The pathogenic yeast Candida parapsilosis degrades various hydroxy derivatives of benzenes and benzoates by the gentisate and 3-oxoadipate pathways. We identified the genes MNX1, MNX2, MNX3, GDX1, HDX1 and FPH1 that code for enzymes involved in these pathways in the complete genome sequence of C. parapsilosis. Next, we demonstrated that MNX1, MNX2, MNX3 and GDX1 are inducible and transcriptionally controlled by hydroxyaromatic substrates present in cultivation media. Our results indicate that MNX1 and MNX2 code for flavoprotein monooxygenases catalysing the first steps in the 3-oxoadipate and gentisate pathways, respectively (i.e. 4-hydroxybenzoate 1-hydroxylase and 3-hydroxybenzoate 6-hydroxylase). Moreover, we found that the two pathways differ by their intracellular localization. The enzymes of the 3-oxoadipate pathway, Mnx1p and Mnx3p, localize predominantly in the cytosol. In contrast, intracellular localization of the components of the gentisate pathway, Mnx2p and Gdx1p, depends on the substrate in the cultivation medium. In cells growing on glucose these proteins localize in the cytosol, whereas in media containing hydroxyaromatic compounds they associate with mitochondria. Finally, we showed that the overexpression of MNX1 or MNX2 increases the tolerance of C. parapsilosis cells to the antifungal drug terbinafine.

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROXl factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using ZacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROXl factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROXl factor.The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADPIATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX%, HEM13 and CYC7 that are negatively regulated by oxygen and heme.In Saccharomyces cerevisiae the mitochondria1 ADP/ATP translocator exists as three isozymes that are encoded by distinct nuclear genes, AACI, AAC2, and AAC3 [l-31. The reason for the existence of multiple genes encoding a protein functioning in ADP/ATP translocation across mitochondrial membrane is not apparent. Gene-disruption experiments have shown that the product of AAC2 gene is the major protein involved in ADP/ATP exchange across the mitochondrial membrane and that it is essential for oxidative phosphorylation [2-41. The disruption of the AACl or AAC3 gene did not yield a distinct phenotype, but the combination of these mutations with other nuclear mutations resulted in phenotypes indicating a vacuolar role for the AACZ product and a role in the anaerobic metabolism for the AAC3 product [3, It has been shown that AAC genes are differentially regulated to express separate isofoms under different physiologi-51. and that a functional ADP/ATP translocator is essential for anaerobic growth of yeast cells [9].The expression of several other yeast genes, TIFSIB (ANBI) [lo, 161, COX% [ll, 121, CYC7 [131, HEM13 [141 and ERG11 [15] is negatively regulated by oxygen. These genes encode proteins functioning in various metabolic pathways including translation initiation (TIFSZB), mitochondrial respiratory chain (CYC7, COX%), heme (HEMZ3) and ergosterol (ERGll) biosynthesis. The oxygen repression in most of these genes appears to be mediated through the action of ROXl factor, the expression of which is heme dependent [ 17, 181. Deletion analysis and site-directed mutagenesis of TIF.51 B promoter pinpointed the upstream sequences which are most probably involved in the negative control of TIFSIB gene [19, 201. Similar sequences are also present in the upstream regions of the o...

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).

The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina, which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are suitable candidates for the missing enzymes of the pathway.