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Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters.

ABSTRACT: In the yeast Saccharomyces cerevisiae, multidrug resistance to unrelated chemicals can result from overexpression of ATP-binding cassette (ABC) transporters such as Pdr5p, Snq2p, and Yor1p. Expression of these genes is under the control of two homologous zinc finger-containing transcription regulators, Pdr1p and Pdr3p. Here, we describe the isolation, by an in vivo screen, of two new Pdr1p-Pdr3p target genes: HXT11 and HXT9. HXT11 and HXT9, encoding nearly identical proteins, have a high degree of identity to monosaccharide transporters of the major facilitator superfamily (MFS). In this study, we show that the HXT11 product, which allows glucose uptake in a glucose permease mutant (rag1) strain of Kluyveromyces lactis, is also involved in the pleiotropic drug resistance process. Loss of HXT11 and/or HXT9 confers cycloheximide, sulfomethuron methyl, and 4-NQO (4-nitroquinoline-N-oxide) resistance. Conversely, HXT11 overexpression increases sensitivity to these drugs in the wild-type strain, an effect which is more pronounced in a strain having both PDR1 and PDR3 deleted. These data show that the two putative hexose transporters Hxt11p and Hxt9p are transcriptionally regulated by the transcription factors Pdr1p and Pdr3p, which are known to regulate the production of ABC transporters required for drug resistance in yeast. We thus demonstrate the existence of genetic interactions between genes coding for two classes of transporters (ABC and MFS) to control the multidrug resistance process.

SUBMITTER: Nourani A

PROVIDER: S-EPMC232394 | biostudies-other | 1997 Sep

REPOSITORIES: biostudies-other

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Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters.

Nourani A A Wesolowski-Louvel M M Delaveau T T Jacq C C Delahodde A A

Molecular and cellular biology 19970901 9

In the yeast Saccharomyces cerevisiae, multidrug resistance to unrelated chemicals can result from overexpression of ATP-binding cassette (ABC) transporters such as Pdr5p, Snq2p, and Yor1p. Expression of these genes is under the control of two homologous zinc finger-containing transcription regulators, Pdr1p and Pdr3p. Here, we describe the isolation, by an in vivo screen, of two new Pdr1p-Pdr3p target genes: HXT11 and HXT9. HXT11 and HXT9, encoding nearly identical proteins, have a high degree ...[more]

PMID: 9271421

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Project description:In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport. Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily. One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new member of this family, HXT3. The inferred amino acid sequence of HXT3 is 87% identical to that of HXT1, 64% identical to that of HXT2, and 32% identical to that of SNF3. Like HXT1 and HXT2, overexpression of HXT3 in snf3 delta cells confers growth on low-glucose or raffinose media. The function of another new member of the HXT superfamily, HXT4 (previously identified by its ability to suppress the snf3 delta phenotype; L. Bisson, personal communication), was revealed in experiments that deleted all possible combinations of the five members of the glucose transporter gene family. Neither SNF3, HXT1, HXT2, HXT3, nor HXT4 is essential for viability. snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells are unable to grow on media containing high concentrations of glucose (5%) but can grow on low-glucose (0.5%) media, revealing the presence of a sixth transporter that is itself glucose repressible. This transporter may be negatively regulated by SNF3 since expression of SNF3 abolishes growth of hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells on low-glucose medium. HXT1, HXT2, HXT3, and HXT4 can function independently: expression of any one of these genes is sufficient to confer growth on medium containing at least 1% glucose. A synergistic relationship between SNF3 and each of the HXT genes is suggested by the observation that SNF2 hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells and snf3 delta HXT1 HXT2 HXT3 HXT4 cells are unable to grow on raffinose (low fructose) yet SNF3 in combination with any single HXT gene is sufficient for growth on raffinose. HXT1 and HXT3 are differentially regulated. HXT1::lacZ is maximally expressed during exponential growth whereas HXT3::lacZ is maximally expressed after entry into stationary phase.

Project description:When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1prom and CCW12prom), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells.ImportanceEthanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.

Project description:BackgroundIn the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose-proton symport.ResultsIntroduction of heterologous glucose- or fructose-proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose-proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose-1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose-proton symporter, fructose-proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent-1) and 46.6% decreased biomass yield on sucrose.ConclusionsThis study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.

Dataset Information

Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters.

Publications

Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters.

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