FIGURE 1.

Overexpression of Ccc1 in Δmrs3mrs4 cells decreases cytosolic iron leading to a poor growth phenotype. A, wild type (WT) and Δmrs3Δmrs4 cells were grown in CM medium with 500 μm iron overnight. Cells were homogenized, vacuoles were isolated, and samples were applied to a SDS-PAGE and examined by Western blot analysis. The blots were probed with a rabbit anti-Ccc1 or mouse anti-carboxypeptidase Y (CPY) followed by a peroxidase-conjugated goat anti-rabbit IgG or peroxidase-conjugated goat anti-mouse IgG. Carboxypeptidase Y was used as a loading control. B, wild type and Δmrs3Δmrs4 cells were grown in CM medium and then incubated with ⁵⁹Fe, as described under “Experimental Procedures.” Vacuoles were isolated, and the amount of radioactivity in isolated vacuoles was determined. The data were normalized to vacuolar protein. C, wild type and Δmrs3Δmrs4 cells transformed with MET3CCC1 plasmid were grown for 2 days on plates with (CCC1 off) or without 10× methionine (CCC1 on). D, wild type and Δmrs3Δmrs4 cells were transformed with a MET3CCC1 plasmid together with a FET3-lacZ reporter plasmid. Cells were grown overnight at 30 °C with or without 10× methionine, and FET3-lacZ activity was measured (left panel). Cells were grown overnight at 30 °C with or without 10× methionine in the absence or presence of varying amounts of ferrous ammonium sulfate, and FET3-lacZ activity was measured (right panel). E, wild type cells or Δmrs3Δmrs4 transformed with a control vector or Δmrs3Δmrs4 cells transformed with a MET3CCC1 plasmid were serial-diluted and grown for 2 days on plates with (CCC1 off) or without 10× methionine (CCC1 on) and with or without five mm ferrous ammonium sulfate. F, wild type and Δmrs3Δmrs4 cells transformed with a MET3CCC1-FLAG plasmid were incubated at 30 °C in the absence of methionine for 6 h. Cells were homogenized with glass beads, cell lysates were applied to SDS-PAGE, and Western blot analysis with either anti-Ccc1 or anti- carboxypeptidase Y was performed as A (left panel). The data were quantified using densitometric analysis (right panel). All experiments were performed a minimum of three times, and error bars represent the S.E.