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. 2014 Oct 1;21(10):1475–1489. doi: 10.1089/ars.2013.5436

FIG. 3.

FIG. 3.

Ccs1p-dependent maturation of Sod1p is necessary for Ycf1p-mediated cadmium resistance. (A) Expression of S. cerevisiae SOD1 or Caenorhabditis elegans wSOD1 rescues Ycf1p function for cadmium resistance in ccs1Δ cells. WT and ccs1Δ cells were co-transformed with YCF1 and SOD1 or wSOD1 expression plasmids and spotted on SC selection media containing indicated CdCl2 concentrations. (B) SOD1 or wSOD1 overexpression rescues lysine (Lys) auxotrophy of ccs1Δ cells. The ccs1Δ cells transformed with an empty vector, SOD1 or wSOD1 expression vector were diluted (OD600=0.5 and 0.1) and spotted on SC media prepared with (+) and without (−) amino acid lysine (Lys). The triangle indicates the 1/5 dilution of cells. (C) Equal amount of lysates prepared from WT, ccs1Δ, and sod1Δ strains with and without expression of SOD1 or wSOD1 were subjected to in-gel superoxide dismutase enzyme assays. Color changes to white on the gel reflect Sod1p activities. (D) YCF1 does not confer cadmium tolerance in sod1Δ cells. The WT and sod1Δ cells transformed with empty vector or YCF1 expression construct were spotted on SC selection media that was supplemented with the indicated CdCl2 concentration. (E) YCF1 knockout in sod1Δ cells does not lead to additional cadmium sensitivity. WT, SOD1 knockout (sod1Δ), and both SOD1 and YCF1 knockout cells (sod1Δycf1Δ) were spotted on SC selection media containing the indicated concentration of CdCl2. (F) Cadmium resistance of sod1Δ cells expressing PCA1. WT and sod1Δ cells transformed with an empty vector or PCA1 expression vector were spotted on SC selection media that was supplemented with CdCl2. (G) WT, ccs1Δ, and sod1Δ cells transformed with an empty vector, or YCF1 expression vector was spotted on SC selection plates that were supplemented with CdCl2. Cells were then cultured under normoxic and oxygen-limited conditions. Cell growth was photographed after 2 days except the right panel of (G), which was photographed in 4 days. Experiments were conducted at least twice with two different clones, and a representative figure is shown.