Figure 2. RIM2 overexpression improves growth of the Δmrs3Δmrs4Δyfh1 triple mutant.

(A) The triple mutant Δmrs3Δmrs4Δyfh1 was maintained anaerobically and transformed with empty plasmid YEp351 or YEp351-RIM2. The WT control strain YPH499 was handled similarly. The transformants were then streaked on to standard defined medium in air. The triple mutant transformed with RIM2 showed enhanced growth compared with empty plasmid (left-hand plate, left side). (B) Rim2 overexpression restores the rate of iron insertion into porphyrin in Δmrs3Δmrs4Δyfh1 mutant mitochondria. Mitochondria (0.5 mg) were treated with 5 mM NADH for 2 min. PPO (2 μM) was added and protoporphyrin IX fluorescence was measured over time (excitation at 410 nm, emission at 632 nm). After 100 s, 1 μM ferrous ammonium sulfate was added and tracing was continued. At the end of the 5 min time trace, fluorescent emissions were scanned from 550 nm to 700 nm (bottom panel). Cells were cultured under low oxygen in argon-bubbled flasks in defined glucose medium and then shifted to air for 4 h in defined raffinose medium. Mitochondria were isolated. white circle, YPH499; black circle, Δmrs3Δmrs4Δyfh1[YEp351-RIM2]; grey circle, Δmrs3Δmrs4Δyfh1. (C) Immunoblot analysis of mitochondrial proteins from: WT YPH499 (lane 1); ΔΔΔ, Δmrs3Δmrs4Δyfh1 (lane 2); ΔΔΔ/Rim2, triple mutant transformed with YEp351-Rim2 (lane 3). Mitochondria (100 μg equivalent) were separated by SDS/PAGE (13% gel) and transferred on to a Protran nitrocellulose membrane for blotting with antibodies as indicated. Aco1, aconitase; Rim2, Yfh1 (yeast frataxin homologue); Cyc1, cytochrome c.