FIG. 6.

Metal specificity of cytoplasmic sequestration of Cuf1-GFP. (A) A cuf1Δ strain of S. pombe which was transformed with either pSP1JB-2283cuf1⁺-GFP or pJBnmt1⁺-cuf1⁺-GFP was grown to mid-logarithmic phase in EMM containing 5 μM thiamine. After two washes, the cells were incubated during the indicated time (0 and 4 h) in EMM without thiamine and in the presence of CuSO₄ (Cu) (100 μM) or BCS (100 μM). Total RNA was prepared and analyzed from culture aliquots. Arrows indicate signals corresponding to ctr4⁺ and act1⁺ mRNA steady-state levels. Results shown are representative of three independent experiments. (B) Strain JSY17 expressing Cuf1-GFP was grown to mid-logarithmic phase in the presence of 5 μM thiamine. Cells were washed twice and then incubated in the presence of CuSO₄ (100 μM), AgNO₃ (2 μM), ZnSO₄ (1 mM), FeCl₃ (100 μM), CdCl₂ (25 μM), or BCS (100 μM). After 4 h treatment, the full-length Cuf1-GFP protein was viewed by direct fluorescence microscopy (GFP). GFP alone expressed under the control of the nmt1⁺ promoter was also examined by direct fluorescence. Corresponding Nomarski images are shown after each GFP panel.