Mitochondrial OXPHOS Biogenesis: Co-Regulation of Protein Synthesis, Import, and Assembly Pathways

Abstract

The assembly of mitochondrial oxidative phosphorylation (OXPHOS) complexes is an intricate process, which-given their dual-genetic control-requires tight co-regulation of two evolutionarily distinct gene expression machineries. Moreover, fine-tuning protein synthesis to the nascent assembly of OXPHOS complexes requires regulatory mechanisms such as translational plasticity and translational activators that can coordinate mitochondrial translation with the import of nuclear-encoded mitochondrial proteins. The intricacy of OXPHOS complex biogenesis is further evidenced by the requirement of many tightly orchestrated steps and ancillary factors. Early-stage ancillary chaperones have essential roles in coordinating OXPHOS assembly, whilst late-stage assembly factors-also known as the LYRM (leucine-tyrosine-arginine motif) proteins-together with the mitochondrial acyl carrier protein (ACP)-regulate the incorporation and activation of late-incorporating OXPHOS subunits and/or co-factors. In this review, we describe recent discoveries providing insights into the mechanisms required for optimal OXPHOS biogenesis, including the coordination of mitochondrial gene expression with the availability of nuclear-encoded factors entering via mitochondrial protein import systems.

Keywords: LYRM proteins; OXPHOS assembly factors; OXPHOS biogenesis; mitochondrial ACP; mitochondrial gene expression; mitochondrial import.

Figure 1

Overview of the main cellular and mitochondrial energy metabolism pathways. Mitochondrial metabolic pathways are fueled by glucose, fatty acids, and amino acids through respective catabolic processes to produce acetyl-CoA, a primary substrate for the TCA cycle, which is also recycled in the fatty acid β-oxidation (FAO) pathway producing NADH and FADH₂ reducing equivalents that feed the ETC CI and CII, respectively. Glucose undergoes glycolysis in the cytoplasm to form pyruvate which is converted into acetyl-CoA in mitochondria by the PDHC. Acetyl-CoA enters a series of enzyme-catalyzed reactions in the TCA cycle producing reduced forms of NADH and FADH₂ required for the transport of electrons to the ETC (the flow of electrons is illustrated by a grey dashed line). CI, CIII and CIV pump protons from the matrix to the IMS, generating a proton-motive force across the IMM, that drives the flux of protons back into the matrix via the ATP synthase to harness energy released in the form of ATP. The oxidation of NADH and FADH₂ via CI and CII replenishes the electron acceptors NAD+ and FAD to maintain TCA cycle activity. Fatty acids are imported into the mitochondria in the form of fatty-acyl-CoA via the carnitine shuttle system (CPT1 and CPT2) to enter the β-oxidation cyclic pathway. Initially, fatty acyl-CoAs undergo dehydrogenation resulting in a production of a FADH₂ reducing equivalent that transfers electrons to the electron transfer protein ETF and subsequently to the ubiquinone pool via the ETF ubiquinone oxidoreductase (ETF-QO), thus facilitating the entry of electrons to CIII. The remaining three catalytic steps involve hydration, second dehydrogenation during which NAD+ is reduced to NADH that is subsequently re-oxidized by CI, and thiolysis. In the final step, two carbons are removed from the fatty acyl-CoA ester, resulting in shortening of the entry product and its recycling via the FAO pathway. In addition, a molecule of acetyl-CoA is generated that contributes to the overall mitochondrial pool of acetyl-CoA used in the TCA cycle. Amino-acid degradation also contributes to the production of pyruvate, acetyl-CoA, or metabolic intermediates to be oxidized in TCA cycle. Abbreviations are as follows: CI: Complex I, CII: Complex II, CIII: Complex III, CIV: Complex IV, CV: Complex V, CoQ: coenzyme Q/ubiquinone, cyt c: cytochrome c, Acetyl-CoA: acetyl coenzyme A, PDHC: pyruvate dehydrogenase complex, CPT1/2: carnitine palmitoyltrasnferase 1/2, ETF: electron transfer flavoprotein, ETF-DH: ETF dehydrogenase, NADH: nicotinamide adenine nucleotide, FADH₂: flavin adenine dinucleotide, ADP: adenosine diphosphate, ATP: adenosine triphosphate.

Figure 2

Models of coordination of human mitochondrial and cytosolic translation programs during OXPHOS assembly. Mitochondrial translation exhibits plasticity in coordination with nuclear gene expression by three possible translational regulatory systems identified in humans. (A) MITRAC15 binds to MT-ND2 in a complex with mitoribosome to promote its translation and assembly into CI. ACAD9, a CI assembly factor encoded by the nuclear genome, associates ND2 polypeptides specifically downstream of MITRAC15 for assembly of the ND2 subunit into CI. (B) C12orf62 and MITRAC12 act specifically on mitoribosomes translating MT-CO1 mRNA and as members of the early COX1 assembly intermediates to promote MT-CO1 translation. The nuclear-encoded COX4 subunit of CIV post-translationally facilitates completion of CIV assembly by binding to the C12orf62/MITRAC12/nascent COX1 protein complex. (C) The translational activator TACO1 is hypothesized to bind 5′ end of mtDNA-encoded MT-CO1 mRNA for the induction of nascent COX1 synthesis to be assembled into CIV. Abbreviations are as follows: SSU: small ribosomal subunit, LSU: large ribosomal subunit, mt-SSU: mitochondrial small ribosomal subunit, mt-LSU: mitochondrial large ribosomal subunit, TACO1: translational activator of cytochrome c oxidase I, COX1: cytochrome c oxidase subunit 1, COX4: cytochrome c oxidase subunit 4, C12orf62: cytochrome c oxidase assembly protein COX14, MITRAC12: mitochondrial translation regulation assembly intermediate of cytochrome c oxidase protein of 12 kDa, ACAD9: Acyl-CoA dehydrogenase family member 9, MITRAC15: mitochondrial translation regulation assembly intermediate of cytochrome c oxidase protein of 15 kDa.

Figure 3

Interactions between the mitochondrial acyl carrier protein and LYRM proteins. The metabolic intermediate acetyl-CoA feeds into two different pathways in the mitochondria: the TCA cycle and mitochondrial type II fatty acid synthesis (mtFAS-II). The mtFAS-II process encompasses stepwise addition of fatty-acyl chain around an ACP scaffold that leads to acylation of ACP. Further elongation of acyl-ACP generates octanoyl-ACP for lipoic acid synthesis. Alternatively, addition of medium- or long-chain fatty acids to acyl-ACP is required for OXPHOS assembly, Fe–S cluster biosynthesis, and mitoribosome assembly through mtACP interaction with LYRM proteins as indicated by the star (★) symbol. The coordination between nuclear and mitochondrial translation required for OXPHOS proteins of dual genetic origin is illustrated within the black dotted box. The majority of nuclear-encoded precursor proteins synthesized by cytosolic ribosomes are imported into the mitochondria by the translocase of the TOM and TIM23 complexes. OXA1L inserts both nuclear- and mitochondrial-encoded proteins into the IMM for the formation of early OXPHOS assembly intermediates (in blue). Abbreviations are as follows: LYRM: leucine–tyrosine–arginine motif, ACN9: acetate non-utilizing protein 9, FMC1: formation of mitochondrial CV assembly factor I homolog, L0R8F8: MIEF1 upstream open reading frame protein, SSU: small ribosomal subunit, LSU: large ribosomal subunit; mt-SSU: mitochondrial small ribosomal subunit, mt-LSU: mitochondrial large ribosomal subunit, MALSU1: mitochondrial assembly of ribosomal large subunit 1, ACP: acyl carrier protein, CoA: coenzyme A, OXA1L: oxidase assembly 1-like protein, TOM: translocase of the outer membrane, TIM23: mitochondrial import inner membrane translocase subunit Tim23.