Figure 3.

Cadmium exposure activates Met4. (A) Wild-type cells were grown in YEPD to OD₆₀₀ = 0.5, incubated with cadmium at the concentrations indicated for 30 min, and cell lysates were analyzed by immunoblotting (gel 7.5%) with polyclonal antibodies directed against Met4 and the proteasome subunit Rpt1 as a loading control. (B) Experimental procedures were as in A, but cells were incubated in YEPD containing 100 μM cadmium for the period indicated. (C) Wild-type cells were grown as in A, incubated in medium containing 1 mM cadmium for 30 min, and expression of Met4-target genes MET3, MET25, and GSH1 was analyzed by real-time RT-PCR. Expression levels were normalized to ACT1 expression. (D) Wild-type cells were grown in YEPD or YEPD supplemented with methionine (1 mM final concentration) to OD₆₀₀ = 0.5, incubated with cadmium at the concentrations indicated for 30 min, and cell lysates were analyzed as described in A. (E) Cells expressing 12myc-Met30 under control of its own promoter (PY1073) were grown in YEPD to an OD₆₀₀ = 0.5 and either exposed or not exposed to 1 mM cadmium for 30 min. Cells expressing untagged Met30 (PY236) were processed in parallel. Cell lysates were immunopurified with anti-myc antibodies and analyzed by immunoblotting with anti-Met4 and anti-Skp1 antibodies.