CodonPlus. A protein of the predicted Mrof 28 kDa was overexpressed (Figure 5) that wascross-reactive with an anti-His6 antibody (datanot shown). Although the protein was expressedin mg quantities, this was largely as inclusionbodies; but small (1–2 mg) quantities of solu-ble protein that retained ATPase activity couldbe obtained from a large-scale (12 l) preparation.The activity, measured as the rate of phosphaterelease, increased in a hyperbolic manner, indi-cating a Km of 240 ± 54 µM and a maximal rateof 0.56 ± 0.04 µM Pi/min, which corresponds toa specific activity of 332 ± 13 nmol Pi/min/mgof protein (Figure 6). The data confirms that Pfr1is an ATPase and, since it can function indepen-dently of any other subunit, that it functions as ahomodimer.

Discussion

Pfr1 is a putative mitochondrial half ABC-trans-porter that belongs to the Mdr/Tap subfamily ofABC-transporters. Consistent with this prediction,the pfr1 gene encodes a protein that has ATPaseactivity; and since the purified NBD of the pro-tein is active, this suggests that Pfr1 operates asa homodimer. Transcription of the pfr1 gene isinduced upon treatment of P. brasiliensis withazole drugs, potentially via a PDR-like transcrip-tion factor, since there is a PDR element in the 5′UTR of the pfr1 gene. Previous studies have estab-lished a connection between mitochondrial functionand pleiotropic drug resistance in S. cerevisiae.For example, mitochondrial dysfunction causes theupregulation of the Pdr3 transcription factor, whichthen upregulates expression of the Pdr5 multidrugtransporter in the plasma membrane (Zhang andMoye-Rowley, 2001); while conversely, overex-pression of Pdr3 can affect mitochondrial func-tion by increasing the expression of the mito-chondrial import machinery (Koh et al., 2001).The high-frequency acquisition of resistance toazole antifungals in the human fungal pathogenCandida glabrata has been linked to the loss of

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

878 C. H. Gray et al.

Figure 6. The ATP concentration dependency of theATPase activity of the purified Pfr1 NBD. The ATPaseactivity of the purified Pfr1 NBD was assayed using amalachite green/ammonium molybdate assay for inorganicphosphate. The protein (10 µg/ml) was incubated withvarying concentrations of ATP; the reaction initiated bythe addition of 2 mM MgCl2; 45 µl samples were removedat 10 min intervals and mixed with 5 µl EDTA in thewell of a microtitre plate to stop the ATPase reaction.Samples were developed by the addition of 100 µl malachitegreen/ammonium molybdate in 6N HCl; the 610 nmabsorbance was determined and the Pi concentrationcalculated from a phosphate standard curve. For each ATPconcentration, the reaction rate was determined by linearregression analysis of the seven time points (0–60 min).The initial rate is plotted as a function of the ATPconcentration; indicating Km and Vmax values of 240 ± 54 µMand 0.56 ± 0.04 µM/min, respectively

mitochondria and the upregulation of the CgCdr1and CgCdr2 ABC multidrug transporters (Sanglardet al., 2001). Our data gives the first indication ofthe converse relationship, that drug treatment caninduce the expression of a mitochondrial ABC-transporter.

The promoter region also contains several GATAand TATCT elements that suggest that it is regu-lated by nitrogen. The P. brasiliensis LON gene,which also encodes a putative mitochondrial pro-tein, also possesses GATA and TATCT elementsin its promoter region (Barros and Puccia, 2001).Previous studies have shown that the expressionof mitochondrial proteins can be regulated in anitrogen-responsive manner, e.g. by the TOR sig-nalling pathway, in response to changes in nitro-gen source (Komeili et al., 2000; Crespo et al.,2002; Cooper, 2002). In particular, when cells aregrown in a poor nitrogen source, the expression ofgenes encoding the TCA cycle enzymes are upreg-ulated, leading to the production of α-ketoglutarate,

which is a precursor of glutamine, a preferrednitrogen source and a key intermediate in nitrogenmetabolism. The TOR kinases bring about theseeffects by activating the Gln3 GATA transcriptionfactor and the Rtg1 and Rtg3 transcription fac-tors, which, interestingly, are also activated bydysfunctional mitochondria during retrograde reg-ulation (Komeili et al., 2000; Epstein et al., 2001;Crespo et al., 2002; Cooper, 2002). Deletion ofthe RTG genes reduces the level of expressionof Pdr3, and consequently of Pdr5 expression anddrug resistance, indicating that the PDR and RTGsignalling pathways are connected (Hallstrom andMoye-Rowley, 2000). However, this may be due topost-translational effects on Pdr3 because the Rtgtranscription factors usually regulate the expres-sion of genes that carry a R-box (i.e. a GTCACsequence element; Jia et al., 1997).

In conclusion, the present investigation pointsto a drug-induced response in P. brasiliensis thatincludes the induction of the expression of a mito-chondrial ABC-transporter. Considering the factthat the expression of Pfr1 is non-constitutive butinduced by specific drugs would argue that it occursas part of a specific protective response rather thana general stress response. It is conceivable thatthe transporter is used to protect the cell fromthe drug by increasing mitochondrial biogenesis.On the other hand, it might play a more directprotective role, by either removing drugs fromthe mitochondria to protect the mitochondria or,in analogy to the role of the vacuole in com-partmentalizing toxic drugs (Theiss et al., 2002),to sequester drugs in the mitochondria to protectthe cell. Interestingly, our transcript analysis indi-cates that drug treatment induces the expression ofa truncated transporter that might lack the MTS,which could be used as a mechanism to retar-get it to the plasma membrane to serve a role incellular drug efflux. We will be able to addresssome of these questions directly using antibod-ies generated against the active NBD to probethe cellular localization of Pfr1 under differentconditions.

Acknowledgements

Our work on Paracoccidioides brasiliensis is supported bythe Wellcome Trust.

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

The ABC-transporter pfr1 from P. brasiliensis 879

References

Aristizabal BH, Clemons KV, Cock AM, Restrepo A, Stevens DA.2002. Experimental Paracoccidioides brasiliensis infection inmice: influence of the hormonal status of the host on tissueresponses. Med Mycol 40: 169–178.

Balzi E, Goffeau A. 1995. Yeast multidrug resistance: the PDRnetwork. J Bioenerg Biomembr 27: 71–76.

Balzi E, Wang M, Leterme S, Van Dyck L, Goffeau A. 1994.PDR5, a novel yeast multidrug resistance conferring transportercontrolled by the transcription regulator PDR1. J Biol Chem 269:2206–2214.

Barros TF, Puccia R. 2001. Cloning and characterization of a LONgene homologue from the human pathogen Paracoccidioidesbrasiliensis . Yeast 18: 981–988.

Bissinger PH, Kuchler K. 1994. Molecular cloning and expressionof the Saccharomyces cerevisiae STS1 gene product. A yeastABC transporter conferring mycotoxin resistance. J Biol Chem269: 4180–4186.

Borges-Walmsley MI, Chen D, Shu X, Walmsley AR. 2002. Thepathobiology of Paracoccidioides brasiliensis . Trends Microbiol10: 80–87.

Borneman AR, Hynes MJ, Andrianopoulos AA. 2002. Basichelix–loop–helix protein with similarity to the fungalmorphological regulators, Phd1p, Efg1p and StuA, controlsconidiation but not dimorphic growth in Penicillium marneffei .Mol Microbiol 44: 621–631.

Brummer E, Castaneda E, Restrepo A. 1993. Paracoccidioidomy-cosis: an update. Clin Microbiol Rev 6: 89–117.

Chiang TY, Marzluf GA. 1994. DNA recognition by the NIT2nitrogen regulatory protein: importance of the number, spacingand orientation of GATA core elements and their flankingsequences upon NIT2 binding. Biochemistry 33: 576–582.

Crespo JL, Powers T, Fowler B, Hall MN. 2002. The TOR-controlled transcription activators GLN3, RTG1 and RTG3 areregulated in response to intracellular levels of glutamine. ProcNatl Acad Sci USA 99: 6784–6789.

Conseil G, Baubichon-Cortay H, Dayan G, et al. 1998. Flavo-noids: a class of modulators with bifunctional interactions atvicinal ATP- and steroid-binding sites on mouse P-glycoprotein.Proc Natl Acad Sci USA 95: 9831–9836.

Cooper TG. 2002. Transmitting the signal of excess nitrogen inSaccharomyces cerevisiae from the Tor proteins to the GATAfactors: connecting the dots. FEMS Microbiol Rev 26: 223–238.

Dean M, Allikmets R, Gerrard B, et al. 1994. Mapping andsequencing of two yeast genes belonging to the ATP-bindingcassette superfamily. Yeast 10: 377–383.

Del Sorbo G, Andrade AC, Van Nistelrooy JG, et al. 1997.Multidrug resistance in Aspergillus nidulans involves novelATP-binding cassette transporters. Mol Gen Genet 254:417–426.

Dutton JR, Johns S, Miller BL. 1997. StuAp is a sequence-specifictranscription factor that regulates developmental complexity inAspergillus nidulans . EMBO J 16: 5710–5721.

Epstein CB, Waddle JA, Hale W IV, et al. 2001. Genome-wideresponses to mitochondrial dysfunction. Mol Biol Cell 12:297–308.

Farnham PJ, Slansky JE, Kollmar R. 1993. The role of E2F in themammalian cell cycle. Biochim Biophys Acta 1155: 125–131.

Fava-Netto C. 1955. Estudos quantitativos sobre a fixacao docomplemento na Blastomicose Sul-Americana com antıgenospolissacarıdicos. Arq Cirurg Clin Exp 18: 197–254.

Folsch H, Gaume B, Brunner M, Neupert W, Stuart RA. 1998. C-to N-terminal translocation of preproteins into mitochondria.EMBO J 17: 6508–6515.

Hallstrom TC, Moye-Rowley WS. 2000. Multiple signals fromdysfunctional mitochondria activate the pleiotropic drugresistance pathway in Saccharomyces cerevisiae. J Biol Chem275: 37 347–37 356.

Harder KW, Owen P, Wong LK, et al. 1994. Characterizationand kinetic analysis of the intracellular domain of humanprotein tyrosine phosphatase β (HPTPβ) using syntheticphosphopeptides. Biochem J 298: 395–401.

Hogue DL, Liu L, Ling V. 1999. Identification and characteriza-tion of a mammalian mitochondrial ATP-binding cassette mem-brane protein. J Mol Biol 285: 379–389.

Komeili A, Wedaman KP, O’Shea EK, Powers T. 2000. Mecha-nism of metabolic control. Target of rapamycin signaling linksnitrogen quality to the activity of the Rtg1 and Rtg3 transcriptionfactors. J Cell Biol 151: 863–878.

Jia Y, Rothermel B, Thornton J, Butow RA. 1997. A basichelix–loop–helix leucine zipper transcription complex in yeastfunctions in a signaling pathway from mitochondria to thenucleus. Mol Cell Biol 17: 1110–1117.

Koh JY, Hajek P, Bedwell DM. 2001. Overproduction of PDR3suppresses mitochondrial import defects associated with aTOM70 null mutation by increasing the expression of TOM72in Saccharomyces cerevisiae. Mol Cell Biol 21: 7576–7586.

Langford CJ, Klinz FJ, Donath C, Gallwitz D. 1984. Pointmutations identify the conserved, intron-contained TACTAACbox as an essential splicing signal sequence in yeast. Cell 36:645–653.

Lapinski PE, Miller GG, Tampe R, Raghavan M. 2000. Pairing ofthe nucleotide binding domains of the transporter associated withantigen processing. J Biol Chem 275: 6831–6840.

Leng P, Lee PR, Wu H, Brown AJ. 2001. Efg1, a morphogeneticregulator in Candida albicans, is a sequence-specific DNAbinding protein. J Bacteriol 183: 4090–4093.

Lill R, Kispal G. 2001. Mitochondrial ABC transporters. ResMicrobiol 152: 331–340.

McEwen JG, Garcia AM, Ortiz BL, Botero S, Restrepo A. 1995.In search of the natural habitat of Paracoccidioides brasiliensis .Arch Med Res 26: 305–306.

Medoff G, Painter A, Kobayashi GS. 1987. Mycelial-to-yeast-phase transitions of the dimorphic fungi Blastomycesdermatitidis and Paracoccidioides brasiliensis . J Bacteriol 169:4055–4060.

Mendes RP. 1994. Treatment and control of cure. In Paracoccid-ioidomycosis, Franco M, et al. (eds). CRC Press: Boca Raton,FL.

Miled C, Mann C, Faye G. 2001. Xbp1-mediated repression ofCLB gene expression contributes to the modifications of yeastcell morphology and cell cycle seen during nitrogen-limitedgrowth. Mol Cell Biol 21: 3714–3724.

Mount SM. 1982. A catalog of splice junction sequences. NucleicAcids Res 10: 459–472.

Prasad R, De Wergifosse P, Goffeau A, Balzi E. 1995. Molecularcloning and characterization of a novel gene of Candidaalbicans, CDR1, conferring multiple resistance to drugs andantifungals. Curr Genet 27: 320–329.

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.

880 C. H. Gray et al.

Pereira M, Felipe MS, Brigido MM, Soares CMA, Azevedo MO.2000. Molecular cloning and characterization of a glucan syn-thase gene from the human pathogenic fungus Paracoccidioidesbrasiliensis . Yeast 16: 451–462.

Petrofeza da Silva S, Borges-Walmsley MI, Pereira IS, et al. 1999.Differential expression of an hsp70 gene during transitionfrom the mycelial to the infective yeast form of the humanpathogenic fungus Paracoccidioides brasiliensis . Mol Microbiol31: 1039–1050.

Restrepo AMcEwen JG, Castaneda E. 2001. The habitat ofParacoccidioides brasiliensis: how far from solving the riddle?Med Mycol 39: 233–241.

Rodero L, Canteros CE, Rivas C, Lee W, Davel G. 1999. In vitrosensitivity of Paracoccidioides brasiliensis to systemically usedantifungal agents. Rev Argent Microbiol 31: 78–81.

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: ALaboratory Manual, 2nd edn. Cold Spring Harbor LaboratoryPress: New York.

San-Blas G, Nino-Veja G. 2001. Paracoccidioides brasiliensis:virulence and host response. In Fungal Pathogenesis: Principlesand Clinical Applications, Cihlar RL, Calderone RA (eds).Marcel Dekker: New York.

San-Blas G, Nino-Veja G, Iturriaga T. 2002. Paracoccidioidesbrasiliensis and paracoccidioidomycosis: molecular approachesto morphogenesis, diagnosis, epidemiology, taxonomy andgenetics. Med Mycol 40: 225–242.

Sanglard D, Ischer F, Bille J. 2001. Role of ATP-binding-cassettetransporter genes in high-frequency acquisition of resistanceto azole antifungals in Candida glabrata . Antimicrob AgentsChemother 45: 1174–1183.

Scazzocchio C. 2000. The fungal GATA factors. Curr OpinMicrobiol 3: 126–131.

Shirihai OS, Gregory T, Yu C, Orkin SH, Weiss MJ. 2000. ABC-me: a novel mitochondrial transporter induced by GATA-1during erythroid differentiation. EMBO J 19: 2492–2502.

Smith PC, Karpowich N, Millen L, et al. 2002. ATP binding tothe motor domain from an ABC transporter drives formation ofa nucleotide sandwich dimer. Mol Cell 10: 139–149.

Theiss S, Kretschmar M, Nichterlein T, et al. 2002. Functionalanalysis of a vacuolar ABC transporter in wild-type Candidaalbicans reveals its involvement in virulence. Mol Microbiol43: 571–584.

Tobin MB, Peery RB, Skatrud PL. 1997. Genes encoding multipledrug resistance-like proteins in Aspergillus fumigatus andAspergillus flavus . Gene 200: 11–23.

Young L, Leonhard K, Tatsuta T, Trowsdale J, Langer T. 2001.Role of the ABC transporter Mdl1 in peptide export frommitochondria. Science 291: 2135–2138.

Zhang S, Varma A, Williamson PR. 1999. The yeast Cryptococcusneoformans uses ‘mammalian’ enhancer sites in the regulationof the virulence gene, CNLAC1. Gene 227: 231–240.

Zhang F, Hogue DL, Liu L, et al. 2000. M-ABC2, a new humanmitochondrial ATP-binding cassette membrane protein. FEBSLett 478: 89–94.

Zhang X, Moye-Rowley WS. 2001. Saccharomyces cerevisiaemultidrug resistance gene expression inversely correlates withthe status of the F(0) component of the mitochondrial ATPase.J Biol Chem 276: 47 844–47 852.

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 865–880.