Cadmium rapidly enhances Pca1 expression by increasing stability.
A, schematic depiction of the Pca1 P1B-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.