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

As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frame YIL120w 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.

基于结构层面的预测，本研究结果显示，酿酒酵母（Saccharomyces cerevisiae）开放阅读框（open reading frame）YIL120w编码的主要促进因子超家族（major facilitator superfamily）成员是一类多药耐药决定因子。Yil120wp与酵母对酮康唑（ketoconazole）及奎尼丁（quinidine）的抗性相关，但与其立体异构体奎宁（quinine）无关，该基因因此被命名为QDR1。实验证实Qdr1p可缓解奎尼丁带来的有害影响：未预适应的酵母群体突然暴露于该药物后会出现细胞活力丧失，而Qdr1p的存在能使细胞在奎尼丁胁迫下更早恢复指数生长。不过，QDR1基因的表达对预先适应奎尼丁的酵母细胞的敏感性无可检测到的显著影响。对活酵母细胞中Qdr1-绿色荧光蛋白（green fluorescent protein, GFP）融合蛋白分布的荧光显微镜观察结果表明，Qdr1p定位于质膜（plasma membrane）。本研究还通过实验证明，酵母在奎尼丁中生长的适应过程涉及预加载该药物的细胞主动外排该药物，尽管这种兼具抗心律失常与抗疟活性的含喹啉环（quinoline ring）药物并不存在于酵母的自然生存环境中。但本研究未能证实Qdr1p直接参与这一外排过程。研究结果明确提示，在缺失QDR1的情况下，酵母还存在其他尚未被鉴定的奎尼丁抗性机制。