FIGURE 5.

The vacuolar copper transporter Ctr6 contributes to production of active SOD1 during mitosis. A, representative in-gel activity assay of S. pombe strain (FY435) harboring a disrupted ctr4Δ allele exhibiting strong reduction of SOD1 activity under low copper conditions (100 μm BCS). Under the same conditions, a ctr4Δ ctr6Δ double mutant strain was devoid of measurable SOD1 activity unless the ctr6⁺ or ctr6⁺-HA₄ allele was returned by integration. Protein extracts prepared from these strains were analyzed for steady-state levels of SOD1 by immunoblotting using anti-SOD1 and anti-α-tubulin antibodies. B, SOD1 activity and steady-state protein levels were determined in the same strains of panel A in the presence of excess CuSO₄ (50 μm). C, cells were analyzed by indirect immunofluorescence microscopy for vacuolar localization of a functional Ctr6-HA₄ fusion protein that was expressed in ctr4Δ ctr6Δ cells grown under copper-limiting conditions (100 μm BCS) (left). FM4–64 staining (right) was visualized by fluorescence microscopy as a marker of vacuolar membranes. Nomarski optics were used to examine cells under fixed (left) and non-fixed (right) conditions. White arrows indicate examples of vacuolar membranes.