A mutation in C2orf64 causes impaired cytochrome c oxidase assembly and mitochondrial cardiomyopathy

The assembly of mitochondrial respiratory chain complex IV (cytochrome c oxidase) involves the coordinated action of several assembly chaperones. In Saccharomyces cerevisiae, at least 30 different assembly chaperones have been identified. To date, pathogenic mutations leading to a mitochondrial disorder have been identified in only seven of the corresponding human genes. One of the genes for which the relevance to human pathology is unknown is C2orf64, an ortholog of the S. cerevisiae gene PET191. This gene has previously been shown to be a complex IV assembly factor in yeast, although its exact role is still unknown. Previous research in a large cohort of complex IV deficient patients did not support an etiological role of C2orf64 in complex IV deficiency. In this report, a homozygous mutation in C2orf64 is described in two siblings affected by fatal neonatal cardiomyopathy. Pathogenicity of the mutation is supported by the results of a complementation experiment, showing that complex IV activity can be fully restored by retroviral transduction of wild-type C2orf64 in patient-derived fibroblasts. Detailed analysis of complex IV assembly intermediates in patient fibroblasts by 2D-BN PAGE revealed the accumulation of a small assembly intermediate containing subunit COX1 but not the COX2, COX4, or COX5b subunits, indicating that C2orf64 is involved in an early step of the complex IV assembly process. The results of this study demonstrate that C2orf64 is essential for human complex IV assembly and that C2orf64 mutational analysis should be considered for complex IV deficient patients, in particular those with hypertrophic cardiomyopathy.     

Mutations in the gene encoding C8orf38 block complex I assembly by inhibiting production of the mitochondria-encoded subunit ND1

The assembly of complex I (NADH-ubiquinone oxidoreductase) is a complicated process, requiring the integration of 45 subunits encoded by both nuclear and mitochondrial DNAs into a structure of approximately 1 MDa. A number of “assembly factors” that aid complex I biogenesis have recently been described, including C8orf38. This protein was identified as an assembly factor by its evolutionary conservation in organisms containing complex I and by a C8orf38 mutation in a patient presenting with Leigh syndrome and isolated complex I deficiency. In this report, we have undertaken the characterization of C8orf38 and its role in complex I assembly. Analysis of mitochondria from fibroblasts of a patient harboring a C8orf38 mutation showed almost undetectable levels of steady-state complex I and defective biogenesis of the mtDNA-encoded subunit ND1. Complementation with wild-type C8orf38 restored the levels of both ND1 and complex I, confirming the C8orf38 mutation as the cause of the complex I defect in the patient. In the absence of ND1 in patient cells, early- and mid-stage intermediate complexes were still formed; however, assembly of late-stage intermediates was impaired, indicating a convergence point in the assembly process. While C8orf38 appears to behave at a step in complex I biogenesis similar to that of the assembly factor C20orf7, complementation studies showed that both proteins are required for ND1 synthesis/stabilization. We conclude that C8orf38 is a crucial factor required for the translation and/or integration of ND1 into an early-stage assembly intermediate and that mutation of C8orf38 disrupts the initial stages of complex I biogenesis.     

Mutation of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease.     

Mutations in mitochondrial ribosomal protein MRPL12 leads to growth retardation,neurological deterioration and mitochondrial translation deficiency

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2–q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent.     

C1orf163/RESA1 Is a Novel Mitochondrial Intermembrane Space Protein Connected to Respiratory Chain Assembly

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60 kDa and 150 kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).     

Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway,causing early-onset fatal hypertrophic cardiomyopathy

Deficiencies in the activity of cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain, are a frequent cause of autosomal recessive mitochondrial disease in infants. These patients are clinically and genetically heterogeneous, and all defects so far identified in this group have been found in genes coding for accessory proteins that play important roles in the assembly of the COX holoenzyme complex. Many patients, however, remain without a molecular diagnosis. We have used a panel of retroviral vectors expressing human COX assembly factors in these patients to identify the molecular basis for the COX deficiency by functional complementation. Here we show that overexpression of COX15, a protein involved in the synthesis of heme A, the heme prosthetic group for COX, can functionally complement the isolated COX deficiency in fibroblasts from a patient with fatal, infantile hypertrophic cardiomyopathy. Mutation analysis of COX15 in the patient identified a missense mutation (C700T) on one allele, changing a conserved arginine to tryptophan (R217W), and a splice-site mutation in intron 3 on the other allele (C447-3G), resulting in a deletion of exon 4. This splicing error introduces a frameshift and a premature stop codon, resulting in an unstable mRNA and, likely, a null allele. Mitochondrial heme A content was reduced in the patient's heart and fibroblast mitochondria, and levels of heme O were increased in the patient's heart. COX activity and the total amount of fully assembled enzyme were reduced by 50%-70% in patient fibroblasts. Expression of COX15 increased heme A content and rescued COX activity. These results suggest that reduced availability of heme A stalls the assembly of COX. This study establishes COX15 as an additional cause, along with SCO2, of fatal infantile, hypertrophic cardiomyopathy associated with isolated COX deficiency.     

A pathogenic 15-base pair deletion in mitochondrial DNA-encoded cytochrome c oxidase subunit III results in the absence of functional cytochrome c oxidase

A 15-base pair, in-frame, deletion (9480del15) in the mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit III (COX III) gene was identified previously in a patient with recurrent episodes of myoglobinuria and an isolated COX deficiency. Transmitochondrial cell lines harboring 0, 97, and 100% of the 9480del15 deletion were created by fusing human cells lacking mtDNA (rho(0) cells) with platelet and lymphocyte fractions isolated from the patient. The COX III gene mutation resulted in a severe respiratory chain defect in all mutant cell lines. Cells homoplasmic for the mutation had no detectable COX activity or respiratory ATP synthesis, and required uridine and pyruvate supplementation for growth, a phenotype similar to rho(0) cells. The cells with 97% mutated mtDNA exhibited severe reductions in both COX activity (6% of wild-type levels) and rates of ATP synthesis (9% of wild-type). The COX III polypeptide in the mutant cells, although translated at rates similar to wild-type, had reduced stability. There was no evidence for assembly of COX I, COX II, or COX III subunits in a multisubunit complex in cells homoplasmic for the mutation, thus indicating that there was no stable assembly of COX I with COX II in the absence of wild-type COX III. In contrast, the COX I and COX II subunits were assembled in cells with 97% mutated mtDNA.     

Functional alteration of cytochrome c oxidase by SURF1 mutations in Leigh syndrome

Subacute necrotising encephalomyopathy (Leigh syndrome) due to cytochrome c oxidase (COX) deficiency is often caused by mutations in the SURF1 gene, encoding the Surf1 protein essential for COX assembly. We have investigated five patients with different SURF1 mutations resulting in the absence of Surf1 protein. All of them presented with severe and generalised COX defect. Immunoelectrophoretic analysis of cultured fibroblasts revealed 85% decrease of the normal-size COX complexes and significant accumulation of incomplete COX assemblies of 90-120 kDa. Spectrophotometric assay of COX activity showed a 70-90% decrease in lauryl maltoside (LM)-solubilised fibroblasts. In contrast, oxygen consumption analysis in whole cells revealed only a 13-31% decrease of COX activity, which was completely inhibited by detergent in patient cells but not in controls. In patient fibroblasts ADP-stimulated respiration was 50% decreased and cytofluorometry showed a significant decrease of mitochondrial membrane potential DeltaPsi(m) in state 4, as well as a 2.4-fold higher sensitivity of DeltaPsi(m) to uncoupler. We conclude that the absence of the Surf1 protein leads to the formation of incomplete COX complexes, which in situ maintain rather high electron-transport activity, while their H(+)-pumping is impaired. Enzyme inactivation by the detergent in patient cells indicates instability of incomplete COX assemblies.     

Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) biogenesis is translationally regulated. Mss51, a specific COX1 mRNA translational activator and Cox1 chaperone, drives the regulatory mechanism. During translation and post-translationally, newly synthesized Cox1 physically interacts with a complex of proteins involving Ssc1, Mss51, and Cox14, which eventually hand over Cox1 to the assembly pathway. This step is probably catalyzed by assembly chaperones such as Shy1 in a process coupled to the release of Ssc1-Mss51 from the complex. Impaired COX assembly results in the trapping of Mss51 in the complex, thus limiting its availability for COX1 mRNA translation. An exception is a null mutation in COX14 that does not affect Cox1 synthesis because the Mss51 trapping complexes become unstable, and Mss51 is readily available for translation. Here we present evidence showing that Cox25 is a new essential COX assembly factor that plays some roles similar to Cox14. A null mutation in COX25 by itself or in combination with other COX mutations does not affect Cox1 synthesis. Cox25 is an inner mitochondrial membrane intrinsic protein with a hydrophilic C terminus protruding into the matrix. Cox25 is an essential component of the complexes containing newly synthesized Cox1, Ssc1, Mss51, and Cox14. In addition, Cox25 is also found to interact with Shy1 and Cox5 in a complex that does not contain Mss51. These results suggest that once Ssc1-Mss51 are released from the Cox1 stabilization complex, Cox25 continues to interact with Cox14 and Cox1 to facilitate the formation of multisubunit COX assembly intermediates.     

A stop-codon mutation in the human mtDNA cytochrome c oxidase I gene disrupts the functional structure of complex IV.

We have identified a novel stop-codon mutation in the mtDNA of a young woman with a multisystem mitochondrial disorder. Histochemical analysis of a muscle-biopsy sample showed virtually absent cytochrome c oxidase (COX) stain, and biochemical studies confirmed an isolated reduction of COX activity. Sequence analysis of the mitochondrial-encoded COX-subunit genes identified a heteroplasmic G-->A transition at nucleotide position 6930 in the gene for subunit I (COX I). The mutation changes a glycine codon to a stop codon, resulting in a predicted loss of the last 170 amino acids (33%) of the polypeptide. The mutation was present in the patient's muscle, myoblasts, and blood and was not detected in normal or disease controls. It was not detected in mtDNA from leukocytes of the patient's mother, sister, and four maternal aunts. We studied the genetic, biochemical, and morphological characteristics of transmitochondrial cybrid cell lines, obtained by fusing of platelets from the patient with human cells lacking endogenous mtDNA (rho0 cells). There was a direct relationship between the proportion of mutant mtDNA and the biochemical defect. We also observed that the threshold for the phenotypic expression of this mutation was lower than that reported in mutations involving tRNA genes. We suggest that the G6930A mutation causes a disruption in the assembly of the respiratory-chain complex IV.     

Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts

Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.     

C6ORF32 is upregulated during muscle cell differentiation and induces the formation of cellular filopodia

We have identified a gene by microarray analysis that is located on chromosome 6 (c6orf32), whose expression is increased during human fetal myoblast differentiation. The protein encoded by c6orf32 is expressed both in myogenic and non-myogenic primary cells isolated from 18-week old human fetal skeletal muscle. Immunofluorescent staining indicated that C6ORF32 localizes to the cellular cytoskeleton and filopodia, and often displays polarized expression within the cell. mRNA knockdown experiments in the C2C12 murine myoblast cell line demonstrated that cells lacking c6orf32 exhibit a myogenic differentiation defect, characterized by a decrease in the expression of myogenin and myosin heavy chain (MHC) proteins, whereas MyoD1 was unaltered. In contrast, overexpression of c6orf32 in C2C12 or HEK293 cells (a non-muscle cell line) promoted formation of long membrane protrusions (filopodia). Analysis of serial deletion mutants demonstrated that amino acids 55-113 of C6ORF32 are likely involved in filopodia formation. These results indicate that C6ORF32 is a novel protein likely to play multiple functions, including promoting myogenic cell differentiation, cytoskeletal rearrangement and filopodia formation.     

Analysis of Synthesis, Stability, Phosphorylation, and Interacting Polypeptides of the 34-Kilodalton Product of Open Reading Frame 6 of the Early Region 4 Protein of Human Adenovirus Type 5

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.     

An RMND1 Mutation Causes Encephalopathy Associated with Multiple Oxidative Phosphorylation Complex Deficiencies and a Mitochondrial Translation Defect

Mutations in the genes composing the mitochondrial translation apparatus are an important cause of a heterogeneous group of oxidative phosphorylation (OXPHOS) disorders. We studied the index case in a consanguineous family in which two children presented with severe encephalopathy, lactic acidosis, and intractable seizures leading to an early fatal outcome. Blue native polyacrylamide gel electrophoretic (BN-PAGE) analysis showed assembly defects in all of the OXPHOS complexes with mtDNA-encoded structural subunits, and these defects were associated with a severe deficiency in mitochondrial translation. Immunoblot analysis showed reductions in the steady-state levels of several structural subunits of the mitochondrial ribosome. Whole-exome sequencing identified a homozygous missense mutation (c.1250G>A) in an uncharacterized gene, RMND1 (required for meiotic nuclear division 1). RMND1 localizes to mitochondria and behaves as an integral membrane protein. Retroviral expression of the wild-type RMND1 cDNA rescued the biochemical phenotype in subject cells, and siRNA-mediated knockdown of the protein recapitulated the defect. BN-PAGE, gel filtration, and mass spectrometry analyses showed that RMND1 forms a high-molecular-weight and most likely homopolymeric complex (∼240 kDa) that does not assemble in subject fibroblasts but that is rescued by expression of RMND1 cDNA. The p.Arg417Gln substitution, predicted to be in a coiled-coil domain, which is juxtaposed to a transmembrane domain at the extreme C terminus of the protein, does not alter the steady-state level of RMND1 but might prevent protein-protein interactions in this complex. Our results demonstrate that the RMND1 complex is necessary for mitochondrial translation, possibly by coordinating the assembly or maintenance of the mitochondrial ribosome.     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.

The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.     

NDUFA2 complex I mutation leads to Leigh disease

Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5. Screening of this gene on genomic DNA revealed a mutation that interferes with correct splicing and results in the skipping of exon 2. Exon skipping was confirmed on the mRNA level. The mutation in this accessory subunit causes reduced activity and disturbed assembly of complex I. Furthermore, the mutation is associated with a mitochondrial depolarization. The expression and activity of complex I and the depolarization was (partially) rescued with a baculovirus system expressing the NDUFA2 gene.     

Solution structure and siRNA‐mediated knockdown analysis of the mitochondrial disease‐related protein C12orf65

Loss of function of the c12orf65 gene causes a mitochondrial translation defect, leading to encephalomyopathy. The C12orf65 protein is thought to play a role similar to that of ICT1 in rescuing stalled mitoribosomes during translation. Both proteins belong to a family of Class I peptide release factors (RFs), all characterized by the presence of a GGQ motif. Here, we determined the solution structure of the GGQ‐containing domain (GGQ domain) of C12orf65 from mouse by NMR spectroscopy, and examined the effect of siRNA‐mediated knockdown of C12orf65 on mitochondria in HeLa cells using flow cytometry. The GGQ domain, comprising residues 60–124 of the 184‐residue full‐length protein, forms a structure with a 3₁₀‐β1‐β2‐β3‐α1 topology that resembles the GGQ domain structure of RF more closely than that of ICT1. Thus, the GGQ domain structures of this protein family can be divided into two types, depending on the region linking β2 and β3; the C12orf65/RF type having a 6‐residue π‐HB turn and the ICT1 type having an α‐helix. Knockdown of C12orf65 resulted in increased ROS production and apoptosis, leading to inhibition of cell proliferation. Substantial changes in mitochondrial membrane potential and mass in the C12orf65‐knockdown cells were observed compared with the control cells. These results indicate that the function of C12orf65 is essential for cell vitality and mitochondrial function. Although similar effects were observed in ICT1‐downregulated cells, there were significant differences in the range and pattern of the effects between C12orf65‐ and ICT1‐knockdown cells, suggesting different roles of C12orf65 and ICT1 in rescuing stalled mitoribosomes. Proteins 2012. © 2012 Wiley Periodicals, Inc.