Contribution of Yap1 towards Saccharomyces cerevisiae adaptation to arsenic-mediated oxidative stress

Abstract

In the budding yeast Saccharomyces cerevisiae, arsenic detoxification involves the activation of Yap8, a member of the Yap (yeast AP-1-like) family of transcription factors, which in turn regulates ACR2 and ACR3, genes encoding an arsenate reductase and a plasma-membrane arsenite-efflux protein respectively. In addition, Yap1 is involved in the arsenic adaptation process through regulation of the expression of the vacuolar pump encoded by YCF1 (yeast cadmium factor 1 gene) and also contributing to the regulation of ACR genes. Here we show that Yap1 is also involved in the removal of ROS (reactive oxygen species) generated by arsenic compounds. Data on lipid peroxidation and intracellular oxidation indicate that deletion of YAP1 and YAP8 triggers cellular oxidation mediated by inorganic arsenic. In spite of the increased amounts of As(III) absorbed by the yap8 mutant, the enhanced transcriptional activation of the antioxidant genes such as GSH1 (gamma- glutamylcysteine synthetase gene), SOD1 (superoxide dismutase 1 gene) and TRX2 (thioredoxin 2 gene) may prevent protein oxidation. In contrast, the yap1 mutant exhibits high contents of protein carbonyl groups and the GSSG/GSH ratio is severely disturbed on exposure to arsenic compounds in these cells. These results point to an additional level of Yap1 contribution to arsenic stress responses by preventing oxidative damage in cells exposed to these compounds. Transcriptional profiling revealed that genes of the functional categories related to sulphur and methionine metabolism and to the maintenance of cell redox homoeostasis are activated to mediate adaptation of the wild-type strain to 2 mM arsenate treatment.

The Yap family and its role in stress response

“…Both proteins constitute a first-line defence, since they facilitate the extrusion of arsenite molecules from the cytoplasm. Yap1 contributes to arsenic stress responses to a lesser extent than Yap8 at at least three different levels: (a) regulating YCF1 expression, which corresponds to a parallel arsenite detoxification system by catalysing the ATP-driven uptake of arsenite (AsIII)-GSH conjugates into the vacuole; (b) contributing to regulated expression of ACR2 and ACR3 (18); and (c) maintaining the redox homeostasis disturbed by arsenic compounds, as we have recently shown [69]. In the absence of Yap1, cells accumulate increased levels of lipid peroxidation, intracellular oxidation and protein carbonylation when cells are subjected to arsenic treatment.…”

“…Yap8 and Yap1 are the master regulators of Acr2/Acr3 and Ycf1, respectively, and their activity is essential to the efficient removal of arsenite from the cytoplasm. Additionally, Yap1 is important to the maintenance of the redox homeostasis disturbed by arsenic compounds (adapted from Menezes et al, 2008) its localization. The discrepancy in these results may be due to methodological reasons and the use of different strains.…”

Insights into the intracellular mechanisms of citronellal in Candida albicans: implications for reactive oxygen species-mediated necrosis, mitochondrial dysfunction, and DNA damage

“…However, Yap1p is not directly oxidized, but needs a glutathione peroxidase (Gpx)-like protein to transduce Yap1p signaling by creating a disulfide bond between Gpx3p and Cys36 (evolutionarily conserved redox-sensitive residues) and Yap1p with Cys598 14 . It is well-known that ROS accretion plays a pivotal role in the necrosis of yeast cells 6 . For instance, natural compounds, such as melittin, silymarin, and silibinin, induce apoptotic pathways via ROS generation [15][16][17] .…”

“…This was achieved using DCFDA, an oxidant-sensitive probe, to assess ROS generation; in this analysis, increased fluorescence within cells indicates enhanced ROS levels 6 . As expected, the fluorescence in Cit-treated cells was considerably higher than that in untreated cells.…”

“…Cells were harvested and washed three times with phosphate-buffered saline (PBS) stained with PI (1µg mL -1 ), an indicator of membrane damage, DCFDA (1µg mL -1 ), an indicator of ROS generation, Rhodamine B (0.5µg mL -1 ), an indicator of mitochondrial membrane potential (∆Ψm), and DAPI (2µg mL -1 ), an indicator of DNA damage, for 20 min in the dark [6][7][8] . For PI experiments, Candida cells were pre-incubated with 5mM NaN 3 for 60 min.…”