NFU1 encodes a mitochondrial matrix protein of the NifU/NFU family that functions as a late-acting [4Fe-4S] cluster carrier in mitochondrial iron-sulfur cluster biogenesis. The protein dimerizes and ligates a bridging [4Fe-4S] cluster that is transferred to specific client proteins. NFU1 receives two [2Fe-2S] units from upstream donors ISCU2 and ISCA1, which are reductively coupled (with electrons from FDX2) to form the [4Fe-4S] cluster. The primary client is lipoyl synthase (LIAS), which requires the auxiliary [4Fe-4S] cluster for its catalytic activity. NFU1 dysfunction therefore impairs lipoylation of pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (OGDH), and the glycine cleavage system, explaining the metabolic features of NFU1 deficiency. Biallelic mutations in NFU1 cause Multiple Mitochondrial Dysfunctions Syndrome type 1 (MMDS1), characterized by early-onset leukoencephalopathy, lactic acidosis, nonketotic hyperglycinemia, and pulmonary hypertension. Alternative splicing produces isoforms with distinct subcellular localizations, with isoform I targeted to mitochondria and isoform II present in cytosol and nucleus.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: NFU1 is established as a mitochondrial protein through multiple lines of evidence. The IBA annotation from phylogenetic analysis is well-supported by direct experimental evidence showing mitochondrial localization of isoform I (PMID:12886008), mitochondrial matrix localization from Reactome pathway analysis, and high-confidence mitochondrial proteomics (PMID:34800366). Reason: Mitochondrial localization is a core feature of NFU1 function. The protein functions specifically in mitochondrial Fe-S cluster biogenesis and is targeted to the mitochondrial matrix (PMID:12886008, Reactome:R-HSA-6793591). This is the primary site of NFU1 function. Supporting Evidence: PMID:12886008 Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. file:human/NFU1/NFU1-deep-research-falcon.md model: Edison Scientific Literature |
| GO:0051604 protein maturation | IBA GO_REF:0000033 | MODIFY | Summary: NFU1 participates in the maturation of [4Fe-4S] cluster-containing proteins by delivering Fe-S clusters to target proteins. The IBA annotation reflects the conserved role of NFU family proteins in Fe-S protein maturation across species. Reason: While NFU1 does participate in protein maturation, the term "protein maturation" (GO:0051604) is too general. The specific role of NFU1 is in iron-sulfur cluster assembly and delivery to target proteins. The more specific process term GO:0016226 (iron-sulfur cluster assembly) better captures NFU1's function (PMID:12886008, PMID:28906594). Proposed replacements: iron-sulfur cluster assembly |
| GO:0051539 4 iron, 4 sulfur cluster binding | IBA GO_REF:0000033 | ACCEPT | Summary: The IBA annotation for [4Fe-4S] cluster binding is strongly supported by direct biochemical evidence. Tong et al. (2003) demonstrated using UV-visible absorption and 57Fe Mossbauer spectroscopy that purified human Nfu assembles approximately one labile [4Fe-4S] cluster per two monomers (PMID:12886008). Wesley et al. (2017) further characterized [4Fe-4S] cluster binding and its disruption by disease-causing mutations (PMID:28906594). Reason: [4Fe-4S] cluster binding is the core molecular function of NFU1. The protein dimerizes and ligates a bridging [4Fe-4S] cluster that is subsequently delivered to target proteins. This has been demonstrated by multiple spectroscopic and biochemical studies. Supporting Evidence: PMID:12886008 The results suggest that Nfu can assemble approximately one labile [4Fe-4S] cluster per two Nfu monomers, and support the proposal that Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. PMID:28906594 iron-sulfur (Fe/S) cluster-containing proteins constitute one of the largest protein classes, with highly varied function...we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (p.Gly189Arg) near the active site of NFU1, an Fe/S scaffold protein. |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: The IEA annotation for iron ion binding is derived from InterPro domain mapping (IPR001075). Direct experimental evidence supports iron binding as part of the [4Fe-4S] cluster (PMID:12886008). Reason: Iron ion binding is inherent to NFU1's function as an Fe-S cluster scaffold. The [4Fe-4S] cluster contains four iron atoms that are coordinated by the protein. While this is a more general term than [4Fe-4S] cluster binding, it is not incorrect and provides complementary information about metal binding capacity. Supporting Evidence: PMID:12886008 Nfu can assemble approximately one labile [4Fe-4S] cluster per two Nfu monomers |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: The IEA annotation from UniProtKB subcellular location mapping is consistent with direct experimental evidence. Multiple IDA annotations also support mitochondrial localization. Reason: While this duplicates the IBA annotation, the evidence source (UniProt subcellular location mapping) provides independent support. Mitochondrial localization is well-established. |
| GO:0005829 cytosol | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Cytosolic localization has experimental support from Tong et al. (2003) who showed that alternative splicing produces isoforms with distinct localizations - isoform II lacks the mitochondrial targeting sequence and localizes to cytosol and nucleus (PMID:12886008). Reason: While cytosolic localization is experimentally validated for isoform II, the primary function of NFU1 in iron-sulfur cluster biogenesis occurs in mitochondria. The cytosolic isoform represents an alternative localization that may have secondary functions. Supporting Evidence: PMID:12886008 Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. |
| GO:0016226 iron-sulfur cluster assembly | IEA GO_REF:0000002 | ACCEPT | Summary: The IEA annotation from InterPro (IPR001075) correctly identifies NFU1's role in iron-sulfur cluster assembly. This is strongly supported by direct experimental evidence (PMID:12886008, PMID:28906594). Reason: Iron-sulfur cluster assembly is the primary biological process in which NFU1 participates. NFU1 functions as a late-acting scaffold/carrier that assembles [4Fe-4S] clusters and delivers them to target proteins including LIAS. Supporting Evidence: PMID:12886008 Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: The IEA annotation from UniProt keyword mapping is very general. NFU1 binds iron as part of iron-sulfur clusters. Reason: While technically correct (NFU1 binds iron ions as part of the [4Fe-4S] cluster), this term is too general and uninformative. The more specific terms GO:0051539 (4 iron, 4 sulfur cluster binding) and GO:0005506 (iron ion binding) already capture this information with greater specificity. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000120 | ACCEPT | Summary: The IEA annotation for general iron-sulfur cluster binding is correct but less specific than the [4Fe-4S] cluster binding term that is also annotated. Reason: This general term is acceptable as NFU1 can bind both [2Fe-2S] and [4Fe-4S] clusters during its function (PMID:27538573 shows [2Fe-2S] binding; PMID:12886008 shows [4Fe-4S]). The more general term captures this broader capacity. Supporting Evidence: PMID:27538573 Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe-2S] cluster. |
| GO:0005515 protein binding | IPI PMID:22190034 Global landscape of HIV-human protein complexes. | MARK AS OVER ANNOTATED | Summary: This annotation is from a large-scale HIV-human interactome study. The biological relevance of interactions detected in such studies for normal NFU1 function is unclear. Reason: The term "protein binding" is uninformative and does not capture the specific functional interactions of NFU1 with Fe-S biogenesis partners (BOLA3, HSPA9, ISCU, ISCA1). Large-scale interactome studies often detect interactions that may not be physiologically relevant. Supporting Evidence: PMID:22190034 Global landscape of HIV-human protein complexes. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MARK AS OVER ANNOTATED | Summary: This annotation is from a proteome-scale human interactome map. While demonstrating NFU1 interacts with other proteins, "protein binding" is uninformative. Reason: The generic "protein binding" term does not capture the biologically meaningful interactions of NFU1. More informative annotations would specify interactions with Fe-S cluster biogenesis components. Supporting Evidence: PMID:25416956 A proteome-scale map of the human interactome network. |
| GO:0005515 protein binding | IPI PMID:27532772 Mitochondrial Bol1 and Bol3 function as assembly factors for... | MODIFY | Summary: This reference (Uzarska et al. 2016, eLife) demonstrates specific interaction between NFU1 and BOLA3 as part of Fe-S cluster biogenesis. This is a biologically meaningful interaction showing BOLA3 preferentially interacts with holo-NFU1. Reason: While the IPI evidence for protein binding is valid, a more informative term could capture the specific functional role. However, without a specific GO term for "BOLA3 binding" or similar, this annotation provides evidence of a physiologically relevant interaction in Fe-S cluster transfer. Proposed replacements: iron-sulfur cluster chaperone activity Supporting Evidence: PMID:27532772 Preferential interaction of BOLA1 with holo-GLRX5 and of BOLA3 with holo-NFU1 |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: This annotation is from a reference map of the human binary protein interactome (HuRI). While detecting interactions, the generic term is uninformative for understanding NFU1 function. Reason: Large-scale interactome data, while valuable for network analysis, produces generic "protein binding" annotations that do not illuminate NFU1's specific role in Fe-S cluster biogenesis. Supporting Evidence: PMID:32296183 Apr 8. A reference map of the human binary protein interactome. |
| GO:0005829 cytosol | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: The IDA from immunofluorescence curation (HPA) detects cytosolic signal. This is consistent with the existence of cytosolic isoform II. Reason: Cytosolic localization is validated but represents a secondary localization. The primary Fe-S cluster assembly function occurs in mitochondria. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-throughput mitochondrial proteomics study confirming NFU1 as a mitochondrial protein. This provides independent high-confidence support for mitochondrial localization. Reason: The quantitative high-confidence human mitochondrial proteome provides strong support for mitochondrial localization of NFU1. Supporting Evidence: PMID:34800366 Epub 2021 Nov 19. Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context. |
| GO:0051537 2 iron, 2 sulfur cluster binding | IDA PMID:27538573 Iron-sulfur cluster exchange reactions mediated by the human... | ACCEPT | Summary: Wachnowsky et al. (2016) demonstrated that human NFU1 can bind a [2Fe-2S] cluster as an intermediate during cluster transfer. The study characterized cluster exchange chemistry and showed NFU1 can accept [2Fe-2S] clusters and transfer them to ferredoxins. Reason: The [2Fe-2S] cluster binding capacity is experimentally demonstrated and represents an intermediate state in the cluster maturation pathway. NFU1 receives [2Fe-2S] units from donors that are subsequently converted to [4Fe-4S] clusters. Supporting Evidence: PMID:27538573 Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe-2S] cluster |
| GO:0051604 protein maturation | IDA PMID:27538573 Iron-sulfur cluster exchange reactions mediated by the human... | MODIFY | Summary: The IDA annotation for protein maturation from this reference reflects NFU1's role in maturing Fe-S proteins by delivering clusters. However, the term is generic. Reason: While NFU1 does contribute to protein maturation, the specific process is iron-sulfur cluster assembly and delivery. The term GO:0016226 (iron-sulfur cluster assembly) more precisely captures NFU1's role. Proposed replacements: iron-sulfur cluster assembly Supporting Evidence: PMID:27538573 Epub 2016 Aug 18. Iron-sulfur cluster exchange reactions mediated by the human Nfu protein. |
| GO:0005515 protein binding | IPI PMID:26702583 Mitochondrial Hspa9/Mortalin regulates erythroid differentia... | MODIFY | Summary: Shan & Cortopassi (2016) demonstrated that HSPA9/Mortalin interacts with NFU1 as part of the mitochondrial Fe-S cluster biogenesis machinery. This is a functionally relevant interaction. Reason: While "protein binding" is too generic, this reference documents a biologically meaningful interaction. HSPA9 is the mitochondrial chaperone that works with the co-chaperone HSC20 in Fe-S cluster transfer. A more specific molecular function term capturing scaffold activity would be more informative. Proposed replacements: iron-sulfur cluster chaperone activity Supporting Evidence: PMID:26702583 HSPA9 interacts with and stabilizes the mitochondrial ISC biogenesis proteins frataxin, Nfs1, ISCU, and Nfu. |
| GO:0016226 iron-sulfur cluster assembly | IDA PMID:28906594 Understanding the molecular basis for multiple mitochondrial... | ACCEPT | Summary: Wesley et al. (2017) characterized NFU1's role in Fe-S cluster assembly and how the disease-causing G189R mutation impairs this function. The study directly demonstrates NFU1's function as an Fe-S scaffold protein. Reason: This is strong direct experimental evidence for NFU1's role in iron-sulfur cluster assembly. The paper shows that pathogenic mutations impair NFU1's ability to receive and transfer Fe-S clusters. Supporting Evidence: PMID:28906594 substitution at position 189 triggers structural changes that increase flexibility, decrease stability, and alter the monomer-dimer equilibrium toward monomer, thereby impairing the ability of the Gly189X derivatives to receive an Fe/S cluster from physiologically relevant sources. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:28906594 Understanding the molecular basis for multiple mitochondrial... | ACCEPT | Summary: Direct experimental evidence from MMDS1 disease characterization confirms [4Fe-4S] cluster binding by NFU1. The pathogenic G189R variant disrupts this capacity. Reason: This provides disease-relevant experimental validation of [4Fe-4S] cluster binding as a core function of NFU1. Supporting Evidence: PMID:28906594 we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (p.Gly189Arg) near the active site of NFU1, an Fe/S scaffold protein. |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-6793591 | ACCEPT | Summary: The Reactome pathway for protein lipoylation places NFU1 in the mitochondrial matrix, where it functions to deliver [4Fe-4S] clusters to LIAS for lipoyl-GCSH synthesis. Reason: Mitochondrial matrix localization is more specific than general mitochondrion annotation and reflects the actual site of NFU1 function in Fe-S cluster delivery to LIAS and other matrix clients. |
| GO:0005829 cytosol | IDA PMID:12915448 The Lafora disease gene product laforin interacts with HIRIP... | KEEP AS NON CORE | Summary: Ganesh et al. (2003) identified NFU1 (as HIRIP5) as a cytosolic protein that interacts with laforin. This reflects the cytosolic isoform. Reason: This provides additional experimental evidence for cytosolic localization of one NFU1 isoform. However, the mitochondrial isoform is the primary functional form for Fe-S cluster biogenesis. Supporting Evidence: PMID:12915448 HIRIP5 encodes a cytosolic protein and is expressed ubiquitously, perhaps reflecting a house-keeping function. |
| GO:0005506 iron ion binding | IDA PMID:12886008 Subcellular compartmentalization of human Nfu, an iron-sulfu... | ACCEPT | Summary: Tong et al. (2003) directly demonstrated iron binding through spectroscopic analysis of purified NFU1 with assembled Fe-S clusters. Reason: Direct experimental evidence from Mossbauer spectroscopy and UV-visible absorption confirms iron binding as part of [4Fe-4S] cluster assembly. Supporting Evidence: PMID:12886008 A combination of biochemical and spectroscopic techniques, including UV-visible absorption and 57Fe Mössbauer spectroscopies, have been used to investigate the ability of purified human Nfu to assemble Fe-S clusters |
| GO:0005634 nucleus | IDA PMID:12886008 Subcellular compartmentalization of human Nfu, an iron-sulfu... | KEEP AS NON CORE | Summary: Tong et al. (2003) showed that isoform II (lacking mitochondrial targeting sequence) localizes to nucleus in addition to cytosol. Reason: Nuclear localization is documented for isoform II but represents a secondary localization. The functional significance of nuclear NFU1 is not well established. Supporting Evidence: PMID:12886008 Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. |
| GO:0005739 mitochondrion | IDA PMID:12886008 Subcellular compartmentalization of human Nfu, an iron-sulfu... | ACCEPT | Summary: Foundational experimental study demonstrating mitochondrial localization of NFU1 isoform I and its ability to assemble [4Fe-4S] clusters. Reason: Primary experimental evidence establishing mitochondrial localization of the functional isoform of NFU1. Supporting Evidence: PMID:12886008 Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. |
| GO:0005829 cytosol | IDA PMID:12886008 Subcellular compartmentalization of human Nfu, an iron-sulfu... | KEEP AS NON CORE | Summary: Tong et al. (2003) demonstrated cytosolic localization of NFU1 isoform II. Reason: Cytosolic localization is documented for isoform II. This represents secondary localization relative to the mitochondrial function. Supporting Evidence: PMID:12886008 Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. |
| GO:0016226 iron-sulfur cluster assembly | IDA PMID:12886008 Subcellular compartmentalization of human Nfu, an iron-sulfu... | ACCEPT | Summary: This is the foundational study establishing NFU1 as an iron-sulfur cluster scaffold protein capable of assembling [4Fe-4S] clusters for delivery to target proteins. Reason: Primary experimental evidence defining NFU1's core biological function in Fe-S cluster assembly. This study established the role of human Nfu as an alternative scaffold in the Fe-S biogenesis pathway. Supporting Evidence: PMID:12886008 support the proposal that Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:12886008 Subcellular compartmentalization of human Nfu, an iron-sulfu... | ACCEPT | Summary: The foundational demonstration that NFU1 binds [4Fe-4S] clusters using Mossbauer spectroscopy and biochemical analysis. Reason: This provides the primary experimental evidence for [4Fe-4S] cluster binding, which is the core molecular function of NFU1. Supporting Evidence: PMID:12886008 The results suggest that Nfu can assemble approximately one labile [4Fe-4S] cluster per two Nfu monomers |
| GO:0140132 iron-sulfur cluster chaperone activity | IDA PMID:12886008 Subcellular compartmentalization of human Nfu, an iron-sulfu... | NEW | Summary: NFU1 functions as an iron-sulfur cluster chaperone, assembling [4Fe-4S] clusters and delivering them to target proteins. This molecular function term captures the core activity of NFU1 better than "protein binding". Reason: This term specifically captures NFU1's role as a late-acting Fe-S cluster chaperone that assembles clusters from [2Fe-2S] intermediates and delivers them to client proteins including LIAS. This is the defining molecular function of NFU1. Supporting Evidence: PMID:12886008 support the proposal that Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. PMID:27538573 Human Nfu is an iron-sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome PMID:28906594 Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1. |
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Download this section (compressed HTML)Q: What is the full spectrum of [4Fe-4S] client proteins for NFU1 beyond LIAS?
Q: Does the cytosolic/nuclear isoform II have a distinct function in cytosolic Fe-S cluster biogenesis?
Q: What determines the specificity of cluster delivery to different client proteins?
Q: How does the ISCA1-NFU1 pathway differ from the GLRX5-BOLA3 pathway in client selectivity?
Experiment: Proteomics identification of all NFU1-dependent [4Fe-4S] client proteins
Experiment: Characterization of cytosolic isoform II function in CIA pathway
Experiment: Structure determination of NFU1 in complex with client proteins
Experiment: In vivo validation of METTL17 as an NFU1 client for mitoribosome biogenesis
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