Fig. 2. Deficiency in Grx3/4 impairs the de novo synthesis of cellular Fe/S clusters and heme.

(A-C) Wild-type (WT) and Grx4-depleted Gal-GRX4 cells overproducing the cytosolic Fe/S proteins Rli1-HA, Dre2 or Nar1 (A), mitochondrial Bio2, Ilv3-Myc (B), or Isu1 (C) were radiolabeled with 10 μCi ⁵⁵Fe for 2 h. The Fe/S proteins were immunoprecipitated and bound ⁵⁵Fe was quantified by scintillation counting. Protein levels were assessed by immunostaining. Porin (Por1) served as a loading control. Gal-GRX4 cells were depleted for Grx4 by growth in SD medium for 40 h, and 64 h (in case of Isu1). (D) Purified apo-Yah1 was incubated under anaerobic conditions in the presence of ⁵⁵Fe and cysteine either alone (−) or with detergent extracts of mitochondria isolated from 40 h or 64 h depleted Gal-GRX4 cells or Gal-GRX4 cells overproducing Grx4 (+Grx4). ⁵⁵Fe/S cluster reconstitution on re-isolated Yah1 was quantified by scintillation counting. (E) WT and Gal-GRX4 cells (40 h depletion) harboring either vector pCM189 (−) or pCM189-GRX4 (+Grx4) were radiolabeled with ⁵⁵Fe. ⁵⁵Fe-heme was extracted with butyl-acetate and quantified by scintillation counting. Error bars indicate the SEM (n ≥ 4).