Yeast response and tolerance to polyamine toxicity involving the drug : H+ antiporter Qdr3 and the transcription factors Yap1 and Gcn4.

The yeast QDR3 gene encodes a plasma membrane drug : H(+) antiporter of the DHA1 family that was described as conferring resistance against the drugs quinidine, cisplatin and bleomycin and the herbicide barban, similar to its close homologue QDR2. In this work, a new physiological role for Qdr3 in polyamine homeostasis is proposed. QDR3 is shown to confer resistance to the polyamines spermine and spermidine, but, unlike Qdr2, also a determinant of resistance to polyamines, Qdr3 has no apparent role in K(+) homeostasis. QDR3 transcription is upregulated in yeast cells exposed to spermine or spermidine dependent on the transcription factors Gcn4, which controls amino acid homeostasis, and Yap1, the main regulator of oxidative stress response. Yap1 was found to be a major determinant of polyamine stress resistance in yeast and is accumulated in the nucleus of yeast cells exposed to spermidine-induced stress. QDR3 transcript levels were also found to increase under nitrogen or amino acid limitation; this regulation is also dependent on Gcn4. Consistent with the concept that Qdr3 plays a role in polyamine homeostasis, QDR3 expression was found to decrease the intracellular accumulation of [(3)H]spermidine, playing a role in the maintenance of the plasma membrane potential in spermidine-stressed cells.

Saccharomyces cerevisiae multidrug resistance transporter Qdr2 is implicated in potassium uptake, providing a physiological advantage to quinidine-stressed cells.

The QDR2 gene of Saccharomyces cerevisiae encodes a putative plasma membrane drug:H(+) antiporter that confers resistance against quinidine, barban, bleomycin, and cisplatin. This work provides experimental evidence of defective K(+) (Rb(+)) uptake in the absence of QDR2. The direct involvement of Qdr2p in K(+) uptake is reinforced by the fact that increased K(+) (Rb(+)) uptake due to QDR2 expression is independent of the Trk1p/Trk2p system. QDR2 expression confers a physiological advantage for the yeast cell during the onset of K(+) limited growth, due either to a limiting level of K(+) in the growth medium or to the presence of quinidine. This drug decreases the K(+) uptake rate and K(+) accumulation in the yeast cell, especially in the Deltaqdr2 mutant. Qdr2p also helps to sustain the decrease of intracellular pH in quinidine-stressed cells in growth medium at pH 5.5 by indirectly promoting H(+) extrusion affected by the drug. The results are consistent with the hypothesis that Qdr2p may also couple K(+) movement with substrate export, presumably with quinidine. Other clues to the biological role of QDR2 in the yeast cell come from two additional lines of experimental evidence. First, QDR2 transcription is activated under nitrogen (NH(4)(+)) limitation or when the auxotrophic strain examined enters stationary phase due to leucine limitation, this regulation being dependent on general amino acid control by Gcn4p. Second, the amino acid pool is higher in Deltaqdr2 cells than in wild-type cells, indicating that QDR2 expression is, directly or indirectly, involved in amino acid homeostasis.

The yeast multidrug transporter Qdr3 (Ybr043c): localization and role as a determinant of resistance to quinidine, barban, cisplatin, and bleomycin.

Saccharomyces cerevisiae ORF YBR043c, predicted to code for a transporter of the major facilitator superfamily required for multiple drug resistance, encodes a plasma membrane protein that confers resistance to quinidine and barban, as observed before for its close homologues QDR1 and QDR2. This ORF was, thus, named the QDR3 gene. The increased expression of QDR3, or QDR2, also leads to increased resistance to the anticancer agents cisplatin and bleomycin. However, no evidence for increased QDR3 expression in yeast cells exposed to all these inhibitory compounds was found. Transport assays support the concept that Qdr3 is involved, even if opportunistically, in the active export of quinidine out of yeast cell. A correlation was established between the efficiency of quinidine active export mediated by Qdr3p, Qdr2p or Qdr1p, and the efficacy of the expression of the encoding genes in alleviating the deleterious action of quinidine, as well as of the other compounds (QDR2>QDR3>>>QDR1).

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

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.