Figure 4.

Iron loading into Rnr2 protein depends on the mitochondrial ISC system and cytosolic Grx3/4 proteins but not the CIA machinery. A and B, wild-type (WT), GalNFS1, GalATM1, GalDRE2, GalCFD1, GalNBP35, GalNAR1, and Gal-GRX4grx3Δ cells were grown in a synthetic iron-free medium containing glucose (GAL-off) for 40 h or 64 h (26) before being radiolabeled with ⁵⁵FeCl₃ for 2 h. After ⁵⁵Fe incorporation, the Rnr2 (A) and Leu1 (B) proteins were immunoprecipitated with specific polyclonal antibodies. Incorporation of ⁵⁵Fe into Rnr2 and Leu1 was quantified by scintillation counting. Values are given as means ± S.D. (n ≥ 3). C, comparison of protein levels of Rnr2, Leu1, as well as individual ISC and CIA components in wild-type (WT), GalNFS1, GalATM1, GalDRE2, GalCFD1, GalNBP35, GalNAR1, and Gal-GRX4grx3Δ mutant cells by Western blot. The protein blot was also probed with anti-Porin as a loading control.