Figure 5.
Role of Atm1 in mitochondrial export of (Fe–S)int to the cytoplasm. A, WT or Atm1-depleted (Atm1↓) mitochondria (200 μg of proteins) were added to WT cytoplasm (200 μg of proteins), and reaction mixtures were incubated with apo-ΔN60 Yah1, [35S]cysteine, nucleotides (1 mm GTP, 2 mm NADH, and 4 mm ATP), and iron (10 μm) at 30 °C for 10–30 min as indicated. After centrifugation, the cytoplasm/supernatant (S) fractions were analyzed. B, mitochondria (WT or Atm1↓; 1× = 200 μg of proteins) were prelabeled by incubating with [35S]cysteine, nucleotides, and iron at 30 °C for 20 min (first step). Mitochondria (35S-PL) were recovered, washed, and then incubated with WT cytoplasm (200 μg of proteins), nucleotides, iron, and apo-ΔN60 Yah1 at 30 °C for 30 min (second step). The samples were centrifuged, and the cytoplasm/supernatant (S) fractions were analyzed. WT mito, WT mitochondria; WT cyto, WT cytoplasm.