Fig 2. Characterization and comparison of frataxin-bypass substitutions of ISU1 amino acid 141: ISU1-Cys, ISU1-Ile, ISU1-Leu and ISU1-Val.

(A) Growth defects in Δyfh1 are corrected by the frataxin-bypass suppressors. The strains YFH1 [ISU1] (Y-M), Δyfh1 [ISU1] (Δ-M), and suppressor strains Δyfh1 [ISU1-Cys] (Δ-C), Δyfh1 [ISU1-Ile] (Δ-I), Δyfh1 [ISU1-Leu] (Δ-L), and Δyfh1 [ISU1-Val] (Δ-V), were grown in an argon-filled chamber, and serial dilutions were spotted on YPAD agar, without or with 4 mM hydrogen peroxide. (B) Mitochondrial protein levels by immunoblotting. Strains were inoculated from the argon-filled chamber into aerobic defined raffinose medium for 16–24 h prior to isolating mitochondria. Mitochondrial proteins (100 μg) were separated by SDS-PAGE and transferred to nitrocellulose. Strips were cut from a single blot and probed with the indicated antibodies against mitochondrial proteins (Aco1, Nfs1, Mir1, Yfh1, and Isu1). Cytochrome c (Cyc1) was analyzed using a duplicate gel. (C) Iron homeostasis in the frataxin-bypass suppressors. The indicated strains were grown in air for six doublings in defined raffinose medium in the presence of 10 μM ⁵⁵Fe ascorbate. Cells were washed free of radioactive iron and separated into mitochondria and post-mitochondrial supernatant. An aliquot of mitochondria was lysed in the presence of 0.1% Triton X-100 for 10 min at room temperature, and supernatant (soluble) and pellet (insoluble) fractions were separated by centrifugation at 20,000 x g for 30 min at 4°C. Iron content was reported for whole cells (pmol per million cells), or various cellular fractions (pmol per micrograms protein).