The ATP Binding Cassette Transporters Pdr5 and Snq2 of Saccharomyces cerevisiae Can Mediate Transport of Steroids in Vivo

Abstract

Multiple or pleiotropic drug resistance in the yeastIn this study, we demonstrate that relief of estradiol toxicity in yeast cells expressing VEO requires functional PDR5 and SNQ2 genes, since a ⌬pdr5 ⌬snq2 double deletion leads to an increased estradiol toxicity. Furthermore, using URA3 as an estradiol-inducible reporter gene, we show that Pdr5 and Snq2, when overexpressed from high-copy plasmids, can reduce the intracellular concentration of estradiol. In contrast, a ⌬pdr5 ⌬snq2 double deletion mutant accumulates almost 30-fold more intracellular estradiol than the isogenic wild type. Indirect immunofluorescence showed that a pdr1-3 mutant massively overexpresses Pdr5 at the plasma membrane, suggesting that estradiol efflux from the cells occurs across the plasma membrane. Our data demonstrate that Pdr5 and Snq2 can transport steroid substrates in vivo and suggest that steroids and/or related membrane lipids could represent physiological substrates for certain yeast ABC transporters, which are otherwise involved in the development of pleiotropic drug resistance. Pleiotropic drug resistance (PDR)1 in yeast is a well documented phenomenon that appears quite similar to P-glycoprotein (Pgp or Mdr1) and MRP-mediated multidrug resistance in mammalian cells (1, 2). For example, two homologous yeast ABC transporter genes, PDR5 (3, 4) and SNQ2 (5), were recently shown to represent functional and structural homologues of mammalian Mdr1 and MRP (6), since their overexpression in yeast is associated with PDR development. Elevated levels of Pdr5 and Snq2 lead to resistance against a variety of structurally unrelated cytotoxic compounds including mycotoxins (3), cycloheximide (7), 4-nitroquinoline N-oxide and sulfomethuron methyl (5). However, each transporter mediates resistance to only a distinct subset of drugs, and there is very little overlap in the substrate specificity of Pdr5 and Snq2 (3,8).Transcription of both PDR5 and SNQ2 is controlled by the transcription regulatory proteins Pdr1 and Pdr3 (4, 9, 10). Loss-of-function mutations such as ⌬pdr1 and ⌬pdr3 deletions result in a dramatic decrease of Pdr5 and Snq2 expression (11) and cause a marked drug hypersensitivity. In turn, several gain-of-function alleles of pdr1 and pdr3 have been isolated by their ability to confer a pleiotropic drug resistance phenotype (12, 13). In such drug-resistant pdr mutants, expression of the drug efflux pumps Pdr5 and Snq2 is dramatically increased (11), and it has been shown that pdr1-mediated cycloheximide resistance requires the presence of functional PDR5 (14).Notably, another pdr1 mutant allele was previously cloned as a suppressor of the estradiol-induced squelching phenomenon caused by a VP16-estrogen receptor fusion protein (VEO) expressed in yeast (15). Induction by estradiol of such a chimeric protein, which has the hormone-binding and the DNAbinding domain of the human estrogen receptor fused to the acidic activation domain of the viral protein VP16, leads to a toxic transcriptional transactivation known as transcriptional...

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

“…Subsequently, the increase in URA1 expression might be related to that of URA3. Of the remaining three genes, PHO5 encodes a secreted acid phosphatase, which is involved in nucleotide-derived phosphate hydrolysis in the periplasmic space; VHR1 encodes a transcriptional activator, required for the induction of vitamin H and biotin intermediate transporters VHT1 and BIO5, respectively (Weider et al, 2006); and PDR5 encodes the plasma membrane ABC-transporter involved in cellular detoxification (Balzi et al, 1994;Mahe et al, 1996).…”

SRE1 and SRE2 are two specific steroid‐responsive modules of Candida drug resistance gene 1 (CDR1) promoter

“…Interestingly, homologues of Cdr1p, e.g. Pdr5p, Snq2p in S. cerevisiae and Mdr1p in humans, have also been shown to transport steroids (Barnes et al, 1996;Mahe et al, 1996).…”

ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein 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 capacity for steroids. We deleted the yeast ABC transporter-encoding genes known or suspected to 1 The abbreviations used are: YOR1, yeast oligomycin resistance; ORF, open reading frame; ABC, ATP-binding cassette; PDR, pleiotropic drug resistance; Pma1p, H ϩ -plasma membrane ATPase; PDRE, Pdr1p/ Pdr3p response element; M-C 6 -NBD-PE, 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; SDC, synthetic complete glucose medium; YD, rich glucose medium; YG, rich glycerol medium; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; TNP, trinitrophenyl; TNP-8-azido-ATP, 2Ј,3Ј-O-(2,4,6-trinitrophenyl)-8-azido-adenosine triphosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); TM, transmembrane; NBF, nucleotide-binding fold; CFTR, cystic fibrosis transmembrane regulator.…”