Mitochondria produce the bulk of the energy used by almost all eukaryotic cells through oxidative phosphorylation (OXPHOS) which occurs on the four complexes of the respiratory chain and the F1–F0 ATPase. Mitochondrial diseases are a heterogenous group of conditions affecting OXPHOS, either directly through mutation of genes encoding subunits of OXPHOS complexes, or indirectly through mutations in genes encoding proteins supporting this process. These include proteins that promote assembly of the OXPHOS complexes, the post-translational modification of subunits, insertion of cofactors or indeed subunit synthesis. The latter is important for all 13 of the proteins encoded by human mitochondrial DNA, which are synthesised on mitochondrial ribosomes. Together the five OXPHOS complexes and the mitochondrial ribosome are comprised of more than 160 subunits and many more proteins support their biogenesis. Mutations in both nuclear and mitochondrial genes encoding these proteins have been reported to cause mitochondrial disease, many leading to defective complex assembly with the severity of the assembly defect reflecting the severity of the disease. This review aims to act as an interface between the clinical and basic research underpinning our knowledge of OXPHOS complex and ribosome assembly, and the dysfunction of this process in mitochondrial disease.

Introduction

Mitochondria are ubiquitous organelles that have their own DNA and translation machinery. The human mitochondrial (mt) DNA is a circular double-stranded molecule containing 16 569 base pairs and 37 genes [1]. Two of these genes encode mitochondrial ribosomal RNAs (mt-rRNA) which are required for the assembly of the mitochondrial ribosome (mitoribosome). Another 22 genes encode mitochondrial transfer RNAs (mt-tRNAs) which are responsible for deciphering RNA sequences during protein translation. The mt-tRNA for valine (tRNAval) is also present as a structural component of the mitoribosome [2–4] (Figure 1, lower inset). The remaining 13 mitochondrial genes encode highly hydrophobic transmembrane proteins that are translated on mitoribosomes and assembled into complexes I, III, IV and V of the oxidative phosphorylation (OXPHOS) system [5–8] (Figure 1, upper inset). Besides the 37 genes encoded on mtDNA, over a thousand other proteins encoded on nuclear DNA (nDNA) are translated in the cytosol and imported into the mitochondria via dedicated protein import machinery [9,10]. Mitochondria are responsible for generating the majority of cellular ATP via OXPHOS, which occurs on the mitochondrial respiratory chain (MRC) and the F1F0-ATPase. The MRC consists of four multiprotein complexes (complexes I–IV) embedded in the inner mitochondrial membrane (IMM) and two electron carriers called coenzyme Q (CoQ, Q) and cytochrome c (Cyt C, C). Complexes I, III and IV are also found together in higher order structures known as supercomplexes or respirasomes [11], though the precise function this coalescence is not fully clear [12]. The MRC as a whole, as well as the two electron carriers are involved in a series of redox reactions that creates a proton gradient across the IMM, which is in turn used by the fifth complex, complex V or the F1F0-ATPase, to synthesise ATP.

The oxidative phosphorylation (OXPHOS) system and the mitoribosome.

Figure 1.
The oxidative phosphorylation (OXPHOS) system and the mitoribosome.

Upper inset, the OXPHOS system consists of complexes I, II, III and IV of the respiratory chain and complex V (the F1Fo-ATP Synthase). Complexes I, III and IV also exist in large assemblies known as supercomplexes (SC). Subunit compositions of individual complexes are indicated. mtDNA, mitochondrial DNA; nDNA, nuclear DNA; Q, Coenzyme Q; C, Cytochrome c. Lower inset, the mitoribosome synthesises the 13 mtDNA-encoded subunits that are present within the OXPHOS complexes. It consists of two major parts, the small subunit (SSU) and large subunit (LSU). The SSU contains one mtDNA-encoded rRNA (12S rRNA) and 30 nDNA encoded proteins. The LSU contains one mtDNA-encoded rRNA (16S rRNA), 52 nDNA encoded proteins and a single mtDNA-encoded tRNAval molecule that has a structural role in mitoribosome. All 22 mtDNA-encoded tRNAs, including tRNAval, are involved in the synthesis of the 13 mtDNA-encoded proteins from 11 mRNAs. Two mRNAs, MT-ND4/MT-ND4L and MT-ATP6/MT-ATP8 share open reading frames and the proteins are transcribed as bicistronic elements. Orange elements in mtDNA represent mtDNA associated protein TFAM. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix. Models for complexes I, II, III, IV, V and mitoribosome generated from PDB: 5LDW, 1ZOY, 1BGY, 5B1A, 5ARE and 3J9M, respectively.

Figure 1.
The oxidative phosphorylation (OXPHOS) system and the mitoribosome.

Upper inset, the OXPHOS system consists of complexes I, II, III and IV of the respiratory chain and complex V (the F1Fo-ATP Synthase). Complexes I, III and IV also exist in large assemblies known as supercomplexes (SC). Subunit compositions of individual complexes are indicated. mtDNA, mitochondrial DNA; nDNA, nuclear DNA; Q, Coenzyme Q; C, Cytochrome c. Lower inset, the mitoribosome synthesises the 13 mtDNA-encoded subunits that are present within the OXPHOS complexes. It consists of two major parts, the small subunit (SSU) and large subunit (LSU). The SSU contains one mtDNA-encoded rRNA (12S rRNA) and 30 nDNA encoded proteins. The LSU contains one mtDNA-encoded rRNA (16S rRNA), 52 nDNA encoded proteins and a single mtDNA-encoded tRNAval molecule that has a structural role in mitoribosome. All 22 mtDNA-encoded tRNAs, including tRNAval, are involved in the synthesis of the 13 mtDNA-encoded proteins from 11 mRNAs. Two mRNAs, MT-ND4/MT-ND4L and MT-ATP6/MT-ATP8 share open reading frames and the proteins are transcribed as bicistronic elements. Orange elements in mtDNA represent mtDNA associated protein TFAM. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix. Models for complexes I, II, III, IV, V and mitoribosome generated from PDB: 5LDW, 1ZOY, 1BGY, 5B1A, 5ARE and 3J9M, respectively.

The MRC, complex V and the mitoribosome are built through a series of orchestrated steps that require the help of other proteins not part of the mature complex, often called assembly factors. Not all assembly factors directly promote assembly, the controlled coalescence of subunits into complexes through stabilisation of assembly intermediates, but many instead provide critical services in the form of maturation and delivery of cofactors such as heme, copper and iron–sulfur clusters (Fe–S), post-translational modification of subunits and regulation of translation. Furthermore, transcription of mtDNA, mitochondrial mRNA processing, mitochondrial tRNA maturation, mitoribosome assembly and translation all directly influence OXPHOS complex biogenesis function [5–8]. Dysfunction in any of the aforementioned processes can lead to mitochondrial disease. Given the central importance of mitochondria to cellular function, disease can affect any tissue in the body, though tissues and organs of high energy demand such as heart, brain and muscle are frequently affected. Furthermore, some patients present with tissue or organ specific phenotypes (e.g. cardiomyopathy) while others exhibit multi-system disorders or delayed onset, reflecting the different requirements for mitochondrial energy production in different cell types, the function of affected protein, their tissue-specific expression or their being encoded on nuclear or mitochondrial DNA [5–8]. There are over 300 known disease genes, located in either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA), implicated in mitochondrial diseases ([5,13], this review). Broadly speaking, these can be separated into two categories: genes that are involved in primary energy generation (i.e. subunits or assembly factors of the OXPHOS system, mitoribosome, involved in transcription, mtDNA homeostasis and related systems) and genes that have a secondary function (i.e. protein quality control, membrane structure and metabolite transport) [5,14,15]. Of the over 190 known primary disease genes, more than half lead to defective assembly of the OXPHOS complexes or the mitoribosome ([5], this review).

The current diagnostic paradigm for mitochondrial disease involves a combination of whole exome (WES) or whole genome (WGS) sequencing, with linkage studies and homozygosity mapping [5,13]. In the case of mtDNA mutations, diagnosis can be even more challenging considering that the mtDNA is present in multiple copies per cell and might contain a heterogenous population of mutated and non-mutated mtDNA, a phenomenon termed heteroplasmy. In the case of mitochondrial disease, the ratio of mutated to non-mutated mtDNA is termed mutation load, which is particularly important in the onset and tissue-specific manifestation of mitochondrial diseases [16]. Diagnostic yield when using strategies combined with WES or WGS is only 30–68% [17–20], suggesting that other novel disease genes and variants remain to be discovered. The principal challenge in the current diagnosis paradigm is validation of variants of uncertain significance (VUS) detected in patients with suspected mitochondrial disease [21]. To address this, investigators often turn to functional studies. Studies such as these, sometimes continuing long after confirmed diagnosis, have been incredibly informative to our understanding of mitochondrial complex assembly. The results of these studies can later provide information invaluable to those attempting to solve new undiagnosed cases. For example, we now know from cultured cell line knockout studies that loss of proteins required for some steps of complex I assembly does not greatly impact assembly and function of the enzyme, whereas loss of proteins required for other steps leads to catastrophic failure in complex biogenesis [22]. Perhaps unsurprisingly, in patients null mutations have been described in genes required for the former step, but not the second (see the below sections for detail). Armed with this information, an investigator may be able to prioritise a novel VUS in a gene associated with one of these steps for follow-up studies. It is, therefore, the intention of this review to present a common ground to both basic researchers interested in the assembly of mitochondrial complexes, as well as more clinically focused audiences, in the hope that both will benefit from each other's ongoing efforts to understand the function and impacts of dysfunction in these complex molecular machines.

Due to the large number of steps in the biogenesis of these complexes, most of the following sections have been separated into sub-sections describing distinct stages of assembly and the impacts of disease on this process. Subunits (i.e. structural proteins found in the fully assembled complex) in the below sections are indicated in bold typeface, whereas assembly factors and other proteins are listed in regular typeface. The nomenclature for some subunits and assembly factors is complicated, different groups may use different symbols and for most complexes the subunit naming convention differs between humans and other organisms. For the purposes of this review, we have chosen to refer to the subunits and assembly factors by their Human Genome Organisation (HUGO) approved gene symbol [23], though have aimed to include the commonly used protein symbol in parenthesis at first mention. Finally, due to the large number of case reports for some disease genes we have chosen to cite the first example for each commonly observed phenotype. Further case reports can be found by referring to the relevant entry in the Online Mendelian Inheritance in Man database (https://www.omim.org), with the OMIM entry number cited for each gene in the relevant table.

Complex I

Mitochondrial complex I (NADH, Ubiquinone reductase) is the first complex in the MRC. The fully assembled complex I is L-shaped with a hydrophobic arm embedded in the IMM and a hydrophilic arm extending into the mitochondrial matrix [24,25]. The latter contains the catalytic N-module, which is involved in oxidation of NADH to NAD+, and the Q-module which transfers electrons via Fe–S clusters to Coenzyme Q [26,27]. This in turn triggers conformational changes leading to pumping of protons across the membrane arm (ND-modules) and creation of the proton-motive force used by complex V [28]. Complex I consists of 45 subunits (44 distinct subunits as one subunit, NDUFAB1 is present in two locations within the complex) [29]. Of the 44 distinct subunits, 14 are classified as core subunits since they possess catalytic function and homologues are present in all organisms with complex I, including bacteria [30,31]. Of the 14 core mammalian subunits, 7 are encoded on mtDNA: ND1, ND2, ND3, ND4, ND4L, ND5 and ND6, all of which are present in the membrane arm, while the remainder are encoded on nuclear DNA. Four are located in the Q-module: NDUFS2, NDUFS3, NDUFS7, NDUFS8; and three in the N-module: NDUFS1, NDUFV1, NDUFV2. The remaining 30 subunits (prefixed NDUFA, NDUFB, NDUFC and NDUFAB1) are known as accessory or supernumerary subunits, with 25 of them being characterised as essential for the assembly and stability of the complex [22]. The assembly of complex I occurs through the sequential addition of modules seeded by core subunits to which accessory subunits are added. Although the order of module coalescence is still not fully understood, the assembly pathway for complex I is one of the most well studied of all mitochondrial complexes and there is an increasingly well accepted model (Figure 2) [22,24,32].

Schematic depicting the complex I assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded subunits underlined and assembly factors in italics.

Figure 2.
Schematic depicting the complex I assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded subunits underlined and assembly factors in italics.

Complex I assembly is characterised by coalescence of distinct modules assembled individually prior to final assembly into large intermediates and eventually the functional complex. The Q-module joins the ND1 module and coalesces with ND2, ND4 and ND5 modules. The N-module (harbouring the site of NADH oxidation) is the last module to join the complex in the assembly pathway. Fe–S, iron–sulfur. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Figure 2.
Schematic depicting the complex I assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded subunits underlined and assembly factors in italics.

Complex I assembly is characterised by coalescence of distinct modules assembled individually prior to final assembly into large intermediates and eventually the functional complex. The Q-module joins the ND1 module and coalesces with ND2, ND4 and ND5 modules. The N-module (harbouring the site of NADH oxidation) is the last module to join the complex in the assembly pathway. Fe–S, iron–sulfur. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Assembly of the Q and ND1 modules

The Q-module subassembly contains nuclear-encoded subunits NDUFA5, NDUFS2, NDUFS3, NDUFS7 and NDUFS8 and appears to be one of the earlier modules to assemble (Figure 2) [22,24,32]. With the exception of NDUFA5, all of the above subunit genes have been associated with Leigh syndrome, leading to early childhood death (Table 1). In line with this, their gene-editing based ablation in the commonly used human embryonic kidney cell (HEK293T) model system leads to complete loss of complex assembly and activity [22]. The assembly factors NDUFAF5 and NDUFAF7 are required for maturation of the Q-module [33–35] and they contain the S-adenosylmethionine (SAM)-dependent methyltransferase domains [33,36] that provide post-translational modifications to subunits within the Q-module. NDUFAF5 is responsible for hydroxylation of an arginine residue in NDUFS7 [34] and requires the assembly factor NDUFAF8 for its stabilisation, though its molecular function in this capacity is not yet clear [37,38]. On the other hand, NDUFAF7 is required for dimethylation of an arginine residue in NDUFS2 [33,35]. While the function of these modifications is unknown, their loss results in complex I dysfunction [39,40]. There have been no patients reported for NDUFAF7, however, a number have been reported for NDUFAF5 and NDUFAF8 (Table 1). For NDUFAF5, the majority of patients harbour missense mutations leading to impaired complex assembly. The phenotype is predominantly Leigh syndrome, however, many patients have survived until adolescence or adulthood (Table 1). NDUFAF8 was only recently reported to be a disease gene, with the patients described having presumably null mutations leading to a classic Leigh syndrome phenotype [38]. The assembly factors NDUFAF3 and NDUFAF4 are required for maturation of the Q-module and have been found in association with both NDUFS3 and NDUFS3 subunits [41] as well as the Q-module intermediate [32]. While the precise molecular function of NDUFAF3 and NDUFAF4 are not known, they appear to be essential for progression to the next assembly stage [22,32,42]. Consistent with this, patients for both have presented with severe infantile disease due to predominantly missense, presumably null variants (Table 1).

Table 1.
Defects affecting biogenesis of the Q and ND1 modules
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
Q-module 
NDUFS2  Various missense leading to uncertain impact or near absence of protein. Leigh syndrome, optic atrophy, hypertrophic cardiomyopathy, elevated blood lactate. [380,381602985 
NDUFS3  Missense leading to uncertain protein impact. Leigh syndrome, optic atrophy. [382603846 
NDUFS7  Predominantly missense but also intronic (nonsense) leading to truncated protein or uncertain impact on protein. Leigh syndrome, hypotonia, may present normal blood lactate and pyruvate. [383,384601825 
NDUFS8  Missense leading to uncertain impact on protein or reduced levels. Ranging from severe Leigh syndrome, elevated blood lactate and pyruvate, hypotonia, early death to slowly progressive neurological disease at the end of first decade of life. [385–387602141 
NDUFAF3  Missense leading to uncertain protein impact. Severe lactic acidosis, optic atrophy, respiratory failure and variable brain involvement including Leigh syndrome, myoclonic seizures, macrocephaly and cavitating leukoencephalopathy. [41,388,389612911 
NDUFAF4  Missense leading to decreased protein levels or uncertain protein impact. Leigh syndrome, hypotonia, elevated blood and cerebrospinal fluid lactate, may present severe infantile cardiomyopathy. [42,390611776 
NDUFAF5  Predominantly missense but also intronic (nonsense) leading to uncertain protein impact. Most common presentation of Leigh syndrome, but also fatal lactic acidosis, hyponatremia, hypotonia and bilateral optic neuropathy. Variable survival depending on variant. Decreased CI activity. [36,391–393612360 
NDUFAF8  Nonsense (frameshift and intronic) and missense leading to uncertain protein impact. Leigh syndrome, may present optic atrophy and elevated blood lactate. [38618461 
ND1 module 
MT-ND1  Multiple, predominantly missense variants with protein levels dependant on variant. and mutant mtDNA load. LHON, MELAS. [43–45516000 
NDUFA1  Missense leading to decreased protein levels. X-linked. Variable presentation from severe Leigh syndrome to developmental delay, hypotonia, elevated blood lactate and myoclonic epilepsy and survival to childhood. [48300078 
NDUFA8  Missense leading to reduced proteins levels. Psychomotor retardation, severe quadriplegia, elevated blood lactate, cerebral atrophy, hypertonia, epilepsy with survival to adulthood. [394603359 
NDUFA13  Missense or nonsense (frameshift) leading to reduced protein levels. Hypotonia, dyskinesia, sensorial impairments or Leigh syndrome and mild hypertrophic cardiomyopathy. [395,396609435 
NDUFAF6  Predominantly missense but also frameshift and intronic leading to decreased protein levels or uncertain protein impact. Leigh syndrome is the most common presentation, but also Fanconi syndrome, lactic acidosis, bilateral striatal necrosis and progressive dystonia in childhood. [47,66,397,398612392 
TIMMDC1  Intronic variant causing frameshift and early termination leading to decreased protein levels. Infantile muscular hypotonia, developmental delay, neurological deterioration. [46615534 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
Q-module 
NDUFS2  Various missense leading to uncertain impact or near absence of protein. Leigh syndrome, optic atrophy, hypertrophic cardiomyopathy, elevated blood lactate. [380,381602985 
NDUFS3  Missense leading to uncertain protein impact. Leigh syndrome, optic atrophy. [382603846 
NDUFS7  Predominantly missense but also intronic (nonsense) leading to truncated protein or uncertain impact on protein. Leigh syndrome, hypotonia, may present normal blood lactate and pyruvate. [383,384601825 
NDUFS8  Missense leading to uncertain impact on protein or reduced levels. Ranging from severe Leigh syndrome, elevated blood lactate and pyruvate, hypotonia, early death to slowly progressive neurological disease at the end of first decade of life. [385–387602141 
NDUFAF3  Missense leading to uncertain protein impact. Severe lactic acidosis, optic atrophy, respiratory failure and variable brain involvement including Leigh syndrome, myoclonic seizures, macrocephaly and cavitating leukoencephalopathy. [41,388,389612911 
NDUFAF4  Missense leading to decreased protein levels or uncertain protein impact. Leigh syndrome, hypotonia, elevated blood and cerebrospinal fluid lactate, may present severe infantile cardiomyopathy. [42,390611776 
NDUFAF5  Predominantly missense but also intronic (nonsense) leading to uncertain protein impact. Most common presentation of Leigh syndrome, but also fatal lactic acidosis, hyponatremia, hypotonia and bilateral optic neuropathy. Variable survival depending on variant. Decreased CI activity. [36,391–393612360 
NDUFAF8  Nonsense (frameshift and intronic) and missense leading to uncertain protein impact. Leigh syndrome, may present optic atrophy and elevated blood lactate. [38618461 
ND1 module 
MT-ND1  Multiple, predominantly missense variants with protein levels dependant on variant. and mutant mtDNA load. LHON, MELAS. [43–45516000 
NDUFA1  Missense leading to decreased protein levels. X-linked. Variable presentation from severe Leigh syndrome to developmental delay, hypotonia, elevated blood lactate and myoclonic epilepsy and survival to childhood. [48300078 
NDUFA8  Missense leading to reduced proteins levels. Psychomotor retardation, severe quadriplegia, elevated blood lactate, cerebral atrophy, hypertonia, epilepsy with survival to adulthood. [394603359 
NDUFA13  Missense or nonsense (frameshift) leading to reduced protein levels. Hypotonia, dyskinesia, sensorial impairments or Leigh syndrome and mild hypertrophic cardiomyopathy. [395,396609435 
NDUFAF6  Predominantly missense but also frameshift and intronic leading to decreased protein levels or uncertain protein impact. Leigh syndrome is the most common presentation, but also Fanconi syndrome, lactic acidosis, bilateral striatal necrosis and progressive dystonia in childhood. [47,66,397,398612392 
TIMMDC1  Intronic variant causing frameshift and early termination leading to decreased protein levels. Infantile muscular hypotonia, developmental delay, neurological deterioration. [46615534 

S., subunit; A.F., assembly factor; LHON, Leber's hereditary optic neuropathy; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes.

Following association with NDUFAF3 and NDUFAF4, the assembled Q-module joins with the mtDNA-encoded ND1 subunit and the assembly factor TIMMDC1 [32]. Numerous mutations in ND1 have been reported leading to Leber hereditary optic neuropathy (LHON) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) (Table 1). Generally, these patients have high mutant loads or are in some cases homoplasmic (100%) for the variants [43–45]. Like most mutations in mtDNA, variants in ND1 generally lead to adult onset disease. TIMMDC1 is only a recently identified disease gene with a single homozygous intronic variant detected, leading to a splicing defect and no detectable protein [46] (Table 1). The assembly factor NDUFAF6 is also required for ND1 biogenesis. While many missense variants in the NDUFAF6 gene lead to Leigh syndrome, it does not appear to be stably associated with an intermediate and its precise function is not yet known [47]. Once the nascent Q–ND1 module is formed, it subsequently incorporates the subunits NDUFA3, NDUFA8 and NDUFA13 [32] to produce the final Q–ND1 intermediate. Both NDUFA8 and NDUFA13 are disease genes with missense variants leading to complex I deficiency, however, the few patients identified have survived into young adulthood (Table 1). The reason for only missense variants leading to reduced protein levels being reported for NDUFA8 and NDUFA13 might be explained by the fact that loss of either protein products in HEK293T cells leads to complete loss of complex assembly and activity [22], suggesting that complete absence of these proteins might be not tolerated. Finally, while NDUFA1 is a subunit of the ND1 module, it is not added to the complex I until late during assembly [32]. Many missense variants in NDUFA1, encoded on the X chromosome, have been linked to complex I deficiency with phenotypes ranging from mild (survival into childhood) to severe (Leigh syndrome and death in early childhood) [48] (Table 1).

Assembly of the ND2 module

In the early steps of the assembly of the ND2 module, the subunits ND2, NDUFC1 and NDUFC2 associate with assembly factors ACAD9, ECSIT, NDUFAF1 and COA1 (Figure 2). To this subcomplex, ND3 and the assembly factors TMEM126B and TMEM186, and subsequently ND4L and ND6 are added [32,49]. The aforementioned assembly factors are also known to form the mitochondrial complex I intermediate assembly (MCIA) complex which is essential for the assembly of the ND2 module [49]. COA1, which was originally suggested to be a complex IV assembly factor [50], has recently been shown to be involved in the early stages of ND2 translation [51]. Patients have been described for all mtDNA-encoded subunits present in this intermediate (ND2, ND3, ND4L and ND6). The phenotype spectrum is broad and depends on both the variant itself and mutant load, however, Leigh syndrome, LHON and associated phenotypes are predominant (Table 2). Although ND4L is transcribed as a biscistronic mRNA with ND4 (of the ND4 module) [52] no variants have been described that affect the both proteins. All assembly factors present in the MCIA complex, with the exception of ECSIT and newly identified members COA1 and TMEM186 [49], are known disease genes (Table 2). The function of NDUFAF1 (formerly CIA30) was first linked to complex I assembly through the use of fungal [53] and then mammalian [54] model systems, and eventually led to the identification of patients presenting with cardiomyopathies [55–57]. Fibroblasts from NDUFAF1 patients have reduced levels of complex I and defective assembly of the ND2 module [55]. ACAD9 was originally thought to be involved in β-oxidation based on sequence homology with other members of the acyl-CoA family as well as its ability to bind acetyl-CoA substrates in vitro [58]. The association with cardiomyopathy [59], demonstration of its interaction with other MCIA members and requirement for complex I assembly [60] solidified its role as an assembly factor. ACAD9 is one of the most common causes of complex I deficiency and while it typically presents as cardiomyopathy, the phenotype can be quite varied with milder presentations such as mild growth retardation and exercise intolerance also noted (Table 2) [61]. Interestingly, while it is now clear that ACAD9 deficient cells lack β-oxidation defects, riboflavin supplementation has been shown to lessen symptoms in patients and improve complex I activity [61]. ACAD9 is a flavoprotein (riboflavin is a precursor to flavin adenine dinucleotide; FAD), however, a catalytically dead mutant retains the ability to rescue complex I assembly, suggesting that ACAD9 has a secondary role in mitochondrial function [62]. TMEM126B was identified as an assembly factor through complexome profiling studies [63] with patients later being identified. The role of TMEM126B in complex I assembly is still not completely clear, although we know this protein has multiple transmembrane domains and may act as an anchor for the other MCIA subunits, despite their levels remaining unaffected by loss of TMEM126B [49]. Patients harbouring mutations in TMEM126B also present milder phenotypes compared with those with mutations in NDUFAF1 and ACAD9 (Table 2).

Table 2.
Defects affecting biogenesis of the ND2 module
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-ND2  Predominantly missense but also nonsense and deletion leading to frameshift and protein impact dependant on variant and mutant mtDNA load. Variable onset and presentation from Leigh syndrome, LHON, mild exercise intolerance, myalgia with survival into late adulthood reported. [171,399,400516001 
MT-ND3  Missense and protein impact dependant on variant and mutant mtDNA load. Variable onset and presentation including severe Leigh syndrome, encephalopathy, lactic acidosis, LHON, hypotonia, ataxia, seizures, dystonia with survival into adulthood reported. [401–406516002 
MT-ND4L  Missense leading to unclear protein impact. LHON. Decreased CI activity. [407,408516004 
MT-ND6  Missense and protein impact dependant on variant and mutant mtDNA load. Most common presentation of LHON and dystonia with variable onset, but also MELAS with survival into childhood and severe Leigh syndrome or adult onset Leigh-like syndrome. [409–413516006 
NDUFA9  Missense leading to decreased protein levels. Variable phenotype of childhood-onset of progressive dystonia developing neuropathy and Leigh syndrome without acidosis in adulthood or severe respiratory and metabolic acidosis, retinitis pigmentosa and early death. [64,69603834 
NDUFA10  Predominantly missense but also insertion leading to decreased protein levels. Leigh syndrome combined with hypertrophic cardiomyopathy, hypotonia. [65,66603835 
NDUFA11  Missense and intronic with unclear protein impact. Variable presentation from mild late-onset myopathy to fatal infantile lactic acidosis, encephalocardiomyopathy, hypotonia, bilateral optic atrophy [67,68612638 
NDUFAF1  Missense leading to decreased or unclear protein levels. Hypertrophic cardiomyopathy, failure to thrive, developmental delay, lactic acidosis, hypotonia, leukodystrophy. [55–57606934 
ACAD9  Predominantly missense but also duplication leading to frameshift with unclear protein impact. Variable phenotype from mild growth retardation, exercise intolerance, cardiac hypertrophy surviving to adulthood to severe hypertrophic cardiomyopathy, encephalopathy and lactic acidosis. [414,415611103 
TMEM126B  Missense but also nonsense (frameshift) leading to unclear protein impact. Most common presentation of myalgia and exercise intolerance with survival to adulthood but also hypertrophic cardiomyopathy, renal tubular acidosis and severe muscle weakness. Decreased CI assembly. [416,417615533 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-ND2  Predominantly missense but also nonsense and deletion leading to frameshift and protein impact dependant on variant and mutant mtDNA load. Variable onset and presentation from Leigh syndrome, LHON, mild exercise intolerance, myalgia with survival into late adulthood reported. [171,399,400516001 
MT-ND3  Missense and protein impact dependant on variant and mutant mtDNA load. Variable onset and presentation including severe Leigh syndrome, encephalopathy, lactic acidosis, LHON, hypotonia, ataxia, seizures, dystonia with survival into adulthood reported. [401–406516002 
MT-ND4L  Missense leading to unclear protein impact. LHON. Decreased CI activity. [407,408516004 
MT-ND6  Missense and protein impact dependant on variant and mutant mtDNA load. Most common presentation of LHON and dystonia with variable onset, but also MELAS with survival into childhood and severe Leigh syndrome or adult onset Leigh-like syndrome. [409–413516006 
NDUFA9  Missense leading to decreased protein levels. Variable phenotype of childhood-onset of progressive dystonia developing neuropathy and Leigh syndrome without acidosis in adulthood or severe respiratory and metabolic acidosis, retinitis pigmentosa and early death. [64,69603834 
NDUFA10  Predominantly missense but also insertion leading to decreased protein levels. Leigh syndrome combined with hypertrophic cardiomyopathy, hypotonia. [65,66603835 
NDUFA11  Missense and intronic with unclear protein impact. Variable presentation from mild late-onset myopathy to fatal infantile lactic acidosis, encephalocardiomyopathy, hypotonia, bilateral optic atrophy [67,68612638 
NDUFAF1  Missense leading to decreased or unclear protein levels. Hypertrophic cardiomyopathy, failure to thrive, developmental delay, lactic acidosis, hypotonia, leukodystrophy. [55–57606934 
ACAD9  Predominantly missense but also duplication leading to frameshift with unclear protein impact. Variable phenotype from mild growth retardation, exercise intolerance, cardiac hypertrophy surviving to adulthood to severe hypertrophic cardiomyopathy, encephalopathy and lactic acidosis. [414,415611103 
TMEM126B  Missense but also nonsense (frameshift) leading to unclear protein impact. Most common presentation of myalgia and exercise intolerance with survival to adulthood but also hypertrophic cardiomyopathy, renal tubular acidosis and severe muscle weakness. Decreased CI assembly. [416,417615533 

S., subunit; A.F., assembly factor; LHON, Leber's hereditary optic neuropathy; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes.

At this stage of complex I biogenesis there appears to be multiple parallel routes to the final complex, either the ND2 module joins with the Q–ND1 intermediate or the ND4 intermediate [32] (Figure 2). Once either the Q–ND1–ND2 subcomplex or ND2–ND4 subcomplex is assembled the ND2 module subunits NDUFA9, or NDUFA10 and NDUFS5 are respectively incorporated. NDUFA11 appears to be added later during assembly once the membrane arm is fully built [32]. Despite the timing of these subunit additions, all are critical for complex I assembly in gene-edited HEK293T cells [22]. Patients have been identified for NDUFA9, NDUFA10 and NDUFA11 (Table 2) and generally present with severe infantile Leigh syndrome or encephalocardiomyopathy and isolated complex I deficiency [64–68], though there have been reports of milder NDUFA9 and NDUFA11 cases presenting with childhood-onset progressive dystonia [69] and late-onset myopathy respectively [68].

Assembly of the ND4 and ND5 modules

The assembly steps of the ND4 and ND5 modules are less well characterised than the other modules, with assembly factors only identified in recent years. In the case of the ND4 module, a subcomplex containing four accessory subunits NDUFB5, NDUFB6, NDUFB10 and NDUFB11 assemble early and are followed by the addition of NDUFB1 and the mtDNA-encoded subunit ND4. Like for NDUFA10 and NDUFS5 (discussed in the ND2 module section), NDUFB4 seems to be incorporated into the ND4 module once the intermediate ND2–ND4 subcomplex is formed [32]. In our HEK293T model system, loss of all nuclear subunits present in the ND4 module leads to turnover of almost all complex I subunits, leaving only an intermediate containing Q/ND1 subunits module intact [22]. As such, defects in genes associated with the ND4 module lead to severe disease (Table 3). ND4, NDUFB10, NDUFB11 are known disease genes and patients with variants in ND4 largely present with similar clinical features to those with mutations in other mtDNA-encoded complex I subunits, with LHON the dominant phenotype but also Leigh syndrome and encephalopathy have been reported. Indeed, one ND4 variant (11778A), accounts for more than half of the reported primary cause of LHON in Caucasian families [70] and over 90% in the Chinese families [71]. Although ND4 and ND4L (found in the ND2 module) are transcribed as a biscistronic mRNA [52], no cases have been described where both proteins are affected. For NDUFB10 there is a single known case that presented with severe neonatal cardiomyopathy leading to infantile death. The patient harboured a compound frame shift variant leading to early termination and a missense (p.C107S) variant leading to a mutation in the conserved CXnC motif important for import of the protein into mitochondria [72]. Consistent with an import defect, tissues had reduced but not absent levels of NDUFB10 and accumulation of complex I intermediates. Similarly, variants in NDUFB11 also typically lead to infantile cardiomyopathy, though the presentation is complicated by the gene being present on the X chromosome (Table 3) [66,73,74]. Interestingly, female patients appear to suffer strong skewing of X-chromosome inactivation toward the mutant allele [73].

Table 3.
Defects affecting biogenesis of the ND4 and ND5 modules
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
ND4 module 
MT-ND4  Missense with unclear protein impact. LHON, early onset Leigh syndrome, late-onset encephalopathy, may present lactic acidosis. Phenotype severity may correlate with mutation load. [418–421516003 
NDUFB10  Missense and nonsense leading to impaired protein import. Fatal lactic acidosis and cardiomyopathy. [72603843 
NDUFB11  Nonsense but also missense and deletion leading to reduced protein levels. X-linked gene. Encephalopathy, cardiomyopathy, MIDAS, LIMD, sideroblastic anaemia [66,73,74300403 
FOXRED1  Missense, frameshift and nonsense leading to unclear protein impact Variable phenotype from severe neonatal lactic acidosis with early death to Leigh syndrome, hypotonia, lactic acidosis, hypertrophic cardiomyopathy and survival into adolescence and adulthood. Decreased CI activity [77,78,422613622 
TMEM70  Clinical cases are presented at the complex V defects section    
ND5 module 
MT-ND5  Predominantly missense but also deletion leading to frameshift with protein impact dependant on variant and mutant mtDNA load Variable onset MELAS, Leigh syndrome, LHON. May present as combination of the previous phenotypes with hypotonia, failure to thrive, cardiomyopathy, renal failure, myopathy. Phenotypes may vary according to age of onset and mutation load [86,87,423–427516005 
NDUFB3  Missense but also nonsense leading to unclear protein impact Variable phenotype from mild short stature, distinctive facial appearance surviving into childhood and decreased CI assembly to severe encephalopathy, myopathy, hypotonia, lactic acidosis, failure to thrive [88–90603839 
NDUFB8  Predominantly missense but also frameshift leading to decreased protein levels Leigh syndrome, fatal infantile lactic acidosis, respiratory failure, cardiac hypertrophy, hypotonia, failure to thrive [91602140 
NDUFB9  Missense leading to decreased protein levels Early onset progressive hypotonia, increased blood lactate [92601445 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
ND4 module 
MT-ND4  Missense with unclear protein impact. LHON, early onset Leigh syndrome, late-onset encephalopathy, may present lactic acidosis. Phenotype severity may correlate with mutation load. [418–421516003 
NDUFB10  Missense and nonsense leading to impaired protein import. Fatal lactic acidosis and cardiomyopathy. [72603843 
NDUFB11  Nonsense but also missense and deletion leading to reduced protein levels. X-linked gene. Encephalopathy, cardiomyopathy, MIDAS, LIMD, sideroblastic anaemia [66,73,74300403 
FOXRED1  Missense, frameshift and nonsense leading to unclear protein impact Variable phenotype from severe neonatal lactic acidosis with early death to Leigh syndrome, hypotonia, lactic acidosis, hypertrophic cardiomyopathy and survival into adolescence and adulthood. Decreased CI activity [77,78,422613622 
TMEM70  Clinical cases are presented at the complex V defects section    
ND5 module 
MT-ND5  Predominantly missense but also deletion leading to frameshift with protein impact dependant on variant and mutant mtDNA load Variable onset MELAS, Leigh syndrome, LHON. May present as combination of the previous phenotypes with hypotonia, failure to thrive, cardiomyopathy, renal failure, myopathy. Phenotypes may vary according to age of onset and mutation load [86,87,423–427516005 
NDUFB3  Missense but also nonsense leading to unclear protein impact Variable phenotype from mild short stature, distinctive facial appearance surviving into childhood and decreased CI assembly to severe encephalopathy, myopathy, hypotonia, lactic acidosis, failure to thrive [88–90603839 
NDUFB8  Predominantly missense but also frameshift leading to decreased protein levels Leigh syndrome, fatal infantile lactic acidosis, respiratory failure, cardiac hypertrophy, hypotonia, failure to thrive [91602140 
NDUFB9  Missense leading to decreased protein levels Early onset progressive hypotonia, increased blood lactate [92601445 

S., subunit; A.F., assembly factor; LHON, Leber's hereditary optic neuropathy; MIDAS, mitochondrial dysfunction-associated senescence; LIMD, lethal infantile mitochondrial disease; MELAS, mitochondrial encephalopathy, lactic Acidosis and stroke-like episodes.

The assembly factors TMEM70, FOXRED1 and ATP5SL are found associated with a near complete ND4 module [22,32,75]. While their function in assembly of this module is not clear yet, all three interact with subunits of the ND4 module and their loss, either in model systems or patient cell lines [22,76] leads to accumulation of membrane assembly intermediates. While no patients have been identified for ATP5SL, both TMEM70 and FOXRED1 are known mitochondrial disease genes (Table 3). FOXRED1 was identified through high-throughput sequencing of a complex I deficient cohort [77] and since then many patients have been characterised [77–79]. The phenotype is variable, with both severe (Leigh syndrome, cardiomyopathy) and mild cases having been identified, presumably dependent on the severity of the particular variant(s). Interestingly, FOXRED is an FAD-dependent oxidoreductase, and although it is not clear if FAD binding is important in its function, Rendon and co-workers [79] identified a patient with relatively mild symptoms (epilepsy and severe psychomotor retardation) that suffered a combined complexes I and II defect, suggesting FOXRED1 may be involved in the biogenesis of multiple flavoprotein containing complexes. TMEM70 is an assembly factor implicated in the assembly of both complexes I and V, with functional studies showing that its loss leads to accumulation of assembly intermediates for both complexes [75,80]. In line with this dual role, patients commonly have cardiac and brain involvement and defects in both OXPHOS complexes [81] or isolated complex I or V deficiencies, varying according to tissue type [81–84] (Tables 3 and 14).

The ND5 module contains the core subunit ND5 and accessory subunits NDUFB2, NDUFB3, NDUFB7, NDUFB8, NDUFB9 and NDUFAB1. NDUFAB1 is unique in that it is found in two pools, a soluble non-complex I associated pool where it associates with many different proteins containing the LYR motif [85] and a complex I associated pool [24]. In the case of the latter it is found twice, associating with the two LYR motif containing complex I subunits, NDUFB7 of this module and NDUFA6 of the N-module (discussed below) (Figure 2). Interestingly, loss of NDUFAB1 in the gene-edited HEK293T model system leads to cell death (whereas loss of the other accessory subunits leads to a presumed shift to glycolysis), and while there is a severe complex I assembly defect the essential role of NDUFAB1 appears to be related to its non-complex I associated pool [22]. Little is known about assembly of the ND5 module with the only intermediate observed containing all known subunits [32]. The recently identified assembly factor DMAC1 [22] is thought to be required for ND5 module assembly as it has been shown to interact with newly synthesised ND5 and other subunits of the module [22], however, its molecular function is not yet known. Interestingly the stability of both the ND2 and ND5 modules appears to rely on the presence of a properly built ND4 module but not vice versa [22]. Defects in ND5 lead to the typical late onset (LHON, MELAS) phenotypes observed in patients with mutations in mtDNA-encoded complex I subunits (Table 3) although there have been many reports of more severe infantile presentations [86,87]. Variants have been identified in NDUFB3, NDUFB8 and NDUFB9. Patients present with typical severe childhood phenotypes underpinned by complex I dysfunction (e.g. encephalopathy, myopathy, hypotonia), dependent on the variants impact on protein function [88–92]. In line with the requirement of these genes for complex I assembly in the HEK293T model system [22], in all patients, residual levels of the presumably semi-functional proteins have been detected [88–92].

Assembly of the N-module

The N-module, which is the site of NADH oxidation, is probably pre-assembled into two subcomplexes containing the NDUFV1 and NDUFV2 subunits and NDUFS1 and NDUFA2 subunits before incorporation with the Q–ND1 subcomplex (Figure 2) [32]. Defects have been identified in all four genes encoding these subunits (Table 4). In line with their central role in complex I enzyme function [24,25], patients present with severe disease, typically Leigh syndrome in infants, although some variants in NDUFA2 which is not thought to be directly involved in NADH catalysis, lead to less severe disease and survival into childhood [93–102]. The assembly factor NUBPL has a CXXC motif that binds iron–sulfur clusters and is thought to aid their delivery to NDUFS1 and NDUFV1. Depletion of NUBPL in model systems leads to turnover of some N-module subunits and accumulation of intermediates [103]. NUBPL was identified in the same high-throughput sequencing study as FOXRED1 [77] and since then a few other patients have been identified [72,77,104]. Patients have milder symptoms than those harbouring mutations in the two known NUBPL substrates, typically presenting with leukoencephalopathy and childhood ataxia followed by other variable but progressive symptoms suggesting that biogenesis of NDUFS1 and NDUFV1 remains partially functional.

Table 4.
Defects affecting biogenesis of the N-module
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
NDUFA2  Majority missense but also intronic and frameshift with unclear protein impact. Variable phenotype from severe Leigh syndrome, hypertrophic cardiomyopathy, severe lactic acidosis to cystic leukoencephalopathy and survival into childhood. Impaired CI assembly/activity. [93,94602137 
NDUFA6  Frameshift, nonsense and missense leading to unclear protein impact. Variable phenotype from optic atrophy, motor regression and survival into childhood to severe fatal lactic acidosis and brain abnormalities, hypotonia, seizures. Decreased CI assembly/activity. [106602138 
NDUFA12  Nonsense leading to complete absence of protein. Leigh syndrome, dystonia, hypotonia, normal hearing and vision. [107614530 
NDUFS1  Predominantly missense but also nonsense with unclear protein impact. Variable severe phenotypes including Leigh syndrome, bilateral optic atrophy, hyperlactatemia, mental retardation, macrocytic anaemia, cavitating leukoencephalopathy, dystonia, hypotonia mostly leading to early death. Decreased CI assembly/activity. [95–98157655 
NDUFS4  Variable. Nonsense, intronic, duplication and deletion causing frameshift. Unclear or undetected protein levels. Variable presentations with predominant Leigh syndrome phenotype but also brain atrophy, cardiac hypertrophy, hypotonia, may present lactic acidosis and decreased CI activity. [108–111602694 
NDUFS6  Intronic, missense and deletion leading to uncertain or decreased protein level. Severe presentation of fatal lactic acidosis, Leigh syndrome. [112–114603848 
NDUFV1  Missense, nonsense and intronic leading to unclear protein impact. Leigh syndrome and may present progressive muscular hypotonia, myoclonic epilepsy, elevated plasma and cerebrospinal fluid lactate. CI deficiency. [95161015 
NDUFV2  Intronic deletion and insertion causing protein frameshift leading to decreased protein levels. Hypertrophic cardiomyopathy and encephalopathy, Leigh syndrome, hypotonia. [100–102600532 
NDUFAF2  Predominantly nonsense but also deletion causing frameshift leading to undetectable protein levels. Severe progressive brain abnormalities distinct from Leigh syndrome, hypotonia, may present apnoea, normal or mildly elevated plasma lactate. [77,116,118,119609653 
NUBPL  Predominantly missense but also frameshift leading to decreased protein levels. Leukoencephalopathy, may present elevated serum and cerebrospinal fluid lactate. [77,104613621 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
NDUFA2  Majority missense but also intronic and frameshift with unclear protein impact. Variable phenotype from severe Leigh syndrome, hypertrophic cardiomyopathy, severe lactic acidosis to cystic leukoencephalopathy and survival into childhood. Impaired CI assembly/activity. [93,94602137 
NDUFA6  Frameshift, nonsense and missense leading to unclear protein impact. Variable phenotype from optic atrophy, motor regression and survival into childhood to severe fatal lactic acidosis and brain abnormalities, hypotonia, seizures. Decreased CI assembly/activity. [106602138 
NDUFA12  Nonsense leading to complete absence of protein. Leigh syndrome, dystonia, hypotonia, normal hearing and vision. [107614530 
NDUFS1  Predominantly missense but also nonsense with unclear protein impact. Variable severe phenotypes including Leigh syndrome, bilateral optic atrophy, hyperlactatemia, mental retardation, macrocytic anaemia, cavitating leukoencephalopathy, dystonia, hypotonia mostly leading to early death. Decreased CI assembly/activity. [95–98157655 
NDUFS4  Variable. Nonsense, intronic, duplication and deletion causing frameshift. Unclear or undetected protein levels. Variable presentations with predominant Leigh syndrome phenotype but also brain atrophy, cardiac hypertrophy, hypotonia, may present lactic acidosis and decreased CI activity. [108–111602694 
NDUFS6  Intronic, missense and deletion leading to uncertain or decreased protein level. Severe presentation of fatal lactic acidosis, Leigh syndrome. [112–114603848 
NDUFV1  Missense, nonsense and intronic leading to unclear protein impact. Leigh syndrome and may present progressive muscular hypotonia, myoclonic epilepsy, elevated plasma and cerebrospinal fluid lactate. CI deficiency. [95161015 
NDUFV2  Intronic deletion and insertion causing protein frameshift leading to decreased protein levels. Hypertrophic cardiomyopathy and encephalopathy, Leigh syndrome, hypotonia. [100–102600532 
NDUFAF2  Predominantly nonsense but also deletion causing frameshift leading to undetectable protein levels. Severe progressive brain abnormalities distinct from Leigh syndrome, hypotonia, may present apnoea, normal or mildly elevated plasma lactate. [77,116,118,119609653 
NUBPL  Predominantly missense but also frameshift leading to decreased protein levels. Leukoencephalopathy, may present elevated serum and cerebrospinal fluid lactate. [77,104613621 

S., subunit; A.F., assembly factor.

The subunits NDUFA6, NDUFA7, NDUFA11, NDUFA12, NDUFS4, NDUFS6, NDUFV3 and the other copy of NDUFAB1, associated with LYR motif containing NDUFA6, seems to be incorporated at later assembly stages of the complex I [32]. Some of these subunits appear to be able to be dynamically exchanged from the assembled complex, possibly to prevent accumulation of oxidatively damaged subunits [105]. Patients have been described for NDUFA6 [106], NDUFA12 [107], NDUFS4 [108–111], and NDUFS6 [112–114]. Patients with mutations in the latter three generally present with severe childhood Leigh syndrome (Table 4). Interestingly, loss of these proteins in the HEK293T model system leads to only mild defects in complex I assembly, turnover of only selected N-module subunits, and mild impacts on complex I function [22]. In line with this, mutations found in these patients generally lead to complete absence of the proteins (Table 4). In contrast, the known patients for NDUFA6, which is required for N-module assembly in model systems [22], present with variable symptoms ranging from severe infantile disease to survival into childhood, presumably due to differing effects on protein stability and function [106]. These examples highlight the delicate balance between the role of the protein in complex assembly and the severity of the variant and presentation of phenotypes in patients.

Finally, the N-module requires the assembly factor NDUFAF2 for its biogenesis (Figure 2). NDUFAF2 is found associated with a complex I intermediate that lacks the N-module, but contains a near complete Q–ND1–ND2–ND4–ND5 subcomplex [22,32,115]. HEK293T cell lines lacking some N-module subunits as well as patients with mutations in NDUFAF2 contain this partially built assembly as a stable intermediate [22,105]. NDUFAF2 is thought to have evolved through duplication of the NDUFA12 gene [116], and the structure of the intermediate isolated from a fungal NDUFS6 knockout model (lacking the N-module) shows NDUFAF2 occupying the site usually taken by NDUFA12 in the mature complex [117], giving rise to the suggestion that the assembly factor caps the near final Q–ND1–ND2–ND4–ND5 intermediate priming it for addition of the N-module. NDUFAF2 patients present with complex I deficiency and severe progressive childhood disease. Most patients present with no detectable NDUFAF2 protein [77,116,118,119], which given the loss of associated N-module in patients is reflected in the similar complex I defect upon loss of NDUFAF2, NDUFA12, NDUFS4 and NDUFS6 in gene-edited model systems [22], as well as the similarity of phenotypes observed in patients for these genes (Table 4).

Complex II

Mitochondrial complex II (or succinate dehydrogenase) is an important enzyme that participates in both central metabolic processes relevant to mitochondrial energy generation: the MRC and the tricarboxylic acid cycle (TCA). This enzyme is responsible for the oxidation of succinate to fumarate with the extracted electrons used to reduce Coenzyme Q [120]. Complex II is the smallest complex in the OXPHOS system and the only one where all subunits are of nuclear origin (SDHA, SDHB, SDHC, SDHD). While all except for SDHC have been linked to mitochondrial disease (Table 5), genes associated with complex II are more commonly known for their association with tumorigenesis, particularly heritable paragangliomas (discussed below). SDHA is the most frequently associated with mitochondrial disease, with multiple variants and presentations leading to complex II deficiency having been described [121–129]. In general, patients carrying mutations in the SDHA locus predicted or shown to lead to reduced protein levels present with classical mitochondrial disease phenotypes including Leigh syndrome (Table 5). There are few examples of SDHB and SDHD patients with phenotypes in line with classical mitochondrial dysfunction. For the former, there are only three known patients [130,131] all harbouring the same homozygous transversion leading to an aspartate to valine substitution, reduced levels of SDHB protein and assembled complex II. Two of these patients presented neurologic impairment, developmental regression and leukoencephalopathy [130,131], but the third, a sibling of one affected individual, was asymptomatic [130] suggesting incomplete penetrance. As such this variant is still classified as a VUS (see OMIM 185470.0020). For SDHD there are only two known patients, both variants leading to reduced levels of protein and impaired complex II assembly, complex II deficiency, however, both presenting with fatal hypertrophic cardiomyopathy or encephalomyopathy [131,132] suggesting this is a genuine mitochondrial disease gene.

Table 5.
Defects affecting biogenesis of complex II
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
SDHA  Predominantly missense but also intronic and nonsense leading to unclear or decreased protein levels. Majority presenting Leigh syndrome and may also present leukodystrophy, dystonia, ataxia, optic atrophy, lactic acidosis, cardiomyopathy. [121–129600857 
SDHB1  Single known homozygous missense variant with possible reduced penetrance but leading to decreased protein levels. Leukoencephalopathy, hypotonia or virtually asymptomatic, minor brain lesions. [127,130185470 
SDHD  Predominantly missense but also disruption of stop codon and protein extension leading unclear or decreased protein levels. Fatal hypertrophic cardiomyopathy or encephalomyopathy, developmental delay and lactic acidosis. Symptoms developed after viral infection. [131,132602690 
SDHAF1  Missense with unclear protein impact. Infantile leukodystrophy, spastic quadriparesis, lactate and succinate accumulation in the brain. Decreased CII activity/assembly. [143612848 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
SDHA  Predominantly missense but also intronic and nonsense leading to unclear or decreased protein levels. Majority presenting Leigh syndrome and may also present leukodystrophy, dystonia, ataxia, optic atrophy, lactic acidosis, cardiomyopathy. [121–129600857 
SDHB1  Single known homozygous missense variant with possible reduced penetrance but leading to decreased protein levels. Leukoencephalopathy, hypotonia or virtually asymptomatic, minor brain lesions. [127,130185470 
SDHD  Predominantly missense but also disruption of stop codon and protein extension leading unclear or decreased protein levels. Fatal hypertrophic cardiomyopathy or encephalomyopathy, developmental delay and lactic acidosis. Symptoms developed after viral infection. [131,132602690 
SDHAF1  Missense with unclear protein impact. Infantile leukodystrophy, spastic quadriparesis, lactate and succinate accumulation in the brain. Decreased CII activity/assembly. [143612848 
1

Possible VUS, see text for detail. S., subunit; A.F., assembly factor.

The assembly of this complex is not well characterised, with the known steps dominated by the insertion of two cofactors, thought to be catalysed by the assembly factors SDHAF1–4 (Figure 3). The first step is likely the flavination of the SDHA subunit by the assembly factor SDHAF2 [133,134]. Although the requirement for SDHAF2 in the flavination of SDHA has been disputed in some cancer cell lines [135,136], evidence exists to the contrary, including both the absence of SDHA flavination in tumours harbouring heterozygous SDHAF2 mutations [133] and a structure of the bacterial homologue of SDHAF2 bound to SDHA [137]. SDHA also interacts with the assembly factor SDHAF4, which is proposed to prevent the generation of reactive oxygen species (ROS) from the oxidation of succinate by unassembled SDHA but has also been suggested to facilitate the interaction of SDHA with SDHB [138]. The other notable step is the insertion of an Fe–S prosthetic group into SDHB, which is thought to be incorporated prior to the formation of an SDHA–SDHB intermediate and be promoted by SDHAF1 and SDHAF3 [139–142]. SDHAF1 is the only assembly factor known to be a mitochondrial disease gene, the two known homozygous missense variants lead to reduced complex II activity [143]. Patients present with infantile leukodystrophy and developmental regression, therefore having similarities to those harbouring the VUS in the associated subunit SDHB, discussed above (Table 5) [131,143]. Little is known about the assembly of the membrane anchored CII subunits, SDHC and SDHD. The heme b group situated at their interface is incorporated is required for their stability [144], but appears to play no catalytic role in the enzymatic function of the complex [145]. Interestingly the presence of both matrix exposed subunits, SDHA and SDHB is required for the stability of the SDHC/SDHD [134,141].

Schematic depicting the complex II assembly pathway showing known mitochondrial disease genes in red and assembly factors in italics.

Figure 3.
Schematic depicting the complex II assembly pathway showing known mitochondrial disease genes in red and assembly factors in italics.

Heme b binds SDHC and SDHD. SDHA is flavinated (FAD) in the presence of SDHAF2. SDHB receives iron–sulfur (Fe–S) clusters through the assistance of SDHAF1 and SDHAF3 and binds SDHA to finalise the assembly of the complex. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Figure 3.
Schematic depicting the complex II assembly pathway showing known mitochondrial disease genes in red and assembly factors in italics.

Heme b binds SDHC and SDHD. SDHA is flavinated (FAD) in the presence of SDHAF2. SDHB receives iron–sulfur (Fe–S) clusters through the assistance of SDHAF1 and SDHAF3 and binds SDHA to finalise the assembly of the complex. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Unlike for the other OXPHOS complexes, the genes encoding succinate dehydrogenase subunits and assembly factors are better known as being implicated in tumorigenesis over mitochondrial disease. Mutations in SDHA [146], SDHB [147], SDHC [148], SDHD [147] and SDHAF2 [133,149] are linked to heritable paragangliomas, gastrointestinal stromal tumours, renal carcinomas and pituitary adenomas [150]. This is likely due to metabolic and epigenetic alterations [151] as unlike the mutations leading to complex II deficiency discussed above, those leading to tumorigenesis are typically heterozygous with modest effects on complex function. The impact of these mutations on complex II assembly and function are discussed in excellent recent reviews from Dalla Pozza et al. [151] and Bezawork-Geleta et al. [152].

Complex III assembly

Complex III or the cytochrome bc1 complex sits at the centre of the MRC, using electrons from complexes I and II via Coenzyme Q to reduce cytochrome c, while also pumping protons into the intermembrane space from the matrix [153]. Complex III forms an obligate homodimer (CIII2), with each monomer being composed of one mtDNA-encoded subunit (CYB) and nine nuclear-encoded subunits, CYC1, UQCRC1, UQCRC2, UQCRFS1, UQCRH, UQCRB, UQCRQ, UQCR10, UQCR11 [154,155]. CYB and the nuclear-encoded cytochrome c1 (CYC1) and UQCRFS1 are the three subunits with electron transfer capabilities [153,156]. Much of the human complex III assembly pathway has been extrapolated from studies of the yeast complex, which presents a similar structure and subunit composition [157–159]. Assembly can be broken down into many discreet steps (Figure 4), which are summarised below.

Schematic depicting the complex III assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded subunits underlined and assembly factors in italics.

Figure 4.
Schematic depicting the complex III assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded subunits underlined and assembly factors in italics.

CYB is joined by UQCRB and UQCRQ. The subcomplex joins with a tetramer of UQCRC1 and UQCRC2, CYC1, UQCRH and UQCR10, resulting in the first dimeric intermediate, pre-CIII2. UQCRFS1 receives the iron–sulfur cluster from the assembly factor LYRM7 and is assembled into the pre-CIII2 structure with the aid of the translocase BCS1L. The incorporation of UQCR11 subunit occurs at later stages of complex III assembly. The assembly factor TTC19 clears proteolytic fragments arising from UQCRFS1 maturation. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Figure 4.
Schematic depicting the complex III assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded subunits underlined and assembly factors in italics.

CYB is joined by UQCRB and UQCRQ. The subcomplex joins with a tetramer of UQCRC1 and UQCRC2, CYC1, UQCRH and UQCR10, resulting in the first dimeric intermediate, pre-CIII2. UQCRFS1 receives the iron–sulfur cluster from the assembly factor LYRM7 and is assembled into the pre-CIII2 structure with the aid of the translocase BCS1L. The incorporation of UQCR11 subunit occurs at later stages of complex III assembly. The assembly factor TTC19 clears proteolytic fragments arising from UQCRFS1 maturation. IMS, intermembrane space; IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Translation of CYB and initial steps of complex III assembly

The assembly of complex III begins with the translation of mtDNA-encoded cytochrome B (CYB) on mitoribosomes and its co-translational insertion into the IMM (Figure 4) [158]. For efficient translation, the mitoribosome must be bound by a dimer of the assembly factors UQCC1 and UQCC2 [160,161]. This dimer interacts with the newly translated polypeptide at the mitoribosome exit tunnel and remains bound to CYB after its incorporation into the IMM [161], which is believed to be aided by the insertase OXA1L [162,163]. Like all other mtDNA protein coding genes, the phenotype and disease onset due to mutations in CYB depend on the variant and mutant load. Though many patients present with relatively mild symptoms (myopathy expressed as exercise intolerance; [164–169]), the classic LHON phenotype is also a common presentation [170–174] (Table 6). Interestingly, for many of these variants, complex III deficiency as well as high levels of mutant load appear to be restricted to muscle tissue. There are two known patients for the assembly factor UQCC2, both with mutations leading to near absence of the protein [160,175] (Table 6). In contrast with those with CYB mutations, the one UQCC2 patient harbouring complete loss of the protein due to defective mRNA splicing showed complex III deficiency in both the expected muscle tissue but also skin fibroblasts [160]. Another noteworthy point is that defects in UQCC2 also lead to a combined complex I defect [160,175], which is likely due to the reliance of complex I on the presence of complex III for its assembly [176].

Table 6.
Defects affecting translation of CYB and initial steps of complex III assembly
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CYB  Predominantly missense but also nonsense leading to reduced levels of protein. Effect is often restricted to muscle tissue. Variable phenotypes and onset depending on variant, spanning from mild exercise intolerance and lactic acidosis, may develop encephalopathy and seizures in adulthood, may include multisystemic involvement (growth retardation, deafness, cognitive dysfunction) to LHON. A single severe case of fatal infantile cardiomyopathy has also been described. [164–167,286,428516020 
UQCC2  Missense or intronic splicing defect, leading to undetectable or very low protein levels. Lactic acidosis, growth retardation, neurological impairment. [160,175614461 
UQCC3  Single patient with missense leading to undetectable protein levels. Lactic acidosis, hypoglycaemia, hypotonia, delayed development. [178616097 
UQCRB  Single patient with deletion leading to C-terminal extension, but with unclear protein impact. Episodic infantile lactic acidosis, hypoglycaemia, transient liver dysfunction, followed by normal psychomotor development in early childhood. Decreased CIII activity. [179191330 
UQCRQ  Missense leading to unclear protein impact. Severe non-lethal psychomotor retardation dystonia, athetosis, ataxia, mildly elevated blood lactate. Decreased CIII activity and variable CI deficiency. [180612080 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CYB  Predominantly missense but also nonsense leading to reduced levels of protein. Effect is often restricted to muscle tissue. Variable phenotypes and onset depending on variant, spanning from mild exercise intolerance and lactic acidosis, may develop encephalopathy and seizures in adulthood, may include multisystemic involvement (growth retardation, deafness, cognitive dysfunction) to LHON. A single severe case of fatal infantile cardiomyopathy has also been described. [164–167,286,428516020 
UQCC2  Missense or intronic splicing defect, leading to undetectable or very low protein levels. Lactic acidosis, growth retardation, neurological impairment. [160,175614461 
UQCC3  Single patient with missense leading to undetectable protein levels. Lactic acidosis, hypoglycaemia, hypotonia, delayed development. [178616097 
UQCRB  Single patient with deletion leading to C-terminal extension, but with unclear protein impact. Episodic infantile lactic acidosis, hypoglycaemia, transient liver dysfunction, followed by normal psychomotor development in early childhood. Decreased CIII activity. [179191330 
UQCRQ  Missense leading to unclear protein impact. Severe non-lethal psychomotor retardation dystonia, athetosis, ataxia, mildly elevated blood lactate. Decreased CIII activity and variable CI deficiency. [180612080 

S., subunit; A.F., assembly factor; LHON, Leber's hereditary optic neuropathy.

Following membrane integration of CYB, the first of two heme b molecules is incorporated at the bL site [158,177], triggering the binding of the assembly factor UQCC3 which based on work on its fungal homologue is thought to promote the incorporation of a second heme b molecule at the bH site [177]. This leads to dissociation of the UQCC1–UQCC2 dimer, which is free to initiate another round of CYB translation [161,177]. There is a single known patient for UQCC3, harbouring a missense variant leading to undetectable protein [178] (Table 6). The patient presented with isolated complex III deficiency (complex I was at the lower end of the control range in muscle), displayed lactic acidosis, hypotonia and delayed development, and fibroblasts exhibited defects in CYB and complex III assembly [178]. Fully hemylated CYB is then stabilised by the binding of the first nuclear-encoded subunits UQCRB and UQCRQ (Figure 4) [177]. Both of these subunits are mitochondrial disease genes, although there are only single variants known for each with limited cases (Table 6). For UQCRB, the single known case harbours a homozygous mutation leading to deletion of the last seven amino acids of the protein and inclusion of a new stretch of 14 amino acids derived from non-coding exons [179]. The protein impact of this mutation is not clear, although the patient, an infant who presented with episodic lactic acidosis, hypoglycaemia and liver dysfunction, had impaired complex III activity in liver, lymphocytes and fibroblasts. Interestingly, by the age of four the patient showed normal growth and psychomotor development. Without further cases or related functional studies, the mechanism underpinning the mild impact of this variant is unknown. For UQCRQ there is a single homozygous missense variant with multiple affected cases, however, these present with a more severe psychomotor retardation phenotype (Table 6). The impact of the mutation on protein function is unknown, although muscle from the patients has only a moderate complex III activity defect [180]. As for most other patients with defects affecting complex III, complex I activity was also moderately impaired in some, but not all of the reported cases.

Assembly of the pre-complex III dimer

Parallel to the coalescence of hemylated CYB with UQCRB and UQCRQ, matrix facing subunits UQCRC1 and UQCRC2 form a separate tetrameric module (i.e. a dimer of each), which is likely incorporated into the CYB-containing module simultaneously with CYC1 and UQCRH to yield the first dimeric intermediate of complex III, the pre-CIII2 (Figure 4) [181]. This step coincides with the dissociation of UQCC3, which may prevent the dimerisation of earlier intermediates [181]. Following dimerisation, the subunit UQCR10 is added to pre-CIII2 [158]. UQCRC2 and CYC1 are mitochondrial disease genes (Table 7). Although there exist only a few patients for both, there is a consistent and similar phenotype and clinical progression to what is seen in the UQCRB patient [179]. Patients present with recurring episodes of metabolic acidosis that largely resolves in childhood or early adulthood [182–184]. Analysis of fibroblasts from the patients have combined complexes I and III defects in assembly, though the complex I enzyme defect is not significant for the CYC1 patients [184]. The mechanism underpinning the similarity in phenotypes between these patients and those with mutations in UQCRB is not yet clear, though suggests a similar impact on complex III assembly and function.

Table 7.
Defects affecting assembly of the pre-complex III dimer
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
CYC1  Missense leading to near absence of protein Episodic severe infantile ketoacidosis, insulin-responsive hyperglycemia, hyperammonemia followed by normal development in childhood or early adulthood. Isolated CIII deficiency [184123980 
UQCRC2  Missense leading to uncertain protein impact Episodic infantile hypoglycaemia, lactic acidosis, ketonuria, variable liver failure followed by normal development in childhood. CIII deficiency and may present combined CI deficiency [182,183191329 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
CYC1  Missense leading to near absence of protein Episodic severe infantile ketoacidosis, insulin-responsive hyperglycemia, hyperammonemia followed by normal development in childhood or early adulthood. Isolated CIII deficiency [184123980 
UQCRC2  Missense leading to uncertain protein impact Episodic infantile hypoglycaemia, lactic acidosis, ketonuria, variable liver failure followed by normal development in childhood. CIII deficiency and may present combined CI deficiency [182,183191329 

S., subunit; A.F., assembly factor.

Biogenesis of UQCRFS1 and final steps of assembly

The Rieske iron–sulfur protein, UQCRFS1, is first imported into the matrix where it is bound by the chaperone LYRM7 which stabilises it and mediates the insertion of a 2Fe–2S cluster (Figure 4) [185,186]. Now-folded and containing an iron–sulfur cluster, UQCRFS1 is thought to be translocated into the IMM by the AAA+ ATPase BCS1L [187]. BCS1L is an inner membrane protein that forms a matrix-sided heptameric ring structure [188], allowing for the translocation of the hydrophilic folded C-terminus of UQCRFS1 across the inner membrane [187–189]. The incorporation of UQCRFS1 marks the important transition to a catalytically active complex III [158]. Both assembly factors and the UQCRFS1 gene itself are linked to mitochondrial disease, with multiple variants described, most of which lead to severely reduced protein levels of the corresponding protein (Table 8). Functional studies in fungal models have indicated that absence of the LYRM7 homologue leads to destabilisation and degradation of UQCRFS1 [185,190,191], although a small amount of UQCRFS1 is still assembled into complex III, which is not seen in cells lacking the homologue of BCS1L [187]. In line with this, patients for all three genes have some similarity in clinical presentation, particularly LYRM7 and UQCRFS1, both of which present severe infantile conditions consistent with complex III deficiency (Table 8) [192–195]. While BCS1L patients can also present with these symptoms, there is considerable symptomatic variability depending on the variant, including hepatic iron overload and the absence of a complex III defect entirely (Table 8) [196–202]. This had led to suggestions that BCS1L may have another yet to be characterised role in mitochondrial function [196].

Table 8.
Defects affecting biogenesis of UQCRFS1 and final steps of CIII assembly
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
UQCRFS1  Missense, intronic, nonsense leading to total absence or decreased protein levels. Severe hypertrophic cardiomyopathy, lactic acidosis, alopecia. Isolated CIII deficiency. Depending on severity of variant survival into childhood with slightly lightly impaired gross and fine motor skills. [195191327 
BCS1L  Predominantly missense but also nonsense leading to decreased protein levels or unclear protein impact. Variable phenotype including i) mild Björnstad syndrome (pili torti, neurosensory deafness), ii) severe Leigh syndrome, tubulopathy, hepatic dysfunction, metabolic acidosis, iii) GRACILE syndrome. May not present CIII deficiency. [196–201603647 
LYRM7  Multiple. Nonsense, intronic, deletion, duplication, missense leading to decreased protein levels. Lactic acidosis, early onset multifocal cavitating leukoencephalopathy, fatal neurologic decompensation. Isolated CIII deficiency. [192–194615831 
TTC19  Predominantly nonsense but also deletions likely leading to absence of protein. Variable onset and neurological phenotypes including psychiatric symptoms, progressive neurodegenerative disorder, developmental delay, ataxia, as well as Leigh syndrome. Isolated CIII deficiency. Onset tends to late childhood with some cases presenting in adulthood. [203,205–208613814 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
UQCRFS1  Missense, intronic, nonsense leading to total absence or decreased protein levels. Severe hypertrophic cardiomyopathy, lactic acidosis, alopecia. Isolated CIII deficiency. Depending on severity of variant survival into childhood with slightly lightly impaired gross and fine motor skills. [195191327 
BCS1L  Predominantly missense but also nonsense leading to decreased protein levels or unclear protein impact. Variable phenotype including i) mild Björnstad syndrome (pili torti, neurosensory deafness), ii) severe Leigh syndrome, tubulopathy, hepatic dysfunction, metabolic acidosis, iii) GRACILE syndrome. May not present CIII deficiency. [196–201603647 
LYRM7  Multiple. Nonsense, intronic, deletion, duplication, missense leading to decreased protein levels. Lactic acidosis, early onset multifocal cavitating leukoencephalopathy, fatal neurologic decompensation. Isolated CIII deficiency. [192–194615831 
TTC19  Predominantly nonsense but also deletions likely leading to absence of protein. Variable onset and neurological phenotypes including psychiatric symptoms, progressive neurodegenerative disorder, developmental delay, ataxia, as well as Leigh syndrome. Isolated CIII deficiency. Onset tends to late childhood with some cases presenting in adulthood. [203,205–208613814 

S., subunit; A.F., assembly factor; GRACILE, growth retardation, amino aciduria, cholestasis, iron overload, lactic acidosis and early death.

The final steps of complex III assembly include the incorporation of the last subunit, UQCR11 [158], followed by transient association of the assembly factor, TTC19. Although with unknown function at the time, TTC19 was identified in patients with progressive neurological phenotypes and isolated complex III deficiency [203]. Functional studies using a mouse knockout model showed that TTC19 is involved in proteolytic clearance of protein fragments derived from the N-terminus of UQCRFS1 that likely inhibit the enzyme [204]. Many other patients and variants have since been reported, generally leading to loss of detectable protein and similar late-onset phenotypes (Table 8) [192–201,205–208]).

Complex IV assembly

Complex IV or cytochrome c oxidase (COX) is the last proton-pumping enzyme in the ETC and displays many interesting features compared with other OXPHOS complexes. For instance, complex IV has the highest ratio of known assembly factors per subunit as well as tissue and developmental specific isoforms which together adds complexity for the diagnosis of mitochondrial diseases [6,209,210]. Mammalian complex IV consists of 14 subunits of which three are core subunits encoded on mtDNA, MT-CO1 (frequently referred to as COX1), MT-CO2 (COX2) and MT-CO3 (COX3) with the remainder encoded by nDNA, COX4 (with 2 possible isoforms encoded on separate genes, COX4I1 and COX4I2), COX5A, COX5B, COX6A (2 possible isoforms, COX6A1-2), COX6B (2 isoforms, COX6B1-2), COX6C, COX7A (3 isoforms, COX7A1-3), COX7B, COX7C, COX8A and COX8C [211–213]. It is generally thought that assembly of complex IV occurs in a modular fashion (Figure 5) [210,214,215], with the three mt-DNA encoded acting as platforms for assembly of nDNA encoded subunits into modules.

Schematic depicting the complex IV assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded proteins underlined and assembly factors in italics.

Figure 5.
Schematic depicting the complex IV assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded proteins underlined and assembly factors in italics.

Complex IV assembly is driven by the coalescence of three distinct modules named after the mtDNA-encoded subunits they contain, COX1, COX2 and COX3. COX1 receives heme and copper (Cu) cofactors and joins COX4 and COX5A to form the COX1 module. COX2 receives copper and is assembled with COX5B, COX6C, COX7B, COX7C and COX8A to form the COX2 module. COX1 and COX2 module are integrated prior to addition of the COX3 module, which is composed of COX6A, COX6B and COX7A. NDUFA4 is the last subunit to be added to form the mature complex IV. IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Figure 5.
Schematic depicting the complex IV assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded proteins underlined and assembly factors in italics.

Complex IV assembly is driven by the coalescence of three distinct modules named after the mtDNA-encoded subunits they contain, COX1, COX2 and COX3. COX1 receives heme and copper (Cu) cofactors and joins COX4 and COX5A to form the COX1 module. COX2 receives copper and is assembled with COX5B, COX6C, COX7B, COX7C and COX8A to form the COX2 module. COX1 and COX2 module are integrated prior to addition of the COX3 module, which is composed of COX6A, COX6B and COX7A. NDUFA4 is the last subunit to be added to form the mature complex IV. IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Assembly of the COX1 module

The assembly of complex IV starts with translation of COX1, which requires a specific translational activator (TACO1) [216,217], and COA3 and COX14, which appear to interact with COX1 [218–220] and suggested to prevent its degradation [218,220]. Another assembly factor, CMC1, has also been implicated in stabilising COX1 at this stage of assembly [221]. At this early step, mutations in TACO1 have been linked to a range of phenotypes, commonly presenting as Leigh syndrome, optic atrophy and muscle involvement with variable survival [216,222,223]. Importantly, TACO1 patients have overlapping phenotypes to those with disease caused by mutations in COX1 (Table 9). Patients with mutations in COX1 (MT-CO1) tend to survive into adulthood and present with phenotypes common to other mtDNA-encoded genes such as Leigh syndrome, lactic acidosis, hearing loss and myopathy, although other presentations including recurrent myoglobinuria and cerebellar ataxia have also been reported, and indeed overlap with patients with mutations in other mtDNA-encoded complex IV subunits [224–226]. Mutations in COA3 have also been identified as the cause of a relatively mild phenotype of peripheral neuropathy, with exercise intolerance and short stature without clear involvement of heart, liver or brain [227], while a homozygous mutation in its assembly partner, COX14, leads to a severe phenotype of respiratory distress, lactic acidosis, hypertrophic cardiomyopathy, brain hypertrophy, microphthalmia and ketonuria [218]. The mild phenotype of the COA3 patient compared with the severe phenotype of COX14 patients might not be explained by residual protein levels as both patients showed almost or totally absent COX14 protein [218,227] and reduced COA3 levels [227]. This might suggest that although these two assembly factors are involved at the same step in complex IV assembly their precise function might differ.

Table 9.
Defects affecting assembly of COX1 module
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CO1 (COX1 Missense, nonsense leading to unclear protein impact. Majority of cases present survival into adulthood and phenotypes of late-onset Leigh syndrome, recurrent myoglobinuria, lactic acidosis, cerebellar ataxia, optic atrophy, hearing loss, myopathy. [224–226516030 
COX4I1  Missense or deletion/insertion leading to unclear or decreased protein levels. Short stature, increased chromosomal breaks, resembling Fanconi anaemia or severe cases of developmental delay, short stature and seizures, resembling Leigh syndrome. [232,233123864 
COX4I2  Missense leading to unclear protein impact. Exocrine pancreatic insufficiency, dyserythropoietic anaemia, calvarial hyperostosis, failure to thrive. [236607976 
COX5A  Missense leading to decreased protein levels. Early-onset pulmonary hypertension, lactic acidosis, heart abnormalities, failure to thrive. [237603773 
COA3  Missense and duplication causing frameshift leading to decreased protein levels. Peripheral neuropathy, exercise intolerance, short stature. Survival to adulthood. [227614775 
COX10  Missense leading to unclear protein impact. Variable severe phenotypes mostly presenting metabolic acidosis and anaemia combined with Leigh syndrome or hypertrophic cardiomyopathy but also tubulopathy, ataxia, hypotonia and early death. [238,239602125 
COX14  Missense leading to undetectable protein levels. Severe lactic acidosis, microphthalmia, ketonuria, hypertrophic cardiomyopathy, respiratory distress, brain hypertrophy. [218614478 
COX15  Missense, intronic and nonsense leading to unclear protein impact. Variable severe phenotypes. Fatal infantile hypertrophic cardiomyopathy, lactic acidosis, seizures, hypotonia or Leigh syndrome, failure to thrive, psychomotor delay, hypotonia, elevated plasma lactate and pyruvate [240,241603646 
SURF1  Over 80 mutations from variable genetic nature. Missense, nonsense, insertions, deletions, intronic. Most common cause of Leigh syndrome associated with CIV deficiency but variable severe phenotypes including Charcot-Marie-Tooth disease, rapidly progressive encephalopathy, ataxia, hypotonia, lactic acidosis and early death. (Reviewed in [242]) 185620 
TACO1  Duplication causing frameshift or missense leading to unclear protein impact. Variable phenotype from mild mental retardation and survival to early adulthood to severe slowly progressive childhood-onset of Leigh syndrome, may present optic atrophy, dystonia, spastic tetraparesis, renal tubulopathy. [216,222,223612958 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CO1 (COX1 Missense, nonsense leading to unclear protein impact. Majority of cases present survival into adulthood and phenotypes of late-onset Leigh syndrome, recurrent myoglobinuria, lactic acidosis, cerebellar ataxia, optic atrophy, hearing loss, myopathy. [224–226516030 
COX4I1  Missense or deletion/insertion leading to unclear or decreased protein levels. Short stature, increased chromosomal breaks, resembling Fanconi anaemia or severe cases of developmental delay, short stature and seizures, resembling Leigh syndrome. [232,233123864 
COX4I2  Missense leading to unclear protein impact. Exocrine pancreatic insufficiency, dyserythropoietic anaemia, calvarial hyperostosis, failure to thrive. [236607976 
COX5A  Missense leading to decreased protein levels. Early-onset pulmonary hypertension, lactic acidosis, heart abnormalities, failure to thrive. [237603773 
COA3  Missense and duplication causing frameshift leading to decreased protein levels. Peripheral neuropathy, exercise intolerance, short stature. Survival to adulthood. [227614775 
COX10  Missense leading to unclear protein impact. Variable severe phenotypes mostly presenting metabolic acidosis and anaemia combined with Leigh syndrome or hypertrophic cardiomyopathy but also tubulopathy, ataxia, hypotonia and early death. [238,239602125 
COX14  Missense leading to undetectable protein levels. Severe lactic acidosis, microphthalmia, ketonuria, hypertrophic cardiomyopathy, respiratory distress, brain hypertrophy. [218614478 
COX15  Missense, intronic and nonsense leading to unclear protein impact. Variable severe phenotypes. Fatal infantile hypertrophic cardiomyopathy, lactic acidosis, seizures, hypotonia or Leigh syndrome, failure to thrive, psychomotor delay, hypotonia, elevated plasma lactate and pyruvate [240,241603646 
SURF1  Over 80 mutations from variable genetic nature. Missense, nonsense, insertions, deletions, intronic. Most common cause of Leigh syndrome associated with CIV deficiency but variable severe phenotypes including Charcot-Marie-Tooth disease, rapidly progressive encephalopathy, ataxia, hypotonia, lactic acidosis and early death. (Reviewed in [242]) 185620 
TACO1  Duplication causing frameshift or missense leading to unclear protein impact. Variable phenotype from mild mental retardation and survival to early adulthood to severe slowly progressive childhood-onset of Leigh syndrome, may present optic atrophy, dystonia, spastic tetraparesis, renal tubulopathy. [216,222,223612958 

S., subunit; A.F., assembly factor.

The insertion of heme A is a two-step process performed by COX10, which converts heme B to heme O, and COX15, which converts heme O to heme A [228]. The heme A group is thought to be delivered to COX1 by SURF1 based on its ability to stoichiometrically bind heme A [229,230]. This step is thought to occur after dissociation of CMC1, which coincides with the binding of nuclear-encoded subunits COX4 and COX5A [221] and the formation of the COX1 module (Figure 5). Patients have been identified harbouring mutations in both COX4 isoform encoding genes, with clinical phenotypes shown to differ depending on which isoform is affected. For example, variants in the ubiquitously expressed COX4I1 [231] cause short stature, increase in chromosomal breaks and a phenotype similar to Fanconi anaemia [232], as well as more severe presentations of developmental delay, short stature and seizures, resembling Leigh syndrome in two siblings [233]. The two severely affected siblings also share compound heterozygous missense variants in MDN1, which is a AAA ATPase involved in cytosolic ribosomal biogenesis [234,235]. Despite the lack of functional studies to confirm pathogenicity of the MDN1 variants, their involvement in the more severe phenotype cannot be discarded and might account for their combined OXPHOS deficiency, especially given that their unaffected sibling has inherited only one MDN1 variant [233]. Likewise, COX4I2 is highly expressed in lungs [231] and in the pancreas [236] with mutations in this gene leading to exocrine pancreatic insufficiency, dyserythropoietic anaemia, calvarial hyperostosis and failure to thrive, without a complex IV defect detected in fibroblasts [236] (Table 9). Taken together, the distinct expression patterns of COX4I1 and COX4I2 seem to correlate with their generalised or tissue-specific presentations. The third COX1 module subunit, COX5A, has also been linked to disease, with mutations in COX5A leading to early onset pulmonary hypertension, brain abnormalities and lactic acidosis [237] (Table 9). Mutations in the assembly factors COX10 and COX15 have been reported to cause variable severe phenotypes including lactic acidosis, Leigh syndrome, hypertrophic cardiomyopathy and hypotonia [238–241], while mutations in SURF1, one of the most common causes of Leigh syndrome (reviewed in [242], has also been associated with other severe phenotypes such as Charcot–Marie–Tooth disease, rapidly progressive encephalopathy, ataxia, hypotonia, lactic acidosis and early death (Table 9). Analysis of patient-derived fibroblasts harbouring null mutation in SURF1 revealed a rapidly degraded monomer and an accumulation of a complex IV subassembly, while a fully assembled complex IV was present in supercomplexes — higher order structures comprised of complexes I, III and IV [11]. Interestingly this phenomenon may be tissue specific, as analysis of different tissues from a patient harbouring truncating mutations in SURF1 revealed decreased levels of fully assembled complex IV in heart, brain and muscle with increased accumulation of complex IV subassemblies in heart and muscle, but absent from brain [243].

The copper biosynthesis and insertion in mammalian cells has mostly been extrapolated from yeast studies. In this pathway, the CuB group is moved by COX17 to COX11, which is a metallochaperone [244,245] containing a conserved copper-binding motif [246]. COX11 also requires another assembly factor, COX19, to maintain its redox state [247] after copper binding. Finally, MITRAC7 is a COX1-specific chaperone that prevents COX1 degradation prior to the fusion of the COX1 and COX2 modules (Figure 5) [248]. Another protein thought to be involved in assembly of the COX1 module is HIGD1A. Based mainly on studies primarily performed in yeast, HIGD1A is thought to bind and stabilise COX4 and COX5A subunits prior to incorporation to COX1 [215] and potentially stay bound to regulate complex IV activity [249]. However, while mammalian HIGD1A associates with COX4 and COX5A [214,215], knockout of HIGD1A in mammalian cells had only a very minor effect on the stability of COX4 and COX5A subunits, or complex IV assembly more broadly [214,250].

Assembly of the COX2 module

The assembly of the COX2 module starts with the translation and membrane insertion of the first transmembrane domain of COX2, which is stabilised by COX20 (Figure 5) [251]. The insertion of the second transmembrane domain also leads to translocation of the globular copper-binding domain which occurs with the assistance of assembly factor COX18 [251]. Patients harbouring mutations in the mtDNA-encoded COX2 (MT-CO2) have been reported to present with phenotypes ranging from mild exercise intolerance and recurrent myoglobinuria or late-onset cerebellar ataxia to severe fatal lactic acidosis, depending on variant and mutant load [252–255]. There are some overlap in phenotypes seen in cases caused by mutations in the assembly factor COX20 [256–259], particularly ataxia and hypotonia with survival into adulthood reported (Table 10).

Table 10.
Defects affecting assembly of COX2 module
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CO2 (COX2)  Missense, nonsense, deletion leading to unclear or decreased protein levels. Variable phenotypes from mild exercise intolerance, recurrent myoglobinuria, late-onset cerebellar ataxia and survival into adulthood to severe cases of lactic acidosis and early death. [252–255516040 
COA6  Mostly missense but also nonsense leading to decreased protein levels. Hypertrophic cardiomyopathy may present lactic acidosis. Decreased CI and CIV in heart but not effect in fibroblasts. [88,271,272614772 
COX7B  Intronic and deletion causing protein frameshift or nonsense leading to unclear protein impact. X-linked gene. Facial dysmorphism, linear skin lesions with survival into childhood, may present tetralogy of Fallot, ventricular hypertrophy. [276,277300885 
COX8A  Intronic leading to unclear protein impact. Leigh-like syndrome, developmental delay, pulmonary hypertension, epilepsy, elevated blood and cerebrospinal fluid lactate. Decreased CIV assembly. [278123870 
COX20  Predominantly missense but also intronic leading to decreased protein levels. Most presentations include ataxia, hypotonia but can also present mild elevation of blood lactate, sensory neuropathy and static encephalopathy with reported survival into adulthood. [256–259614698 
PET100  Nonsense or missense abolishing first methionine leading to unclear protein impact. Variable severe phenotypes. Fatal infantile lactic acidosis, brain abnormalities, severe coagulopathy or Leigh syndrome, elevated blood lactate, seizures, hypotonia. but Decreased CIV assembly/activity. [274,279614770 
PET117  Nonsense leading to unclear protein impact. Brain and motor development regression with survival into adulthood. Decreased CIV activity/assembly. [280614771 
SCO1  Predominantly missense or frameshift may lead to decreased protein levels. Liver failure, encephalopathy, hypotonia, metabolic acidosis, may present cardiac hypertrophy or respiratory distress. [266,267603644 
SCO2  Mostly missense but also nonsense and duplication leading to unclear or reduced protein levels. Fatal infantile hypertrophic cardiomyopathy, encephalopathy, elevated blood lactate, respiratory distress. [268–270604272 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CO2 (COX2)  Missense, nonsense, deletion leading to unclear or decreased protein levels. Variable phenotypes from mild exercise intolerance, recurrent myoglobinuria, late-onset cerebellar ataxia and survival into adulthood to severe cases of lactic acidosis and early death. [252–255516040 
COA6  Mostly missense but also nonsense leading to decreased protein levels. Hypertrophic cardiomyopathy may present lactic acidosis. Decreased CI and CIV in heart but not effect in fibroblasts. [88,271,272614772 
COX7B  Intronic and deletion causing protein frameshift or nonsense leading to unclear protein impact. X-linked gene. Facial dysmorphism, linear skin lesions with survival into childhood, may present tetralogy of Fallot, ventricular hypertrophy. [276,277300885 
COX8A  Intronic leading to unclear protein impact. Leigh-like syndrome, developmental delay, pulmonary hypertension, epilepsy, elevated blood and cerebrospinal fluid lactate. Decreased CIV assembly. [278123870 
COX20  Predominantly missense but also intronic leading to decreased protein levels. Most presentations include ataxia, hypotonia but can also present mild elevation of blood lactate, sensory neuropathy and static encephalopathy with reported survival into adulthood. [256–259614698 
PET100  Nonsense or missense abolishing first methionine leading to unclear protein impact. Variable severe phenotypes. Fatal infantile lactic acidosis, brain abnormalities, severe coagulopathy or Leigh syndrome, elevated blood lactate, seizures, hypotonia. but Decreased CIV assembly/activity. [274,279614770 
PET117  Nonsense leading to unclear protein impact. Brain and motor development regression with survival into adulthood. Decreased CIV activity/assembly. [280614771 
SCO1  Predominantly missense or frameshift may lead to decreased protein levels. Liver failure, encephalopathy, hypotonia, metabolic acidosis, may present cardiac hypertrophy or respiratory distress. [266,267603644 
SCO2  Mostly missense but also nonsense and duplication leading to unclear or reduced protein levels. Fatal infantile hypertrophic cardiomyopathy, encephalopathy, elevated blood lactate, respiratory distress. [268–270604272 

S., subunit; A.F., assembly factor.

The insertion of the copper centre (CuA) in COX2 requires five known assembly factors: COX17, SCO1, SCO2, COA6 and COX16 (Figure 5). COX17 donates copper to SCO1 [260], a metallochaperone that delivers the copper to COX2. Copper delivery to COX2 also requires SCO2, another metallochaperone that reduces the disulfide bonds in COX2 to allow the copper insertion [261]. COA6 and COX16 also interact with COX2, probably at the time of copper insertion, and promote the function of SCO1 and SCO2 [262–264]. COX16 has also been shown to act in the recruitment of the COX1 module to the COX2 module in the next step of assembly, in a COX2-dependent manner [264]. Another putative assembly factor, TMEM177, was shown to associate with the COX2/COX20/SCO1/SCO2 intermediate [265], and while TMEM177 is thought to regulate COX20 levels, loss of TMEM177 does not impair complex IV assembly in mammalian HEK293T cells [265]. Of the proteins involved in copper assembly, mutations have been identified in SCO1, SCO2 and COA6, all leading to severe phenotypes and commonly linked to fatal infantile cardiomyopathy, with shared clinical presentations of encephalopathy, liver failure, respiratory distress and metabolic acidosis [88,266–272] (Table 10).

The timing of the incorporation of the mammalian subunits COX5B, COX6C, COX7B, COX7C and COX8A to the COX2 module remain unclear as well as the precise functions of associated assembly factors PET100, PET117 and MR-1S [215,273–275]. Despite their unclear roles, functional studies suggest the interaction between PET117, MR-1S and complex IV subunits are mediated by PET100 [215]. At this step, mutations in COX7B and COX8A and the genes encoding assembly factors PET100 and PET117 have been linked to diseases with variable presentations [274,276–280] (Table 10). COX7B is an X-linked gene, and while many variants have been described, all known cases are in females who present with facial dysmorphism, linear skin defects, short stature with variable presentation of tetralogy of Fallot and ventricular hypertrophy [276,277]. For COX8A, a homozygous intronic mutation has been shown to cause a more severe phenotype of Leigh-like syndrome, developmental delay, pulmonary hypertension, epilepsy and elevated lactate in blood and cerebrospinal fluid [278]. For PET100 and PET117 it is useful to consider the fungal studies where much more is known about their function. The yeast homologue pPet100 was found in an assembly intermediate containing pCox7, pCox7a and pCox8 (human COX7A, COX6C and COX7C) and loss of pPet100 leads to its accumulation together with another subcomplex composed of Cox5a and Cox6 (human COX4I1 and COX5A), thus preventing the assembly of the mature complex IV [273]. Consistently, the patients reported to harbour PET100 mutations show undetectable [279] or residual mature complex IV [274], with the former displaying a more severe phenotype and neonatal death [279]. In the case of pPet117, yeast studies have shown that it stabilises Cox15 oligomers and might function in heme A synthesis and/or transfer to pCox1, and is also required for complex IV assembly [275]. In contrast with this, mammalian studies have shown that PET117 interacts with PET100, MR-1S and other subunits of the COX1 and COX2 modules [215], suggesting that mammalian PET117 could have a distinct function to its yeast counterpart. Despite unknown effect of PET117 mutation on protein levels, the patients reported with a homozygous nonsense mutation display decreased complex IV assembly and a milder phenotype [280] when compared with PET100 patients [274,279].

Assembly of the COX3 module

The COX3 module is the last module to be added to the now complete COX1 and COX2 modules in the nascent complex IV assembly (Figure 5). This module consists of the core subunit COX3 and the nuclear-encoded subunits COX6A, COX6B and COX7A. Even though complex IV displays the highest assembly factor to subunit ratio amongst other OXPHOS complexes, it was surprising that no assembly factors have been found to be required for building the COX3 module. Although COX3 is not directly involved in electron transport, it is thought to play a regulatory role in enzyme function [211,281]. HIGD2A was previously thought to act as an assembly factor for the complexes I, III, IV supercomplex [282] but recently shown to be required for the assembly of the COX3 module [214]. While HIGD2A appears to be needed for the early steps of COX3 biogenesis, it is unclear if it is involved in COX3 translation, membrane integration or its integration with the nascent complex IV assembly. Following coalescence of the COX1, COX2 and COX3 modules, NDUFA4, formerly thought to be a complex I subunit [212], is likely the last subunit to be incorporated to form the mature complex IV [210].

Similar to other mitochondrially encoded genes, mutations in MT-CO3 (COX3) cause a variety of phenotypes and onsets including Leigh-like syndrome, myopathy, lactic acidosis, MELAS, LHON and recurrent myoglobinuria with survival into adulthood reported [172,283–286] (Table 11). Interestingly, the phenotype of recurrent myoglobinuria is also found in patients harbouring mutations in COX1 (MT-CO1) [225] and COX2 (MT-CO2) [252]. In the case of COX6A1 and COX6A2, mutations have been reported to cause different clinical presentations. While a homozygous deletion in COX6A1 was shown to cause a neuromuscular disease called Charcot–Marie–Tooth disease [287], mutations in COX6A2 lead to a muscle-specific presentation of myopathy and cardiomyopathy [288]. As COX6A2 is exclusively expressed in heart and skeletal muscle [289], the mutations reported in COX6A2 were shown to only affect complex IV activity in differentiated muscle and not in undifferentiated myoblasts [288]. For the only hydrophilic extramembrane subunit of complex IV facing the IMS, COX6B1 [290], pathogenic variants have been shown to cause severe infantile encephalomyopathy, cardiomyopathy and lactic acidosis. The last subunit to be assembled into complex IV, NDUFA4, has been reported to cause Leigh syndrome, congenital lactic acidosis and variable presentation of dystonia due to a homozygous intronic variant. The intronic variant leads to undetectable NDUFA4 protein levels via western blot analysis and was shown to not impair the assembly of the other 13 complex IV subunits [291].

Table 11.
Defects affecting assembly of COX3 module
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CO3 (COX3)  Missense, nonsense, insertion, deletion leading to unclear or decreased protein levels depending on variant and mutation load. Variable phenotype, including Leigh-like syndrome, myopathy, lactic acidosis, MELAS, LHON, recurrent myoglobinuria with cases of survival into adulthood. [172,283–286516050 
COX6A1  Deletion leading to decreased protein levels. Recessive axonal type of Charcot-Marie-Tooth with survival into adulthood. [287602072 
COX6A2  Missense leading to decreased protein levels. Congenital myopathy, cardiomyopathy, isolated CIV deficiency in muscle and absent from undifferentiated myoblast. [288602009 
COX6B1  Missense leading to decreased protein levels. Severe infantile encephalomyopathy, cardiomyopathy, lactic acidosis. CIV deficiency detected in muscle and mildly in fibroblasts. [429,430124089 
NDUFA4  Intronic leading to undetectable protein levels. Leigh syndrome, congenital lactic acidosis, may present dystonia with survival into adulthood. [291603833 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-CO3 (COX3)  Missense, nonsense, insertion, deletion leading to unclear or decreased protein levels depending on variant and mutation load. Variable phenotype, including Leigh-like syndrome, myopathy, lactic acidosis, MELAS, LHON, recurrent myoglobinuria with cases of survival into adulthood. [172,283–286516050 
COX6A1  Deletion leading to decreased protein levels. Recessive axonal type of Charcot-Marie-Tooth with survival into adulthood. [287602072 
COX6A2  Missense leading to decreased protein levels. Congenital myopathy, cardiomyopathy, isolated CIV deficiency in muscle and absent from undifferentiated myoblast. [288602009 
COX6B1  Missense leading to decreased protein levels. Severe infantile encephalomyopathy, cardiomyopathy, lactic acidosis. CIV deficiency detected in muscle and mildly in fibroblasts. [429,430124089 
NDUFA4  Intronic leading to undetectable protein levels. Leigh syndrome, congenital lactic acidosis, may present dystonia with survival into adulthood. [291603833 

S., subunit; A.F., assembly factor; LHON, Leber's hereditary optic neuropathy; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes.

Although the exact mechanism in which COA7 is involved in complex IV assembly remains unclear, mutations in COA7 have been shown to cause leukoencephalopathy or spinocerebellar ataxia and axonal neuropathy with variable onset and survival into late adulthood is reported (Table 12). A patient reported with undetectable COA7 levels via western blot analysis also showed decreased levels of COX2 and COX3 subunits, and decreased assembly of complex IV [292], providing insights into the stage of assembly of which COA7 may be involved.

Table 12.
Defects affecting unknown steps of complex IV assembly or function
Gene (alias)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
COA7  Predominantly missense but also intronic and deletion causing protein frameshift and undetectable protein levels Variable phenotypes and onset with survival into late adulthood presenting leukoencephalopathy or spinocerebellar ataxia with axonal neuropathy [292,431615623 
Gene (alias)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
COA7  Predominantly missense but also intronic and deletion causing protein frameshift and undetectable protein levels Variable phenotypes and onset with survival into late adulthood presenting leukoencephalopathy or spinocerebellar ataxia with axonal neuropathy [292,431615623 

S., subunit; A.F., assembly factor.

Complex V assembly

Complex V, also known as FoF1-ATPase, is the last enzyme in the OXPHOS system, utilising the proton gradient generated by complexes I, III and IV to power ATP synthesis. Complex V is composed of a membrane-embedded Fo section connected by an external stalk to a matrix soluble F1 section containing the ATPase domains [293]. Like complexes I, III and IV, complex V is under dual genetic control, with two of the membrane subunits encoded on mtDNA (ATP6, MT-ATP6; ATP8, MT-ATP8) and the remaining 16 encoded on nDNA [294]. Much of what is known for complex V is extrapolated from studies in fungal models, though in recent years there is an increasing amount of literature documenting the assembly in mammals. The nomenclature for nuclear complex V genes has also undergone a recent overhaul [23], now all being prefixed ATP5 (e.g. ATP5F1A). Typically, the corresponding proteins are represented by single Greek and Latin letters (e.g. α or α-subunit for ATP synthase subunit alpha, which is the recommended name for the protein product of ATP5F1A) though there are some inconsistencies. As for other complexes, we have chosen to refer to the subunits and assembly factors by their gene name using the aforementioned formatting (bold typeface for subunit, regular typeface for assembly factor) though have included the commonly used protein symbol on the first instance.

Assembly of the F1 catalytic module and central stalk

The assembly of complex V starts with the oligomerisation of three ATP5F1A (α-subunit) and three ATP5F1B (β-subunit) subunits into an alternating hexamer, in a series of assembly steps mediated by the assembly factors ATPAF1 and ATPAF2 respectively (Figure 6) [295]. These proteins likely act as placeholders to prevent the formation of homomeric complexes [296,297] and their loss in fungal models leads to aggregation of the subunits into large insoluble complexes [298]. In general, there are few reported cases for patients harbouring mutations affecting assembly or function of this module (Table 13). For ATP5F1A there are two known variants [299,300] with cases for both presenting with severe encephalopathy or microcephaly, followed by early infantile death. Tissue and fibroblast material from affected patients had reduced but not absent levels of ATP5F1A protein as well as lower levels of other subunits, suggesting stability of the F1 module is compromised [299]. For ATPAF2, there is a single known example [301]. In line with the requirement for this protein in assembly of the F1 module, the patient presented with similar symptoms to those with mutations in ATP5F1A, degenerative encephalopathy, severe developmental delay and death in early childhood (Table 13). The next step in the assembly pathway is the incorporation of the subunits belonging to the central stalk, ATP5F1C (γ-subunit), ATP5F1D (δ-subunit) and ATP5F1E (ε-subunit). Functional studies have shown that loss of any of these subunits leads to similar defects in complex V assembly, lower levels of mature complex and turnover of subunits for the F1 module [302]. ATP5F1C is the key structural molecule connecting the F1 catalytic module to the F0 module and through structural similarities of its coiled–coiled tail with the C-terminal regions of ATPAF1 and ATPAF2, likely displaces the assembly factors during module biogenesis [303,304]. There are no known patients for ATP5F1C, although a few cases have been reported for ATP5F1D and ATP5F1E [305,306]. Although patients present with similar, relatively mild phenotypes in line with expectations based on functional studies (Table 13), the molecular underpinnings are different. In the case of ATP5F1D, the patient had normal levels of the mutant protein, whereas other subunits of the F1 module were destabilised [305]. In the case of ATP5F1E, the patient had reduced levels of protein, though retained a fully assembled complex (including the mutant protein) albeit at lower levels than controls [306]. Both patients had similar net effects on total levels of assembled Complex V and function (Table 13).

Schematic depicting the complex V (F1F0-ATPase) assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded proteins underlined and assembly factors in italics.

Figure 6.
Schematic depicting the complex V (F1F0-ATPase) assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded proteins underlined and assembly factors in italics.

To aid correlation with disease genes, subunits have been labelled according to their gene name. The commonly used protein names are in parenthesis as follows: three copies of the ATP5F1A (α-subunit) and ATP5F1B (β-subunit) are assembled with the aid of the chaperones ATPAF1 and ATPAF2 with later binding of the subunits ATP5F1C1 (γ-subunit), ATP5F1D (δ-subunit) and ATP5F1E (ε-subunit). The membrane ring composed of ATP5MC1 (c-subunits; also encoded by ATP5MC2 and ATP5MC2) subunits is assembled and joins the pre-complex V prior to the addition of the subunits ATP5PB (b-subunit), ATP5PD (d-subunit), ATP5PF (F6-subunit) and ATP5PO (OSCP). The assembly pathway is followed by integration of ATP5ME (e-subunit), ATP5MF (f-subunit) and ATP5MG (g-subunit) and then by the mtDNA-encoded ATP6 (subunit 6) and ATP8 (subunit 8). The last subunits ATP5MPL (MP68) and ATP5MD (DAPIT) are added to complete the assembly of complex V. IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Figure 6.
Schematic depicting the complex V (F1F0-ATPase) assembly pathway showing known mitochondrial disease genes in red, mtDNA-encoded proteins underlined and assembly factors in italics.

To aid correlation with disease genes, subunits have been labelled according to their gene name. The commonly used protein names are in parenthesis as follows: three copies of the ATP5F1A (α-subunit) and ATP5F1B (β-subunit) are assembled with the aid of the chaperones ATPAF1 and ATPAF2 with later binding of the subunits ATP5F1C1 (γ-subunit), ATP5F1D (δ-subunit) and ATP5F1E (ε-subunit). The membrane ring composed of ATP5MC1 (c-subunits; also encoded by ATP5MC2 and ATP5MC2) subunits is assembled and joins the pre-complex V prior to the addition of the subunits ATP5PB (b-subunit), ATP5PD (d-subunit), ATP5PF (F6-subunit) and ATP5PO (OSCP). The assembly pathway is followed by integration of ATP5ME (e-subunit), ATP5MF (f-subunit) and ATP5MG (g-subunit) and then by the mtDNA-encoded ATP6 (subunit 6) and ATP8 (subunit 8). The last subunits ATP5MPL (MP68) and ATP5MD (DAPIT) are added to complete the assembly of complex V. IMM, inner mitochondrial membrane; Matrix, mitochondrial matrix.

Table 13.
Defects in the F1 catalytic module and central stalk
Gene (protein1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
ATP5F1A (α-subunit)  Missense leading to unclear protein impact. Fatal neonatal encephalopathy, lung hypoplasia or hypertension, may present seizures, heart failure. Decreased CV assembly/activity. [299,300164360 
ATPAF2 (Atp12)  Single known missense leading to unclear protein impact. Degenerative encephalopathy, severe developmental delay, death in early childhood. Decreased CV assembly/activity. [301608918 
ATP5F1D (δ-subunit)  Single known missense leading to normal protein levels.  Hyperammonemia, lactic acidosis or ketoacidosis, may present cardiomyopathy, delayed speech with survival into childhood. Decreased CV assembly/activity. [305603150 
ATP5F1E (ε-subunit)  Single known missense leading to reduced protein levels. Neonatal onset lactic acidosis, mild mental retardation, severe peripheral neuropathy with survival into adulthood. Reduced levels of fully assembled CV. [306606153 
Gene (protein1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
ATP5F1A (α-subunit)  Missense leading to unclear protein impact. Fatal neonatal encephalopathy, lung hypoplasia or hypertension, may present seizures, heart failure. Decreased CV assembly/activity. [299,300164360 
ATPAF2 (Atp12)  Single known missense leading to unclear protein impact. Degenerative encephalopathy, severe developmental delay, death in early childhood. Decreased CV assembly/activity. [301608918 
ATP5F1D (δ-subunit)  Single known missense leading to normal protein levels.  Hyperammonemia, lactic acidosis or ketoacidosis, may present cardiomyopathy, delayed speech with survival into childhood. Decreased CV assembly/activity. [305603150 
ATP5F1E (ε-subunit)  Single known missense leading to reduced protein levels. Neonatal onset lactic acidosis, mild mental retardation, severe peripheral neuropathy with survival into adulthood. Reduced levels of fully assembled CV. [306606153 
1

Commonly used protein name; S., subunit; A.F., assembly factor.

Assembly of the c-ring

The membrane-embedded c-ring and the central stalk are components of the rotor part of the Complex V and therefore essential for ATP synthesis. In humans, the c-ring consists of eight c-subunits encoded by ATP5MC1, ATP5MC2 and ATP5MC3 (Figure 6). Interestingly, all three genes encode the same mature protein, the proteins only differing in the sequence of their cleaved mitochondrial targeting signals [307,308]. There are no reported cases of mitochondrial disease linked to mutations in these genes, however, many cases and >5 different variants have been linked to defects in the assembly factor TMEM70 (Table 14) which has been linked with its biogenesis. TMEM70 has been suggested to be required for assembly of the c-subunits into the c-ring [80]. Although this has been argued in the literature [75], it is clear from these studies that TMEM70 is required for the joining of the c-ring to the F1 module, and its absence leads to a severe assembly defect. As a disease gene, TMEM70 is further complicated by variable presentations, depending on the variant patients either present with isolated or combined Complex V or Complex I defects, as well as severe cardiac and neuronal phenotypes (Table 14) [81–84]. Although the precise involvement in Complex I assembly is even less clear, recent studies in gene-edited cells completely lacking TMEM70 protein [75] are consistent with suggesting it has a dual role in assembly of both complexes.

Table 14.
Defects affecting the c-ring
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
TMEM70  Multiple. Intronic, insertion, deletion, duplication, nonsense, missense leading to unclear or absent protein levels. Variable phenotype including hypertrophic cardiomyopathy, lactic acidosis, hyperammonemia, persistent pulmonary hypertension, encephalocardiomyopathy, neonatal hypotonia or hypertonia, facial dysmosphism, bilateral cataracts, leukoencephalopathy. Presentations vary from normal to defective isolated (CI or CIII) or combined (CI + CIII) OXPHOS activities in muscle or fibroblasts. [81,82,84,432612418 
GeneS.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
TMEM70  Multiple. Intronic, insertion, deletion, duplication, nonsense, missense leading to unclear or absent protein levels. Variable phenotype including hypertrophic cardiomyopathy, lactic acidosis, hyperammonemia, persistent pulmonary hypertension, encephalocardiomyopathy, neonatal hypotonia or hypertonia, facial dysmosphism, bilateral cataracts, leukoencephalopathy. Presentations vary from normal to defective isolated (CI or CIII) or combined (CI + CIII) OXPHOS activities in muscle or fibroblasts. [81,82,84,432612418 

S., subunit; A.F., assembly factor.

Assembly of the peripheral stalk and F0 module

The peripheral stalk assembles with the F1 subcomplex in a two-step manner. The first four subunits to be incorporated are ATP5PB (b-subunit), ATP5PD (d-subunit), ATP5PF (F6-subunit), ATP5PO (OSCP) followed by the membrane-associated subunits ATP5ME (e-subunit), ATP5MF (f-subunit) and ATP5MG (g-subunit) [294,309,310] (Figure 6). In the absence of the c-ring, an intermediate complex V is assembled containing the F1 catalytic module, the peripheral stalk and the membrane subunits ATP5ME, ATP5MF and ATP5MG [311].

Once the previously formed subcomplex containing the F1 module and peripheral stalk join with the c-ring, this provides the scaffold necessary for the incorporation of the two mitochondrially-encoded subunits ATP6 (subunit 6) and ATP8 (subunit 8) (Figure 6) [309]. Mutations in both lead to disease (Table 15). More than 200 ATP6 cases have been reported with 19 different underlying mutations and a large variability in mutant mtDNA load (reviewed in [312]). Phenotypes are highly variable and both severe infantile Leigh syndrome as well adult onset disease has been reported (Table 15), and as such there is no clear assembly phenotype underpinning mutations in this gene. There are comparatively few reported patients for ATP8, however, like for ATP8 these cases report with a different phenotype likely underpinned by differences in the variant and mutant load. Phenotypes include cardiomyopathy and neuropathy [313] as well as seizures and neuropsychologic decline [314] as well as defects in complex V activity (Table 15). In the case of the former, the fibroblasts from the patient accumulated subcomplexes of unassembled complex V, including a free catalytically active F1 domain, suggesting the protein may be structurally important [313]. Interestingly, ATP6 and ATP8 are transcribed as a polycistronic mRNA and there are a few cases known to likely affect both proteins [315,316]. Patients present with severe early onset cardiomyopathy or neurological symptoms, although there have has been one case of a kindred with adult onset cerebellar ataxia and peripheral neuropathy (Table 15). Aside from defects in complex V function, the molecular impacts in these cases are unknown.

Table 15.
Defects in the Fo module
Gene (protein1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-ATP6 (subunit 6)  Several missense variants with protein levels dependant on variant and mutant mtDNA load. Most commonly presented as Leigh syndrome, NARP, spinocerebellar ataxia but also Charcot-Marie-Tooth, hypertrophic cardiomyopathy, lactic acidosis. Higher heteroplasmic levels correlate with earlier-onset phenotypes. Often but not always show ATP synthesis rate. Reviewed in [312516060 
MT-ATP8 (subunit 8)  Missense or nonsense leading to unclear protein impact. Hypertrophic cardiomyopathy and neuropathy with survival into adolescence. Decreased CV assembly/activity. Alternately has presented with reversible cognitive dysfunctions with seizures and brain pseudoatrophy. [313–316,433516070 
ATP5MD (DAPIT)  Single known intronic variant leading to undetectable protein levels. Leigh syndrome, developmental regression after febrile illness. Survival into childhood. Decreased proportion of CV dimer. [317615204 
Gene (protein1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-ATP6 (subunit 6)  Several missense variants with protein levels dependant on variant and mutant mtDNA load. Most commonly presented as Leigh syndrome, NARP, spinocerebellar ataxia but also Charcot-Marie-Tooth, hypertrophic cardiomyopathy, lactic acidosis. Higher heteroplasmic levels correlate with earlier-onset phenotypes. Often but not always show ATP synthesis rate. Reviewed in [312516060 
MT-ATP8 (subunit 8)  Missense or nonsense leading to unclear protein impact. Hypertrophic cardiomyopathy and neuropathy with survival into adolescence. Decreased CV assembly/activity. Alternately has presented with reversible cognitive dysfunctions with seizures and brain pseudoatrophy. [313–316,433516070 
ATP5MD (DAPIT)  Single known intronic variant leading to undetectable protein levels. Leigh syndrome, developmental regression after febrile illness. Survival into childhood. Decreased proportion of CV dimer. [317615204 
1

Commonly used protein name; S., subunit; A.F., assembly factor; NARP, neuropathy, ataxia and retinitis pigmentosa.

Finally, the subunit ATP5MPL (MP68), which is required for ATP6 and ATP8 stability is incorporated, followed by ATP5MD (DAPIT) [309]. A single variant of the latter is known to cause Leigh syndrome with childhood onset [317]. Fibroblasts from patients had no detectable protein and reduced ATP synthesis. Fully assembled complex V has been shown to assemble into dimers [318,319] and, more recently, in tetramers [293]. Interestingly, fibroblasts from ATP5MD patients had markedly reduced dimerisation [317], suggesting the protein is involved in this process.

Mitochondrial translation and the mitoribosome

Mitochondrial protein synthesis is a complex process that has its own components such as a mitochondria-specific genetic code, an exclusive set of tRNA and tRNA synthetases and its own ribosome known as the mitoribosome [320]. The composition of ribosomes has considerably changed over the course of evolution, specially the RNA to protein ratio which has reversed from 1 : 2 protein : RNA in bacteria and cytosolic ribosomes to 2 : 1 protein : RNA in the mammalian mitoribosome. This explains why almost half of the mitoribosome proteins are mitochondrion-specific and absent from the bacterial ancestor [4]. Like other ribosomes, the mammalian mitoribosome is composed of two subunits, the small (mtSSU) and the large (mtLSU). The mRNA engages with the mtSSU while the mtLSU can anchor itself to the IMM and catalyse translation [321]. The mitoribosome has a total sedimentation coefficient of 55S, composed of the 28S for the isolated mtSSU and the 39S for the mtLSU [322]. The mtSSU consists of the 12S rRNA encoded by the mtDNA, and 30 MRP mitoribosomal proteins encoded by the nDNA [323]. The mtLSU consists of a tRNA valine (tRNAval) [2] and a 16S rRNA, both encoded by the mtDNA, and 52 nuclear-encoded mitoribosomal proteins. The peptidyl transferase centre (PTC) catalyses the formation of the peptide bonds of nascent polypeptides [324] and is located in the internal part of the mitoribosome formed exclusively by the 12S and 16S rRNAs [325,326]. The polypeptide exit tunnel (PET) starts at the PTC and ends at the polypeptide exit site (PES) where the translated polypeptide leaves the mitoribosome [327]. The polypeptide tunnel is an important region that is targeted in bacteria as a binding site for antibiotics [321]. The PET is surrounded by ring composed of bacterial conserved proteins bL17m (encoded by the gene MRPL17), uL22m (MRPL22), uL23m (MRPL23), uL24m (MRPL24), uL29m (MRPL47) and the mitochondrial specific mL45 (MRPL45) which promotes the tethering of the mitoribosome to the IMM [321]. The translocation of mitochondrial proteins is mediated by OXA1, which binds newly synthesised polypeptides [162,163] and the mitoribosome [328,329]. The nomenclature for mitoribosomal proteins has recently been overhauled for consistency between cytosolic and mitochondrial ribosomes found across different organisms [330]. To assist readers linking phenotype to function we have chosen to refer to the protein using their HUGO assigned gene name and have listed the revised protein name in parenthesis at first mention.

Assembly of the mtSSU

Even though the mitoribosome has unarguable importance, its assembly pathway has not been studied in as much detail as the assembly of OXPHOS complexes [331]. Recent research has begun to solve the timing of incorporation of mitoribosome protein using pulse and pulse-chase stable isotope labelling with amino acids in cell culture (SILAC) proteomics approaches [331,332], suggesting that the mtSSU displays an early and a late class of incorporation of mitoribosomal proteins, while the mtLSU has an additional intermediate class (Figure 7).

Figure 7.

Schematic model depicting the mitoribosome assembly pathway based on data from [331].

To aid correlation with disease genes, subunits have been labelled according to their gene name (outlined in 332). The incorporation stage of MRPS6 (bS6M), MRPS18C (bS18M), MRPS37 (mS37), MRPS38 (mS38), MRPL36 (bL36m) and MRPL52 (ml52) remains unclear. Known mitochondrial disease genes in red, mtDNA-encoded rRNAs and tRNA underlined, assembly factors in italics. The mtSSU proteins are incorporated at early or late stage in the assembly pathway while the mtLSU proteins are incorporated at early, intermediate or late stages. Mitoribosome small subunit, mtSSU; mitoribosome large subunit, mtLSU.

Figure 7.

Schematic model depicting the mitoribosome assembly pathway based on data from [331].

To aid correlation with disease genes, subunits have been labelled according to their gene name (outlined in 332). The incorporation stage of MRPS6 (bS6M), MRPS18C (bS18M), MRPS37 (mS37), MRPS38 (mS38), MRPL36 (bL36m) and MRPL52 (ml52) remains unclear. Known mitochondrial disease genes in red, mtDNA-encoded rRNAs and tRNA underlined, assembly factors in italics. The mtSSU proteins are incorporated at early or late stage in the assembly pathway while the mtLSU proteins are incorporated at early, intermediate or late stages. Mitoribosome small subunit, mtSSU; mitoribosome large subunit, mtLSU.

The mitoribosome biogenesis has been suggested to start at or near the mitochondrial nucleoid and present subclusters of assembly proteins for each incorporation class or ‘module’ in the terminology used for the OXPHOS complexes [331]. The 12S rRNA is stabilised by the RNA chaperone ERAL1 [333,334] and metal-dependent endoribonuclease YBEY [335], and methylated by two proteins, the adenine dimethyltransferase TFB1M [336] and the cytosine methyltransferase NSUN4 [337]. The early mitochondrial proteins participating in mtSSU assembly are mainly localised at the top and bottom of the 12S rRNA core and seem to be grouped into three different clusters [331]. One cluster contains MRPS5 (uS5m), MRPS16 (bS16m), MRPS22 (mS22), MRPS27 (mS27), MRPS34 (mS34) and MRPS18B (mS40). A second assembly cluster contains MRPS7 (uS7m), MRPS9 (uS9m), MRPS29 (mS29), MRPS31 (mS31), MRPS35 (mS35) and MRPS39 (mS39). A third cluster of SSU early binding proteins consists of MRPS2 (uS2m), MRPS23 (mS23) and MRPS28 (bS1m). The remaining proteins involved in the early assembly of the mtSSU are MRPS11 (uS11m), MRPS12 (uS12m) and MRPS17 (uS17m) appear to incorporate in an independent manner, with recent studies suggesting that YBEY may incorporate MRPS11 (uS11m) to the nascent mtSSU [335]. The early assembly of mitoribosomal proteins seem to be facilitated by the GTPase NOA1/C4orf14, which was shown to interact with several early assembled mtSSU proteins and the mitochondrial nucleoid [338]. In addition to that, METTL15 was shown to methylate the 12S rRNA at position C839 and be required for the biogenesis of mtSSU with its absence affecting both early assembled proteins MRPS12 (uS12m), MRPS17 (uS17m) and late assembled protein MRPS15 (uS15m), as well as MRPS38 (mS38) [339] which has not yet been assigned an incorporation class [331].

For the later incorporated mtSSU proteins, two assembly clusters seem to be present. One cluster consists of MRPS10 (uS10m), MRPS14 (uS14m), MRPS24 (uS3m) and MRPS33 (mS33) located at the top of the 12S rRNA, and another assembly cluster comprised of MRPS15 (uS15m), MRPS25 (mS25) and MRPS26 (mS26) locating at the bottom of the 12S rRNA with the remaining proteins incorporating in an independent manner [331].

To date, mutations in eleven components and in one assembly factor of the mtSSU have been identified, causing a range of phenotypes, onset and variable survival (Table 16). Interestingly, majority of the disease genes participate in the early stages of the mtSSU assembly (Figure 7) and patients tend to have a combined OXPHOS deficiency depending on tissue type and mutation [340–345]. Several cases of mutations in MT-RNR1 (12S rRNA) have been reported to cause hearing loss induced by aminoglycoside exposure (Table 16) with one family also presenting cardiomyopathy [346]. While mutations in ERAL1, the RNA chaperone shown to stabilise 12S rRNA [333,334], have also been shown to cause sensorineural deafness, it has not been linked to aminoglycoside exposure as have mutations in MT-RNR1 [347].

Table 16.
Defects in the assembly of the mtSSU
Gene (RNA/protein name1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-RNR1 (12S rRNA)  Transition, transversion and deletion/insertion. Aminoglycoside-induced hearing loss, incomplete penetrance reported, may include cardiomyopathy. [346,434,435], and reviewed in [436561000 
ERAL1  Missense leading to decreased protein levels. Perrault syndrome expressed as sensorineural hearing loss and ovarian dysgenesis with survival into late adulthood. [347607435 
MRPS2 (uS2m)  Missense leading to decreased protein levels. Sensorineural hearing impairment, mild developmental delay and hypoglycaemia. Variable OXPHOS deficiency depending on the tissue type. Survival into childhood [344611971 
MRPS7 (uS7m)  Missense leading to decreased protein levels. Congenital sensorineural hearing impairment, lactic acidosis, hypoglycaemia. Variable OXPHOS deficiency depending on the tissue type. Survival into childhood. [345,348611974 
MRPS14 (uS14m)  Missense leading to increased protein levels. Lactic acidosis, hypertrophic cardiomyopathy, hypotonia with survival into childhood. Unaffected mitoribosome assembly but decreased assembly of CI, CIII, CIV and CV suggesting incorporation of the mutant protein causes impaired mitochondrial translation. [340611978 
MRPS16 (bS16m)  Nonsense leading to unclear protein impact but decreased mitochondrial translation. Fatal lactic acidosis, tubulopathy, hypotonia. Variable OXPHOS deficiency depending on the tissue type. [342609204 
MRPS22 (mS22)  Missense or duplication causing frameshift leading to unclear or decreased protein levels. Variable phenotypes including fatal lactic acidosis, hypertrophic cardiomyopathy, tubulopathy, dysmorphic features, hypotonia, Leigh-like lesions, hypergonadotropic hypogonadism. Variable OXPHOS deficiency depending on the tissue type. [349–351605810 
MRPS23 (mS23)  Missense leading to unclear protein impact. Hepatic disease and hypoglycaemia. Combined CI and CIV deficiencies in fibroblasts. [66611985 
MRPS25 (mS25)  Missense leading to decreased protein levels. Multiple presentations including encephalopathy, short stature, muscle fatigue, dystonia, mild elevation of plasma lactate with survival into adulthood. [343611987 
MRPS28 (bS1m)  Missense and deletion causing frameshift and early termination leading to decreased protein levels. IUGR, cerebellar atrophy, microcephaly, hyperlactatemia, developmental delay, sensorineural deafness, failure to thrive with survival into adulthood. CIV deficiency in muscle, fibroblasts and liver. [352,353611990 
MRPS34 (mS34)  Intronic, nonsense, missense leading to decreased protein levels. Leigh or Leigh-like syndrome, mild or hyperlactatemia, may present microcephaly, optic atrophy. Variable OXPHOS defect and survival depending on tissue type and mutation. [341611994 
PTCD3/MRPS39(mS39)  Intronic and insertion causing frameshift leading to decreased protein levels. Severe Leigh syndrome, optic atrophy, hearing loss. Decreased CI and CIV in fibroblasts. [354614918 
Gene (RNA/protein name1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-RNR1 (12S rRNA)  Transition, transversion and deletion/insertion. Aminoglycoside-induced hearing loss, incomplete penetrance reported, may include cardiomyopathy. [346,434,435], and reviewed in [436561000 
ERAL1  Missense leading to decreased protein levels. Perrault syndrome expressed as sensorineural hearing loss and ovarian dysgenesis with survival into late adulthood. [347607435 
MRPS2 (uS2m)  Missense leading to decreased protein levels. Sensorineural hearing impairment, mild developmental delay and hypoglycaemia. Variable OXPHOS deficiency depending on the tissue type. Survival into childhood [344611971 
MRPS7 (uS7m)  Missense leading to decreased protein levels. Congenital sensorineural hearing impairment, lactic acidosis, hypoglycaemia. Variable OXPHOS deficiency depending on the tissue type. Survival into childhood. [345,348611974 
MRPS14 (uS14m)  Missense leading to increased protein levels. Lactic acidosis, hypertrophic cardiomyopathy, hypotonia with survival into childhood. Unaffected mitoribosome assembly but decreased assembly of CI, CIII, CIV and CV suggesting incorporation of the mutant protein causes impaired mitochondrial translation. [340611978 
MRPS16 (bS16m)  Nonsense leading to unclear protein impact but decreased mitochondrial translation. Fatal lactic acidosis, tubulopathy, hypotonia. Variable OXPHOS deficiency depending on the tissue type. [342609204 
MRPS22 (mS22)  Missense or duplication causing frameshift leading to unclear or decreased protein levels. Variable phenotypes including fatal lactic acidosis, hypertrophic cardiomyopathy, tubulopathy, dysmorphic features, hypotonia, Leigh-like lesions, hypergonadotropic hypogonadism. Variable OXPHOS deficiency depending on the tissue type. [349–351605810 
MRPS23 (mS23)  Missense leading to unclear protein impact. Hepatic disease and hypoglycaemia. Combined CI and CIV deficiencies in fibroblasts. [66611985 
MRPS25 (mS25)  Missense leading to decreased protein levels. Multiple presentations including encephalopathy, short stature, muscle fatigue, dystonia, mild elevation of plasma lactate with survival into adulthood. [343611987 
MRPS28 (bS1m)  Missense and deletion causing frameshift and early termination leading to decreased protein levels. IUGR, cerebellar atrophy, microcephaly, hyperlactatemia, developmental delay, sensorineural deafness, failure to thrive with survival into adulthood. CIV deficiency in muscle, fibroblasts and liver. [352,353611990 
MRPS34 (mS34)  Intronic, nonsense, missense leading to decreased protein levels. Leigh or Leigh-like syndrome, mild or hyperlactatemia, may present microcephaly, optic atrophy. Variable OXPHOS defect and survival depending on tissue type and mutation. [341611994 
PTCD3/MRPS39(mS39)  Intronic and insertion causing frameshift leading to decreased protein levels. Severe Leigh syndrome, optic atrophy, hearing loss. Decreased CI and CIV in fibroblasts. [354614918 
1

Commonly used protein name; S., subunit; A.F., assembly factor; IUGR, intrauterine growth restriction.

Both MRPS2 and MRPS7 encode proteins assembled early in the mtSSU (uS2m and uS7m, respectively) and mutations in these genes have been reported to cause sensorineural hearing impairment and hypoglycaemia but with survival into adulthood [344,345,348] (Table 16). On the other hand, mutations in MRPS16 (bS16m) and MRPS22 (mS22) commonly lead to clinical presentations of fatal lactic acidosis and tubulopathy, with MRPS22 mutations also leading to hypertrophic cardiomyopathy, Leigh-like brain lesions and delayed sexual development [342,349–351]. Interestingly, a homozygous missense mutation in MRPS23 leads to hypoglycaemia and hepatic disease [66], sharing partial phenotypic similarities with other genes encoding early assembled proteins, MRPS2, MRPS7, MRPS16 and MRPS22 (Table 16) (Figure 7). Comparably, mutations in MRPS28 (bS1m) were shown to present before birth as intrauterine growth retardation (IUGR) and progress with multisystemic involvement including sensorineural deafness, brain abnormalities, hyperlactatemia and failure to thrive with survival into adulthood [352,353]. The last two genes linked to mitochondrial disease participating in the early assembly of the mtSSU are MRPS34 and PTCD3. Mutations in both genes have been shown to cause Leigh or Leigh-like syndrome with variable presentations of hyperlactatemia, optic atrophy, hearing loss, microcephaly and variable OXPHOS defects and survival depending on tissue type and mutation [341,354] (Table 16).

While the majority of mutations in the mtSSU lead to decreased protein levels (Table 16), a missense homozygous mutation was identified in MRPS14 (uS14m) leading to increased levels of corresponding protein, and clinical presentation of lactic acidosis, hypertrophic cardiomyopathy and hypotonia [340]. The unaffected assembly of the mitoribosome suggests that the incorporation of the mutant protein causes impaired mitochondrial translation which was predicted to disrupt the mitoribosome mRNA channel [340]. The other late assembled mtSSU protein linked to disease, MRPS25 (mS25), assembles in a different cluster to MRPS14 (uS14m) [331] and has been linked to a different phenotype including encephalopathy, short stature, muscle fatigue, dystonia, mild elevation of plasma lactate [343].

Assembly of the mtLSU

The assembly of the mtLSU seems to present an early, an intermediate and a late class of incorporation of proteins (Figure 7) with several subclusters of proteins present at each stage [331]. Assembly factors involved in the biogenesis of the mtLSU have been identified mostly at late stages. MALSU1 is a member of the DUF143 family of proteins of conserved ribosomal silencing factors and is required for mtLSU biogenesis and translation [355–357] (Figure 7). Recently, the structure of an mtLSU intermediate containing MALSU1 was solved [358] revealing the involvement of two other proteins, NDUFAB1, a subunit of complex I with an essential secondary role in mitochondrial function (discussed above) [22], and L0R8F8, the product of a bicistronic transcript that also encodes MID51, a mitochondrial protein involved in morphology [85]. While the function of these new assembly factors is not yet known, they have been suggested to act as caps to prevent premature association of the nascent mtLSU with the mtSSU [358]. At least five other assembly factors have been characterised to be involved in late mtLSU assembly and the formation of the 55S monosome. Three of these are quality control GTPases proposed to prevent premature monosome formation: GTPBP5 (MTG2) [359], GTPBP7/MTG1 [360] and GTPBP10 [361,362], and two others form a heterodimeric complex comprised of NSUN4 and MTERF4 assembly factors which facilitates the monosome formation and enables mtDNA translation [337,363,364].

Once the 55S monosome is assembled, the tRNAval is nestled between two groups of proteins: the early assembled MRPL40 (mL40), MRPL46 (mL46) and MRPL48 (mL48) proteins in one side and the intermediate assembled proteins MRPL18 (mL18), MRPL38 (mL38) and MRPL27 (bL27m) on the other side [4,331]. Although several components required for the assembly of the human mitoribosome have been identified over the years, the lack of assembly factors characterised for the different incorporation clusters and independently incorporated proteins for both mtSSU and mtLSU might suggest that some assembly factors are yet to be discovered.

Even though the mtLSU contains more structural proteins than the mtSSU, there are only four nuclear disease genes associated with the mtLSU, as well as 2 mtDNA-encoded rRNAs, with majority of these participating in the early assembly steps (Figure 7). The majority of mutations associated with defects in the mtLSU are missense mutations leading to a variable phenotype and OXPHOS defect, depending on mutation and tissue type analysed (Table 17). Mutations identified in the 16SrRNA (MT-RNR2) have been reported to cause hypertrophic cardiomyopathy and myopathy with survival into adulthood [365,366], while mutations in the tRNAval (MT-TV) appear to cause a broader spectrum of phenotypes including muscle weakness, hearing and visual loss, MELAS, Leigh syndrome and hypertrophic cardiomyopathy [367–372] (Table 17). It is not clear if mutations in tRNAval lead to combined defects in mitoribosome assembly and defective polypeptide elongation, although it should be noted that mitoribosomes are able to incorporate tRNAphe when the levels of tRNAval are severely decreased [373]. Despite this implying that the phenotypes are due to defective elongation, the adaptative switch in tRNA composition still leads to impaired mitochondrial translation [374] hinting at a combined defect. Severe phenotypes leading to early death have been associated with MRPL3 (uL3m) and MRPL12 (bL12m) suggesting these proteins play an essential for mitochondrial translation. In fact, uL3m, encoded by MRPL3 displays extensive binding contact with the 16S rRNA and is one of the earliest assembled proteins, providing an anchor to MRPL39 (mL39) followed by MRPL45 (mL45) [331], the latter known to tether the mitoribosome at the IMM for translation [321]. Recent structural studies also revealed that MRPL12 (bL12m) plays an important role in interacting with mitochondrial elongation factor (EF-G1mt) [375] and promoting tRNA translocation on the mitoribosome during translation [376]. On the other hand, mutations in MRPL24 (uL24m) and MRPL44 (mL44) have been associated with severe to mild phenotypes with survival into adolescence and early adulthood [377–379] (Table 17). MRPL24 is a late assembled protein that lacks extensive contact with early assembled proteins and is involved in the formation of the PET [331]. Despite its involvement with the PET, a teenager was recently reported harbouring a homozygous missense variant leading to cerebellar atrophy, choreoathetosis, increased lactate in blood and tachycardia (Wolff–Parkinson–White syndrome) [377]. Finally, four patients have been reported harbouring missense mutations in the gene encoding early assembled protein MRPL44 (mL44). The patients commonly presented with hypertrophic cardiomyopathy with majority presenting stabilisation of their phenotype over the years [378,379].

Table 17.
Defects in the assembly of the mtLSU
Gene (RNA/protein name1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-RNR2 (16S rRNA)  Transition leading to unclear RNA impact. Myopathy, hypertrophic cardiomyopathy. Survival into adulthood reported. Decreased ATP production. [365,366561010 
MT-TV (tRNAval Transition leading to unclear or decreased tRNAval levels. Variable phenotype and onset depending on mutation and mutant load with survival into adulthood reported. Predominantly presenting ataxia, hearing and/or visual loss, bilateral cataracts or migraines and muscle weakness, MELAS severe Leigh or Leigh-like syndrome, hypertrophic cardiomyopathy. [367–372590105 
MRPL3 (uL3m)  Missense or deletion leading to unclear or decreased protein levels. Early onset severe cardiomyopathy, psychomotor retardation, failure to thrive, lactic acidosis. Variable OXPHOS defect depending on tissue type and mutation. [437,438607118 
MRPL12 (bL12m)  Missense leading to decreased protein levels. Antenatal hypotrophy, tonic seizures, ataxia, hyperlactatemia, failure to thrive. Variable OXPHOS defect depending on tissue type and mutation. [439602375 
MRPL24 (uL24m)  Missense leading to decreased protein levels. Cerebellar atrophy, choreoathetosis of limbs and face, Wolff–Parkinson–White syndrome, increased lactate in blood with survival into adolescence. Combined CI and CIV defect in muscle. [377611836 
MRPL44 (mL44)  Missense leading to unclear or decreased protein levels. Predominantly hypertrophic cardiomyopathy, hepatopathy, but also muscle weakness, granular pigmentation of retina, metabolic acidosis. Survival into early adulthood reported. Variable OXPHOS defect depending on tissue type and mutation. [378,379611849 
Gene (RNA/protein name1)S.A.F.Types of genetic variants and protein impactClinical presentations and relevant informationRef.OMIM
MT-RNR2 (16S rRNA)  Transition leading to unclear RNA impact. Myopathy, hypertrophic cardiomyopathy. Survival into adulthood reported. Decreased ATP production. [365,366561010 
MT-TV (tRNAval Transition leading to unclear or decreased tRNAval levels. Variable phenotype and onset depending on mutation and mutant load with survival into adulthood reported. Predominantly presenting ataxia, hearing and/or visual loss, bilateral cataracts or migraines and muscle weakness, MELAS severe Leigh or Leigh-like syndrome, hypertrophic cardiomyopathy. [367–372590105 
MRPL3 (uL3m)  Missense or deletion leading to unclear or decreased protein levels. Early onset severe cardiomyopathy, psychomotor retardation, failure to thrive, lactic acidosis. Variable OXPHOS defect depending on tissue type and mutation. [437,438607118 
MRPL12 (bL12m)  Missense leading to decreased protein levels. Antenatal hypotrophy, tonic seizures, ataxia, hyperlactatemia, failure to thrive. Variable OXPHOS defect depending on tissue type and mutation. [439602375 
MRPL24 (uL24m)  Missense leading to decreased protein levels. Cerebellar atrophy, choreoathetosis of limbs and face, Wolff–Parkinson–White syndrome, increased lactate in blood with survival into adolescence. Combined CI and CIV defect in muscle. [377611836 
MRPL44 (mL44)  Missense leading to unclear or decreased protein levels. Predominantly hypertrophic cardiomyopathy, hepatopathy, but also muscle weakness, granular pigmentation of retina, metabolic acidosis. Survival into early adulthood reported. Variable OXPHOS defect depending on tissue type and mutation. [378,379611849 
1

Commonly used protein name; S., subunit; A.F., assembly factor; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes.

Conclusion

In this review, we have attempted to catalogue the breadth of genetic and clinical phenotypes associated with impaired assembly of mitochondrial OXPHOS complexes and the mitoribosome. Along with highlighting the intricacy of this system, we hope to have demonstrated the high heterogeneity in clinical presentations that challenges the diagnosis of new patients and the validation of novel disease genes linked to dysfunction in this critical process.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

We acknowledge funding from the National Health and Medical Research Council (NHMRC Fellowship 1140851 to DAS). DHH is supported by a Melbourne International Research Scholarship and the Mito Foundation PhD Top-up Scholarship.

Open Access

Open access for this article was enabled by the participation of The University of Melbourne in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

Acknowledgements

We apologise to those colleagues whose work we could not cite due to space restrictions. We thank Alison Compton, David Thorburn and all members of the Stroud lab for their input.

Abbreviations

     
  • COX

    cytochrome c oxidase

  •  
  • IMM

    inner mitochondrial membrane

  •  
  • LHON

    Leber hereditary optic neuropathy

  •  
  • MCIA

    mitochondrial complex I intermediate assembly

  •  
  • MRC

    mitochondrial respiratory chain

  •  
  • mt

    mitochondrial

  •  
  • nDNA

    nuclear DNA

  •  
  • OXPHOS

    oxidative phosphorylation

  •  
  • PTC

    peptidyl transferase centre

  •  
  • VUS

    variants of uncertain significance

  •  
  • WES

    whole exome

  •  
  • WGS

    whole genome

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