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Pdr5 is the most abundant ABC transporter in Saccharomyces cerevisiae and plays a major role in the pleiotropic drug resistance (PDR) network, which actively prevents cell entry of a large number of structurally unrelated compounds. Due to a high level of asymmetry in one of its nucleotide binding sites (NBS), Pdr5 serves as a perfect model system for asymmetric ABC transporter such as its medical relevant homologue Cdr1 from Candida albicans. In the past 30 years, this ABC transporter was intensively studied in vivo and in plasma membrane vesicles. Nevertheless, these studies were limited since it was not possible to isolate and reconstitute Pdr5 in a synthetic membrane system while maintaining its activity. Here, the functional reconstitution of Pdr5 in a native‐like environment in an almost unidirectional inside‐out orientation is described. We demonstrate that reconstituted Pdr5 is capable of translocating short‐chain fluorescent NBD lipids from the outer to the inner leaflet of the proteoliposomes. Moreover, this transporter revealed its ability to utilize other nucleotides to accomplish transport of substrates in a reconstituted system. Besides, we were also able to estimate the NTPase activity of reconstituted Pdr5 and determine the kinetic parameters for ATP, GTP, CTP, and UTP. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, the Rpd3L complex includes the histone deacetylase Rpd3 and the DNA binding proteins Ume6 and Ash1 and serves as a transcriptional silencer or enhancer. In S. cerevisiae, the transcription of PDR5, which encodes a major drug efflux pump, and pleiotropic drug resistance (PDR) are hyperactivated by the transcription factor Pdr3 in ρ0/− cells, which lack mitochondrial DNA. We previously showed that RPD3 and UME6 are required for the activation of PDR5 transcription and PDR in S. cerevisiae ρ0 cells. Here, using real-time PCR analysis, we revealed that RPD3 and UME6 are responsible for the activated basal expression of the ABC transporter-encoding genes SNQ2, PDR15, and PDR5 in S. cerevisiae ρ0 cells. Furthermore, using real-time PCR analysis and a spot dilution assay, we found that Ume6 increases the basal expression of PDR5 and PDR15 and induces PDR in a manner dependent on RPD3 and PDR3 in ρ0 cells. This finding may contribute to the elucidation of the relationships between the molecules required for the activation of ABC transporter genes in S. cerevisiae ρ0/− cells and in pathogenic Candida species.

In Saccharomyces cerevisiae, the Rpd3L complex includes the histone deacetylase Rpd3 and the DNA binding proteins Ume6 and Ash1 and serves as a transcriptional silencer or enhancer. In S. cerevisiae, the transcription of PDR5, which encodes a major drug efflux pump, and pleiotropic drug resistance (PDR) are hyperactivated by the transcription factor Pdr3 in ρ0/− cells, which lack mitochondrial DNA. We previously showed that RPD3 and UME6 are required for the activation of PDR5 transcription and PDR in S. cerevisiae ρ0 cells. Here, using real-time PCR analysis, we revealed that RPD3 and UME6 are responsible for the activated basal expression of the ABC transporter-encoding genes SNQ2, PDR15, and PDR5 in S. cerevisiae ρ0 cells. Furthermore, using real-time PCR analysis and a spot dilution assay, we found that Ume6 increases the basal expression of PDR5 and PDR15 and induces PDR in a manner dependent on RPD3 and PDR3 in ρ0 cells. This finding may contribute to the elucidation of the relationships between the molecules required for the activation of ABC transporter genes in S. cerevisiae ρ0/− cells and in pathogenic Candida species. [ABSTRACT FROM AUTHOR]

In Saccharomycescerevisiae, the Rpd3L complex contains a histone deacetylase, Rpd3, and the DNA binding proteins, Ume6 and Ash1, and acts as a transcriptional repressor or activator. We previously showed that RPD3 and UME6 are required for the activation of PDR5, which encodes a major efflux pump, and pleiotropic drug resistance (PDR) in ρ0/− cells, which lack mitochondrial DNA. However, there are inconsistent reports regarding whether RPD3 and UME6 are required for Pdr5-mediated PDR in ρ+ cells with mitochondrial DNA. Since PDR5 expression or PDR in the ρ+ cells of the rpd3Δ and ume6Δ mutants have primarily been examined using fermentable media, mixed cultures of ρ+ and ρ0/− cells could be used. Therefore, we examined whether RPD3 and UME6 are required for basal and drug-induced PDR5 transcription and PDR in ρ+ cells using fermentable and nonfermentable media. UME6 suppresses the basal transcription levels of the ABC transporters, including PDR5, and drug resistance in ρ+ cells independent of the carbon source used in the growth medium. In contrast, RPD3 is required for drug resistance but did not interfere with the basal PDR5 mRNA levels. UME6 is also required for the cycloheximide-induced transcription of PDR5 in nonfermentable media but not in fermentable media. [ABSTRACT FROM AUTHOR]

Analysis of synonymous mutations established that although the primary amino acid sequence remains unchanged, alterations in transcription and translation can result in significant phenotypic consequences. We report the novel observation that a series of nonsynonymous mutations in an unconserved stretch of amino acids found in the yeast multidrug efflux pump Pdr5 increases expression, thus enhancing multidrug resistance. Cycloheximide chase experiments ruled out the possibility that the increased steady-state level of Pdr5 was caused by increased protein stability. Quantitative-RT PCR experiments demonstrated that the mutants had levels of PDR5 transcript that were two to three times as high as in the isogenic wild-type strain. Further experiments employing metabolic labeling of mRNA with 4-thiouracil followed by uracil chasing showed that the half-life of PDR5 transcripts was specifically increased in these mutants. Our data demonstrate that the nucleotides encoding unconserved amino acids may be used to regulate expression and suggest that Pdr5 has a newly discovered RNA stability element within its coding region.

A yeast estrogen screening (YES) assay was improved to increase sensitivity for detection of phytoestrogens. New yeast strains minus one or the other of transporters Pdr5 or Snq2 and harboring yEGFP as a reporter gene were developed. The new strains showed 2-100-fold improvement in sensitivity for detection of standard estrogens and antiestrogens. In addition, the assay time (1 h) using the newly developed strains was shorter than that (4 h) previously reported. Furthermore, the snq2-minus strains were most effective for detection of estrogenic activity while the pdr5-minus strains were most effective for detection of antiestrogenic activity. The efficacy of the new methods was evaluated and confirmed by testing with 23 Thai medicinal plant species. The new strains were also tested for detection of xenoestrogens. The results revealed that the newly developed YES methods were specific and rapid and suitable for simple high-throughput screening or detection of estrogen-like compounds. [ABSTRACT FROM AUTHOR]

ABSTRACT This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

In Saccharomycescerevisiae, the Rpd3L complex contains a histone deacetylase, Rpd3, and the DNA binding proteins, Ume6 and Ash1, and acts as a transcriptional repressor or activator. We previously showed that RPD3 and UME6 are required for the activation of PDR5, which encodes a major efflux pump, and pleiotropic drug resistance (PDR) in ρ0/− cells, which lack mitochondrial DNA. However, there are inconsistent reports regarding whether RPD3 and UME6 are required for Pdr5-mediated PDR in ρ+ cells with mitochondrial DNA. Since PDR5 expression or PDR in the ρ+ cells of the rpd3Δ and ume6Δ mutants have primarily been examined using fermentable media, mixed cultures of ρ+ and ρ0/− cells could be used. Therefore, we examined whether RPD3 and UME6 are required for basal and drug-induced PDR5 transcription and PDR in ρ+ cells using fermentable and nonfermentable media. UME6 suppresses the basal transcription levels of the ABC transporters, including PDR5, and drug resistance in ρ+ cells independent of the carbon source used in the growth medium. In contrast, RPD3 is required for drug resistance but did not interfere with the basal PDR5 mRNA levels. UME6 is also required for the cycloheximide-induced transcription of PDR5 in nonfermentable media but not in fermentable media.

Analysis of synonymous mutations established that although the primary amino acid sequence remains unchanged, alterations in transcription and translation can result in significant phenotypic consequences. We report the novel observation that a series of nonsynonymous mutations in an unconserved stretch of amino acids found in the yeast multidrug efflux pump Pdr5 increases expression, thus enhancing multidrug resistance. Cycloheximide chase experiments ruled out the possibility that the increased steadystate level of Pdr5 was caused by increased protein stability. Quantitative-RT PCR experiments demonstrated that the mutants had levels of PDR5 transcript that were two to three times as high as in the isogenic wild-type strain. Further experiments employing metabolic labeling of mRNA with 4-thiouracil followed by uracil chasing showed that the half-life of PDR5 transcripts was specifically increased in these mutants. Our data demonstrate that the nucleotides encoding unconserved amino acids may be used to regulate expression and suggest that Pdr5 has a newly discovered RNA stability element within its coding region. [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) in tumors and pathogens remains a major problem in the efficacious treatment of patients by reduction of therapy options and subsequent treatment failure. Various mechanisms are described to be involved in the development of MDR with overexpression of ATP-binding cassette (ABC) transporters reflecting the most extensively studied. These membrane transporters translocate a wide variety of substrates utilizing energy from ATP hydrolysis leading to decreased intracellular drug accumulation and impaired drug efficacy. One treatment strategy might be inhibition of transporter-mediated efflux by small molecules. Isocoumarins and 3,4-dihydroisocoumarins are a large group of natural products derived from various sources with great structural and functional variety, but have so far not been in the focus as potential MDR reversing agents. Thus, three natural products and nine novel 3,4-dihydroisocoumarins were designed and analyzed regarding cytotoxicity induction and inhibition of human ABC transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) in a variety of human cancer cell lines as well as the yeast ABC transporter Pdr5 in Saccharomyces cerevisiae. Dual inhibitors of P-gp and BCRP and inhibitors of Pdr5 were identified, and distinct structure-activity relationships for transporter inhibition were revealed. The strongest inhibitor of P-gp and BCRP, which inhibited the transporters up to 80 to 90% compared to the respective positive controls, demonstrated the ability to reverse chemotherapy resistance in resistant cancer cell lines up to 5.6-fold. In the case of Pdr5, inhibitors were identified that prevented substrate transport and/or ATPase activity with IC50 values in the low micromolar range. However, cell toxicity was not observed. Molecular docking of the test compounds to P-gp revealed that differences in inhibition capacity were based on different binding affinities to the transporter. Thus, these small molecules provide novel lead structures for further optimization. [ABSTRACT FROM AUTHOR]

Studies in the yeast Saccharomyces cerevisiae have provided much of the basic detail underlying the organization and regulation of multiple or pleiotropic drug resistance gene network in eukaryotic microbes. As with many aspects of yeast biology, the initial observations that drove the eventual molecular characterization of multidrug resistance gene were provided by genetics. This review focuses on contributions from the laboratory of Dr. André Goffeau that uncovered key aspects of the transcriptional regulation of these multidrug resistance genes. André's group made many seminal discoveries that helped lead to the current picture we have of how eukaryotic microbes respond to and deal with a variety of antifungal agents. The importance of the transcriptional contribution to antifungal drugs is illustrated by the large number of drug resistant mutants found in several yeast species that lead to increased activity of transcriptional regulators. The characterization of the Saccharomyces cerevisiaePDR1 gene by the Goffeau group provided the first molecular basis explaining the link between this hyperactive transcription factor and drug resistance. [ABSTRACT FROM AUTHOR]

Pdr5 is a founding member of a large (pdr) subfamily of clinically and agriculturally significant fungal ABC transporters. The tremendous power of yeast genetics combined with biochemical and structural approaches revealed the astonishing asymmetry of this efflux pump. Asymmetry is manifested in Pdr5′s ATP-binding sites, drug binding sites, signal transformation interface, and molecular exit gate. Even its mode of conformational switching is asymmetric with one half of the protein remaining nearly stationary. In the case of its ATP-binding sites, asymmetry is created by replacing a set of highly conserved residues with a characteristic set of deviant ones. This contrasts with the asymmetry of the molecular gate. There, a full complement of canonical residues is present, but structural features in the vicinity prevent some of these from forming a molecular plug during closure. Compared to their canonical-functioning counterparts, the deviant ATP site and these gating residues have different, essential functions. In addition to its remarkable asymmetry, the surprising observation that Pdr5 is a drug / proton co-transporter shines a new light on this remarkable protein. [ABSTRACT FROM AUTHOR]

Abstract In animals, cigarette smoke may alter pharmacokinetics by altering activity and expression of ABC drug transporters. We previously demonstrated that cigarette smoke condensate (CSC) impairs activity and expression of several hepatic ABC drug transporters which mediate toxicant efflux. However, CSC effects on efflux transporters are still unknown in Saccharomyces cerevisiae which resists diverse chemical stresses, by inducing pleiotropic drug resistance (PDR) genes among others. The yeast ABC transporters are functionally and structurally homologous to the mammalian ones. In this study, Saccharomyces cerevisiae exposure to CSC for 15 min caused a dose-dependent inhibition of rhodamine 123 efflux, whereas a longer exposure (3 h) induced mRNA expression of the ABC PDR efflux pumps Pdr5, Snq2, Pdr 10 and Pdr15, and of Tpo1, a member of the major facilitator superfamily (MFS). CSC also increased toxicity of caffeine, which is handled by two PDR transporters, Pdr5 and Snq2. Taken together, these data demonstrated that yeast efflux transporters are targets of cigarette smoke chemicals, and that Saccharomyces cerevisiae may cope with CSC-induced stress, including the initial efflux inhibition, by induction of the mRNA of several plasma membrane PDR and MFS efflux transporters. Saccharomyces cerevisiae is therefore a valid model to investigate pollutant effects on ABC and MFS transporters. [ABSTRACT FROM AUTHOR]

Honokiol (HNK), an important medicinal component of Magnolia officinalis, is reported to possess pharmacological activities against a variety of diseases. However, the molecular mechanisms of HNK medicinal functions are not fully clear. To systematically study the mechanisms of HNK action, we screened a yeast mutant library based on the conserved nature of its genes among eukaryotes. We identified genes associated with increased resistance or sensitivity to HNK after mutation. After functional classification of these genes, we found that most HNK‐resistant strains in the largest functional category were petites with mutations in mitochondrial genes, indicating that mitochondria were related to HNK resistance. Additional analysis showed that resistance of petite mutants to HNK was associated with upregulation of the ATP‐binding cassette transporter Pdr5, which pumps out HNK. We also found that several HNK‐sensitive mitochondria mutants were not petites, and had larger lipid droplets (LDs). Furthermore, HNK treatment on wild‐type yeast cells seemed to disrupt mitochondrial morphology, induced triacylglycerol synthesis, and generated supersized LDs surrounded by mitochondria and endoplasmic reticulum (ER). These changes are also applied to atp7Δ mutant if no carbon resource was available. These results suggested that HNK treatment partly impaired normal mitochondrial function to form larger LDs by altering lipid metabolism. The pharmacological agent honokiol (HNK) possesses pharmacological activities against a variety of diseases with unclear molecular mechanisms. We screened a yeast mutant library and found that most HNK‐resistant strains were petites with mutations in mitochondrial genes and with increased expression of the ATP‐binding cassette (ABC) transport, Pdr5, to pump out HNK. HNK treatment on wild‐type yeast cells seemed to disrupt mitochondria morphology, induced triacylglycerol synthesis, and generated supersized lipid droplets (LDs) surrounded by mitochondria and endoplasmic reticulum, suggesting that HNK treatment partly impaired mitochondrial function to form larger LDs. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae has sophisticated nutrient-sensing programs for responding to harsh environments containing limited nutrients. As a result, yeast cells can live in diverse environments, including animals, as a commensal or a pathogen. Because they live in mixed populations with other organisms that excrete toxic chemicals, it is of interest to know whether yeast cells maintain functional multidrug resistance mechanisms during nutrient stress. We measured the activity of Pdr5, the major Saccharomyces drug efflux pump under conditions of limiting nutrients. We demonstrate that the steady-state level of this transporter remains unchanged during growth in low concentrations of glucose and nitrogen even though two-dimensional gel electrophoresis revealed a decrease in the level of many proteins. We also evaluated rhodame 6G transport and resistance to three xenobiotic agents in rich (synthetic dextrose) and starvation medium. We demonstrate that Pdr5 function is vigorously maintained under both sets of conditions. [ABSTRACT FROM AUTHOR]

Honokiol (HNK), one of the main medicinal components in Magnolia officinalis , possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5 , a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5 Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae , particularly in inducing iron starvation to inhibit cell growth. [ABSTRACT FROM AUTHOR]

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity. [ABSTRACT FROM AUTHOR]

Background trans -Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum and a weaker antimicrobial potency than other available antibiotics. When combined with polygodial, nagilactone E, and n -dodecanol, anethole has been shown to exhibit synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae , and a human opportunistic pathogenic yeast, Candida albicans . However, the mechanism underlying this synergistic effect of anethole has not been characterized. Methods We studied this mechanism using dodecanol-treated S. cerevisiae cells and focusing on genes related to multidrug efflux . Results Although dodecanol transiently reduced the number of colony forming units, this recovered to levels similar to those of untreated cells with continued incubation beyond 24 h. Reverse transcription polymerase chain reaction analysis revealed overexpression of an ATP-binding cassette (ABC) transporter gene, PDR5 , in addition to a slight increase in PDR11 , PDR12 , and PDR15 transcriptions in dodecanol-treated cells. In the presence of anethole, these effects were attenuated and the fungicidal activity of dodecanol was extended. Dodecanol showed longer lasting fungicidal activity against a Δ pdr5 . In addition, Δ pdr3 and Δ lge1 , lack transcription factors of PDR5 and PDR3 , were partly and completely susceptible to dodecanol, respectively. Furthermore, combination of anethole with fluconazole was also found to exhibit synergy on C. albicans . Conclusions These results indicated that although anethole reduced the transcription of several transporters, PDR5 expression was particularly relevant to dodecanol efflux. General significance Anethole is expected to be a promising candidate drug for the inhibition of efflux by reducing the transcription of several ABC transporters. [ABSTRACT FROM AUTHOR]

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

Asymmetric ABC (ATP-binding cassette) transporters make up a significant proportion of this important superfamily of integral membrane proteins. These proteins contain one canonical (catalytic) ATP-binding site and a second atypical site with little enzymatic capability. The baker's yeast (Saccharomyces cerevisiae) Pdr5 multidrug transporter is the founding member of the Pdr subfamily of asymmetric ABC transporters, which exist only in fungi and slime moulds. Because these organisms are of considerable medical and agricultural significance, Pdr5 has been studied extensively, as has its medically important homologue Cdr1 from Candida albicans. Genetic and biochemical analyses of Pdr5 have contributed important observations that are likely to be applicable to mammalian asymmetric ABC multidrug transporter proteins, including the basis of transporter promiscuity, the function of the non-catalytic deviant ATP-binding site, the most complete description of an in vivo transmission interface, and the recent discovery that Pdr5 is a molecular diode (one-way gate). In the present review, we discuss the observations made with Pdr5 and compare them with findings from clinically important asymmetric ABC transporters, such as CFTR (cystic fibrosis transmembrane conductance regulator), Cdr1 and Tap1/Tap2. [ABSTRACT FROM AUTHOR]

Multiple drug resistance pumps are potential drug targets. Here we asked whether the lipophilic cation dodecyltriphenylphosphonium (C 12 TPP) can interfere with their functioning. First, we found that suppression of ABC transporter gene PDR5 increases the toxicity of C 12 TPP in yeast. Second, C 12 TPP appeared to prevent the efflux of rhodamine 6G – a fluorescent substrate of Pdr5p. Moreover, C 12 TPP increased the cytostatic effects of some other known Pdr5p substrates. The chemical nature of C 12 TPP suggests that after Pdr5p-driven extrusion the molecules return to the plasma membrane and then into the cytosol, thus effectively competing with other substrates of the pump. [ABSTRACT FROM AUTHOR]

The use of statins is well established in human therapy, and model organisms such as Saccharomyces cerevisiae are commonly used in studies of drug action at molecular and cellular levels. The investigation of the resistance mechanisms towards statins may suggest new approaches to improve therapy based on the use of statins. We investigated the susceptibility to lovastatin of S. cerevisiae strains deleted for PDR genes, responsible for exporting hydrophobic and amphiphilic drugs, such as lovastatin. Strains deleted for the genes tested, PDR1, PDR3, PDR5 and SNQ2, exhibited remarkably different phenotypes, with deletion of PDR5 causing the highest sensitivity to lovastatin. The study helped clarifying which pdr mutants to use in studies of physiological actions of statins in yeast. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A subset of the family of ATP-binding cassette (ABC) transporters has been in focus owing to their involvement in conferring multidrug resistance in cancer cells and among immune compromised individuals. Saccharomyces cerevisiae is protected against xenobiotics by similar machineries that are part of the pleitropic drug resistance (PDR) network. The ABC transporter Pdr5 is an important member of this PDR network in yeast and is involved in cellular detoxification by the efflux of a wide variety of drugs and substrates. In this review, we focus on the aspects of detergent effects and the degeneracy in conserved sequences that is observed in the nucleotide binding domains of Pdr5 and discuss their functional relevance. [ABSTRACT FROM AUTHOR]

Multidrug resistance is a major challenge in the therapy of cancer and pathogenic fungal infections. More than three decades ago, P-glycoprotein was the first identified multidrug transporter. It has been studied extensively at the genetic and biochemical levels ever since. Pdr5, the most abundant ATP-binding cassette transporter in Saccharomyces cerevisiae, is highly homologous to azole-resistance-mediating multidrug transporters in fungal pathogens, and a focus of clinical drug resistance research. Despite functional equivalences, P-glycoprotein and Pdr5 exhibit striking differences in their architecture and mechanisms. In this minireview, we discuss the mechanisms of substrate selection and multidrug transport by comparing the fraternal twins P-glycoprotein and Pdr5. We propose that substrate selection in eukaryotic multidrug ATP-binding cassette transporters is not solely determined by structural features of the transmembrane domains but also by their dynamic behavior. [ABSTRACT FROM AUTHOR]

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Although it has been reported that TBT induces apoptotic cell death in mammalian, the action of TBT on eukaryotic microorganisms has not yet been fully investigated. In this study we examined the mechanism involved in cell death caused by TBT exposure in Saccharomyces cerevisiae. The median lethal concentration of TBT was 10 μM for the parent strain BY4741 and 3 μM for the pdr5Δ mutant defective in a major multidrug transporter, respectively. Fluorescence microscopic observations revealed nuclear condensation and chromatin fragmentation in cells treated with TBT indicating that cells underwent an apoptosis-like cell dearth. TBT-induced cell death was suppressed by deletion of the yca1 gene encoding a homologue of the mammalian caspase. In parallel, reactive oxygen species (ROS) were produced by TBT. These results suggest that TBT induces apoptosis-like cell death in yeast via an Yca1p-dependent pathway possibly downstream of the ROS production. This is the first report on TBT-induced apoptotic cell death in yeast. [ABSTRACT FROM AUTHOR]

Abstract: BcatrA was cloned from the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) and sequenced. Sequence analysis revealed that BcatrA encodes a protein composed of 1562 amino acid residues displaying high similarity with various fungal ATP-binding cassette (ABC) transporters having the (NBF-TM6)2 topology. Expression of BcatrA is barely detectable during normal vegetative growth in liquid substrates. Transcript levels of BcatrA are enhanced in a dose- and time-dependent manner after treatment with cycloheximide or catechol, but not by a number of other drugs or fungicides, including fludioxonil, fenarimol, imazalil, and the plant defense compounds pisatin and resveratrol. Quantitative analysis of BcatrA during the synchronized infection of bean leaves revealed an overaccumulation of the gene transcript at 6, 12 and 24h post-inoculation, suggesting an involvement of the gene in the first steps of pathogenesis. Functional analysis of BcatrA was performed by targeted gene replacement in a wild-type strain of the fungus, and by overexpression in a mutant of Saccharomyces cerevisiae carrying multiple non-functional multidrug-resistance genes. BcatrA replacement mutants did not show any significant increase in sensitivity to drugs, including inducers of BcatrA transcription, and displayed an unaltered virulence on several common host plants of B. cinerea. However, when expressed in the heterologous system, BcatrA reduced sensitivity to cycloheximide and catechol, thus indicating the ability of the BcatrA product to function as a multidrug transporter. [Copyright &y& Elsevier]

The PDR5 gene encodes the major multidrug resistance efflux pump in Saccharomyces cerevisiae. In drug-resistant cells, the hyperactive Pdr1p or Pdr3p transcriptional activators are responsible for the PDR5 upregulation. In this work, it is shown that the RPD3 gene encoding the histone deacetylase that functions as a transcriptional corepressor at many promoters and the ROM2 gene coding for the GDP/GTP exchange protein for Rho1p and Rho2p participating in signal transduction pathways are required for PDR5 transcription under cycloheximide-induced and noninduced conditions. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3Δ and rom2Δ mutants, the level of PDR5 mRNA and the rate of rhodamine 6G efflux were reduced. Unlike rpd3Δ, in rom2Δ mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by PDR1 or PDR3 overexpressed from heterologous promoters and by the hyperactive pdr3-9 mutant allele. The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p participating in the cell integrity pathway are involved in the control of PDR5 expression and modulation of multidrug resistance in yeast. [ABSTRACT FROM AUTHOR]

Abstract: In order to get deeper insights into oxidative degradation of the hydrophobic substrates (HS) triglycerides and alkanes by yeasts, tagged mutants affected in these pathways were generated by random insertion of a mutagenesis cassette MTC into the genome of Yarrowia lipolytica. About 9.600 Ura+ transformants were screened in plate tests for utilization of alkanes (C10, C16), oleic acid and tributyrin. HS degradation mutants were recovered as unable to grow on alkane or on intermediates of the pathway (AlkA–AlkE phenotype classes). To identify the disrupted genes, insertion points of the MTC were sequenced using convergent and divergent PCR. Sequence analysis evidenced both known and new genes required for HS utilization, e.g. for AlkD/E mutants MTC insertion had occurred in genes of thioredoxin reductase, peroxines PEX14 and PEX20, succinate-fumarate carrier SFC1, and isocitrate lyase ICL1. Several mutants were affected in alkane utilization depending on chain length. Mutant Z110 (AlkAb: C10− C16+) was shown to be disrupted for ANT1 encoding a peroxisomal membrane localized adenine nucleotide transporter protein, providing ATP for the activation of short-chain fatty acids by acyl-CoA synthetase II in peroxisomes. Mutants N046 and B095 (AlkAc: C10+ C16−) were disrupted for the ABC transporter encoded by ABC1 gene, thus providing first evidence for its participation in chain length dependent alkane transport processes. [Copyright &y& Elsevier]

Abstract: Multidrug transporters are of considerable importance because they present problems in the treatment of infectious disease and cancer. A central issue is the ability of efflux pumps to recognize an astounding array of structurally diverse compounds. The yeast Pdr5p efflux pump, which is a member of the ATP-binding cassette superfamily, has at least 3 substrate-binding sites, each of which appears to use different chemical properties to transport compounds. All Pdr5p substrates, however, have a size requirement that is independent of hydrophobicity. [Copyright &y& Elsevier]

The expression of the transcription regulator Pdr1p and its target genes PDR5 and TPO1 is required for Saccharomyces cerevisiae adaptation and resistance to artesunate, a promising antimalarial drug, also active against tumour cells and viruses. PDR5 and TPO1 encode plasma membrane multidrug transporters of the ATP-binding cassette and the major facilitator superfamilies, respectively. The transcriptional activation of TPO1 (10-fold) and PDR5 (13-fold) was registered after 30 min of exposure of the unadapted yeast population to acute artesunate-induced stress, being significantly reduced in the absence of Pdr1p and abolished in the absence of Pdr1p and Pdr3p. Maximum TPO1 mRNA levels were rapidly reduced to basal values following adaptation of the yeast population to artesunate, while high PDR5 levels were maintained during drug-stressed exponential growth. [ABSTRACT FROM AUTHOR]

Abstract: Communication between the mitochondria and the nucleus is essential to ensure correct metabolic coordination of the cell. Signaling pathways leading from the mitochondria to the nucleus are referred to as retrograde signaling and were first discovered in the yeast Saccharomyces cerevisiae. Cells that lack their mitochondrial genome (ρ0 cells) trigger expression of the nuclear CIT2 gene in order to ensure adequate amino acid biosynthesis. More recently, it has been found that a different set of genes involved in multidrug resistance in S. cerevisiae is strongly induced in ρ0 cells. During a search for negative regulators of the ATP-binding cassette (ABC) transporter-encoding gene PDR5, it was observed that ρ0 mutants exhibited dramatic up-regulation of the transcript of this gene. This induction was due to the post-translational activation of a direct upstream regulator of PDR5 that was designated Pdr3p. Loss of the LGE1 gene led to a block in ρ0-mediated induction of PDR5 expression. Lge1p has been observed by others to be involved in histone H2B ubiquitination along with the ubiquitin-conjugating enzyme Rad6p and the ubiquitin ligase Bre1p. Our studies provide evidence that Lge1p has another function unique from H2B ubiquitination that is required for retrograde regulation of PDR5 transcription. We have also found that the Pdr pathway regulates expression of several genes involved in sphingolipid biosynthesis. These findings suggest that the physiological role of the PDR genes might be to regulate membrane homeostasis and ρ0-triggered changes in this parameter may be the signal controlling PDR gene expression. [Copyright &y& Elsevier]

Summary PDR5-like proteins represent one group of the ABC superfamily of transporters. Members of this group are present in plants and, due to the function of PDR5-related proteins in fungi in the excretion of xenobiotics (including antifungal agents), it has been proposed that they might play a similar role in plants in the response to and detoxification of herbicides and fungicides. However, until now no functional data has been presented showing an altered plant response to any herbicide or fungicide as a result of manipulating the expression of a PDR5-like gene in plants. In this paper, we show that the plant SpTUR2 PDR5-like ABC transporter is localised to the plasma membrane and that expression of this protein in Arabidopsis leads to the acquisition of resistance to the diterpenoid antifungal agent sclareol. These data both define a possible endogenous substrate for this transporter and highlight the potential of manipulating plant chemical resistance via modulating the expression of specific PDR5-like transporters. [ABSTRACT FROM AUTHOR]

The transcription regulator Pdr1p is a determinant of Saccharomyces cerevisiae resistance to 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The Pdr1p-regulated genes, TPO1 and PDR5, encoding putative multidrug transporters belonging to the major facilitator superfamily (MFS) and to the ATP-binding cassette (ABC) superfamily, respectively, are required for yeast resistance to sudden exposure to these herbicides. A rapid and transient activation of TPO1 (sixfold) and PDR5 (twofold) transcription takes place during the adaptation period preceding cell division under MCPA or 2,4-D moderate stress. These activations are mediated by both Pdr1p and Pdr3p and, as soon as adapted cells start duplication under herbicide stress, mRNA levels are drastically reduced to basal values. The longer duration of the adaptation period, observed for the Δpdr1 population, may involve the abolishment of the Pdr1p-mediated transcriptional activation of TPO1 and PDR5 genes, whose expression is critical to surpass the viability loss during the initial period of adaptation to the herbicides. [Copyright &y& Elsevier]