Loss of COX4I1 Leads to Combined Respiratory Chain Deficiency and Impaired Mitochondrial Protein Synthesis

Abstract

The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.

Complexome Profiling: Assembly and Remodeling of Protein Complexes

“…The aim of each complexome profiling experiment is to gain information of native protein complexes and to compare the appearance and abundance of assemblies under different conditions or in disease states. Biological samples for CP range from bacteria [39,40], yeast [41] or cell culture [4,[17][18][19]21,36,[42][43][44][45][46][47][48][49][50][51], tissue or organ specimens from patients and animals [17,19,37,38,52,53] to plants [54][55][56][57][58][59][60][61][62]. Biochemical preparations of subcellular fractions (e.g., mitochondria, microsomes) reduce sample complexity for a more in depth complexome analysis [17,51].…”

“…Assembly of protein complexes [70] or even further association of several protein complexes require a coordinated process with the help of chaperons also called assembly factors. In recent years, classical CP became a very popular tool to study the significance of individual subunits and assembly factors of the OXPHOS complexes in bacteria [39,40,61] and mitochondria from mammals [17,21,22,27,28,36,[41][42][43][44][45][46][47][48][49][50]53,[64][65][66] and plants [54][55][56][57]60,62].…”

“…Profiles from patients mainly show typical accumulation of assembly intermediates that can be used for the interpretation of the significance of a subunit, an assembly factor in the assembly pathway or a ribosomal protein [36,[44][45][46][47][48][49][50]64,71]. In contrast to the de novo synthesis of OXPHOS-complexes upon treatment with a translation inhibitor [42], patients with defects in the NDUFA6 [45], NDUFC2 [36], and COX4I1 [43] showed, under steady state levels, a clear association of stalled assembly intermediates with other respiratory chain complexes, suggesting that completion of individual complexes is not a prerequisite for supercomplex formation [48].…”

Redox-Mediated Regulation of Mitochondrial Biogenesis, Dynamics, and Respiratory Chain Assembly in Yeast and Human Cells

“…Then again, mammalian CI shows a high interdependence with the other components of the ETC. Strong CIII 2 and CIV assembly defects produce a secondary decline in CI amounts ( Diaz et al, 2006 ; Protasoni et al, 2020 ; Čunátová et al, 2021 ). This was explained by the existence of an active degradation of fully assembled CI in response to RET-mediated ROS production and oxidative damage ( Guarás et al, 2016 ).…”

NDUFS3 depletion permits complex I maturation and reveals TMEM126A/OPA7 as an assembly factor binding the ND4-module intermediate

“…Contrariwise, mutations causing a strong impairment in CIII 2 biogenesis are often associated with secondary loss of CI and CIV activities (Acin-Perez and Enriquez, 2014;Carossa et al, 2014;Protasoni et al, 2020). In some cases, severe defects in CIV biogenesis also have an effect on CI assembly or stability ( Cuná tová et al, 2021;Diaz et al, 2006). In human cells, the near totality (90%-95%) of CI appears associated with SCs when the mitochondria are solubilized with a mild detergent, such as digitonin, whereas approximately 50% of CIII 2 is inside SCs and 90% of CIV is in the ''free'' monomeric form (Lobo-Jarne and Ugalde, 2018).…”