Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System*

Background: The mitochondrial sulfhydryl oxidase Erv1 is implicated in cytosolic iron-sulfur protein maturation and iron regulation. Results: An erv1 yeast strain used in previous iron metabolism studies is glutathione-deficient, and erv1/mia40 mutants with sufficient glutathione do not exhibit iron-related defects. Conclusion: Iron homeostasis is independent of Erv1/Mia40 function. Significance: The unexplained role of Erv1 in yeast iron metabolism is resolved. The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.