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

Abstract

Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.Iron-sulfur cluster proteins are an important class of proteins that contain iron-sulfur clusters in a variety of configurations, including [2Fe-2S] and [4Fe-4S] (1, 2). The distinctive redox and biophysical properties of these clusters make them suitable for a range of functions. Consequently, iron-sulfur proteins have diverse roles and participate in a broad range of biologic processes, including Krebs cycle reactions, oxidative phosphorylation, translation, iron regulatory pathways, and purine metabolism. Notably, these proteins exist in many cellular locales, including the cytosol, mitochondria, and nucleus.How iron-sulfur cluster proteins are assembled in different cellular compartments has been an area of active investigation (3, 4). Iron-sulfur cluster assembly for all compartments originates in the mitochondria, and the initial step is catalyzed by a cysteine desulfurase that removes elemental sulfur from cysteine. The incorporation of this sulfur into iron-sulfur clusters then occurs by mechanisms that depend, in part, on cell compartment-specific machineries. Such a pathway exists, for example, for cytosolic iron-sulfur proteins. Important insights into this pathway, the cytosolic iron-sulfur protein assembly (CIA) 2 pathway, have been obtained from studies in Saccharomyces cerevisiae (3, 5). These studies have revealed a series of proteins that, in a stepwise fashion, assemble and deliver ironsulfur clusters to target proteins. A current model is that the proteins Tah18 and Dre2 provide reducing equivalents for the assemb...

IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

“…In contrast, yeast studies show that Mms19 is not essential and that Nar1 is essential in yeast cells, suggesting that Nar1 has more important roles in the CIA pathway compared with Mms19. However, recent studies using knock-out mice have shown that both IOP1 and MMS19 are essential in higher eukaryotes (33,36,37). The role of IOP1 and MMS19 in the vertebrate CIA machinery may be different from that of yeast orthologs.…”

“…5A). As IOP1 functions in the CIA pathway (36,37) and it is a putative Fe-S cluster donor, Fe-S clusters might be further transferred from IOP1 to the target proteins by the full complex. However, studies in yeast show that Nar1 has already been matured in the early phase of the CIA pathway.…”

Mammalian iron–sulphur proteins: novel insights into biogenesis and function

“…As summarized recently 9 , this pathway is postulated to depend on export of a special form of sulphur from mitochondria, which is transferred to a tetrameric scaffold composed of two members of the P-loop NTPase family -cytosolic Fe-S cluster-deficient 1 (Cfd1; NUBP2 (also known as CFD1) in humans) 64 and nucleotide-binding protein 35 (Nbp35; NUBP1 (also known as NBP35) in humans) 65 -which ligate bridging [4Fe-4S] clusters. These clusters obtain electrons from Tah18 (NADPH-dependent diflavi n oxidoreductase 1 (NDOR1) in humans) and Dre2 (CIAPIN1 (also known as anamorsin) in humans) 66 , and are transferred to nuclear architecture-related 1 (Nar1; IOP1 (also known as NARFL) in humans) 67 , which is thought to mediate interactions between early and late steps of the CIA transfer machinery. Nar1 is then proposed to transfer its Fe-S clusters to targeting complexes composed of Cia1 (also known as Asf1), Fam96b (also known as Cia2B) 68 and methyl methanesulphonatesensitivity 19 (Mms19) 5 , which in turn transfer Fe-S clusters to proteins involved in DNA replication, repair and glyco sylation, as well as chromosomal segregation and telomeric stability.…”

A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation

“…To evaluate IRP ubiquitination state, cells were lysed as described (6) with the addition of 10 mM N-ethylmaleimide, 3 mM iodoacetamide, and 0.5 mM dithiothreitol to inhibit deubiquitination (44,45). IRP1 was immunoprecipitated from 0.5 mg of cell lysate by incubation with 5 g of anti-Myc monoclonal antibody (9E10) for 12 h at 4°C with orbital shaking.…”