scholarly journals Resistance and Adaptation to Quinidine in Saccharomyces cerevisiae: Role of QDR1 (YIL120w), Encoding a Plasma Membrane Transporter of the Major Facilitator Superfamily Required for Multidrug Resistance

2001 ◽  
Vol 45 (5) ◽  
pp. 1528-1534 ◽  
Author(s):  
Patrı́cia A. Nunes ◽  
Sandra Tenreiro ◽  
Isabel Sá-Correia
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Multidrug Resistance ◽  
Fluorescent Protein ◽  
Resistance Mechanisms ◽  
Major Facilitator Superfamily ◽  
Yeast Cells ◽  
Detectable Effect ◽  
Protein Fusion ◽  
Major Facilitator

ABSTRACT As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frameYIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1.

scholarly journals EmrE, a Small Escherichia coli Multidrug Transporter, Protects Saccharomyces cerevisiae from Toxins by Sequestration in the Vacuole

1999 ◽  
Vol 181 (3) ◽  
pp. 949-956 ◽  
Author(s):  
Rodrigo Yelin ◽  
Dvir Rotem ◽  
Shimon Schuldiner
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Methyl Viologen ◽  
Fluorescent Protein ◽  
Copper Ions ◽  
Yeast Cells ◽  
Multidrug Transporter ◽  
Uptake System ◽  
Bacterial Cells

ABSTRACT In this report we describe the functional expression of EmrE, a 110-amino-acid multidrug transporter from Escherichia coli, in the yeast Saccharomyces cerevisiae. To allow for phenotypic complementation, a mutant strain sensitive to a series of cationic lipophilic drugs was first identified. A hemagglutinin epitope-tagged version of EmrE (HA-EmrE) conferring resistance to a wide variety of drugs, including acriflavine, ethidium, methyl viologen, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), was functionally expressed in this strain. HA-EmrE is expressed in yeast at relatively high levels (0.5 mg/liter), is soluble in a mixture of organic solvents, and can be functionally reconstituted in proteoliposomes. In bacterial cells, EmrE removes toxic compounds by active transport through the plasma membrane, lowering their cytosolic concentration. However, yeast cells expressing HA-EmrE take up 14C-methyl viologen as well as control cells do. Thus, we investigated the basis of the enhanced resistance to the above compounds. Using Cu2+ ions or methylamine, we could selectively permeabilize the plasma membrane or deplete the proton electrochemical gradients across the vacuolar membrane, respectively. Incubation of yeast cells with copper ions caused an increase in 14C-methyl viologen uptake. In contrast, treatment with methylamine markedly diminished the extent of uptake. Conversely, the effect of Cu2+ and methylamine on a plasma membrane uptake system, proline, was essentially the opposite: while inhibited by the addition of Cu2+, it remained unaffected when cells were treated with methylamine. To examine the intracellular distribution of HA-EmrE, a functional chimera between HA-EmrE and the green fluorescent protein (HA-EmrE-GFP) was prepared. The pattern of HA-EmrE-GFP fluorescence distribution was virtually identical to that of the vacuolar marker FM 4-64, indicating that the transporter is found mainly in this organelle. Therefore, HA-EmrE protects yeast cells by lowering the cytoplasmic concentrations through removal of the toxin to the vacuole. This novel way of detoxification has been previously suggested to function in organisms in which a large vacuolar compartment exists. This report represents the first molecular description of such a mechanism.

scholarly journals Dtr1p, a Multidrug Resistance Transporter of the Major Facilitator Superfamily, Plays an Essential Role in Spore Wall Maturation in Saccharomyces cerevisiae

2002 ◽  
Vol 1 (5) ◽  
pp. 799-810 ◽  
Author(s):  
Thomas Felder ◽  
Edith Bogengruber ◽  
Sandra Tenreiro ◽  
Adi Ellinger ◽  
Isabel Sá-Correia ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Multidrug Resistance ◽  
De Novo ◽  
Physiological Role ◽  
Spore Wall ◽  
Major Facilitator Superfamily ◽  
Spore Surface ◽  
Prospore Membrane ◽  
A Genome ◽  
Major Facilitator

ABSTRACT The de novo formation of multilayered spore walls inside a diploid mother cell is a major landmark of sporulation in the yeast Saccharomyces cerevisiae. Synthesis of the dityrosine-rich outer spore wall takes place toward the end of this process. Bisformyl dityrosine, the major building block of the spore surface, is synthesized in a multistep process in the cytoplasm of the prospores, transported to the maturing wall, and polymerized into a highly cross-linked macromolecule on the spore surface. Here we present evidence that the sporulation-specific protein Dtr1p (encoded by YBR180w) plays an important role in spore wall synthesis by facilitating the translocation of bisformyl dityrosine through the prospore membrane. DTR1 was identified in a genome-wide screen for spore wall mutants. The null mutant accumulates unusually large amounts of bisformyl dityrosine in the cytoplasm and fails to efficiently incorporate this precursor into the spore surface. As a result, many mutant spores have aberrant surface structures. Dtr1p, a member of the poorly characterized DHA12 (drug:H+ antiporter with 12 predicted membrane spans) family, is localized in the prospore membrane throughout spore maturation. Transport by Dtr1p may not be restricted to its natural substrate, bisformyl dityrosine. When expressed in vegetative cells, Dtr1p renders these cells slightly more resistant against unrelated toxic compounds, such as antimalarial drugs and food-grade organic acid preservatives. Dtr1p is the first multidrug resistance protein of the major facilitator superfamily with an assigned physiological role in the yeast cell.

scholarly journals The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

2009 ◽  
Vol 418 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Fernando Teijeira ◽  
Ricardo V. Ullán ◽  
Susana M. Guerra ◽  
Carlos García-Estrada ◽  
Inmaculada Vaca ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Gene Replacement ◽  
Major Facilitator Superfamily ◽  
Cephalosporin C ◽  
Protein Fusion ◽  
Reading Frame ◽  
Peroxisomal Targeting ◽  
Major Facilitator ◽  
Green Fluorescent ◽  
Targeted Inactivation

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM–GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1–CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.

scholarly journals Chemosensitization of Fluconazole Resistance in Saccharomyces cerevisiae and Pathogenic Fungi by a d-Octapeptide Derivative

2004 ◽  
Vol 48 (4) ◽  
pp. 1256-1271 ◽  
Author(s):  
K. Niimi ◽  
D. R. K. Harding ◽  
R. Parshot ◽  
A. King ◽  
D. J. Lun ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Efflux Pumps ◽  
Major Facilitator Superfamily ◽  
Yeast Cells ◽  
Multidrug Efflux ◽  
Atpase Inhibitor ◽  
Multidrug Efflux Pumps

ABSTRACT Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5+) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 μg ml−1), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 μg ml−1). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer d-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted d-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC50], 4 μM) which chemosensitized AD/PDR5+ to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC50, 30 μM) and Cdr2p (IC50, 2 μM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter BenRp, or the FLC drug target lanosterol 14α-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC50, 1 μM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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