The ATP‐binding cassette multidrug transporter Snq2 of <i>Saccharomyces cerevisiae</i>: a novel target for the transcription factors Pdr1 and Pdr3

Mahé, Yannick; Parle‐McDermott, Anne; Nourani, Amine; Delahodde, Agnès; Lamprecht, Andrea; Kuchler, Karl

doi:10.1111/j.1365-2958.1996.tb02493.x

Cited by 110 publications

(125 citation statements)

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“…Although the PDR5 and SNQ2 promoters each have three PDREs (12,31,32), PDR5-lacZ expression was enhanced 5 times by the wildtype Pdr1p while the expression of the SNQ2-lacZ fusion was increased only 1.4 times upon the addition of the wild-type PDR1 allele. Under the same conditions, the induction of the other translational lacZ fusions showing 1 or 2 putative PDREs varied between 1.4 (YOR1-lacZ) and 2.2 (PDR10-lacZ).…”

Section: Resultsmentioning

confidence: 99%

“…The method takes advantage of point mutations in the transcription factorencoding genes PDR1/PDR3, which activate the transcription of their target genes. The target gene promoters contain typical binding sequences called PDREs (for PDR response elements) which correspond to the 5Ј-TCCG(C/T)GGA-3Ј consensus sequence (12,31,32) (Table III). An inherent problem to this approach is the simultaneous overexpression of several Pdr1p-regulated proteins, including other ABC transporters.…”

Section: Resultsmentioning

confidence: 99%

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ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p

Decottignies¹,

Grant²,

Nichols³

et al. 1998

Journal of Biological Chemistry

362

334

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The Saccharomyces cerevisiae genome encodes 15 fullsize ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were upregulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes. The yeast YOR11 gene confers oligomycin resistance on overexpression in a 2-m plasmid (1). Its nucleotide sequence reveals an ORF of 1477 amino acids encoding an ABC protein highly homologous to mammalian transporters such as the multidrug resistance-conferring enzyme MRP (BLAST (see Ref. 2) sequence homology score: p ϭ e Ϫ228 ), the organic anion transporter cMOAT (p ϭ e Ϫ216 ), the sulfonylurea receptor (p ϭ e Ϫ164 ), and the cystic fibrosis transmembrane conductance regulator CFTR (p ϭ e Ϫ132 ). Yor1p is a "full-size" ABC transporter with the topology (TM-NBF) 2 (3, 4). It consists of two homologous halves, with each containing a putative ATP-binding domain (NBF) and a transmembrane domain of six membrane spans (TM). Cui et al. (5) showed that Yor1p confers resistance to a series of drugs including reveromycin A and suggested that Yor1p may be involved in the cellular efflux of organic anions including the fluorescent dye rhodamine B. They also showed that incubation with reveromycin A increases the YOR1 mRNA level. The transcription of YOR1 is controlled by the homologous pair of transcription factors Pdr1p/Pdr3p. The level of YOR1 transcription is decreased by the deletion of either PDR1 or PDR3 and increased in the presence of the gain-of-function PDR1 alleles (1).In this paper, we have investigated the transport activity of Yor1p. Building on previous studies, which indicated that the (TM-NBF) 2 -type Yor1p, together with the (NBF-TM) 2 -type Pdr5p and Snq2p ABC transporters, are overexpressed in the PDR1-3 mutant plasma membrane (6 -8), the PDR1-3 mutant has been used as a tool that enhances the Yor1p protein level. As another investigative tool, we constructed a set of isogenic strains, in the PDR1-3 mutant, with multiple deletions of homologous ABC genes since, in situations where two or more proteins located in the same subcellular compartment share a common substrate, a clear phenotype is only seen when all the corresponding genes are deleted, as illustrated by the work of Mahé et al. (9), who showed that Pdr5p and Snq2p have an overlapping transport ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p

Decottignies¹,

Grant²,

Nichols³

et al. 1998

Journal of Biological Chemistry

362

334

View full text Add to dashboard Cite

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“…In summary, our studies strongly suggest that Rdr1p acts through Pdr1p/Pdr3p binding sites. However, many genes (11,(13)(14)(15)(16)(17) (like SNQ2) that have been shown to contain PDREs and to be regulated by Pdr1p/ Pdr3p are not affected by removal of Rdr1p. Thus, the effect of Rdr1p appears to be mediated by a subset of the target genes of FIG.…”

Section: Discussionmentioning

confidence: 99%

Zinc Cluster Protein Rdr1p Is a Transcriptional Repressor of the PDR5 Gene Encoding a Multidrug Transporter

Hellauer¹,

Akache²,

MacPherson³

et al. 2002

Journal of Biological Chemistry

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The yeast PDR5 gene encodes an efflux pump that confers multidrug resistance. Expression of PDR5 is positively regulated by the transcription factors Pdr1p and Pdr3p that recognize the same pleiotropic drug resistance elements (PDREs) in the PDR5 promoter. Pdr1p and Pdr3p belong to the Gal4p family of zinc cluster proteins. The function of RDR1 (YOR380W), which also encodes a member of this family, is unknown. To identify target genes for Rdr1p, we have performed wholegenome analysis of gene expression with DNA microarrays. Our results show that Rdr1p is a transcriptional repressor of five genes, including PDR5. A ⌬rdr1 strain has increased resistance to cycloheximide, as expected from the overexpression of PDR5. In addition, the activity of a PDR5-lacZ reporter is increased in a ⌬rdr1 strain. All (but one) genes affected by removal of Rdr1p contain PDREs in their promoters. We tested if the effect of Rdr1p is mediated through PDREs by inserting this DNA element in front of a minimal promoter. Activity of this reporter was increased in a ⌬rdr1 strain. Moreover, mutations known to reduce binding of Pdr1/ Pdr3p abolished the induction observed in the ⌬rdr1 strain. Thus, we have identified a transcriptional repressor involved in the control of multidrug resistance.Multidrug resistance is a widespread phenomenon that allows organisms ranging from bacteria to humans to defend themselves against a variety of toxic compounds. Drug resistance is mediated through membrane-bound transporters that act as drug efflux pumps. This process has been extensively studied in the yeast Saccharomyces cerevisiae. Two major classes of multidrug transporters have been identified: the major facilitator superfamily (MFS) 1 (1) and the ABC (ATP binding cassette) family of transporters (2-4). ABC transporters act in an ATP-dependent manner, and their overexpression leads to increased drug resistance. Members of the family of ABC transporters include Pdr5p, Snq2p, and Yor1p (2).The transcriptional activators Pdr1p and Pdr3p control the expression of many ABC transporters (3, 4). These activators belong to a family of transcriptional regulators called zinc cluster or binuclear cluster proteins (5-7). Members of this family contain six highly conserved cysteines that coordinate binding to two zinc atoms to allow proper folding of the DNA binding domain. The cysteine-rich region (or zinc finger) is usually followed by a short linker sequence that bridges the zinc finger to a dimerization domain (6). This domain is involved in recognition of specific DNA sequences through interaction of the zinc finger with DNA (for references see Ref. 8). Pdr1p and Pdr3p both recognize CGG triplets oriented in opposite directions (CCGCGG) to form an everted repeat (9). This motif is found in the target genes of Pdr1p and Pdr3p (10 -17). For example, three binding sites called PDREs (pleiotropic drug response elements) for Pdr1p and Pdr3p have been mapped in the PDR5 promoter (10, 12). Thus, Pdr1p and Pdr3p recognize the same DNA sequences in target promoters f...

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“…However, as with the stable overexpression of the ABC transporters CDR1 and CDR2, the genetic basis for the constitutive activation of the MDR1 gene in such strains has not been elucidated. In Saccharomyces cerevisiae several regulatory proteins (Pdr1p, Pdr3p, and Yap1p) controlling expression of multiple drug resistance genes of the ABC transporter and major-facilitator superfamilies are known (1,3,5,11,12,15,16,20,28,30), and mutations in these regulators that result in upregulation of their respective target genes have been identified (4,19). Recently, functional homologues of these regulators have also been found in C. albicans (1,27); however, conflicting data about the roles of these transcriptional regulators have been obtained.…”

Section: Discussionmentioning

confidence: 99%

Activation of the Multiple Drug Resistance Gene MDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in a trans -Regulatory Factor

et al. 2000

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Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA R developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazoleresistant isolates, resulting in deregulated, constitutive MDR1 expression.Candida albicans is an important opportunistic fungal pathogen of humans and is the major cause of oropharyngeal candidiasis (OPC) in patients with AIDS (21). The azole antifungal agent fluconazole is a widely used compound to treat OPC. In recent years, however, the incidence of treatment failures has been rising. Especially in patients with AIDS who have recurrent OPC and who are receiving prolonged fluconazole therapy, treatment failures are due to the emergence of fluconazole-resistant strains (10, 22). Resistant C. albicans isolates frequently exhibit reduced intracellular drug accumulation that correlates with enhanced expression of certain multiple drug resistance genes, the ATP-binding cassette (ABC) transporters CDR1 and CDR2, and the major facilitator MDR1 (8,14,24,25,29). Fluconazole resistance is usually a stable phenotype that is maintained in the absence of selection pressure by the drug. This implies that genetic alterations have occurred in the resistant isolates that result in a constitutive overexpression of the drug efflux pumps. The MDR1 gene is not detectably expressed in vitro in fluconazole-susceptible C. albicans isolates but is strongly activated in many strains after the development of fluconazole resistance. The molecular chang...

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The ATP‐binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3

Cited by 110 publications

References 39 publications

ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p

ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p

Zinc Cluster Protein Rdr1p Is a Transcriptional Repressor of the PDR5 Gene Encoding a Multidrug Transporter

Activation of the Multiple Drug Resistance Gene MDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in a trans -Regulatory Factor

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