In Saccharomyces cerevisiae, the simultaneous resistance to various cytotoxic compounds known as multidrug resistance (MDR) is caused by overexpression of membrane efflux pumps under the control of two main transcriptional activators Pdr1p and Pdr3p. In this work we describe the results of functional analysis of a single Kluyveromyces lactis homolog of the PDR1 gene, which encodes a zinc finger Zn(2)Cys(6)-containing transcription factor. The KlPDR1 deletion generated a strain hypersusceptible to oligomycin, antimycin A and azole antifungals. Overexpression of KlPDR1 from a multicopy plasmid in the Klpdr1Delta mutant strain increased the tolerance of transformants to all the drugs tested (oligomycin, antimycin A and azole antifungals). The plasmid-borne KlPDR1 gene was able to complement drug hypersensitivity of the S. cerevisiae pdr1Deltapdr3Delta mutant strain. The KlPDR1 was found to be necessary for upregulation of the ATP-binding cassette transporter encoded by the KlPDR5 gene and rhodamine 6G efflux out of the cells. The KlPDR5 and some other K. lactis pleiotropic drug resistance (PDR) orthologues were found to contain putative PDR-responsive elements in their promoters. These results demonstrate that KlPdr1p is involved in K. lactis MDR and is required for cell's tolerance to various cytotoxic compounds.
Saccharomyces cerevisiae pell and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL) as well as Kluyveromyces lactis mutants impaired in the respiratory chain function (RCF) containing dysfunctional mitochondria show altered sensitivity to metabolic inhibitors. The S. cerevisiae pell mutant displayed increased sensitivity to cycloheximide, chloramphenicol, oligomycin and the cell-wall perturbing agents caffeine, caspofungin and hygromycin. On the other hand, the pel1 mutant was less sensitive to fluconazole, similarly as the K. lactis mutants impaired in the function of mitochondrial cytochromes. Mitochondrial dysfunction resulting either from the absence of PG and CL or impairment of the RCF presumably renders the cells more resistant to fluconazole. The increased tolerance of K. lactis respiratory chain mutants to amphotericin B, caffeine and hygromycin is probably related to a modification of the cell wall.
The b-Zip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the KNQ1 gene, encoding a multidrug transporter of the major facilitator superfamily in Kluyveromyces lactis and containing two potential Yap1p response elements in its promoter, is a putative transcriptional target of KlYap1p, the structural and functional homologue of ScYap1p. In this work, we provide evidence that KlYAP1 controls the expression of the KNQ1 gene. Using a P(KNQ1)-gusA fusion construct we showed that the expression of KNQ1 is induced upon cell treatment with the oxidizing agents H2O2 and menadione and that this induction is mediated by KlYap1p. These results were confirmed by Northern-blot analysis showing that the expression of KNQ1 is responsive to hydrogen peroxide and dependent on the presence of KlYap1p. The role of KlYAP1 in the control of KNQ1 expression was further demonstrated by EMSA experiments and drug resistance assays. These results clearly demonstrate the involvement of the KlYap1p transcription factor in the control of KNQ1 gene expression.
Decreased susceptibility of K. lactis mutants impaired in the function of cytochrome c, cytochrome c1 and cytochrome-c oxidase to fluconazole, bifonazole and amphotericin B in comparison with the isogenic wild-type strain was observed. Flow cytometry with rhodamine 6G did not show any changes in the accumulation of the dye in the mutant cells compared with the corresponding wild-type strain. Sterol analysis showed similar overall amount of sterols in both wild-type and mutant cells. Taking into account the increased amphotericin B resistance and significantly diminished susceptibility of mutant cells to lyticase digestion, the cell wall structure and/or composition may probably be responsible for the observed changes in the susceptibility of mutants to the antifungal compounds used.
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