FIGURE 5.

Deficient Fe-S cluster synthesis in Δpos5 mitochondria persists even under reduced oxidative stress conditions. Mitochondria were isolated from WT and Δpos5 cells grown under low oxygen conditions. A, aconitase activity (top panel) and protein (bottom panel) levels were determined as in Fig. 4A. B, mitochondria were supplemented with ATP (4 mm), GTP (1 mm), and [³⁵S]cysteine (10 μCi). Following addition of NAD⁺ and/or ferrous ascorbate as indicated, reaction mixtures were incubated at 30 °C for 60 min under low O₂ conditions. Samples were analyzed by native PAGE followed by autoradiography. C, mitochondria were supplemented with ATP, GTP, NAD⁺, and ferrous ascorbate as indicated. Following addition of [³⁵S]cysteine (10 μCi), samples were incubated at 30 °C for different time periods under reduced O₂ conditions and analyzed. The right panel shows quantitation of data presented on the left panel. The intensity of Aco1p (Fe-³⁵S) in WT mitochondria at the 30-min time point (lane 5) was considered 100%.