Figure 4.

Requirement of mitochondrial iron for cytoplasmic iron–sulfur cluster assembly. A, WT mitochondria (200 μg of proteins) were incubated with WT cytoplasm (200 μg of proteins), apo-ΔN60 Yah1, [³⁵S]cysteine, and nucleotides (1 mm GTP, 2 mm NADH, and 4 mm ATP) at 30 °C for 30 min, in the absence or presence of added iron (Fe²⁺; 10 μm ferrous ascorbate). After centrifugation, the cytoplasm/supernatant (S) fractions were analyzed. B, WT mitochondria (400 μg of proteins) were prelabeled by incubating with [³⁵S]cysteine and nucleotides at 30 °C for 20 min, in the absence or presence of iron (10 μm) (first step). Mitochondria (³⁵S-PL) were recovered, washed, and then mixed with WT cytoplasm (200 μg of proteins), nucleotides, and as indicated, apo-ΔN60 Yah1. The samples were incubated at 30 °C for 30 min, with or without added iron (10 μm) (second step). After centrifugation, the cytoplasm/supernatant (S) fractions were analyzed. C, increasing concentrations of WT mitochondria (1× = 200 μg of proteins) were incubated with iron (10 μm) at 30 °C for 20 min, in the absence or presence of added nucleotides (first step). Mitochondria were recovered, washed, and mixed with WT cytoplasm (200 μg of proteins), [³⁵S]cysteine, nucleotides, and apo-ΔN60 Yah1. No iron was added at this stage. The samples were incubated at 30 °C for 30 min (second step). After centrifugation, the cytoplasm/supernatant (S) fractions were analyzed. WT mito, WT mitochondria; WT cyto, WT cytoplasm.