FIGURE 4.

In vivo expression of Pos5p restores Fe-S cluster biogenesis in Δpos5 mitochondria. A, WT, Δpos5, and Δpos5 with reintroduced Pos5p (Δpos5 + Pos5p) cells were grown under aerobic conditions, and mitochondria were isolated. Aconitase activity in these mitochondria (top panel) was determined by a native in-gel assay as described previously (28). Identical samples were also analyzed by SDS-PAGE followed by immunoblotting using anti-Aco1p (middle panel) and anti-Tom40p (bottom panel) antibodies. B, mitochondria were supplemented with ferrous ascorbate (10 μm) and [³⁵S]cysteine (10 μCi). Reaction mixtures were incubated at 30 °C for 30 min, with or without added nucleotides (4 mm ATP, 1 mm GTP, and 5 mm NADH). Samples were analyzed by native PAGE followed by autoradiography.