The absence of Zip1 does not result in sulfur amino-acid auxotrophy. Wild-type, zip1⁺-deleted and met5-1 mutant cells were streaked on minimal medium whose sulfur sources are provided from only Na₂SO₄ (middle) or methionine (right) and incubated at 26°C for 5 days. Some growth of met5-1 in the presence of Na₂SO₄ is most likely ascribable to the existence of residual methionine pool inside met5-1 cells.

A model for the function and regulation of Zip1. Zip1 is constitutively produced, but its cellular levels are kept low under normal, noncadmium, growing conditions (left). This is achieved by constitutive SCF^Pof1-dependent ubiquitylation and proteasome-mediated degradation. Binding between Pof1 and Zip1 is dependent upon Zip1 phosphorylation. Under cadmium stress, some switch triggers an increase in Zip1 protein levels and its transcriptional activity. This results in an increased production of glutathione coupled with an arrest of cell growth, thereby ensuring high cell viability during cadmium exposure. The mechanism of this switch is unknown, but the accumulation of phosphorylated Zip1 species during cadmium exposure suggests that the phosphorylation of Zip1 could play some role in transcriptional activity.

Ubiquitylation of Zip1. (A) Zip1 is polyubiquitylated in vivo. Plasmids containing 6His-ubiquitin (His-Ubi, lanes 1 and 3) or empty vector (lanes 2 and 4) were introduced into the mts3-1 mutant containing Zip1-HA. After 1 h incubation at 36°C, ubiquitylated proteins were purified with Ni²⁺-NTA beads (lanes 1 and 2). Total extracts (60 μg) are also run (lanes 3 and 4). Immunoblotting was performed with anti-HA antibody. (B) Synthetic phenotype between pof1-6 and mts3-1. mts3-1 single (left) or mts3-1 pof1-6 double mutants (right) containing Zip1-HA were streaked on rich medium and incubated at 26°C for 3 days (upper). Cell morphology for mts3-1 pof1-6 is also shown (lower). The bar indicates 10 μm.

Zip1 is required for maintenance of viability during cadmium exposure. (A) Viability under cadmium stress. Wild-type (triangles), pof1-6 (squares) or zip1-deleted cells (circles) were treated with 0.5 mM cadmium sulfate at 26°C and cell viability was examined at every 2 h interval. At each time point, 2 × 200 cells were plated onto rich medium (lacking cadmium) and incubated at 26°C for 5 days. The number of viable colonies was counted and viability was calculated by dividing this number by 200 and calculating an average. (B) Cell size change upon cadmium exposure. The cell size was measured and plotted (n=100 per time point). (C) Cell morphology under cadmium stress. Phase-contrast micrographs of wild-type cells before (left) or after cadmium treatment (6 h, right) are shown. The bar indicates 10 μm. (D) Percentage of septated cells.

Comparison of Zip1-target and cadmium-specific genes and accumulation of Zip1 upon cadmium exposure. (A) Summary of DNA microarray analysis. Genes induced by cadmium stress in a Zip1-dependent manner and those induced in a cadmium stress-specific manner (Chen et al, 2003) are compared. (B) Increase of Zip1 upon cadmium exposure. Exponentially growing wild-type (lanes 2) or pof1-6 cells containing Zip1-HA (lane 7) were shifted from 26 to 36°C and incubated for 1 h (lanes 3 and 8). Then, 0.5 mM cadmium sulfate was added, followed by protein extracts at 0, 30, 90 and 180 min time points (lanes 3–6 and 8–11). Immunoblotting was performed with anti-HA (upper) or anti-Cdc2 antibody (lower). Untagged wild type was used as a negative control (lane 1). (C) Quantification. Zip1-HA levels shown in (B) are quantified using Cdc2 as a control.

Complex formation between Pof1 and cullin-1 and creation of ts pof1 mutants. (A) Interaction between Pof1 and cullin-1. Protein extracts were prepared from a single tagged (pcu1⁺-13myc, lanes 1, 2, 4 and 5), or double-tagged strain (pof1⁺-GST pcu1⁺-13myc, lanes 3 and 6), and immunoprecipitation was performed with anti-GST antibody (lanes 4–6). After running on SDS–PAGE, immunoblotting was performed with anti-Myc (upper) or anti-GST antibody (lower). (B) Structure of Pof1 and mutation sites in pof1-6 and pof1-12. Pof1-6 contains two amino-acid replacements at P109S and S118P, while Pof1-12 carries K246E and S566G mutations. F-box motif and WD40 repeats are shown with boxes. (C) Cell morphology. A pof1-6 strain was grown at 26°C (left), shifted to 36°C and incubated for 6 h (middle). Phase-contrast microscopy images are shown. In addition, average cell length (n=200) was plotted at each time point. The bar indicates 10 μm. (D) DNA content. A pof1-6 strain was grown at 26°C (left) or 36°C (right) and samples were collected for flow cytometry at 2 h intervals. Note that a small G1 (1C) peak appeared at 6 and 8 h at 36°C.

Identification of zip1 as an extragenic suppressor locus of pof1 mutants. (A) Cadmium hypersensitivity of sup9 (zip1). Wild-type (top row), sup9 (=zip1, middle) or pap1-deleted cells (bottom), which are defective in oxidative stress response (Toone and Jones, 1998), were spotted onto rich plates in the absence (left) or presence (right) of 0.5 mM cadmium sulfate (10⁵ cells in the far-left spots for each plate and then diluted 10-fold in each subsequent spot rightwards) at 26°C and incubated for 4 days. (B) Schematic structure of Zip1. The position of the nonsense mutation in sup9 (from glycine to TGA) and the C-terminal ZIP region are shown. (C) Suppression of pof1-6 by zip1 deletion. Four strains (wild type, pof1-6, zip1 deletion and pof1-6 zip1) were streaked on rich medium and incubated at 26 or 36°C. (D, E) Toxicity of overexpressed zip1⁺. Plasmids containing zip1⁺ under the thiamine repressible mild nmt41 promoter (D) or strongest nmt1 promoter (E) were transformed into pof1-6 (D) or wild-type cells (E), respectively, streaked on minimal plates in the absence (upper) or presence (lower) of thiamine and incubated at 26°C for 3 days. Cell morphology of pof1-6 or wild-type cells containing zip1⁺ plasmids under each condition is shown in the right panels together with morphology of cells containing empty plasmids. The bar indicates 10 μm.

Stabilization of Zip1 in pof1 mutants and binding between Pof1 and phosphorylated Zip1. (A) Accumulation and partial stabilization of Zip1 in pof1-6. Exponentially growing wild-type pof1⁺-GFP (upper gel) or pof1-6-GFP (lower) cells containing Zip1-HA were shifted to 36°C for 2 h and CHX (100 μg/ml) was then added (time 0). Protein samples were prepared at indicated time points. Immunoblotting was performed with anti-HA (top), anti-GFP (middle) or anti-Cdc2 antibody (bottom). Samples from untagged strains were also run as a negative control (lane 1). Nonspecific band is marked with asterisk. (B) Quantification of Zip1 levels. Zip1-HA amount shown in (A) was quantified. Intensities of upper (squares), or lower bands (triangles) of Zip1-HA in pof1-6 and a lower band (diamonds) in wild type were measured. (C) Phosphorylation of Zip1. pof1-6 mutant cells containing Zip1-HA were shifted to 36°C for 4 h and immunoprecipitation was performed with mock (lane 3) or anti-HA antibody (lanes 4 and 5). Immunoprecipitates were treated with λ-protein phosphatase for 30 min at 30°C (lane 5). Total extract (30 μg) was also run (lane 2). As a negative control, protein samples prepared from untagged pof1-6 were also run (lane 1). Immunoblotting was performed with anti-HA antibody. Three Zip1-HA bands are marked. (D, E) Binding between Zip1 and Pof1. Immunoprecipitation was performed with anti-HA (lanes 4 and 6), anti-GFP antibody (lanes 5 and 7) or mock (lane 3 in D), using protein extracts prepared from mts3-1 (D and lanes 2, 4 and 5 in E) or pof1-6 mutants containing Pof1-GFP Zip1-HA (lanes 3, 6 and 7 in E), which were incubated at 36°C for 1 h. Total extracts from a tagged (lane 2) or untagged strain (lane 1) were also run. Immunoblotting was performed with anti-GFP (upper) or anti-HA antibody (lower).