FIGURE 8.

Pos5p overexpression enhances Fe-S cluster biogenesis in wild-type mitochondria. A, wild-type strain, called WT (Pos5p-TAP), expressed Pos5p-TAP from the POS5 promoter in the genome. This strain was transformed with a plasmid for overexpression of Pos5p-TAP from the GAPDH promoter (Pos5p-TAP▴). Mitochondria isolated from these strains were evaluated for Pos5p-TAP protein levels by immunoblotting using affinity-purified rabbit IgG as the first antibody. B, aconitase activity (top panel) was determined in WT and wild-type with overexpressed and untagged Pos5p (WT with Pos5p▴) mitochondria by a native in-gel assay (28). Identical samples were also analyzed by SDS-PAGE followed by immunoblotting using anti-Aco1p (middle panel) and anti-Tom40p (bottom panel) antibodies. C, mitochondria were supplemented with ATP (4 mm) and GTP (1 mm). As indicated, reaction mixtures also contained NADH (5 mm) and/or ferrous ascorbate (10 μm). Following addition of [³⁵S]cysteine, samples were incubated at 30 °C for 60 min and analyzed by native PAGE and autoradiography. The intensity of Aco1p (Fe-³⁵S) in WT with Pos5p▴ mitochondria with both NADH and iron added (lane 8) was considered 100%. D, mitochondria were supplemented with ATP, GTP, NADH, ferrous ascorbate, and [³⁵S]cysteine as in C. Samples were analyzed after incubation at 30 °C for different time periods. The right panel shows quantitation of the autoradiogram presented on the left panel. The intensity of Aco1p (Fe-³⁵S) in WT with Pos5p▴ mitochondria at the 60-min time point (lane 12) was considered 100%.