FIG. 9.

Cu-mediated inhibition of nuclear import of Cuf1 by its C-rich motif. (A) S. pombe JSY8 cells were cotransformed with an empty control plasmid and GFP alone, an empty vector and ¹cuf1⁶¹-GFP, ¹cuf1⁶¹-GFP and ⁶²cuf1⁴¹⁰-FLAG₂, or ¹cuf1⁶¹-GFP and ⁶²cuf1-M6⁴¹⁰-FLAG₂. To examine GFP fluorescence, the cotransformed cells specified above were grown to A₆₀₀ of 1.0 in the presence of 5 μM thiamine, at which step the cultures were washed twice. Cultures were divided for their respective treatments (100 μM CuSO₄ or 100 μM BCS) and grown in selective media lacking thiamine to induce protein synthesis. After 4 h, cells were subjected to fluorescence microscopy to visualize the ¹Cuf1⁶¹-GFP fusion protein (GFP). Cell morphology was also examined through Nomarski optics (Nomarski). (B) Whole-cell extracts were prepared from aliquots of cultures described above for panel A and analyzed by immunoblotting using either anti-GFP or anti-FLAG antibody (a). For simplicity, results shown are samples that were analyzed from copper-replete cells since the expression level of proteins detected from copper-deficient cells were virtually identical. As a control, total extract preparations were probed with anti-PCNA antibody. WB, Western blot.