Fast adaptive coevolution of nuclear and mitochondrial subunits of ATP synthetase in orangutan

Nuclear and mitochondrial genomes have to work in concert to generate a functional oxidative phosphorylation (OXPHOS) system. We have previously shown that we could restore partial OXPHOS function when chimpanzee or gorilla mitochondrial DNA (mtDNA) were introduced into human cells lacking mtDNA. However, we were unable to maintain orangutan mitochondrial DNA in a human cell. We have now produced chimpanzee, gorilla, orangutan, and baboon cells lacking mtDNA and attempted to introduce mtDNA from different apes into them. Surprisingly, we were able to maintain human mtDNA in an orangutan nuclear background, even though these cells showed severe OXPHOS abnormalities, including a complete absence of assembled ATP synthetase. Phylogenetic analysis of complex V mtDNA-encoded subunits showed that they are among the most evolutionarily divergent components of the mitochondrial genome between orangutan and the other apes. Our studies showed that adaptive coevolution of nuclear and mitochondrial components in apes can be fast and accelerate in recent branches of anthropoid primates.     

Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I

Complex I of the respiratory chain is composed of at least 45 subunits that assemble together at the mitochondrial inner membrane. Defects in human complex I result in energy generation disorders and are also implicated in Parkinson's disease and altered apoptotic signaling. The assembly of this complex is poorly understood and is complicated by its large size and its regulation by two genomes, with seven subunits encoded by mitochondrial DNA (mtDNA) and the remainder encoded by nuclear genes. Here we analyzed the assembly of a number of mtDNA- and nuclear-gene-encoded subunits into complex I. We found that mtDNA-encoded subunits first assemble into intermediate complexes and require significant chase times for their integration into the holoenzyme. In contrast, a set of newly imported nuclear-gene-encoded subunits integrate with preexisting complex I subunits to form intermediates and/or the fully assembly holoenzyme. One of the intermediate complexes represents a subassembly associated with the chaperone B17.2L. By using isolated patient mitochondria, we show that this subassembly is a productive intermediate in complex I assembly since import of the missing subunit restores complex I assembly. Our studies point to a mechanism of complex I biogenesis involving two complementary processes, (i) synthesis of mtDNA-encoded subunits to seed de novo assembly and (ii) exchange of preexisting subunits with newly imported ones to maintain complex I homeostasis. Subunit exchange may also act as an efficient mechanism to prevent the accumulation of oxidatively damaged subunits that would otherwise be detrimental to mitochondrial oxidative phosphorylation and have the potential to cause disease.     

Diseases caused by defects of mitochondrial carriers: a review

A strikingly large number of mitochondrial DNA (mtDNA) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. These mitochondrial disorders were the first to be investigated after the small mtDNA had been sequenced in the 80s. Only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. Among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. Mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, HHH syndrome, aspartate/glutamate isoform 2 deficiency, Amish microcephaly, and neonatal myoclonic epilepsy; these disorders are characterized by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabolism. Defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phosphate and ADP, are responsible for diseases characterized by defective energy production. Herein, all the mitochondrial carrier-associated diseases known to date are reviewed for the first time. Particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders.     

Suppression mechanisms of COX assembly defects in yeast and human: insights into the COX assembly process

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin whose assembly is intricate and highly regulated. In addition to the structural subunits, a large number of accessory factors are required to build the holoenzyme. The function of these factors is required in all stages of the assembly process. They are relevant to human health because devastating human disorders have been associated with mutations in nuclear genes encoding conserved COX assembly factors. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to attain the current state of knowledge, even if still fragmentary, of the COX assembly process. After the identification of the genes involved, the isolation and characterization of genetic and metabolic suppressors of COX assembly defects, reviewed here, have become a profitable strategy to gain insight into their functions and the pathways in which they operate. Additionally, they have the potential to provide useful information for devising therapeutic approaches to combat human disorders associated with COX deficiency.     

Defects in mitochondrial respiratory complexes III and IV,and human pathologies

Here, relationships between alterations in tissue-specific content, protein structure, activity, and/or assembly of respiratory complexes III and IV induced by mutations in corresponding genes and various human pathologies are reviewed. Cytochrome bc(1) complex and cytochrome c oxidase (COX) deficiencies have been detected in a heterogeneous group of neuromuscular and non-neuromuscular diseases in childhood and adulthood, presenting a number of clinical phenotypes of variable severity. Such disorders can be caused by mutations located either in mitochondrial genes or in nuclear genes encoding structural subunits of the complexes or corresponding assembly factors/chaperones. Of the defects in mitochondrial DNA genes, mutations in cytochrome b subunit of complex III, and in structural subunits I-III of COX have been described to date. As to defects in nuclear DNA genes, mutations in genes encoding the complexes assembly factors such as the BCS1L protein for complex III; and SURF-1, SCO1, SCO2, and COX10 for complex IV have been identified so far.     

CYTOCHROME C OXIDASE ACTIVITY IN INTERPOPULATION HYBRIDS OF A MARINE COPEPOD: A TEST FOR NUCLEAR-NUCLEAR OR NUCLEAR-CYTOPLASMIC COADAPTATION

The respiratory enzyme cytochrome c oxidase (COX) is composed of subunits encoded by both nuclear and mitochondrial genes; thus, COX activity reflects, to some extent, the coordinated function of the two genomes. Because extensive mtDNA differentiation exists between populations of the copepod Tigriopus californicus, we hypothesized that laboratory hybridizations that disrupt natural combinations of nuclear and mitochondrial genes might negatively impact COX activity. Although experimental results varied greatly among different crosses, replicate sets of crosses between two particular populations showed consistent evidence for nuclear-cytoplasmic coadaptation.     

Biochemical and molecular diagnosis of mitochondrial respiratory chain disorders

Biochemical diagnosis of mitochondrial respiratory chain disorders requires caution to avoid misdiagnosis of secondary enzyme defects, and can be improved by the use of conservative diagnostic criteria. Pathogenic mutations causing mitochondrial disorders have now been identified in more than 30 mitochondrial DNA (mtDNA) genes encoding respiratory chain subunits, ribosomal- and t-RNAs. mtDNA mutations appear to be responsible for most adult patients with mitochondrial disease and approximately a quarter of paediatric patients. A family history suggesting maternal inheritance is the exception rather than the norm for children with mtDNA mutations, many of whom have de novo mutations. Prenatal diagnosis and pre-implantation genetic diagnosis can be offered to some women at risk of transmitting a mtDNA mutation, particularly those at lower recurrence risk. Mutations in more than 30 nuclear genes, including those encoding for respiratory chain subunits and assembly factors, have now been shown to cause mitochondrial disorders, creating difficulties in prioritising which genes should be studied by mutation analysis in individual patients. A number of approaches offer promise to guide the choice of candidate genes, including Blue Native-PAGE immunoblotting and microarray expression analysis.     

Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

The mitochondrial respiratory chain consists of multi-subunit protein complexes embedded in the inner membrane. Although the majority of subunits are encoded by nuclear genes and are imported into mitochondria, 13 subunits in humans are encoded by mitochondrial DNA. The coordinated assembly of subunits encoded from two genomes is a poorly understood process, with assembly pathway defects being a major determinant in mitochondrial disease. In this study, we monitored the assembly of human respiratory complexes using radiolabeled, mitochondrially encoded subunits in conjunction with Blue Native polyacrylamide gel electrophoresis. The efficiency of assembly was found to differ markedly between complexes, and intermediate complexes containing newly synthesized mitochondrial DNA-encoded subunits could be observed for complexes I, III, and IV. In particular, we detected human cytochrome b as a monomer and as a component of a novel approximately 120 kDa intermediate complex at early chase times before being totally assembled into mature complex III. Furthermore, we show that this approach is highly suited for the rapid detection of respiratory complex assembly defects in fibroblasts from patients with mitochondrial disease and, thus, has potential diagnostic applications.     

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Cytochrome c oxidase deficiency: Patients and animal models

Cytochrome c oxidase (COX) deficiencies are one of the most common defects of the respiratory chain found in mitochondrial diseases. COX is a multimeric inner mitochondrial membrane enzyme formed by subunits encoded by both the nuclear and the mitochondrial genome. COX biosynthesis requires numerous assembly factors that do not form part of the final complex but participate in prosthetic group synthesis and metal delivery in addition to membrane insertion and maturation of COX subunits. Human diseases associated with COX deficiency including encephalomyopathies, Leigh syndrome, hypertrophic cardiomyopathies, and fatal lactic acidosis are caused by mutations in COX subunits or assembly factors. In the last decade, numerous animal models have been created to understand the pathophysiology of COX deficiencies and the function of assembly factors. These animal models, ranging from invertebrates to mammals, in most cases mimic the pathological features of the human diseases.     

The FANCM family Mph1 helicase localizes to the mitochondria and contributes to mtDNA stability

Mitochondria are membrane-bound organelles found in eukaryotic cells where they generate energy through the respiratory chain. They contain their own genome that encodes genes critical to the mitochondrial function, but most of their protein content is synthetized from nuclear encoded genes. Damages to the mtDNA can cause mutations and rearrangements with an impact on the respiratory functions of the cell. DNA repair factors are able to localize to mitochondria to restore mtDNA integrity and ensure its proper inheritance. We describe in this article the mitochondrial localization of the Mph1/FANCM helicase that serves critical roles in nuclear DNA repair processes. Mph1 localizes to mitochondria and its functions contribute to the mtDNA integrity under mtDNA damaging conditions.     

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.     

Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress

Mitochondrial biogenesis and mitochondrial DNA (mtDNA) maintenance depend on coordinated expression of genes in the nucleus and mitochondria. A variety of intracellular and extracellular signals transmitted by hormones and second messengers have to be integrated to provide mammalian cells with a suitable abundance of mitochondria and mtDNA to meet their energy demand. It has been proposed that reactive oxygen species (ROS) and free radicals generated from respiratory chain are involved in the signaling from mitochondria to the nucleus. Increased oxidative stress may contribute to alterations in the abundance of mitochondria as well as the copy number and integrity of mtDNA in human cells in pathological conditions and in aging process. Within a certain level, ROS may induce stress responses by altering expression of specific nuclear genes to uphold the energy metabolism to rescue the cell. Once beyond the threshold, ROS may cause oxidative damage to mtDNA and other components of the affected cells and to elicit apoptosis by induction of mitochondrial membrane permeability transition and release of pro-apoptotic proteins such as cytochrome c. On the basis of recent findings gathered from this and other laboratories, we review the alterations in the abundance of mitochondria and mtDNA copy number of mammalian cells in response to oxidative stress and the signaling pathways that are involved.     

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.     

The mitochondrial respiratory chain and ATP synthase complexes: Composition,structure and mutational studies

The oxidative phosphorylation process is dependent on the assembly of both the respiratory chain that generates the electrochemical potential of the mitochondrial inner membrane and the ATP synthase complex which uses this membrane potential to drive ATP synthesis. The five respiratory enzymes involved in this process, complexes I to V, are composed of multiple subunits, some of which are synthesized on mitochondrial ribosomes, whereas others are a product of the nucleocytoplasmic genetic system. The mitochondrial genome has a limited coding capacity and the co-ordinate expression of all the subunits forming these complexes has been shown to be under nuclear control. Present knowledge of complexes I to V mainly comes from studies of bovine and fungal mitochondria. If beef heart mitochondria represent a choice material for studying the composition and structure of these complexes, Saccharomyces cerevisiae and Neurospora crassa and their numerous respiratory mutants, are ideal organisms for investigating the co-ordination of nuclear and mitochondrial genomes in their assembly. The major reason for the interest in respiratory complexes and ATP synthase from the mitochondrial inner membrane in Homo sapiens and in higher plants is the relationship between enzyme deficiencies and human diseases and ageing on one hand, and such plant phenotypic abnormalities as cytoplasmic male sterility on the other.     

Molecular genetic and clinical aspects of mitochondrial disorders in childhood

Mitochondrial OXPHOS disorders are caused by mutations in mitochondrial or nuclear genes, which directly or indirectly affect mitochondrial oxidative phosphorylation (OXPHOS). Primary mtDNA abnormalities in children are due to rearrangements (deletions or duplications) and point mutations or insertions. Mutations in the nuclear-encoded polypeptide subunits of OXPHOS result in complex I and II deficiency, whereas mutations in the nuclear proteins involved in the assembly of OXPHOS subunits cause defects in complexes I, III, IV, and V. Here, we review recent progress in the identification of mitochondrial and nuclear gene defects and the associated clinical manifestations of these disorders in childhood.     

Diseases caused by mutations in mitochondrial DNA

Mitochondrial diseases associated with mutations within mitochondrial genome are a subgroup of metabolic disorders since their common consequence is reduced metabolic efficiency caused by impaired oxidative phophorylation and shortage of ATP. Although the vast majority of mitochondrial proteins (approximately 1500) is encoded by nuclear genome, mtDNA encodes 11 subunits of respiratory chain complexes, 2 subunits of ATP synthase, 22 tRNAs and 2 rRNAs. Up to now, more than 250 pathogenic mutations have been described within mtDNA. The most common are point mutations in genes encoding mitochondrial tRNAs such as 3243A-->G and 8344T-->G that cause, respectively, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) or MIDD (maternally-inherited diabetes and deafness) and MERRF (myoclonic epilepsy with ragged red fibres) syndromes. There have been also found mutations in genes encoding subunits of ATP synthase such as 8993T-->G substitution associated with NARP (neuropathy, ataxia and retinitis pigmentosa) syndrome. It is worth to note that mitochondrial dysfunction can also be caused by mutations within nuclear genes coding for mitochondrial proteins.