Figure 2.

Mitochondrial ISC components involved in cytoplasmic iron–sulfur cluster assembly. A, WT or nfs1-14 mitochondria (200 μg of proteins) were added to WT cytoplasm (200 μg of proteins). Apo-ΔN60 Yah1 protein (1 μg) was added as indicated. The samples were then incubated with [³⁵S]cysteine (10 μCi), nucleotides (1 mm GTP, 2 mm NADH, and 4 mm ATP), and iron (10 μm) at 30 °C for 30 min. After centrifugation, the cytoplasm/supernatant (S) fractions were analyzed. B, WT or Isu1-depleted (Isu1↓, Isu2 absent) mitochondria were added to WT cytoplasm, and reaction mixtures were incubated with apo-ΔN60 Yah1, [³⁵S]cysteine, nucleotides, and iron at 30 °C for 10–30 min as indicated. After centrifugation, the cytoplasm/supernatant (S) fractions were analyzed. C, WT mitochondria or mitochondria with the Isu1 (M141E) mutant protein (WT Isu1 depleted, Isu2 absent, plasmid YCplac22-Isu1 (M141E)) were added to WT cytoplasm, and assays were performed as in B. D, WT mitochondria or Ssq1-depleted (Ssq1↓) mitochondria were added to WT cytoplasm, and assays were performed as in B. WT mito, WT mitochondria; WT cyto, WT cytoplasm.