A Role for IOP1 in Mammalian Cytosolic Iron-Sulfur Protein Biogenesis

Song, Daisheng; Lee, Frank S.

doi:10.1074/jbc.m708077200

Cited by 70 publications

(85 citation statements)

References 33 publications

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“…Protein concentration measurements and siRNA transfections were performed as described (12). Lipofectamine 2000 (Invitrogen) was employed for siRNA and transient transfections.…”

Section: Methodsmentioning

confidence: 99%

“…Current evidence indicates an important role for human Nbp35 in cytosolic iron-sulfur protein maturation (11). We have recently provided evidence that the human homologue of Nar1, IOP1, plays a role in cytosolic iron-sulfur protein maturation (12). IOP1 is homologous to iron-only hydrogenases, enzymes found in anaerobic bacteria that generate hydrogen gas from reducing equivalents.…”

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confidence: 99%

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

Song

Lee

2009

Journal of Biological Chemistry

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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...

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“…Protein concentration measurements and siRNA transfections were performed as described (12). Lipofectamine 2000 (Invitrogen) was employed for siRNA and transient transfections.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Song

Lee

2009

Journal of Biological Chemistry

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“…In fact, seven mammalian relatives of yeast ISC and CIA proteins have been experimentally studied to date and shown to serve a similar function in the cell as their yeast counterparts [17][18][19][20][21][22][23] . The crucial importance of Fe/S proteins and their assembly machineries has been recognized by virtue of the association of lesions in ISC machinery genes with three human diseases (for a review, see refs.…”

Section: Introductionmentioning

confidence: 99%

Analysis of iron–sulfur protein maturation in eukaryotes

2009

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“…IOP1 was first assumed to modulate the activity of hypoxia-inducible factor 1R that represents the global mediator of the mammalian response to hypoxia. Recently, depletion of the protein was shown to be essential for the formation of cytosolic Fe/S proteins identifying IOP1 as a functional Nar1 orthologue (18). Depletion of IOP2 (also termed NARF) was without effects on these Fe/S proteins, suggesting an alternative cellular function.…”

Section: Discussionmentioning

confidence: 99%

“…Its essential role for cell growth and in Fe/S protein biogenesis has been established in yeast and human cells (14,18). The related plant Nar1 protein is crucial for growth under normoxic but not under hypoxic conditions (19).…”

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confidence: 99%

Crucial Role of Conserved Cysteine Residues in the Assembly of Two Iron−Sulfur Clusters on the CIA Protein Nar1

et al. 2009

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Iron-sulfur (Fe/S) protein maturation in the eukaryotic cytosol and nucleus requires conserved components of the essential CIA machinery. The CIA protein Nar1 performs a specific function in transferring an Fe/S cluster that is assembled de novo on the Cfd1-Nbp35 scaffold to apoproteins. Here, we used systematic site-directed mutagenesis and a combination of in vitro and in vivo studies to show that Nar1 holds two Fe/S clusters at conserved N-and C-terminal cysteine motifs. A wealth of biochemical studies suggests that the assembly of these Fe/S clusters on Nar1 cannot be studied in Escherichia coli, as the recombinant protein does not contain the native Fe/S clusters. We therefore followed Fe/S cluster incorporation directly in yeast by a 55 Fe radiolabeling method in vivo, and we measured the functional consequences of Nar1 mutations in the assembly of cytosolic Fe/S proteins. We find that both Fe/S clusters are essential for Nar1 function and cell viability. Molecular modeling using a structurally but not functionally related bacterial iron-only hydrogenase as a template provided compelling structural explanations for our mutational data. The C-terminal Fe/S cluster is stably buried within Nar1, whereas the N-terminal one is exposed at the protein surface and hence may be more easily lost. Insertion of an Fe/S cluster into the C-terminal location depends on the N-terminal motif, suggesting the participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centers suggests a close functional cooperation during cytosolic Fe/S protein maturation.Iron-sulfur (Fe/S) 1 clusters are inorganic cofactors of many proteins found in nearly all prokaryotic and eukaryotic organisms. Fe/S proteins play important roles in many cellular processes, including electron transport, enzyme catalysis, and regulation of gene expression. Cells have developed dedicated systems for synthesizing Fe/S clusters and insert them into apoproteins. In Saccharomyces cerevisiae, three different machineries encompassing more than 20 proteins were identified with mature Fe/S proteins located in mitochondria, cytosol, and nucleus (1, 2). The mitochondrial ISC (Fe/S cluster) assembly machinery is needed for the biosynthesis of all cellular Fe/S proteins, whereas the ISC export and CIA machineries are specifically required for the biogenesis of cytosolic and nuclear Fe/S proteins. A central component of the ISC assembly system is the cysteine desulfurase complex Nfs1-Isd11 (3-5) which converts cysteine to alanine and transfers the sulfur to Isu1 and Isu2 which serve as scaffold proteins for Fe/S cluster assembly (6). Thereafter, a dedicated chaperone system is needed for the transfer of the Fe/S cluster from Isu1 and Isu2 to apoproteins. This system includes a mitochondrial Hsp70 protein (Ssq1), its cochaperone Jac1, and nucleotide exchange factor Mge1 (7). The mitochondrial ISC assembly machinery is also required for biogenesis of extra-mitochondrial Fe/S proteins. According to a working ...

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A Role for IOP1 in Mammalian Cytosolic Iron-Sulfur Protein Biogenesis

Cited by 70 publications

References 33 publications

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

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

Analysis of iron–sulfur protein maturation in eukaryotes

Crucial Role of Conserved Cysteine Residues in the Assembly of Two Iron−Sulfur Clusters on the CIA Protein Nar1

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