Search

The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H(+)-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme beta-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased [(3)H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.

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.

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.