Ycf1p attenuates basal level oxidative stress response in Saccharomyces cerevisiae

Abstract

Ycf1p function is regulated by casein kinase 2α, Cka1p, via phosphorylation of Ser251. Cka1p-mediated phosphorylation of Ycf1p is attenuated in response to high salt stress. Previous results from our lab suggest a role for Ycf1p in cellular resistance to salt stress. Here, we show that Ycf1p plays an important role in cellular resistance to salt stress by maintaining the cellular redox balance via glutathione recycling. Our results suggest that during acute salt stress increased Sod1p, Sod2p and Ctt1p activity is the main compensatory for the loss in Ycf1p function that results from reduced Ycf1p-dependent recycling of cellular GSH levels.

Figure 1. Deletion of ycf1Δ increases intracellular NaCl-mediated ROS and oxidative stress

A) ROS (panel A), MDA formation (panel B) and total protein carbonylation (panel C) was measured in WT and ycf1Δ cells after treatment with 0.5 M NaCl by DCF fluorescence as described in the Materials and Methods. Statistical analysis was carried-out in Prism Graphpad by One-Way and Two-Way ANOVA analysis with Bonferroni post tests (*=p<0.05, **=p<0.01, all statistics shown are with respect to untreated WT). All experiments were carried out with an n≥3. Blots shown here are representative of our findings which show that ycf1Δ has increased basal carbonylation as compared to WT which directly affects carbonylation in NaCl and H₂O₂ treated cells as dose increases.

Figure 2. NaCl treatment dramatically reduces intracellular GSH in ycf1Δ cells

Free intracellular GSH (panel A) and the intracellular [GSH]/[GSSG] ratio (panel B) was determined in untreated (black bars) H₂O₂ treated (grey bars),and NaCl treated (hatched bars) WT and ycf1Δ cells, as described in the Materials and Methods. Statistical analysis was carried-out in Prism Graphpad by One-Way and Two-Way ANOVA analysis with Bonferroni post tests (*=p<0.05, **=p<0.01, all statistics shown are with respect to untreated WT).

Figure 3. The oxidative stress response systems are upregulated in response to NaCl stress in ycf1Δ cells

Activities of Sod1p (panel A), Sod2p (panel B), Ctt1p (panel E) and GPx (panel F) were measured in untreated (black bars), H₂O₂ treated (grey bars) and NaCl treated (hatched bars) WT and ycf1Δ cells, as described in the Materials and Methods. Expression of Sod1p (panel C), Sod2p (panel D), Ctt1p (panel G), Gpx1p and Gpx2p (panel H) was measured by Western blot in untreated (black bars), H₂O₂ treated (grey bars) and NaCl treated (hatched bars) WT and ycf1Δ cells, as described in the Materials and Methods. Statistical analysis was carried-out in Prism Graphpad by Two-Way ANOVA analysis with Bonferroni post tests (*=p<0.05, **=p<0.01) where untreated WT and ycf1Δ served as controls for their respective treatment conditions, either NaCl or H₂O₂.

Figure 4. Deletion of Ycf1p in Sod1p and Ctt1p deletion strains increases cellular resistance to NaCl and H2O2

WT, ycf1Δ , sod1Δ, sod1Δ ycf1Δ, sod2Δ, sod2Δ ycf1Δ, ctt1Δ, ctt1Δ ycf1Δ, gpx1Δ, gpx1Δ ycf1Δ, and gpx2Δ, gpx2Δ ycf1Δ were tested for growth by spot test on plates containing vehicle alone,5 mM H₂O₂, and 0.5 M NaCl as described in the materials and methods..