Accessory subunits are integral for assembly and function of human mitochondrial complex I

Abstract

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome

“…Figure 5C and Movie S1 show the changes in protein abundance between subject 1 and control individuals mapped to the human mitoribosome structure, as we have done previously for complex I. 42 These representations clearly illustrate the marked decrease in small mitoribosomal subunit proteins in subject 1 compared to control individuals, as well as the relative sparing of the large subunit, consistent with destabilization of the small mitoribosomal subunit. Mapping of protein levels to the structures of OXPHOS Isolated mitochondria were subject to BN-PAGE, following which the complexes were visualized by autoradiography.…”

“…We recently demonstrated the utility of this profiling technique for identifying proteomic signatures of expression changes in response to knockout of individual subunits of OXPHOS complex I in gene-edited cell lines relative to isogenic controls. 42 Here, we further demonstrate that it can also serve as an effective tool for detecting specific proteomic signatures in fibroblasts from subjects relative to fibroblast controls from varied genetic backgrounds. Indeed, in the absence of any prior knowledge of disease causation, quantitative proteomic analysis of fibroblasts from subject 1 would have identified MRPS34 as the most reduced mitochondrial protein.…”

“…We have previously shown that defective incorporation of mtDNA-encoded subunits into complexes I and IV can preclude assembly of these complexes, resulting in a general destabilization and turnover of unincorporated subunits. 40,42 Most of the mtDNA-encoded subunits of complexes I and IV that we detected in proteomic analyses of MRPS34-deficient fibroblasts appeared to be decreased. This was not the case for the MT-CYB and MT-ATP8 subunits of complexes III and V, suggesting that the lesser impact on complexes III and V may relate to longer half-lives of these proteins or their stabilization by association with other polypeptides within the assembled complex.…”

“…42 Fibroblasts from three separate control subjects and two subcultures of subject 1 cultured as above were solubilized in 1% w/v sodium deoxycholate, 100 mM TrisHCl (pH 8.1) prior to incubation at 99 C for 10 min with vortexing. Samples were further incubated for 10 min at 60 C in a sonicator waterbath, followed by the addition of 5 mM Tris(2-carboxyethy)phosphine (TCEP), 20 mM chloroacetamide and incubation for 5 min at 99 C with vortexing.…”

“…42,47 For mitoribosome subunit mapping, the human mitoribosome structure (PDB: 3J9M) was used. 48 For complexes III and IV, homologous human subunits were mapped to the bovine structures PDB: 1BGY and 5B1A, respectively, 49,50 whereas for complex II homologous human subunits were mapped to those found in the porcine structure (PDB: 1ZOY).…”