Yannick Mahé

The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast

Exposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP‐binding cassette (ABC) transporter Pdr12. In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels. However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane. Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Δpdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels. Moreover, active benzoate efflux is severely impaired in Δpdr12 cells. Hence, Pdr12 confers weak acid resistance by mediating energy‐dependent extrusion of water‐soluble carboxylate anions. The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH. This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts. The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.

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

Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 and Snq2 ATP binding cassette multidrug transporters. Expression of Pdr5 and Snq2 is regulated by the two transcription factors Pdr1 and Pdr3, as multidrug-resistant pdr1 and pdr3 gain-of-function mutants overexpress both drug efflux pumps. One such pdr1 mutant allele was previously cloned in a genetic screen by its ability to suppress the squelching toxicity mediated by an estradiol-inducible chimeric VP16-human estrogen receptor (VEO) expressed in yeast (Gilbert, D. M., Heery, D. M., Losson, R., Chambon, P., and Lemoine, Y. (1993) Mol. Cell. Biol. 13, 462-472). In this study, we demonstrate that relief of estradiol toxicity in yeast cells expressing VEO requires functional PDR5 and SNQ2 genes, since a Δpdr5 Δsnq2 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 Δpdr5 Δsnq2 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.

Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.

Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter. We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development. Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min. A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation. Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole.

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

Pleiotropic drug resistance (PDR) in the yeast Saccharomyces cerevisiae can arise from overexpression of ATP‐binding cassette (ABC) efflux pumps such as Pdr5 and Snq2. Mutations in the transcription factor genes PDR1 and PDR3 are also associated with PDR. We show here that a pdr1–3 mutant exhibits a PDR phenotype, including elevated resistance to the mutagen 4‐nitroquinoline‐N‐oxide, a known substrate for Snq2 but not for Pdr5. Northern analysis and immunoblotting demonstrated that the SNQ2 gene is 10‐fold overexpressed in a pdr1–3 gain‐of‐function mutant strain, whereas Snq2 expression is severely reduced in a Δpdr1 deletion strain, and almost abolished in a Δpdr1Δpdr3 double disruptant when compared to the PDR1 strain. However, expression of the Ste6 a‐factor pheromone transporter, another yeast ABC transporter not associated with PDR, is unaffected in pdr1–3 mutant cells and in strains carrying Δpdr1, Δpdr3, or Δpdr1Δpdr3 deletions. Finally, DNA footprint analysis revealed that the SNQ2 promoter contains three binding sites for Pdr3. Our results identify SNQ2 as a novel target for both Pdr1 and Pdr3, and demonstrate that the PDR phenotype of a pdr1–3 mutant strain results from overexpression of more than one ABC drug‐efflux pump.

The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo‐ and heterodimers in vivo

The transcription factors Pdr1p and Pdr3p from Saccharomyces cerevisiae mediate pleiotropic drug resistance (PDR) by controlling expression of ATP‐binding cassette (ABC) transporters such as Pdr5p, Snq2p and Yor1p. Previous in vitro studies demonstrated that Pdr1p and Pdr3p recognize so‐called pleiotropic drug resistance elements (PDREs) in the promoters of target genes. In this study, we show that both Pdr1p and Pdr3p are phosphoproteins; Pdr3p isoforms migrate as two bands in gel electrophoresis, reflecting two distinct phosphorylation states. Most importantly, native co‐immunoprecipitation experiments, using functional epitope‐tagged Pdr1p/Pdr3p variants, demonstrate that Pdr1p and Pdr3p can form both homo‐ and heterodimers in vivo. Furthermore, in vivo footprinting of PDRE‐containing promoters demonstrate that Pdr1p/Pdr3p constitutively occupy both perfect and degenerate PDREs in vivo. Thus, in addition to interaction with other regulators, differential dimerization provides a plausible explanation for the observation that Pdr3p and Pdr1p can both positively and negatively control PDR promoters with different combinations of perfect and degenerate PDREs.

The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15 are novel targets for the Pdr1 and Pdr3 transcriptional regulators.

The yeast transcription factors Pdr1 and Pdr3 control pleiotropic drug resistance (PDR) development, since they regulate expression of ATP‐binding cassette (ABC) drug efflux pumps through binding to cis‐acting sites known as PDREs ( R esponsive lements). In this report, we show by Northern blotting, gel shift mobility assays and DNase I footprinting that transcription of the ABC genes PDR10 and PDR15 is also controlled by Pdr1 and Pdr3. In addition, in vitro band shift assays demonstrate that a GST‐Pdr1 fusion protein can bind to the PDREs of PDR10 and PDR15. DNase I footprinting allowed the identification of the precise PDRE binding motifs, indicating the presence of a novel slightly degenerate PDRE motif in the PDR15 promoter. Finally, PDR10 and PDR15 mRNA levels vary dramatically in abundance in isogenic yeast strains carrying either Δpdr1, Δpdr3 and Δpdr1 Δpdr3 deletions or pdr1‐3 and pdr3‐2 gain‐of‐function mutations, demonstrating that both PDR10 and PDR15 are new members of the yeast PDR network.

Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family

Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two‐hybrid assays, in vitro, and in pull‐down experiments performed with SK‐BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.

ATP binding cassette transporters in yeast: From mating to multidrug resistance

Publisher Summary ATP binding cassette (ABC) transporters comprise a novel superfamily of membrane transport proteins that are found operating from bacteria to humans. More than 150 ABC sequences are deposited in the National Center for Biotechnology Information (NCBI) databank and their number keeps growing rapidly. The discovery that the Ste6 a-factor pheromone transporter represents a close homolog of the mammalian multidrug resistance P-glycoproteins motivated many laboratories to search for other yeast genes that are functionally or structurally related to STE6 . The joint efforts of many laboratories to systematically sequence the yeast genome has led to the identification of additional novel yeast ABC proteins. Therefore, the functional diversity of bacterial ABC transporters implies that even more ABC proteins can be discovered in both yeast and in mammalian systems.

The Molecular Basis for Pleiotropic Drug Resistance in the Yeast Saccharomyces Cerevisiae: Regulation of Expression, Intracellular Trafficking and Proteolytic Turnover of ATP Binding Cassette (ABC) Multidrug Resistance Transporters

The major research goal of our laboratory is to understand the function mechanism of certain eukaryotic ABC transporters, including those from yeast as well as selected mammalian ABC proteins of medical importance. ABC transporters comprise the largest membrane transport protein family known to date, as more than 200 different ABC transporters have been identified operating from bacteria to man (Higgins, 1992; Kuchler and Thorner, 1992). The hallmark characteristics of all ABC-proteins include the presence of two highly conserved domains for ATP-binding (ABC), and two membrane domains each containing several predicted membrane-spanning α-helices (TMS). These four domains are normally arranged in an (TMS-ABC)2 or (ABC-TMS)2 configuration, although “half-size” transporters with an TMS-ABC or ABC-TMS topology and other topologies are also frequently found (Figure 1).