NDUFAF4 (formerly C6orf66/HRPAP20) is a nuclear-encoded mitochondrial assembly factor for NADH:ubiquinone oxidoreductase (respiratory chain complex I). It is not a structural subunit of the holoenzyme and has no catalytic activity; instead it acts transiently during complex I biogenesis, working together with its tight interaction partner NDUFAF3 (and, in the same early membrane-arm intermediate, TIMMDC1) to build and anchor an early membrane-arm intermediate of the complex. The protein is N-myristoylated at the N-terminal glycine and is peripherally associated with the mitochondrial inner membrane. Loss-of-function variants cause autosomal recessive isolated mitochondrial complex I deficiency (nuclear type 15), with clinical presentations including Leigh syndrome, leukodystrophy, encephalopathy, and cardiomyopathy.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetically inferred mitochondrial localization, consistent with direct experimental evidence (PubMed:18179882) and with the protein's role as a mitochondrial complex I assembly factor. Correct but a more specific location (mitochondrial inner membrane) is better. |
| GO:0032981 mitochondrial respiratory chain complex I assembly | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetically inferred core biological process. This is the established, well-supported function of NDUFAF4 and matches the experimental IMP annotation (PubMed:18179882). Core function. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping from the UniProt Subcellular Location "Mitochondrion". Consistent with experimental IDA evidence (PubMed:18179882). Correct; a more specific inner-membrane term is available. |
| GO:0016020 membrane | IEA GO_REF:0000044 | REMOVE | Summary: Over-general electronic mapping from the UniProt "Membrane; Lipid-anchor" subcellular-location keyword. Although NDUFAF4 is membrane-associated (N-myristoylated, peripheral on the mitochondrial inner membrane), the bare "membrane" term is uninformative and superseded by the specific mitochondrial inner membrane (GO:0005743) and mitochondrial membrane (GO:0031966) annotations. Removing an over-propagated electronic inference. |
| GO:0032981 mitochondrial respiratory chain complex I assembly | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the NDUFAF4 family signature (IPR009622) to the complex I assembly process. This is the correct core process and is well supported by experimental evidence. Accept. |
| GO:0005515 protein binding | IPI PMID:19688755 LC-MS/MS as an alternative for SDS-PAGE in blue native analy... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a blue-native LC-MS/MS complex-profiling study that co-migrated C6ORF66 (NDUFAF4) with complex I subunits. The term is uninformative (does not name a specific molecular function) and derives from a high-throughput screen. Per policy, IPI protein-binding is not removed; marking as over-annotated. The underlying biology (association with complex I assembly) is captured by the assembly-process annotations. |
| GO:0005515 protein binding | IPI PMID:24344204 TIMMDC1/C3orf1 functions as a membrane-embedded mitochondria... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from interaction proteomics of complex I assembly factors (TIMMDC1/MCIA network). Uninformative term from a high-throughput dataset; not removed per IPI policy but marked over-annotated. Supports the assembly-factor role rather than a distinct molecular function. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a proteome-scale binary interactome (Y2H) map. Uninformative term from a high-throughput screen; retained per IPI policy but marked over-annotated. |
| GO:0005515 protein binding | IPI PMID:25910212 Widespread macromolecular interaction perturbations in human... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a high-throughput interactome-perturbation study. Uninformative term; retained per IPI policy but marked over-annotated. |
| GO:0005515 protein binding | IPI PMID:27499296 Mitochondrial Protein Interaction Mapping Identifies Regulat... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a mitochondrial affinity-enrichment MS interaction map (Floyd et al.). Uninformative term from a high-throughput screen; retained per IPI policy but marked over-annotated. Consistent with a respiratory-chain assembly context. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a reference human binary interactome (HuRI) map. Uninformative term from a high-throughput screen; retained per IPI policy but marked over-annotated. |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a neurodegenerative-disease interactome mapping study (interactions with WFS1 and GRN). Uninformative term from a high-throughput screen; retained per IPI policy but marked over-annotated. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a cell-specific proteome-scale interactome (BioPlex) study capturing the NDUFAF4-NDUFAF3 interaction. Uninformative term from a high-throughput screen; retained per IPI policy but marked over-annotated. |
| GO:0005515 protein binding | IPI PMID:40205054 Multimodal cell maps as a foundation for structural and func... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a multimodal cell-map / interactome study. Uninformative term from a high-throughput dataset; retained per IPI policy but marked over-annotated. |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | ACCEPT | Summary: Mitochondrial localization from curated immunofluorescence (HPA). Consistent with all other evidence for mitochondrial localization. Accept. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: Mitochondrial localization from a high-throughput high-confidence mitochondrial proteome study. Corroborates the mitochondrial localization; accept as supporting evidence for the well-established mitochondrial location. Supporting Evidence: PMID:34800366 Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context. |
| GO:0005515 protein binding | IPI PMID:33635491 The host cellular protein Ndufaf4 interacts with the vesicul... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" capturing the interaction between NDUFAF4 and the vesicular stomatitis virus matrix (M) protein (UniProtKB:P03519). A specific, experimentally validated interaction (yeast two-hybrid, cotransformation, GST pulldown), but the GO term itself is uninformative. Retained per IPI policy; marked over-annotated. The biological consequence is captured by the defense-response-to-virus annotation. |
| GO:0051607 defense response to virus | IMP PMID:33635491 The host cellular protein Ndufaf4 interacts with the vesicul... | KEEP AS NON CORE | Summary: NDUFAF4 binds the VSV matrix (M) protein; overexpression inhibited and shRNA knockdown promoted VSV propagation, independent of the type I interferon response. A single low-throughput study describing a plausible moonlighting/host-restriction activity distinct from the protein's core mitochondrial assembly role. Keeping as a non-core annotation. Supporting Evidence: PMID:33635491 Overexpression of Ndufaf4 inhibited VSV propagation, and knockdown of Ndufaf4 by short hairpin RNA (shRNA) markedly promoted VSV replication. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-6799178 | ACCEPT | Summary: Author-stated (Reactome "Complex I biogenesis") localization to the mitochondrial inner membrane, where NDUFAF4 acts on the membrane-arm assembly intermediate. Specific and correct location; accept. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-6799179 | ACCEPT | Summary: Reactome-curated inner-membrane localization within the complex I biogenesis pathway. Correct and specific; accept. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-6799191 | ACCEPT | Summary: Reactome-curated inner-membrane localization within the complex I biogenesis pathway. Correct and specific; accept. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-6799196 | ACCEPT | Summary: Reactome-curated inner-membrane localization within the complex I biogenesis pathway (dissociation of assembly factors from the near-mature complex). Correct and specific; accept. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-6799197 | ACCEPT | Summary: Reactome-curated inner-membrane localization within the complex I biogenesis pathway. Correct and specific; accept. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-6799202 | ACCEPT | Summary: Reactome-curated inner-membrane localization within the complex I biogenesis pathway. Correct and specific; accept. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-6799203 | ACCEPT | Summary: Reactome-curated inner-membrane localization; this reaction is where the IP subcomplex binds NDUFAF3, NDUFAF4, and TIMMDC1 to form an early membrane-arm intermediate ("Intermediate 1"). Directly on the core assembly step; accept. |
| GO:0005739 mitochondrion | IDA PMID:18179882 C6ORF66 is an assembly factor of mitochondrial complex I. | ACCEPT | Summary: Direct experimental evidence that the C6ORF66/NDUFAF4 protein is mitochondrial, from the study establishing it as a complex I assembly factor. Core localization evidence; accept (inner-membrane is the more specific location). |
| GO:0031966 mitochondrial membrane | IDA PMID:19463981 Mutations in NDUFAF3 (C3ORF60), encoding an NDUFAF4 (C6ORF66... | ACCEPT | Summary: Direct experimental evidence for mitochondrial-membrane localization, from the study characterizing the NDUFAF3-NDUFAF4 interaction. Consistent with the more specific inner-membrane annotations; accept. |
| GO:0005515 protein binding | IPI PMID:19463981 Mutations in NDUFAF3 (C3ORF60), encoding an NDUFAF4 (C6ORF66... | MARK AS OVER ANNOTATED | Summary: IPI to NDUFAF3 (UniProtKB:Q9BU61) - the functionally central interaction partner of NDUFAF4 in complex I assembly. Although the biology is core, the GO term "protein binding" is uninformative (no specific molecular function). Per policy, IPI protein-binding is not removed; marking over-annotated. A more informative statement of the NDUFAF3 partnership is captured in core_functions and the assembly-process annotations. Supporting Evidence: PMID:19463981 NDUFAF3 tightly interacts with NDUFAF4 (C6ORF66), a protein previously implicated in complex I deficiency |
| GO:0032981 mitochondrial respiratory chain complex I assembly | IMP PMID:18179882 C6ORF66 is an assembly factor of mitochondrial complex I. | ACCEPT | Summary: Core, experimentally established function. A conserved-residue mutation in C6ORF66 caused isolated complex I deficiency with reduced levels of the fully assembled complex, and transfection of patient fibroblasts with wild-type cDNA restored complex I activity, demonstrating a direct role in complex I assembly. Supporting Evidence: PMID:18179882 Transfection of the patients' fibroblasts with wild-type C6ORF66 cDNA restored complex I activity. These data suggest that C6ORF66 is an assembly factor of complex I. |
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Download this section (compressed HTML)Q: Does NDUFAF4 have any specific molecular activity beyond scaffolding/adapter binding of NDUFAF3 and nascent complex I subunits (e.g. a defined chaperone or membrane-anchoring function)?
Q: Is the antiviral effect against VSV a genuine physiological moonlighting function of NDUFAF4, or an indirect consequence of altered mitochondrial/metabolic state?
Experiment: Structural or crosslinking-MS characterization of the NDUFAF3-NDUFAF4(-TIMMDC1) assembly intermediate to define NDUFAF4's molecular role and interaction surfaces.
Hypothesis: NDUFAF4 acts as a scaffold/anchor that positions NDUFAF3 and nascent subunits on the membrane arm during complex I assembly.
Experiment: Domain-mapping and mutagenesis (including the N-myristoylation site and disease variants A3P and L65P) to determine how membrane anchoring and specific residues contribute to complex I assembly.
Hypothesis: N-myristoylation and the conserved disease-associated residues are required for NDUFAF4's membrane association and assembly-factor function.
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