FIGURE 1.

Cadmium rapidly enhances Pca1 expression by increasing stability. A, schematic depiction of the Pca1 P_1B-type ATPase. Gray and black rectangles represent the N-terminal cysteine-rich cytosolic domain encompassing the first 392 amino acids and eight predicted transmembrane domains, respectively. B, cadmium resistance in cells expressing Pca1 alleles. Pca1, N-terminal GFP-fused Pca1 (GFP-Pca1), three tandem HA epitope-tagged Pca1 (HA-Pca1), and Pca1 deleted of the N-terminal cysteine-rich domain (Pca1Δ392) with or without N-terminal GFP fusion were expressed in a BY4741 yeast strain in which endogenous Pca1 is nonfunctional (30). The cells (5 μl, A₆₀₀ = 1.0) were spotted on SC solid medium containing the indicated concentration of CdCl₂, and then cell growth was assessed after 2 days. C–E, HA-Pca1 expression was detected by Western blotting using anti-HA antibodies. The same blot was probed for PGK to determine equal loading. C, cycloheximide chase of yeast cells expressing HA-Pca1. Exponentially growing cells were co-cultured with cycloheximide (100 μg/ml) and then collected at the indicated time points for Western blot analysis. D, rapid up-regulation of Pca1 in response to cadmium. Cadmium (50 μm CdCl₂) was added to exponentially growing yeast cultures, and then the cells were collected into ice-cold kill buffer (15 mm NaN₃ PBS) at the indicated time points. E, cadmium-induced stabilization of Pca1 determined by cycloheximide chase. Yeast cells precultured for 15 min without (NT) or with cadmium (20 μm CdCl₂) were collected at the indicated time points. Cycloheximide (100 μg/ml) was added to the culture medium after collection of time zero. Total protein extracts were subjected to Western blot analysis.