FIGURE 6.

Deletion of abc3⁺ leads to vacuolar accumulation of ZnMP. The indicated isogenic yeast strains were precultured in the presence of Dip (50 μm) or FeCl₃ (Fe, 100 μm), and ALA (200 μm). Cells were washed and incubated in ALA-free medium and treated with Dip (250 μm) or FeCl₃ (100 μm) for 6 h. Monochlorobimane (100 μm) was then added for the last 3 h of culture. Subsequently, ZnMP (2 μm) was added for 30 min. A, iron-starved (Dip) cells were analyzed by fluorescence microscopy for accumulation of fluorescent ZnMP (far left) and bimane-GS (center left). The merged images are shown in the center right panels. Normarski optics (far right) was used to ascertain cell morphology. B, as negative controls, iron-treated strains are shown because shu1⁺ and abc3⁺ genes are known to be repressed under iron-replete conditions. White arrows indicate examples of vacuoles where bimane-GS was sequestered. Yellow arrows depict cytoplasmic accumulation of ZnMP. Results of microscopy are representative of five independent experiments.