FIGURE 3.

Addition of NADH stimulates Fe-³⁵S labeling of imported ferredoxin in wild-type mitochondria. A, precursor form of ferredoxin was expressed in bacteria, and the protein was solubilized from inclusion bodies with 8 m urea. Wild-type mitochondria (200 μg of proteins) were supplemented with ATP, GTP, NADH, and ferrous ascorbate 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 different time periods and diluted with buffer A, and mitochondria were reisolated by centrifugation. Samples were analyzed by native PAGE followed by autoradiography. B, wild-type mitochondria were supplemented with ATP (4 mm) and GTP (1 mm) and incubated at 30 °C for 15 min with or without added unlabeled Yah1p precursor protein (200 ng). Valinomycin (5 μm) was added to stop any further import, and reaction mixtures were supplemented with [³⁵S]cysteine (10 μCi). Samples were incubated at 30 °C for 30 min with or without added NADH (2 mm) and analyzed as in A.