FIGURE 6.

Impaired [2Fe-2S] cluster biogenesis in Δpos5 mitochondria. Mitochondria were isolated from WT and Δpos5 cells grown under reduced O₂ conditions. A, precursor form of ferredoxin with a C-terminal His₆ tag was expressed in bacteria in the presence of [³⁵S]methionine, solubilized with 8 m urea, and purified by Ni-NTA chromatography. The radiolabeled protein (100 ng) was added to mitochondria supplemented with ATP (4 mm) and GTP (1 mm), and incubated at 30 °C for 15 min. Following import, samples were treated with trypsin (0.2 mg/ml) as indicated and analyzed by SDS-PAGE followed by autoradiography. The precursor and mature forms of Yah1p are indicated by p and m, respectively. Std indicates 50% of the precursor protein used for import experiments. B, mitochondria were supplemented with ATP, GTP, and NADH. Ferrous ascorbate was included as indicated. Following addition of [³⁵S]cysteine (10 μCi) and unlabeled ferredoxin (Yah1p) precursor protein (200 ng), reaction mixtures were incubated at 30 °C for 30 min and diluted with buffer A, and mitochondria were reisolated by centrifugation. Samples were analyzed by native PAGE followed by autoradiography.