Immunocapture of human Complex I followed by 1D/2D gel electrophoresis, MALDI-TOF peptide mass fingerprinting and nanoflow LC-MS/MS resolved 42 polypeptides, matching the subunit inventory of the better-characterized bovine complex; NDUFA2/B8 is among them.

The concordance between human and bovine Complex I isolated by different procedures gives confidence that the identified polypeptides really are constituents of the complex rather than co-purifying contaminants.

An inducible NDUFS3-GFP system in HEK293 cells resolved six distinct NDUFS3-GFP-containing subcomplexes on blue-native blots, also seen in wild-type mitochondria, giving direct evidence that Complex I assembles stepwise.

Differential solubilization and heat incubation distinguished genuine assembly intermediates from products of instability and breakdown, so the composition analysis that places NDUFA2 in the complex reports assembly rather than degradation.

Human complex I comprises 45 subunits, of which only 14 core subunits are shared with bacteria and essential for enzymatic function; the role of the remaining 31 accessory subunits, NDUFA2 among them, was previously unclear.

Knockout cell lines were generated by gene editing for every accessory subunit and profiled by quantitative proteomics; 25 accessory subunits proved strictly required for assembly of a functional complex.

Loss of any given subunit destabilized the other subunits of the same structural module - the observation that assigns each accessory subunit, NDUFA2 included, to a module of the complex, and the experimental basis for the UniProt FUNCTION statement.

Cryo-EM of the human respiratory megacomplex I2III2IV2 resolved 140 subunits together with associated cofactors, providing the structural context in which individual Complex I subunits were placed.

The structure gives a precise individual assignment of the subunits of human Complex I, the basis for the ComplexPortal IDA/IPI annotations placing NDUFA2 in Complex I in the inner membrane.

In the OXPHOS complexes the catalytic work is done by a set of core proteins, while the numerous supernumerary subunits - the class NDUFA2 belongs to - serve assembly, regulation and stability roles. This is the explicit division of labour that makes a direct catalytic MF inappropriate for NDUFA2.

Complexes I, III and IV associate into respiratory supercomplexes or respirasomes, the higher-order context in which Complex I subunits such as NDUFA2 operate.

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; NDUFA2's membership supports its mitochondrial localization.

The dataset is quantitative and spans six orders of magnitude of abundance, but it is an organelle-level inventory that does not resolve submitochondrial compartment, so it cannot support the inner-membrane term.

Complex I is located in the inner mitochondrial membrane and its main function is the transport of electrons from NADH to ubiquinone, accompanied by translocation of protons from the matrix to the intermembrane space - the complex-level process to which NDUFA2 contributes structurally.

The paper reports cDNA sequences for the eight remaining uncharacterized nuclear-encoded subunits, completing the inventory of the 41 then-known human Complex I proteins; it is a sequence-characterization study and assays no enzymatic activity of any individual subunit.

Reactome describes Complex I as an L-shaped 45-subunit enzyme whose IP and FP fractions protrude into the matrix while the HP arm lies within the inner mitochondrial membrane, with FMN and eight Fe-S clusters carrying electrons from NADH to ubiquinone - the catalytic machinery NDUFA2 does not possess.

The hydrophobic protein (HP) fraction is anchored to the inner mitochondrial membrane by the Intermediate 1 assembly factors NDUFAF3, NDUFAF4 and TIMMDC1 to form Intermediate 2.

The peripheral (flavoprotein) arm subunits join the 815 kDa intermediate to form the 980 kDa complex - the arm of the complex in which NDUFA2 resides.

The 315 kDa intermediate is centred on the core iron-sulfur subunits NDUFS2 and NDUFS3, 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 dissociate from the 980 kDa complex to leave mature Complex I - the distinction between transient assembly factors and true subunits such as NDUFA2, which is retained.

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.

The 315 kDa and 370 kDa subcomplexes associate to form the 550 kDa complex, an intermediate step of the inner-membrane Complex I biogenesis pathway.

LONP1 binds, unfolds and degrades several mitochondrial inner-membrane proteins, binding substrate in an ATP-independent manner before ATP-dependent translocation to the protease site. NDUFA2 enters this Reactome reaction as a substrate, which is a statement about its turnover rather than about its function.

The companion degradation step: LONP1 binds and degrades several mitochondrial inner membrane proteins, cleaving preferentially next to hydrophobic residues. Again NDUFA2 appears as the substrate, so the reaction supports its localization but not any molecular activity of its own.