FIGURE 8.

Derepression of sulfate assimilation increases oxidative stress and the NADP⁺/NADPH ratio in zinc-limited cells. A, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were inoculated at low density (A₆₀₀ = 0.1) in high or low zinc media as described in Fig. 6C in the presence of the indicated concentration of H₂O₂. Cell densities were then measured after 38 h of culturing at 30 °C. Cell densities are reported as percent of the corresponding untreated controls. B, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were grown in high or low zinc media as described in Fig. 6C and then assayed for reactive oxygen species using the fluorescent probe DCFH-DA. The values are means of three independent experiments, and the error bars represent ± S.D. C, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were grown in low zinc medium (LZM + 1 μm ZnCl₂) prior to assay of total glutathione (Total GSH), reduced glutathione (GSH), and oxidized glutathione (GSSG). The ratios of GSSG/GSH ratios are shown in the right panel. The plotted values are the means of 12 independent cultures, and the error bars represent ±1 S.E. D, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were grown in high or low zinc media as described in Fig. 6C prior to analysis of total NADP(H), reduced NADPH, and oxidized NADP⁺. The NADP⁺/NADPH ratio is reported in the right panel. The data plotted are the means of six independent cultures, and the error bars indicate 1 S.D. p values were determined using the paired, two-tailed Student's t test.