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Background: The precise molecular function of Isa1 and Isa2 proteins in Fe/S protein biogenesis is unknown. Results: The Isa1-Isa2 complex binds iron in vivo and acts late in the synthesis of 4Fe-4S clusters. Conclusion: Mitochondria possess specialized factors for maturation of 4Fe-4S proteins. Significance: This study defines the role of Isa proteins in mitochondrial Fe/S protein biogenesis. Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial 4Fe-4S proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial 2Fe-2S proteins and cytosolic 4Fe-4S proteins. Targeting of bacterial 2Fe-2S and 4Fe-4S ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the 2Fe-2S cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial 4Fe-4S protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of 4Fe-4S clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial 4Fe-4S proteins.
Mühlenhoff et al. (Tue,) studied this question.