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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 ⌬qdr2 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 ⌬qdr2 cells than in wild-type cells, indicating that QDR2 expression is, directly or indirectly, involved in amino acid homeostasis.

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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 ⌬qdr2 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.The therapeutic potential of drugs is seriously limited by the manifestation of cellular drug resistance (11). The yeast Saccharomyces cerevisiae is an easy-to-manipulate experimental eukaryotic model useful to unveil novel determinants and mechanisms underlying the apparently conserved multidrug resistance (MDR) phenomenon in more complex and less genetically accessible eukaryotes. The present knowledge on the poorly characterized families of putative yeast drug:H ϩ antiporters belonging to the major facilitator superfamily (MFS) was driven by postgenomic yeast research (21). The great majority of the approximately 23 yeast genes encoding proteins of the MFS (15,18,21), putatively or proven to be involved in multidrug resistance, escaped characterization before the release of the genome sequence. These proteins comprise 12 and 14 predicted membrane-spanning segments and were included in the so-called DHA12 and DHA14 drug efflux families (18).In the present work, we carried out a functional analysis of S. cerevisiae open reading frame (ORF) YIL121w, encoding a member of the DHA12 family and a close homologue to the previously characterized QDR1 gene (ORF YIL120w), required for yeast resistance to quinidine and ketoconazole (17). Qdr1p and Yil121wp, with 64% sequence identity, belong to the same cluster I in the classification of Nelissen (15), who distinguished two clusters in the DHA12 drug efflux family. Like Qdr1p, Yil121wp was localized at the plasma membrane, and its increased expression led to increased tolerance to quinidine. ORF YIL121w was thus named the QDR2 gene. Other proteins of the...

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. INTRODUCTIONMultidrug efflux transporters are found in all living cells and are thought to recognize a wide variety of structurally and pharmacologically unrelated drugs. They are proposed to actively extrude or compartmentalize drugs and other xenobiotics, thus providing protection from these compounds (Jungwirth & Kuchler, 2006;Paulsen, 2003;Prasad et al., 2002;Roepe et al., 1996;. The activity of these proteins underlies the manifestation of cellular drug resistance, seriously limiting the therapeutic potential of drugs (Hayes & Wolf, 1997). The in silico analysis of the yeast genome revealed the existence of 23 putative drug : H + antiporters of the multiple drug resistance (MDR) family of the major facilitator superfamily (MFS). Although many of these proteins have been shown to confer resistance to a wide variety of drugs and chemicals (Paulsen et al., 1998;, the molecular mechanisms behind their apparent promiscuity remain elusive and a topic of debate (Jungwirth & Kuchler, 2006;Paulsen, 2003;Prasad et al., 2002;Roepe et al., 1996;.In the present work, we have further examined the biological function of the MFS-MDR transporter encoded by the QDR3 gene, which is present in the plasma membrane of Saccharomyces cerevisiae (Huh et al., 2003;Tenreiro et al., 2005). Qdr3 belongs to cluster I of the DHA1 drug efflux family, including putative drug : H + antiporters with 12 predicted membrane-spanning segments (Paulsen et al., 1998), and confers yeast resistance against the antiarrhythmic and antimalarial drug quinidine, the herbicide barban and the antitumour agents bleomycin and cisplatin (Tenreiro et al., 2005). Through genome-wide screenings, QDR3 gene deletion was also found to increase yeast susceptibility towards ma...