FIG. 5.

Roles for genes encoding antioxidant in Ycf1p-mediated cadmium resistance. (A) WT and antioxidant gene deletion strains were transformed with an empty vector or a YCF1 expression plasmid. Cells were spotted on SC selection media containing indicated CdCl₂ concentrations. (B) Limited oxygen rescues Ycf1p functions for cadmium resistance in glr1Δ cells. WT and glr1Δ cells transformed with empty vector or YCF1 expression vector were spotted on SC selection that was supplemented with CdCl₂. Cells were then cultured under normal and limited oxygen conditions. (C) The glr1Δ cells display higher Sod1p enzyme activities. Sod1p activities in cell lysates of the indicated yeast strains were detected by in-gel assays. (D) The glr1Δ cells transformed with empty vector or an expression construct of GFP-fused functional Ycf1p were co-cultured with CdCl₂ (15 μM, 9 h) and then subjected to confocal microscopy. Co-culture of cells with FM4-64 at the last 30 min of cadmium exposure stained the vacuolar membrane. (E) GSH levels and (F) GSH/GSSG ratio in WT control, ccs1Δ, and sod1Δ cells with and without YCF1 expression. GSH and GSSG levels were measured using deproteinated lysates of the cells that were cultured without (black bars) or with (gray bars) CdCl₂ (15 μM CdCl₂, 9 h). The results were normalized to protein concentrations of cell lysates before deproteination and presented as relative levels to those of WT expressing empty vector. Each datum represents the average±SD of four experiments. All other experiments were conducted at least twice with two different clones, and a representative figure is shown. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars