Saccharomyces cerevisiae multidrug transporter Qdr2p (Yil121wp): localization and function as a quinidine resistance determinant

Abstract

This work reports the functional analysis of Saccharomyces cerevisiae open reading frame YIL121w, encoding a member of a family of drug:H(+) antiporters with 12 predicted membrane-spanning segments (DHA12 family). Like its close homologue Qdr1p, Yil121wp was localized at the plasma membrane, and its increased expression also led to increased tolerance to the antiarrhythmia and antimalarial drug quinidine. The quinidine resistance phenotype was confirmed for different yeast strains and growth media, including a prototrophic strain, and YIL121w was named the QDR2 gene. Both QDR1 and QDR2 were also implicated in yeast resistance to the herbicide barban (4-chloro-2-butynyl [3-chlorophenyl] carbamate), and the genes are functionally interchangeable with respect to both resistance phenotypes. The average intracellular pH of a yeast population challenged with quinidine added to the acidic growth medium was significantly below the intracellular pH of the unstressed population, suggesting plasma membrane permeabilization by quinidine with consequent increase of the H(+) influx rate. For the same extracellular quinidine concentration, internal acidification was more intense for the Deltaqdr2 deletant compared with the parental strain. Although QDR2 transcription was not enhanced in response to quinidine, the results confirmed that Qdr2p is involved in the active export of quinidine out of the cell, thus conferring resistance to the drug.

FIG. 1.

ORF YIL121w is a determinant of yeast resistance to quinidine and provides slight protection against ketoconazole. The susceptibility to the drugs, at the indicated concentrations, of the S. cerevisiae W303.1b parental strain and the deletion mutant Δyil121w harboring either recombinant plasmid pYCG_YIL121w, with ORF YIL121w inserted into pFL38, or the cloning vector was compared by spot assays in MM2 (without uracil) agar plates. The cell suspensions used to prepare the spots in b and c were 1:2 and 1:4 dilutions of the cell suspension used in a, respectively.

FIG. 2.

Comparison of the susceptibility to quinidine and barban of the S. cerevisiae BY4741 parental strain (▪, •) and the deletion mutant Δyil121w (□, ○), (A) by spot assays (3.6 g of quinidine per liter or 0.09 mM barban) or (B) by cultivation in MM4 liquid medium (▪, □) or in this medium supplemented with 4.0 g of quinidine per liter or 0.08 mM barban (•, ○). Cell suspensions used as inocula were exponential-phase cells cultivated in the absence of chemical stress.

FIG. 3.

Comparison of the susceptibility to barban and quinidine, assessed by spot assays in MM4-U medium, of the S. cerevisiae BY4741 parent strain and the deletion mutants Δqdr1 and Δqdr2 harboring either recombinant plasmid pYCG_YIL120w or pYCG_YIL121w, which have the QDR1 and QDR2 gene inserted into pFL38, respectively, or the cloning vector. The cell suspensions used to prepare the spots in b and c were 1:2 and 1:4 dilutions of the cell suspension used in a, respectively.

FIG. 4.

Comparison of the susceptibility, in minimal medium MM5, to 2.5 g of quinidine per liter or 0.09 mM barban of the prototrophic strain 23344c harboring either recombinant plasmid pYCG_QDR2, with the QDR2 gene inserted into pFL38, or the cloning vector. The cell suspensions used to prepare the spots in b and c were 1:2 and 1:4 dilutions of the cell suspension used in a, respectively.

FIG. 5.

Northern blot hybridization experiments with a DNA fragment of QDR2 as the probe. Ethidium bromide-stained rRNA was used as a loading control. Total RNA was extracted from (A) cells of BY4741 grown in MM4 medium (with 1.4% ethanol) (1 and 2) or in this medium supplemented with 3.6 g of quinidine per liter (3 to 5) or (B) cells of BY4741 grown in MM4 medium (with 0.2% acetone), in the absence (6 and 7) or presence of 0.08 mM of barban (8 to 10).

FIG. 6.

Fluorescence of exponential-phase cells of S. cerevisiae BY4741 harboring (A) expression vector pMET25_GFP (control cells) or the multicopy plasmid pMET25_QDR2-GFP (B, C, and D), indicating that the Qdr2-Gfp fusion protein is localized at the plasma membrane.

FIG. 7.

Comparison of the average pHi of cell populations of the BY4741 parental strain (solid and striped symbols) or the Δqdr2 mutant (open and dotted symbols) grown to the exponential phase (at the standardized culture OD₆₀₀ of 0.6) in the presence (striped and dotted symbols) or the absence (solid and open symbols) of 3.2 g of quinidine (qd) per liter.

FIG. 8.

Time course of the expulsion of [9-³H]quinidine accumulated beforehand in cells of BY4741 (▴, •) or the Δqdr2 mutant (▵, ○) (as shown in a) after transfer to TM buffer (pH 5.5) (▴, ▵) or to the same buffer supplemented with glucose (•, ○). Extracellular quinidine concentrations are average values from at least two independent transport assays with a standard deviation below 0.8. In the inset(a) is shown the time course of [9-³H]quinidine accumulation in deenergized cells of parental strain BY4741 (▪) and deletion mutant Δqdr2 (□) grown in the absence of quinidine and resuspended in TM buffer without glucose. The values shown in the inset are also average values for at least two independent experiments used to obtain a standard deviation below 0.4.