Novel target genes of the yeast regulator Pdr1p: a contribution of the TPO1 gene in resistance to quinidine and other drugs.

The yeast transcription factor Pdr1p regulates the expression of a number of genes, several of which encode ATP-driven transport proteins involved in multiple drug resistance. Among 20 genes containing binding consensus sequences for the transcription factor Pdr1p in their promoter, we studied more particularly the regulation and function of PDR16 (involved in phospholipid synthesis), TPO1 (involved in vacuolar transport of polyamines), YAL061W (homologous to polyol dehydrogenases) and YLR346C (unknown function). We found that the regulation of these four genes depends on Pdr1p, since promoter activities studied by lacZ fusion analysis and mRNA levels studied by Northern blotting analysis changed upon deletion or hyperactivation by the pdr1-3 mutant of this transcription factor. The drug sensitivity of the strains deleted for these genes revealed that TPO1, a gene previously found to be involved in spermidine resistance and vacuolar polyamine transport, is a determinant of multidrug transporter since it also mediates growth resistance to cycloheximide and quinidine. This resistance pattern overlapped with that of YOR273C, a homolog of TPO1. These two homologous transporters are thus bona fide members of the phylogenetic subfamily DHA1 (drug/proton antiport TC 2.A.1. 2) of the major facilitator superfamily. Both YOR273C and TPO1 as well as at least one other determinant involved in the yeast pleiotropic drug resistance network contribute to resistance to a quinoline-containing antimalarial drug.

Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae.

Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p. Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin beta-lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p. Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae.

The pleitropic drug ABC transporters from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains 16 genes encoding full-size ABC transporters. Each comprises two nucleotide binding folds (NBF) alternating with transmembrane domains (TM). We have studied in detail three plasma membrane multidrug exporters: Pdr5p (TC3.A.1.205.1) and Snq2p (TC3.A.1.205.2) which share NBF-TM-NBF-TM topology as well as Yor1p (TC3.A.1.208.3) which exhibits the reciprocal TM-NBF-TM-NBF topology. The substrate specificity of Pdr5p, Snq2p and Yor1p are largely, but not totally, overlapping as shown by screening the growth inhibition by 349 toxic compounds of combinatorial deletants of these three ABC genes. Multiple deletion of 7 ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15) and of two transcription activation factors (PDR1 and PDR3) renders the cell from 2 to 200 times more sensitive to numerous toxic coumpounds including antifungals used in agriculture or medicine. The use of the pdr1-3 activating mutation and when necessary of the PDR5 promoter in appropriate multideleted hosts allow high levels of expression of Pdr5p, Snq2p or Yor1 p. These overexpressed proteins exhibit ATPase activity in vitro and confer considerable multiple drug resistance in vivo. The latter property can be used for screening specific inhibitors of fungal and other ABC transporters.

Coordinate control of sphingolipid biosynthesis and multidrug resistance in Saccharomyces cerevisiae.

Multiple or pleiotropic drug resistance often occurs in the yeast Saccharomyces cerevisiae through genetic activation of the Cys(6)-Zn(II) transcription factors Pdr1p and Pdr3p. Hyperactive alleles of these proteins cause overproduction of target genes that include drug efflux pumps, which in turn confer high level drug resistance. Here we provide evidence that both Pdr1p and Pdr3p act to regulate production of an enzyme involved in sphingolipid biosynthesis in S. cerevisiae. The last step in formation of the major sphingolipid in the yeast plasma membrane, mannosyldiinositol phosphorylceramide, is catalyzed by the product of the IPT1 gene, inositol phosphotransferase (Ipt1p). Transcription of the IPT1 gene is responsive to changes in activity of Pdr1p and Pdr3p. A single Pdr1p/Pdr3p response element is present in the IPT1 promoter and is required for regulation by these factors. Loss of IPT1 has complex effects on drug resistance of the resulting strain, consistent with an important role for mannosyldiinositol phosphorylceramide in normal plasma membrane function. Direct assay for lipid contents of cells demonstrates that changes in sphingolipid composition correlate with changes in the activity of Pdr3p. These data suggest that Pdr1p and Pdr3p may act to modulate the lipid composition of membranes in S. cerevisiae through activation of sphingolipid biosynthesis along with other target genes.

Antibiotic efflux pumps.

Active efflux from procaryotic as well as eucaryotic cells strongly modulates the activity of a large number of antibiotics. Effective antibiotic transport has now been observed for many classes of drug efflux pumps. Thus, within the group of primary active transporters, predominant in eucaryotes, six families belonging to the ATP-binding cassette superfamily, and including the P-glycoprotein in the MDR (Multi Drug Resistance) group and the MRP (Multidrug Resistance Protein), have been recognized as being responsible for antibiotic efflux. Within the class of secondary active transporters (antiports, symports, and uniports), ten families of antibiotic efflux pumps have been described, distributed in five superfamilies [SMR (Small Multidrug Resistance), MET (Multidrug Endosomal Transporter), MAR (Multi Antimicrobial Resistance), RND (Resistance Nodulation Division), and MFS (Major Facilitator Superfamily)]. Nowadays antibiotic efflux pumps are believed to contribute significantly to acquired bacterial resistance because of the very broad variety of substrates they recognize, their expression in important pathogens, and their cooperation with other mechanisms of resistance. Their presence also explains high-level intrinsic resistances found in specific organisms. Stable mutations in regulatory genes can produce phenotypes of irreversible multidrug resistance. In eucaryotes, antibiotic efflux pumps modulate the accumulation of antimicrobials in phagocytic cells and play major roles in their transepithelial transport. The existence of antibiotic efflux pumps, and their impact on therapy, must now be taken fully into account for the selection of novel antimicrobials. The design of specific, potent inhibitors appears to be an important goal for the improved control of infectious diseases in the near future.

Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants.

The cDNA from activated mutants of the homologous transcription factors Pdr1p and Pdr3p was used to screen DNA microarrays of the Saccharomyces cerevisiae complete genome. Twenty-six overexpressed targets of the PDR1-3 and/or PDR3-7 mutants were identified. Twenty-one are new targets, the majority of which are of unknown function. In addition to well known ABC transporters, these targets appear to be involved in transport or in membrane lipids and cell wall biosyntheses. Several of the targets seem to contribute to the cell defence against a variety of stresses. Pdr1p and Pdr3p do not act similarly on all targets. Unexpectedly, the expression of 23 other genes appeared to be repressed in the PDR1-3 and/or PDR3-7 mutants. In contrast to the majority of the activated genes, none of the repressed genes contains pleiotropic drug resistance binding sites in their promoter.

Molecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genes.

Mutations at the yeast PDR1 transcriptional regulator locus are responsible for overexpression of the three ABC transporter genes PDR5, SNQ2 and YOR1, associated with the appearance of multiple drug resistance. The nucleotide sequences of 13 alleles of PDR1, comprising 6 multidrug resistance mutants, 1 intragenic suppressor and 6 wild types, have been determined. Single amino acid substitutions were shown to result from the mutations pdr1-2 (M308I), pdr1-3 (F815S), pdr1-6 (K302Q), pdr1-7 (P298A) and pdr1-8 (L1036 W), whereas the intragenic suppressor mutant pdr1-100 is deleted for the two amino acids L537 and A538. An isogenic series of strains was constructed containing the mutant alleles pdr1-3, pdr1-6 and pdr1-8 integrated into the genome. We found that the levels of resistance to cycloheximide, oligomycin, 4-nitroquinoline-N-oxide and ketoconazole were increased in all three mutants. The increase was more pronounced in the pdr1-3 than in the pdr1-6 and pdr1-8 mutants. Studies of the activity of the promoters of the ABC genes PDR5, SNQ2 and YOR1 demonstrated that the combination of the PDR5 promoter and the pdr1-3 mutation resulted in the highest level of promoter induction. Concomitantly, the level of PDR5 mRNA, of Pdr5p protein, and of its associated nucleoside triphosphatase activity, was strongly increased in the plasma membranes of the PDR1 mutants. Again, the pdr1-3 allele was associated with a stronger effect than the pdr1-8 and pdr1-6 alleles. The locations of the mutations in the PDR1 gene indicate that at least three different regions distributed throughout the Pdr1p transcription factor may be mutated to generate a Pdr1p with considerably increased transcriptional activation potency. These gain-of-function mutations support the concept, recently proposed, that in members of the large family of yeast Zn2Cys6 transcription factors a central inhibitory domain exists (delineated by the pdr1-7, pdr1-6 and pdr1-2 mutations). This domain may interact in a locked conformation with a putative, more C-terminally located inhibitory domain (mutated in pdr1-3), and with the putative activation domain (mutated in pdr1-8).

Multidrug resistance in Aspergillus nidulans involves novel ATP-binding cassette transporters.

Two single-copy genes, designated atrA and atrB (ATP-binding cassette transporter A and B), were cloned from the filamentous fungus Aspergillus nidulans and sequenced. Based on the presence of conserved motifs and on hydropathy analysis, the products encoded by atrA and atrB can be regarded as novel members of the ATP-binding cassette (ABC) superfamily of membrane transporters. Both products share the same topology as the ABC transporters PDR5 and SNQ2 from Saccharomyces cerevisiae and CDR1 from Candida albicans, which are involved in multidrug resistance of these yeasts. Significant homology also occurs between the ATP-binding cassettes of AtrA and AtrB, and those of mammalian ABC transporters (P-glycoproteins). The transcription of atrA and, in particular, atrB in mycelium of A. nidulans is strongly enhanced by treatment with several drugs, including antibiotics, azole fungicides and plant defense toxins. The enhanced transcription is detectable within a few minutes after drug treatment and coincides with the beginning of energy-dependent drug efflux activity, reported previously in the fungus for azole fungicides. Transcription of the atr genes has been studied in a wild-type and in a series of isogenic strains carrying the imaA and/or imaB genes, which confer multidrug resistance to various toxic compounds such as the azole fungicide imazalil. atrB is constitutively transcribed at a low level in the wild-type and in strains carrying imaA or imaB. Imazalil treatment enhances transcription of atrB to a similar extent in all strains tested. atrA, unlike atrB, displays a relatively high level of constitutive expression in mutants carrying imaB. Imazalil enhances transcription of atrA more strongly in imaB mutants, suggesting that the imaB locus regulates atrA. Functional analysis demonstrated that cDNA of atrB can complement the drug hypersensitivity associated with PDR5 deficiency in S. cerevisiae.

Identification and characterization of SNQ2, a new multidrug ATP binding cassette transporter of the yeast plasma membrane.

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

Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals.

By functional complementation of a PDR5 null mutant of Saccharomyces cerevisiae, we have cloned and sequenced the multidrug-resistance gene CDR1 of Candida albicans. Transformation by CDR1 of a PDR5-disrupted host hypersensitive to cycloheximide and chloramphenicol resulted in resistance to cycloheximide, chloramphenicol and other drugs, such as the antifungal miconazole, with collateral hypersensitivity to oligomycin, nystatin and 2,4 dinitrophenol. Our results also demonstrate the presence of several PDR5 complementing genes in C. albicans, displaying multidrug-resistance patterns different from PDR5 and CDR1. The nucleotide sequence of CDR1 revealed that, like PDR5, it encodes a putative membrane pump belonging to the ABC (ATP-binding cassette) superfamily. CDR1 encodes a 1501-residue protein of 169.9 kDa whose predicted structural organization is characterized by two homologous halves, each comprising a hydrophobic region with a set of six transmembrane stretches, preceded by a hydrophilic nucleotide binding fold.

Yeast multidrug resistance: the PDR network.

This minireview describes a network of genes involved in multiple drug resistance of the yeast S. cerevisiae. The transcription regulators, PDR1, PDR3, PDR7, and PDR9 control the expression of the gene PDR5, encoding a membrane protein of the ATP-binding-cassette superfamily and functioning as a drug extrusion pump. Next to PDR5, several other target genes, encoding membrane pumps of the ABC type, such as SNQ2, STE6, PDR10, PDR11, YOR1, but also other membrane-associated (such as GAS1, D4405) or soluble proteins (such as G3PD), involved or not in multidrug resistance, are found to be controlled by PDR1. On another side, the PDR3 regulator participates with its homolog PDR1 to co- and auto-regulation circuits of yeast multidrug resistance.

Solubilization and characterization of the overexpressed PDR5 multidrug resistance nucleotide triphosphatase of yeast.

A 160-kDa plasma membrane protein of the yeast Saccharomyces cerevisiae was overexpressed by mutating the PDR1 or the PDR3 transcription factor gene. The protein is the membrane-bound ATP binding cassette transporter PDR5 (Balzi, E., Wang, M., Leterme, S., Van Dyck, L., and Goffeau, A. (1994) J. Biol. Chem. 269, 2206-2214). PDR5 was solubilized with n-dodecyl-beta-D-malto-side and separated from the PMA1 plasma membrane H(+)-ATPase by glycerol gradient centrifugation. The PDR5 protein hydrolyzes nucleoside diphosphates and triphosphates. This activity is sensitive to low concentrations of vanadate, of oligomycin, and of a variety of hydrophobic compounds. Many of these properties liken PDR5 to the purified mammalian P-glycoprotein responsible for multidrug resistance.

PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1.

The complete sequence of the pleiotropic drug resistance gene PDR5 from Saccharomyces cerevisiae is reported and analyzed. PDR5 encodes a 160-kDa protein with a predicted duplicated six membrane-span domain and a repeated putative ATP-binding domain. PDR5 shares this structural feature with the mammalian multidrug resistance pumps as well as the functional capacity of conferring resistance to various inhibitors upon amplification (Leppert, G., McDevitt, R., Falco, S. C., Van Dyk, T. K., Ficke, M. B., and Golin, J. (1990) Genetics 125, 13-20). The yeast PDR5 is thus a new member of the ABC (ATP-binding cassette) protein superfamily. Mutations in another yeast pleiotropic drug resistance gene, PDR1, encoding a putative transcription regulator (Balzi, E., Chen, W., Ulaszewski, S., Capieaux, E., and Goffeau, A. (1987) J. Biol. Chem. 262, 16871-16879), increase markedly the mRNA levels of the PDR5 and STE6 genes. The multidrug resistance mutations pdr1-3 and pdr1-6 also lead to considerable overexpression of the PDR5 plasma membrane protein.

Interaction of the yeast pleiotropic drug resistance genes PDR1 and PDR5.

The network of genes which mediates multiple drug resistance in yeast includes, among others, the PDR1 gene, which encodes a putative regulator of gene expression, and PDR5, a locus whose amplification leads to resistance. We demonstrate that disruption of PDR5 causes marked hypersensitivity not only to cycloheximide but also to sulphometuron methyl and the mitochondrial inhibitors chloramphenicol, lincomycin, erythromycin and antimycin. Genetic analysis of double mutants containing an insertion in PDR5 (pdr5:Tn5), which renders cells hypersensitive to cycloheximide, and a pdr1 mutation, which confers resistance to this inhibitor, indicates that the expression of resistance requires a functional PDR5 gene. The same interdependency is observed for chloramphenicol, but not for oligomycin, lincomycin, erythromycin or sulphometuron methyl. Northern analysis of PDR1 and PDR5 transcripts reveals that the 5.2 kbp PDR5 transcript is overexpressed in pdr1 (resistant) mutants, but underexpressed in a disruption of PDR1. These observations provide strong experimental support for our former proposal that the PDR5 gene is a target for regulation by the PDR1 gene product.

Physical, transcriptional and genetical mapping of a 24 kb DNA fragment located between the PMA1 and ATE1 loci on chromosome VII from Saccharomyces cerevisiae.

A physical map of a contiguous DNA fragment of 60 kb, extending from the centromere to TRP5 on the left arm of the chromosome VII of Saccharomyces cerevisiae, strain IL125-2B, was established. Within a 31 kb region from PMA1 towards TRP5, a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified in cells grown exponentially on rich medium. Near 87% of the DNA investigated was transcribed and on average one transcript, of 2.3 kb average length, was detected every 2.7 kb of DNA. The physical and genetical distances between the markers CEN7, pma1, leu1, pdr1 and trp5 were compared. A recombination frequency of 1 cM corresponds to an average distance of 3.3 kb between alleles in this region of chromosome VII.