Homozygosity mapping and sequencing in five complex I-deficient patients from three unrelated families identified mutations in NDUFAF3 (C3ORF60) in all of them, and pathogenicity was confirmed by NDUFAF3-GFP baculovirus complementation of patient fibroblasts.

NDUFAF3 is a genuine mitochondrial complex I assembly protein that interacts with complex I subunits - the primary evidence for the core biological process and for the assembly-factor (rather than subunit) role.

NDUFAF3 tightly interacts with NDUFAF4 (C6ORF66), a protein already implicated in complex I deficiency; UniProt records the interaction set as NDUFAF4, NDUFS2 and NDUFS3 from this same study.

Gene-conservation analysis links NDUFAF3 to the bacterial membrane-insertion gene cluster SecF/SecD/YajC and to C8ORF38, suggesting that several complex I disease genes encode proteins that cooperate during assembly.

Label-free semi-quantitative LC-MS/MS across blue-native gel slices yields an abundance profile for each protein, allowing potential interaction partners to be identified by protein correlation profiling rather than by a directed binding assay.

Profiling against the complex I core subunits NDUFS2, NDUFS3, NDUFS7 and NDUFS8 identified C3ORF60 (NDUFAF3) as a novel candidate complex I biogenesis protein, alongside the already-implicated chaperone C6ORF66 (NDUFAF4).

Interaction proteomics on 15 complex I core subunits and assembly factors previously linked to human CI deficiency produced a network of 101 proteins and 335 interactions, the source of the NDUFAF3-NDUFAF4 IntAct edge recorded here.

TIMMDC1 associates reciprocally with MCIA complex members and core CI subunits and localizes to the mitochondrial inner membrane; its depletion reduces CI activity and respiration, placing NDUFAF3's partners in a membrane-embedded assembly module.

A systematic map of roughly 14,000 high-quality human binary protein-protein interactions, generated without hypothesis-driven selection; the NDUFAF3 hits it contributes (SNRPA, RBCK1) are screen-derived and lack mitochondrial context.

Condition-specific protein-protein interactions were assessed for 50 mitochondrial proteins of unclear function (MXPs) by affinity-enrichment mass spectrometry, connecting them to respiratory chain processes; this is the assay behind the NDUFAF3-NDUFAF4 edge from this paper.

The same approach validated C17orf89 (NDUFAF8) as a complex I assembly factor whose disruption markedly reduced CI activity, demonstrating that the map can identify genuine CI assembly factors - though it did not do so for NDUFAF3, already known.

HuRI is a systematic all-by-all yeast two-hybrid screen of about 90% of the protein-coding genome, yielding 52,569 verified binary PPIs among 8,275 proteins; this is the source of the many non-mitochondrial NDUFAF3 partners.

The authors note that the cellular function of most individual PPIs in the map remains to be elucidated, which is why a HuRI-derived 'protein binding' annotation conveys no molecular function for NDUFAF3.

BioPlex 3.0 was built by affinity purification of 10,128 human proteins in 293T cells, giving 118,162 interactions among 14,586 proteins; the NDUFAF3-NDUFAF4 edge recorded here comes from this network.

Comparison across the 293T and HCT116 networks shows that shared interactions reside in core complexes and involve essential proteins, which is consistent with the NDUFAF3-NDUFAF4 pair being a stable functional module.

More than 8,000 proteins were classified in mitochondrial preparations of human cells to define MitoCoP, a high-confidence mitochondrial proteome of over 1,100 proteins; NDUFAF3's membership supports its mitochondrial localization.

The study explicitly reports interactors of respiratory chain and ATP synthase assembly factors, the functional class NDUFAF3 belongs to, but it remains an organelle-level inventory and does not resolve submitochondrial compartment.

A global map of human subcellular architecture was built by jointly measuring biophysical interactions and immunofluorescence images for over 5,100 proteins in U2OS cells, resolving 275 molecular assemblies; the NDUFAF3-NDUFAF4 edge recorded here comes from this resource.

Reactome names NDUFAF3 (C3orf60), together with NDUFAF4 (C6orf66) and TIMMDC1 (C3orf1), as the Intermediate 1 assembly factors that anchor the hydrophobic protein (HP) fraction to the inner mitochondrial membrane to form Intermediate 2.

The peripheral (flavoprotein) arm subunits join the 815 kDa intermediate to form the 980 kDa complex - a later step of the same inner-membrane Complex I biogenesis pathway in which NDUFAF3 acts earlier.

The 315 kDa intermediate is centred on the core iron-sulfur subunits NDUFS2 and NDUFS3 - the very subunits NDUFAF3 interacts with - and the IP subcomplex is anchored to the inner mitochondrial membrane by MT-ND1.

In the final step of Complex I biogenesis the MCIA complex and the assembly factors NDUFAF2-7 and TIMMDC1 all dissociate from the 980 kDa complex to leave mature Complex I, confirming that NDUFAF3 is transient and not a holoenzyme subunit.

The distal membrane-arm subunits MT-ND4 and MT-ND5 associate with the 550 kDa complex to form the 815 kDa complex, requiring DMAC1 and DMAC2 - a downstream step of the same inner-membrane pathway.

The 315 kDa and 370 kDa subcomplexes associate to form the 550 kDa complex, an intermediate step between the NDUFAF3-dependent nucleation and the completion of the membrane arm.

Complex I assembly begins with a 315 kDa subcomplex centred on the core iron-sulfur subunits NDUFS2 and NDUFS3; the IP subcomplex then binds NDUFAF3, NDUFAF4 and TIMMDC1 to form Intermediate 1. This is the step in which NDUFAF3 itself acts, and it matches the UniProt SUBUNIT record (NDUFAF4, NDUFS2, NDUFS3).