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Multidrug resistance (MDR), a ubiquitous phenomenon conserved from bacteria to humans, causes serious problems in the treatment of human cancers and infections of bacterial and fungal origin. The development of MDR in yeast is frequently associated with gain-of-function mutations in the Zn 2 Cys 6 transcription factors activating the expression of several plasma membrane exporters. In the aerobic yeast Kluyveromyces lactis the Zn 2 Cys 6 transcription factor Kl Pdr1p is involved in the control of multidrug resistance. The aim of the present study was to identify the changes in K. lactis proteome of the Klpdr1Δ deletion mutant compared with the wild-type expressing the KlPDR1 gene from a multicopy plasmid. A total of 15 differentially expressed proteins, out of 20 spots with different intensities detected, were identified. In the Klpdr1Δ deletion mutant, the increase in the abundance of proteins involved in carbohydrate metabolism (mainly glycolysis/gluconeogenesis) was observed. Most of the proteins overexpressed in the wild type strain containing the KlPDR1 gene on multicopy plasmid were involved in the stress defence and redox homeostasis. The results indicate a close connection between MDR and oxidative stress response associated with the post-translational mechanisms regulating the levels of active forms of proteins involved in K. lactis MDR.

The KlYAP1 and KlPDR1 genes encode two main transcriptional regulators involved in the control of multidrug resistance in Kluyveromyces lactis. Deletion of KlPDR1 or KlYAP1 genes in K. lactis generated strain hypersusceptible to diamide, benomyl, fluconazole and oligomycin. Overexpression of genes KlPDR1 or KlYAP1 from a multicopy plasmid in the Klpdr1Δ mutant strain increased the tolerance of transformants to all the drugs tested. YRE response elements were found in the promoter of the KlPDR1 gene. Gel retardation assays confirmed the binding of KlYap1p to the YREs in the KlPDR1 gene promoter indicating that KlYap1p can control the KlPDR1 gene expression.

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.

Wolfram syndrome 1 (WFS1) is a genetic disorder which has been associated both with impaired early brain development and neurodegeneration. Recent studies have suggested regulation of Ca2+ homeostasis by wolframin (Wfs1) and have demonstrated the involvement of endoplasmatic reticulum (ER) stress in Wfs1 deficiency. Despite the ER dysfunction WFS1 shows several characteristics of pathologies related to mitochondrial dynamics. Therefore our aim was to examine the hypothesis that Wfs1 deficiency could disturb mitochondrial dynamics contributing to impaired neuronal functioning. First we show that Wfs1 deficiency induces mild ER stress leading to Inositol 1,4,5-Trisphosphate Receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn alters mitochondrial trafficking, inhibits mitochondrial fusion and augments mitophagy. The overexpression of the active IP3R fragment restores IP3R-mediated Ca2+ release and corrects all perturbations in mitochondrial dynamics suggesting that these events are causally linked. We further demonstrate that suppressing the expression of two Parkinson disease-related proteins, Pink1 and Parkin, leads to reduced Wfs1 deficiency-induced mitophagy and also to the correction of the fusion-fission dynamics and mitochondrial motility. These data suggest that Wfs1 deficiency may over-activate Pink1 and Parkin pathways. Our most important discovery is that Wfs1 deficiency delays neuronal development and axonal growth in primary rat cortical neurons. According to our data, the link between Wfs1 deficiency and delayed neuronal development appears to be mediated by impaired mitochondrial dynamics because suppression of the Pink1-Parkin pathway corrected also the developmental delay. Our data shed light on the mechanisms of neuronal abnormalities in WFS1 and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.