Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Abstract

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.Iron-sulfur proteins play essential roles in pathways that include the Krebs cycle, oxidative phosphorylation, gene regulation, and purine metabolism (1, 2). Their assembly presents particular challenges in that iron is readily oxidized and can generate free radicals. Hence, there are specific pathways that participate in the assembly of iron-sulfur clusters. In bacteria such as Escherichia coli, there is a pathway dedicated to the assembly of iron-sulfur clusters (3). The isc operon is the central genetic locus for this pathway, and it contains genes that encode for the proteins that include IscS, IscU, and IscA. The key elements of this pathway include a mechanism for obtaining sulfur through the enzymatic activity of a cysteine desulfurase, a mechanism for combining sulfur with iron on scaffold proteins, and a means for delivery of the resultant iron-sulfur clusters to target apoproteins. It appears that these key elements have been conserved through evolution. Thus, the E. coli cysteine desulfurase IscS has homologues in Saccharomyces cerevisiae and mammalian cells, indicative of a central conserved function. Moreover, homologues of IscU and IscA also exist in yeast and mammalian cells. The exact function of these is less certain. For example, IscU has been proposed to be a scaffold of iron-sulfur cluster assembly in E. coli, S. cerevisiae, and mammals (4 -6). IscA has been proposed to serve as a scaffold for delivering iron-sulfur clusters to select target proteins in E. coli and S. cerevisiae or, alternatively, as an iron donor in E. coli (7-10); its role in mammalian cells is not known.In eukaryotic cells, the situation is made more complex by the necessity of having to assemble both mitochondrial and cytosolic iron-sulfur clusters. In S. cerevisiae, there are proteins dedicated...

The Role of Mitochondria in Cellular Iron-Sulfur Protein Biogenesis: Mechanisms, Connected Processes, and Diseases

“…For all mitochondrial [4Fe-4S] proteins, on the other hand, cofactor insertion must be preceded or accompanied by conversion of the [2Fe-2S] cluster that has been synthesized on Isu1 and transferred by Grx5. This reaction is accomplished by the A-type ISC proteins Isa1 and Isa2 (human ISCA1 and ISCA2) and the tetrahydrofolate-binding protein Iba57 (Mühlenhoff et al , 2011Gelling et al 2008;Song et al 2009;Long et al 2011;Sheftel et al 2012). The three proteins functionally interact with each other, and deletion of the individual genes elicits highly similar phenotypes indicating that they cooperate in the same reaction (Gelling et al 2008;Waller et al 2010;Müh-lenhoff et al 2011;Sheftel et al 2012).…”

Mouse Knock-out of IOP1 Protein Reveals Its Essential Role in Mammalian Cytosolic Iron-Sulfur Protein Biogenesis

“…A number of studies in recent years have pointed to a role for IOP1 in the mammalian CIA pathway (16,18). We report here the results of knocking out Iop1 in the mouse.…”

“…The supernatant from this centrifugation was designated as the mitochondrial fraction. For MEFs, cytosolic and mitochondrial extracts were prepared as described previously (16,18). Protein concentrations of extracts were measured using a DC protein assay kit (Bio-Rad).…”

“…Aconitase, lactate dehydrogenase, citrate synthase, and xanthine oxidase activities were measured as described (16,18). An MTT assay was performed using a kit from ATCC (catalogue number 30-1010K).…”

“…These clusters are then delivered to Nar1, which in conjunction with Cia1 then facilitates the insertion of the iron-sulfur clusters into appropriate apoproteins (11,12). It should also be recognized that there is a role for other proteins, including proteins that have the following characteristics: they participate in iron-sulfur cluster biogenesis in the mitochondria, are predominantly mitochondrial in localization, but are also present at low levels in the cytosol (4,(13)(14)(15)(16)(17).…”